Procreation is essential for survival of species. Not surprisingly, complex neuronal networks have evolved to mediate the diverse internal and external environmental inputs that regulate reproduction in vertebrates. Ultimately, these regulatory factors impinge, directly or indirectly, on a final common pathway, the neurons producing the gonadotropin-releasing hormone (GnRH), which stimulates pituitary gonadotropin secretion and thereby gonadal function. Compelling evidence, accumulated in the last few years, has revealed that kisspeptins, a family of neuropeptides encoded by the Kiss1 gene and produced mainly by neuronal clusters at discrete hypothalamic nuclei, are pivotal upstream regulators of GnRH neurons. As such, kisspeptins have emerged as important gatekeepers of key aspects of reproductive maturation and function, from sexual differentiation of the brain and puberty onset to adult regulation of gonadotropin secretion and the metabolic control of fertility. This review aims to provide a comprehensive account of the state-of-the-art in the field of kisspeptin physiology by covering in-depth the consensus knowledge on the major molecular features, biological effects, and mechanisms of action of kisspeptins in mammals and, to a lesser extent, in nonmammalian vertebrates. This review will also address unsolved and contentious issues to set the scene for future research challenges in the area. By doing so, we aim to endow the reader with a critical and updated view of the physiological roles and potential translational relevance of kisspeptins in the integral control of reproductive function.
I. INTRODUCTION: THE REPRODUCTIVE BRAIN
Reproduction is an indispensable function for the perpetuation of the species and, as such, is under the control of a sophisticated network of regulatory signals. While different reproductive strategies have been co-opted during evolution, in mammals and other species the above regulatory factors mainly originate and/or integrate at the so-called hypothalamic-pituitary-gonadal (HPG; also termed gonadotropic) axis (126, 397, 457). The function of this neurohormonal system relies primarily on the dynamic interaction of three major groups of signals arising from 1) the hypothalamus, where a scarce group of scattered neurons (∼1,000 in higher mammals) synthesize and release the decapeptide, gonadotropin-releasing hormone (GnRH); 2) the anterior pituitary, where gonadotropes, which account for <10% of all pituitary cells, secrete the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH); and 3) the gonads that, in addition to generating gametes, are responsible for the synthesis and release of sex steroid and peptide hormones (126, 397, 457). These major components of the HPG axis are connected via feed-forward loops whereby GnRH stimulates the secretion of gonadotropins and these, in turn, promote gonadal maturation and function (FIGURE 1). In addition, feedback regulatory loops operate also within this axis, which facilitate the homeostatic regulation of the system in different physiological conditions.
Given its central position in such neuroendocrine network, GnRH neurons are considered a major hierarchical element of this system, and GnRH operates as the final output signal for the hypothalamic regulation of the downstream elements of the HPG axis. Adequate pulsatile secretion of GnRH is mandatory for proper attainment and maintenance of reproductive function (26, 212, 223, 390). The synchronized release of GnRH bursts is the result of the integral function of the so-called GnRH pulse generator, an hypothalamic network, originally proposed by Knobil (222), that encompasses GnRH neurons as well as other afferents and enables the pulsatile secretion of GnRH. The anatomy of such pulse generator has been the subject of active investigation, and compelling evidence in different species, including rodents and primates, suggests that GnRH secretory patterns are not solely dictated by the intrinsic activity of GnRH neurons, mainly located in the preoptic area (POA), but requires also the contribution of additional hypothalamic afferents (259, 462). Among those, classical deafferentation experiments revealed the key role of the mediobasal hypothalamus (MBH) (159). Nonetheless, several in vitro and ex vivo studies have also documented the ability of GnRH neurons per se to generate secretory pulses (259, 462), and recent neuroanatomical analyses have revealed the existence of dendro-dendritic bundling and shared synapses between GnRH neurons (44), features that likely contribute to synchronize GnRH secretory patterns.
Besides its dynamic regulation in adulthood, the reproductive axis undergoes significant maturational and functional changes during fetal and postnatal development (457), which include the process of sexual differentiation of the brain and the attainment of reproductive capacity at puberty. Importantly, the above phenomena display sexual dimorphism, and substantial differences are detected between males and females in relation to the development of reproductive brain circuits, the timing of puberty, and the function of the HPG axis in adulthood (126, 168, 397). Clear examples of the latter are the female-specific functional changes of the gonadotropic axis during the ovarian cycle, pregnancy, and lactation (380, 397, 430). In addition, reproductive capacity is closely linked to other essential body functions, such as general welfare, immune/inflammatory state, and energy homeostasis (123, 452, 467), and is sensitive to a diversity of environmental cues, including photoperiods and stress conditions (218, 247, 413). Accordingly, attainment and maintenance of reproductive capacity critically depend on the adequate interplay, along the lifespan, of a large diversity of endogenous and exogenous regulators, including circulating hormones, neuropeptides, neurotransmitters, and metabolic products, which, at least partially, impinge on the GnRH pulse generator to conduct their regulatory actions (126, 168, 397).
Recently, our knowledge about the nature, developmental maturation, and major regulatory mechanisms of the neuroendocrine pathways responsible for the control of GnRH secretion has expanded significantly. Compelling evidence accumulated in the last two decades has established that GnRH neurons are controlled by a variety of interacting trans-synaptic and glial inputs (168, 319–322). Recognition of the latter was particularly relevant as glial cells were long thought to be devoid of any regulatory role on neuronal function. Yet, they are now recognized as a source of critical facilitatory signals for puberty onset and adult reproduction, such as glial-derived growth factors and glutamate, with stimulatory effects of GnRH release (319, 320, 322). Nonetheless, neuronal afferents are crucial components of the mechanisms for the synchronized triggering of pulsatile GnRH secretion (168, 321). The nature of such neuronal transmitters had been partially elucidated in the last decades, with the recognition of the roles, among others, of glutamate and norepinephrine as major excitatory signals, and GABA and endogenous opioids as key inhibitory factors (320). Yet, GABA can also directly excite GnRH neurons under specific conditions (170), thus illustrating the complexity of the system. More recent evidence has documented the participation in these regulatory networks of additional factors, such as 1) members of the RF-amide superfamily, which include not only kisspeptins but also 26/43RFa, gonadotropin-inhibiting hormone (GnIH), and its orthologs, RF-releasing peptides (RFRP) (63, 303, 419); 2) metabolic neuropeptides, such as neuropeptide Y (NPY) and nesfatin-1 (133, 351); and 3) tachykinins, including neurokinin B (NKB) (241). As stated above, the generation of GnRH pulses is certainly not driven by the isolated action of single molecules but rather by the dynamic balance between excitatory and inhibitory signals (88, 319, 320). Yet, within this circuitry, the relative importance of those regulatory signals is considerably different, which allows us to highlight the major, pivotal elements of the neuronal afferents governing GnRH secretion.
II. THE KISS1/GPR54 SYSTEM
A major advance in our understanding of the neuronal mechanisms controlling GnRH secretion, and therefore gonadal function, came with the identification of the physiological roles of kisspeptins and their receptor, GPR54. This has revolutionized the field of reproductive physiology and has fuelled an escalating number of studies in this and related areas (91, 317, 374, 448). The impact of the disclosure of the reproductive functions of kisspeptins is illustrated not only by the exponential amount of original articles and reviews which have appeared on this topic in recent years (>370 in PubMed since January 2009) but also by the collective attention attracted by this family of peptides, even among scientists not directly working in reproduction. As a means of introduction, in this section we will describe the major elements of the so-called Kiss1/GPR54 system, its identification as an essential player in the control of puberty and reproduction, and the nomenclature adopted for referring to kisspeptins and their receptor in this review.
Kisspeptins are a family of structurally related peptides, encoded by the KISS1/Kiss1 gene, that act through binding and subsequent activation of the G protein-coupled receptor GPR54. The elements (genes and peptides) of this ligand-receptor system were sequentially identified between 1996 and 2001, but their close association with reproductive physiology was not recognized at that time (317, 374, 448). In fact, KISS1 and its products, kisspeptins, were originally catalogued as metastasis suppressors. Thus, by the use of subtractive hybridization in melanoma cell lines with different metastatic capacity, KISS1 mRNA was identified in 1996 as a selectively overexpressed transcript in tumor cells with low invasiveness (237). This initial finding was followed by further characterization of the antimetastatic potential of the KISS1 transcript (238, 239) and the cloning and chromosomal localization of human KISS1 gene (496). However, it was not until 2001 when three independent studies provided full characterization of the peptide products of the KISS1 gene (228, 292, 318). Based on structural similarities and its common origin as KISS1-derived peptides, the term kisspeptins was coined to globally define this family (228), a name that has become popular within the field and has displaced the initial terminology of metastin.
Kisspeptins are derived from the differential proteolytic processing of a single precursor. In the human, the kisspeptin precursor comprises 145 amino acids, with a putative 19-amino acid signal sequence, two potential dibasic cleavage sites (at amino acids 57 and 67), and one site for terminal cleavage and amidation (at amino acids 121–124) (228, 292, 318), which generates the biologically active kisspeptins (FIGURE 2). Indeed, proteolysis of prepro-kisspeptin gives rise to a 54-amino acid peptide (kisspeptin-54), initially termed metastin because of its capacity to inhibit tumor metastasis, which has been considered the major product of the KISS1 gene (318). In addition, other peptide fragments of the kisspeptin precursor have been identified, such as kisspeptin-14, kisspeptin-13, and kisspeptin-10 (30, 228), that share the COOH-terminal region of the kisspeptin-54 molecule, where they harbor an Arg-Phe-NH2 motif characteristic of the RF-amide peptide family. This family is composed by a number of neuroactive peptides, which in mammals include also the neuropeptides FF and AF, prolactin-releasing peptide (PrRP), 26/43RFa (also termed QRFP43), and RF-related peptides (RFRP-1 and RFRP-3) (35, 63). Of note, not only kisspeptins, but also RFRPs, 26RFa, and PrRP have been shown to modulate gonadotropin secretion in various mammalian species (35, 63, 303, 436).
Some controversy persists on whether the smaller peptides of the kisspeptin family are endogenously generated by differential proteolytic processing of the kisspeptin precursor or may arise from unspecific degradation of the major product, kisspeptin-54; in fact, there are no consensus cleavage sites upstream the sequences of the smaller peptides in mammals. Similarly, it is yet to be defined which are the predominant forms of kisspeptin produced in different tissues, although kisspeptin-54 has been abundantly detected in placental extracts (318). In the rat and mouse, the largest proteolytic product of the kisspeptin precursor is composed of 52 amino acids (kisspeptin-52), and the terminal RF-amide signature is substituted by an Arg-Tyr-NH2 motif (461).
Cloning of the canonical receptor for kisspeptins preceded the characterization of this family of peptides and took place in a totally unrelated context. Thus Gpr54 was identified in the rat brain in 1999 as an orphan receptor with a significant sequence similarity (>40%) with the transmembrane regions of galanin receptors (236). Subsequently, the human ortholog of GPR54 was cloned, and named AXOR12 or hOT7T175 (292, 318). It was not until 2001 when GPR54 was catalogued as putative receptor for KISS1-derived peptides (292, 318). In fact, assessment of the functional features of GPR54 conducted at that time using heterologous cell systems (CHO K1 cells stably expressing this receptor) conclusively demonstrated that all kisspeptins efficiently activate GPR54, with the shorter 10-amino acid fragment (kisspeptin-10) retaining maximal activity in terms of receptor activation (228).
Of note, the above description applies to the elements of the Kiss1/GPR54 system (genes and peptides) in placental mammals. However, comparative studies in nonplacental mammals, such as monotremes, and various nonmammalian vertebrates, including fish, amphibians, and reptiles, have underscored a greater level of molecular diversity for the genes encoding both the ligands and receptor, for which additional isoforms have now been documented (5, 481). For further details regarding gene structure, molecular evolution, and comparative endocrinology of Kiss/kisspeptins and GPR54, see section XV.
B. Discovery of the Reproductive Roles of Kisspeptins and GPR54
Although the elements of the Kiss1 system had been already identified in 2001, their reproductive dimension remained unsuspected until the end of 2003. In that year, two independent reports (95, 401) documented the presence of deletions and inactivating mutations of the GPR54 gene in patients suffering familial or sporadic forms of idiopathic (or isolated) hypogonadotropic hypogonadism (iHH), a rare condition of impuberism, defective gonadotropin secretion, and infertility of central origin. These observations, from the teams of de Roux and co-workers in Paris (95) and Seminara and collaborators in Boston and Cambridge (401), were the first to highlight the indispensable roles of GPR54 and its ligands in the control of key aspects of reproductive function. Such seminal findings in humans were reinforced by the simultaneous report that mice engineered to lack functional Gpr54 were a complete phenocopy of affected patients (132, 401), a condition that was later confirmed in Kiss1 null mice (93, 234), even though Kiss1 KO animals appear to have a milder reproductive impact than Gpr54-deficient mice (72). This combination of genetic (human) and functional genomic (mouse) studies paved the way for the analysis of the physiological relevance and underlying mechanisms of the reproductive actions of kisspeptins, which have progressed rapidly and have resulted, in a rather short period of time, in the elucidation of basic aspects of the molecular biology, neuroanatomy, physiology, pharmacology, and pathophysiology of this neuroendocrine system.
C. Consensus Nomenclature
There have been quite some differences/inconsistencies regarding the nomenclature of the elements of the kisspeptin system. Admittedly, this review has as a major aim to provide an integral review of the physiology of kisspeptins and, as such, does not attempt to propose a specific nomenclature. Nonetheless, efforts have been paid to sum up and integrate different terminologies used in the field during the last years. Whereas the general guidelines of the Human Genome Organization Gene Nomenclature Committee (HGNC) and the International Union of Pharmacology (IUPHAR) have been considered (148), the conventional terminology among the scientific community has been also taken into account, as a means to maintain coherence with the authoritative literature in this field. Some considerations for the nomination of genes, mRNA transcripts, and proteins will be briefly made below.
Concerning the ligands, we have adopted the proposal for nomenclature by Gottsch et al. (148). According to this proposal, which adheres to the general indications of the HGNC, KISS1 and Kiss1 are used to name the human and non-human (primate and non-primate) genes, respectively. A similar terminology is adopted for the corresponding mRNA transcripts. Of note, this gene/mRNA was initially termed KiSS-1, as a way to recognize the origin of the research team that identified this factor, from Pennsylvania, the homeland of the famous Hershey Kiss chocolates, and its metastasis suppressor (sequence) activity; hence, the use of “SS.” Yet, the use of the hyphenation and mixture of upper and lowercase letters has been progressively eliminated from the literature. In addition, the peptide products of KISS1/Kiss1 are globally termed kisspeptins, following the original proposal of Kotani et al. (228), with the abbreviation Kp followed by a numeric extension to indicate the amino acid length (e.g., Kp-10), if relevant. Even though the term metastin was originally coined to name Kp-54 (318), we will not systematically use it herein, in keeping with current literature (148).
Regarding the receptor, suggestions have been made recently to use the terms KISS1R and Kiss1r to refer to GPR54 in human and non-human species, respectively, following again the indications of HGNC (148). While we acknowledge that such terminology is fully valid and has gained a considerable number of users recently, we believe there is still a case for the use of the term GPR54 that, despite not being an orphan receptor since 2001, has been systematically employed to refer to the kisspeptin receptor over the last years. The use of GPR54 is further supported by the discrepancies between the proposals of HGNC and IUPHAR regarding its nomenclature (148) and the fact that kisspeptins might activate other receptors (258), or vice versa. In addition, the use of nonconventional nomenclature on the basis of a solid scientific tradition has been common for other “neuroendocrine” factors. In any event, in an attempt to make terminology more homogeneous, whenever possible, GPR54 and Gpr54 have been used to refer to human and non-human genes/mRNAs, respectively, whereas nonitalicized terms were used to name the corresponding receptor proteins.
Finally, since most of the experimental data obtained so far come from studies in non-human species, the term Kiss1 (or Kisspeptin) will be used hereafter for global reference to the ligand-receptor system, whereas GPR54 will be used to refer to the receptor, when no single species is alluded to. In addition to those general guidelines, which apply to the mammalian Kiss1 system, some specific comments on the proposed nomenclature for Kiss and GPR54 genes in non-mammalian species is provided in section XV of this review.
III. KISSPEPTIN SIGNALING VIA GPR54
Although the mapping of the signaling pathways employed by GPR54 was initiated already in 2001 by the use of different heterologous cell systems (228, 292, 318), this aspect has received far less attention than the characterization of other facets of kisspeptin physiology and remains to date partially unfolded (51). To ease the understanding of later sections of this review, this section recapitulates our current knowledge of the mechanisms of GPR54 signaling and its potential regulation. Some of this information is also schematically summarized in FIGURE 3. Whereas some reference to specific features in reproductive cells/functions will be made herein, this section intends to provide a more general biochemical overview of the signaling characteristics of GPR54, and their putative involvement in the complete repertoire of biological functions of this receptor system.
A. Intracellular Mediators and Signaling Pathways
Dissection of the signaling pathways operated by GPR54 was initiated by the use of different heterologous cell models (such as CHO-K1, HEK203, and B16-BL6) expressing the rat or human receptors (228, 292, 318). These studies set the contention that all kisspeptins can bind and activate GPR54, which in turn was shown to be devoid of detectable binding affinity to galanin ligands, despite the partial similarity with galanin receptors (236). Furthermore, conventional biochemical characterization demonstrated that GPR54 is a seven transmembrane domain, Gq/11-coupled receptor, whose activation leads to increases in intracellular Ca2+ levels ([Ca2+]i) in a pertussis toxin-independent manner, without detectable changes in intracellular cAMP levels, therefore suggesting the lack of association with Gs and/or Gi/o proteins (228, 292). This increase in [Ca2+]i is caused by the activation of phospholipase C (PLC), with the subsequent stimulation of the hydrolysis of phosphatidylinositol bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3), which in turn evokes the mobilization of this ion from intracellular stores. Such an increase in phosphatidylinositol turnover has been demonstrated for both human and mouse GPR54 (228, 434). In addition, the rise of PIP2 hydrolysis following kisspeptin stimulation leads to diacylglycerol (DAG) formation and, thereby, protein kinase C (PKC) activation (373). In turn, activated PKC is thought to cause phosphorylation of mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38, which have been also involved in this signaling cascade (228) (see below). In addition, activation of GPR54 has been reported to increase arachidonic acid release in CHO-K1 cells stably expressing this receptor (228). From a physiological perspective, it is worth noting that studies using hypothalamic explants and isolated GnRH neurons have fully confirmed the importance of the above PLC-Ca2+ pathway in mediating the biological effects of kisspeptins in a more relevant cellular context in terms of control of reproductive function, such as hypothalamic explants and GnRH neurons (57, 250).
The above signaling features do not only have implications in terms of regulation of hormone secretion and neuroendocrine function, but are also the basis for additional biological actions of kisspeptins, such as the control of cell proliferation and migration. Thus, as mentioned above, activation of GPR54 leads to phosphorylation of different MAPK, which might contribute to the antimetastatic and/or antiproliferative effects of kisspeptins (51). However, the subset of intracellular kinases activated upon kisspeptin stimulation appears to be, at least partially, dependent on the cellular context. For example, initial studies in CHO-K1 cells stably expressing GPR54 demonstrated sustained phosphorylation of ERK1 and ERK2, together with weaker stimulation of p38 MAPK phosphorylation following exposure to kisspeptin, whereas no activation was observed for stress-activated protein kinase/c-Jun-terminal kinase (JNK) (228). In contrast, in anaplastic thyroid cancer cells that endogenously express GPR54, kisspeptin induced phosphorylation of ERK1/2 but not of p38 MAPK or PKB/Akt (373). Likewise, studies in pancreatic cancer cell lines expressing GPR54 revealed that, whereas kisspeptin stimulated ERK1 phosphorylation in two different cell lines (AsPC-1 and PANC-1 cells), it only activated p38 in PANC-1 cells (267). Altogether, these observations bring a call of caution when generalizing signaling mechanisms by extrapolation of results from one cell type to another, and emphasize the need for complementary physiological studies. In this context, analyses using hypothalamic explants suggested that the stimulatory effects of kisspeptins in this tissue are mediated via both ERK1/2 and p38 MAPK (57).
In addition, kisspeptins have been shown to cause phosphorylation of focal adhesion kinase and paxillin in mouse melanoma cells stably expressing GPR54, a phenomenon which may contribute to their antimetastatic functions (318). In addition, kisspeptins can interact with specific chemokine signaling routes, such as that of the receptor CXCR4, which may also contribute to their function in prevention of tumor spread. In this pathway, kisspeptin partially inhibits CXCR4 signaling, probably by blocking the ability of SDF-1/CXCL12 (ligand of CXCR4) to stimulate the rise in [Ca2+]i and Akt phosphorylation (309); direct Akt activation by tyrosine kinase receptors can be also abolished by GPR54 signaling (308). On the other hand, KISS1 overexpression interferes with NFκB signaling in HT-1080 cells (506), likely via accumulation of cytoplasmic IκB-α. The resulting reduction of NFκB binding to the matrix metalloproteinase (MMP)-9 promoter and the subsequent decrease of MMP-9 expression may contribute to the prevention of tumor spread (506). Finally, kisspeptins appear to induce apoptosis in certain cell types, but the consistency of such observation and the underlying molecular mechanisms are yet to be fully defined (25, 228, 307, 308).
B. Additional Signaling Partners, Regulatory Mechanisms, and Desensitization
Besides its major signaling mechanisms, other important facets of GPR54 function have begun to be unfolded recently. These include additional signaling conduits (mainly G protein independent) as well the mechanisms for receptor desensitization and regulated expression. Notwithstanding these recent efforts, little is still known about these key aspects that are likely to attract considerable attention in the near future.
Recent biochemical studies have demonstrated that the COOH-terminal tail of GPR54 is able to physically interact with the catalytic subunit of the ubiquitous Ser/Thr protein phosphatase PP2A (117). While the functional relevance of this interaction in terms of signaling is still unclear, it has been proposed that such a GPR54/PP2A complex may participate in the dephosphorylation of Akt, thereby contributing to mediate some of the anti-metastatic function of kisspeptins (117). In addition, it was very recently shown that GPR54 signaling is also modulated by β-arrestins (326). Thus activated GPR54 recruited β-arrestin-1 and -2 to the plasma membrane, via interactions with sequences of the second intracellular loop and the cytoplasmic tail, as demonstrated in HEK293 cells. Moreover, β-arrestin-2 is required for proper GPR54 signaling to ERK1/2, whose phosphorylation following Kp-10 stimulation was severely reduced in MDA-MB-231 breast cancer cells with suppressed β-arrestin-2 expression (326). In contrast, β-arrestin-1 appears to inhibit GPR54 signaling to ERK (439). Altogether, these findings underscore additional, G protein-independent signaling pathways for GPR54, whose physiological relevance is yet to be established. Nonetheless, the importance of the second intracellular loop for proper GPR54 signaling had been previously unveiled by the observation that a point L148S mutation in this region causes iHH in humans (401), and severely blunts (>90% reduction) the ability of GPR54 to induce PI hydrolysis and to activate ERK1/2 upon Kp-10 stimulation in vitro (487). Indeed, the second intracellular loop was proposed as the site for GPR54 binding to Gαq (487).
Another aspect of GPR54 that has received attention recently was the biochemical mechanism for receptor desensitization. In this sense, while robust electrophysiological and hormonal data evidenced that GPR54 undergoes desensitization upon continuous ligand exposure, the molecular basis for this important phenomenon has remained largely unattended. As is the case for many GPCR, GPR54 has been shown to rapidly internalize, via a clathrin-mediated mechanism, following ligand binding (326), yet a substantial fraction of internalized receptors seems to be recycled back to the membrane rather than degraded (29). It has been also suggested that the GPCR serine/threonine kinase (GRK), GRK-2, is involved in the process of homologous desensitization. Thus GRK2 is able to interact with sequences of the second intracellular loop and the cytoplasmic tail of GPR54, and overexpression of GRK2 enhances the homologous desensitization of the receptor (326). Yet the mechanism whereby GRK2 conducts this effect appears to be largely phosphorylation-independent and does not seem to involve a reduction of GPR54 expression or an increase in receptor internalization, but rather the uncoupling of GPR54 from its effector systems (326). The basis for receptor internalization following kisspeptin exposure remains to be elucidated. Worthy to note, studies in HEK293 cells evidenced a high rate of ligand-independent GPR54 internalization as well, with an abundant intracellular pool of the receptor. Again, the eventual physiological relevance of this phenomenon, if any, awaits further investigation.
Another potential source for signaling plasticity and regulation is the possibility of receptor homo- or heteromerization, as has been illustrated for numerous GPCRs. This aspect, however, remains scarcely evaluated for GPR54, as to our knowledge only one report has documented its potential hetero-oligomerization with the GnRH receptor (354). In this sense, recent electrophysiological studies have characterized the autoregulatory actions of GnRH on GnRH neurons in mice (162). Whether the proposed heteromerization between GPR54 and GnRH receptors has any physiological role in this phenomenon warrants specific investigation. Similarly, the potential interactions of GPR54 with itself (homodimerization) or additional receptor partners have not been documented to date.
Finally, while considerable efforts have been paid to unravel the major signals and molecular mechanisms responsible for the control of Kiss1 expression in different tissues and cell types, our knowledge on analogous aspects of GPR54 expression is still very limited. Although this point will be partially addressed at other sections of this review, it is worth noting here that recent studies, involving sequence and functional analyses of GPR54 promoter, have initiated the characterization of the potential molecular mechanisms regulating GPR54 gene expression (98). Thus three consensus SP1 sites, conserved in human, rat, and mouse genes, have been found in the 5' end of the GPR54 promoter. The functional relevance of these sites as activators of mouse Gpr54 expression was documented by experiments involving their deletion/mutation, which resulted in reduced transcription (98). In addition, a conserved partial estrogen-responsive element (ERE) was also found in GPR54 promoter. Deletion of this site resulted in enhanced promoter activity, suggesting that estrogen could operate as transcriptional repressor of GPR54 expression via this site (98).
IV. THE KISS1 SYSTEM AS GATEKEEPER OF REPRODUCTION: GENERAL ROLES AND NEUROANATOMY
As highlighted above, kisspeptins are now recognized as essential elements of the reproductive brain. As introduction to later sections of this review, we provide here an overview of the proposed roles and primary sites of action of kisspeptins, as outlined by the original observations in humans and rodents with inactivating mutations of GPR54 or Kiss1. In addition, a comprehensive description of the anatomical distribution of Kiss1 neurons within the mammalian brain will also be included in this section.
A. General Roles of Kisspeptins in Reproduction: Lessons From Mutant Models/Patients
The original studies on patients with inactivating mutations and deletions of GPR54 gene not only allowed to unveil the unsuspected reproductive dimension of kisspeptins but permitted also to initiate the characterization of the major physiological features of this system in the control of the HPG axis. Moreover, the generation of Gpr54 or Kiss1 null mutants provided an excellent mechanistic complement to the human studies. A brief review of those findings is not only interesting from an historical perspective but also quite illustrative on the importance of bidirectional bedside-to-bench approaches when tackling relevant biomedical questions.
Neuroendocrine characterization of humans with inactivating mutations/deletions of the GPR54 gene evidenced that, despite their striking phenotype in terms of sexual immaturity, failure of gonadal function, and hypogonadotropism, the affected individuals retained their capacity to respond to exogenous GnRH (401). This feature excluded a primary defect at the pituitary level as cause for their HH, in contrast to previously reported cases of inactivating mutations of the GnRH receptor (96). Even more interestingly, analyses of Gpr54 null mice demonstrated that the hypothalamic content of GnRH was preserved (401). This observation ruled out the possibility of a defective migration of GnRH neuron precursors from the olfactory placode, as had been proven the case for other monogenic forms of HH, such as those associated with mutations in KAL1 or FGF receptor 1 (FGFR1 or KAL2) genes (6, 105, 400). In spite of some phenotypic variability among mouse models, this feature (i.e., conserved GnRH migration/content in the hypothalamus) was later confirmed in Kiss1 knockout mice (93).
The lack of overt defects in GnRH neuronal migration, GnRH synthesis, or pituitary responsiveness to GnRH in individuals with impaired kisspeptin signaling led to the proposal that the Kiss1/GPR54 system is an essential, excitatory upstream regulator of GnRH neurons so that absence of kisspeptin signaling would result in suppressed GnRH secretion. This hypothesis, which set the ground for subsequent experimental analyses of the roles of kisspeptins in the control of the HPG axis, was proposed before key findings in the field, such as the demonstrations of 1) the actual existence of Kiss1 neurons at the hypothalamus, 2) the extraordinarily potent effects of kisspeptins on GnRH/gonadotropin secretion, and 3) the expression of GPR54 in GnRH neurons. Yet, the above assumptions turned out to be fully valid, thus allowing the rapid progress of our knowledge of the major features of the hypothalamic Kiss1 system as key regulator of the gonadotropic axis (FIGURE 4). As an illustration of such a fast progress, a timeline of major developments in kisspeptin physiology can be found in TABLE 1; for additional details, see also Reference 454.
B. Neuroanatomy of Kiss1 Neurons as Key Elements of the Reproductive Brain
While the existence of specific populations of Kiss1 neurons in the hypothalamus was first recognized in 2005 during the course of initial studies on the reproductive actions of kisspeptins, previous screening analyses, using of RT-PCR assays on tissue fragments from different brain areas, had already demonstrated the expression of KISS1 and GPR54 genes in different regions of the human central nervous system (CNS). For instance, the scattered presence of KISS1 mRNA was documented in the human brain in 2001, with prominent signals in the basal ganglia and the hypothalamus (292, 318). Simultaneously, GPR54 gene expression was also found in the spinal cord and different brain areas, such as basal ganglia, amygdala, substantia nigra, hippocampus, and hypothalamus (292, 318). In addition, mapping of the brain distribution of kisspeptins was also initiated shortly after the disclosure of their reproductive roles. Thus one study in early 2005 documented the presence of kisspeptin-like immunoreactivity (IR) in a wide diversity of brain regions in the rat, with the strongest signals at the hypothalamic dorsomedial (DMN), ventromedial (VMN) and arcuate (ARC) nuclei, as well as at the nucleus of the solitary tract and caudal ventrolateral medulla (38). Likewise, fibers with clear-cut kisspeptin-IR were described in many different areas of the rat telencephalon, diencephalon, mesencephalon, metencephalon, and myelencephalon (38). As a general call of caution, however, results from that study could be partially biased by the lack of complete specificity of the kisspeptin antibody used, which has been later demonstrated to react with other RFamide peptides, such as RFRP-1 and RFRP-3 (282).
The disclosure of the reproductive dimension of kisspeptins brought considerable efforts into the characterization of the neuroanatomical features of this system, which allowed the optimization of more powerful analytical tools, such as in situ hybridization assays for Kiss1 (and GPR54) mRNA, as well as the generation of more reliable and specific antisera against kisspeptins for the conduction of detailed immunohistochemical (IHC) mapping. As a result, the patterns of expression of Kiss1/Kisspeptin within the hypothalamus have been thoroughly characterized in the last few years, with commonalities and differences being recognized across different mammalian species. Rodents (rat, mouse, hamster) are by far the best characterized in terms of neuroanatomical distribution of Kiss1 neurons within the hypothalamus (12, 66, 282, 422, 423), but mRNA and/or peptide location data have been also reported in a wide variety of mammals, including porcine, ovine, equine, and primate species (97, 130, 362, 383, 420, 468), with the latter involving also studies in humans (180, 383). As a whole, these studies have conclusively demonstrated that a prominent population of Kiss1 neurons resides in the ARC, or the equivalent infundibular region in primates, as documented in the mouse, rat, hamster, guinea pig, shrew, sheep, horse, pig, monkey, and human, either in terms of expression of Kiss1 mRNA and/or kisspeptin-IR (12, 28, 34, 66, 97, 116, 130, 145, 180, 185, 186, 260, 383, 408, 422, 423). The phenotype of these ARC neurons has been actively investigated recently, and neuroanatomical studies in different species, such as the sheep, mouse, monkey, and human, have demonstrated that in addition to kisspeptins, they express NKB and its putative receptor, NK3R, as well as dynorphyn (Dyn), therefore leading the proposal of the term KNDy to name this neuronal population (241).
In addition to the ARC/infundibular region, studies in rodents, involving mRNA and peptide/IHC analyses, have proven the presence of another population of Kiss1 neurons in the anteroventral periventricular nucleus (AVPV) of the hypothalamus (2, 66, 69, 422, 423). Furthermore, the existence of a scarce group of kisspeptin neurons at the DMN was suggested on the basis of IHC data (66, 282), but its nature remains contentious, as Kiss1 RNA expression has not been consistently detected at this area and the abundance of RFRPs in this nucleus might have yielded false positives in some IHC approaches (369). Regarding the periventricular area, elegant neuroanatomical analyses in mice have suggested that rather than being restricted to the AVPV, kisspeptin neurons at this site constitute a continuum including also the contiguous periventricular preoptic area; the so-called rostral periventricular area of the third ventricle (RP3V) (66, 69). Yet, for sake of homogeneity with consensus nomenclature, we will use the term AVPV to refer to this specific set of Kiss1 neurons. The AVPV population displays a striking sexual dimorphism at puberty and adulthood, with female rodents consistently having larger numbers of Kiss1 neurons at this site than males (2, 207). As is the case for the ARC, very recent studies suggest that Kiss1 neurons in the AVPV likely coexpress other neuropeptides, including galanin and Met-enkephalin, whose physiological roles in this neuronal population remain unfolded (201, 350).
Recent studies in mice, using transgenic approaches, have refined our knowledge of the patterns of distribution of Kiss1 neurons initially defined by in situ hybridization. Thus a recent study from Cravo et al. (77) employed a genetically engineered Kiss1-Cre mouse line to target green fluorescent protein (GFP) or β-galactosidase activity to Kiss1 neurons. In the same line, a Kiss1-CreGFP knock-in mouse line has been recently reported by Gottsch et al. (151). Neuroanatomical studies in those mouse lines have roughly replicated previous data of Kiss1 mRNA distribution in the mouse brain, with prominent expression in the ARC and preoptica area (74, 151), as well as in different extrahypothalamic sites (74). The latter is partially in keeping with previous data showing the presence of additional groups of Kiss1 neurons in brain areas other than the hypothalamic ARC and AVPV, such the medial amygdala, the anterodorsal preoptic nucleus, and the bed nucleus of the stria terminalis, although the physiological roles of such Kiss1 neuronal populations remain largely unknown (149). Yet, recent evidence suggests that, as is the case in the hypothalamus, sex steroids participate also in the regulation of Kiss1 expression at some of these extrahypothalamic sites, such as the medial amygdala (214). Moreover, additional populations of Cre-positive cells that do not apparently express Kiss1 mRNA in adulthood were also identified in the study of Cravo et al. All in all, the initial results from models of Kiss1-driven Cre expression clearly demonstrate the power of this genetic approach to dissect out essential aspects of kisspeptin distribution and function. However, these data stress also the importance of cautious interpretation of results from these models, where issues such as promoter specificity of Cre expression, especially when using BAC transgenic approaches (77), and/or developmental timing of Kiss1 expression in distinct neuronal lineages, must be carefully considered.
An intriguing feature of the AVPV population of Kiss1 neurons, which is prominently detected in female rodents, is whether it is also present in other mammalian species. Indeed, initial studies suggested that this population might be scarce, if not totally absent, in sheep, horse, and primate (97, 116, 362, 383, 408), but present in porcine and guinea pig hypothalamus (34, 468). The lack of an AVPV population of Kiss1 neurons in the above species appeared to fit well with the fact that, contrary to rodents (88), this area had not been linked in other mammals to the positive feedback of estrogen and the generation of the preovulatory surges, a phenomenon in which Kiss1 neurons at the AVPV have been proposed to play an essential role in rodents. Notwithstanding this, the presence of discernible kisspeptin-positive cells also in the periventricular area, embedded in a dense plexus of fibers with strong kisspeptin-IR, has been recently documented in humans (180). Similarly, studies in the sheep have demonstrated the expression of Kiss1 mRNA and kisspeptin immunoreactivity in the POA, in addition to the ARC (19, 20, 130). Therefore, it remains possible that a population of Kiss1 neurons in a region equivalent to the AVPV, but probably in a more lateral location, exists also in larger mammals, including sheep and primates, albeit at much less abundance than in rodents. The physiological relevance of such a rostral periventricular population in these species is yet to be fully defined.
In addition to the obvious differences in terms of location, there seem to be also important anatomical differences as to how the ARC and AVPV populations of Kiss1 neurons engage into the neural circuits afferent to GnRH neurons. Thus direct appositions between Kiss1 and GnRH neurons were initially reported only for those originating in the AVPV, as documented rodents (69). In the same vein, studies in the sheep suggested that ARC Kiss1 neurons do not significantly project to the ventromedial POA, but rather that kisspeptin afferents to GnRH neurons in this species originate from the population located at the POA (19). Nonetheless, recent detailed neuroanatomical studies in the mouse, using anterograde and retrograde tracing techniques, have revealed that Kiss1 neurons in the rostral portion of the ARC, which may account for ∼20% of the total ARC population, do project to the rostral POA, thus making highly plausible their direct interaction with GnRH neurons (510). Of note, detailed IHC studies in rodents and primates strongly suggest that the number of synaptic contacts between Kiss1 and GnRH neurons is surprisingly low, which might imply the existence of interneuronal or even nonsynaptic communication between these two populations (182, 362, 480), a contention that has been also proposed in ovine species recently (269).
Besides studies addressing the location of Kiss1 neuron cell bodies, recent efforts have been devoted to map the pattern of distribution of kisspeptin-positive fibers in the hypothalamus, in an attempt to validate initial results using less reliable antibodies. In this context, a detailed mapping of kisspeptin-IR fibers in the female mouse brain has been recently reported (66). This study documented the presence of abundant fibers within the hypothalamus, including prominently the AVPV, and adjacent areas, as well as the ARC. Elsewhere within the hypothalamus, kisspeptin-IR fibers were found in a diversity of nuclei, including the supraoptic (SON), paraventricular (PVN) and DMN, but were absent in the suprachiasmatic nucleus (SCN) and VMN. In addition, a considerable number of fibers appeared to stream from the periventricular area into the lateral septum. Of note, in that study kisspeptin fibers were not detectable at the external zone of the mouse median eminence but present in its internal zone and lateral margins (66). Abundant presence of kisspeptin-positive axons in the internal (and to a much lesser extent, the external) zone of the median eminence has been also documented recently in the male monkey and the adult cyclic female rat (99, 362), by the use of other polyclonal antibodies. This reinforces the specificity of the above observations, which are further supported by the lack of detectable staining in brains from Kiss1 KO mice (66).
Notably, a recent study systematically compared the patterns of distribution of kisspeptin-IR fibers revealed by two different antibodies, designed to identify Kp-10 or Kp-52, in the brain of the adult female rat (99). While the patterns of kisspeptin-IR raised by the two antisera perfectly matched in most regions, selective staining with the Kp-10 antibody was detected in some areas, including the lateral septum and bed nucleus of stria terminalis. This might suggest a differential processing of shorter and larger kisspeptins in different brain areas. Also of interest, this study disclosed that, despite abundant expression of Kiss1 mRNA, the number of kisspeptin-IR cells in the AVPV is surprisingly low in the adult female rat (99), in contrast to previous reports in mice (66, 69). This might be indicative of species differences in the kinetics of kisspeptin processing at this nucleus, whose physiological relevance is yet to be defined.
In addition, recent tracing studies in the mouse have demonstrated that the patterns of projections of ARC and AVPV Kiss1 neuronal populations are considerably different, as ARC Kiss1 neurons innervate a wide number of hypothalamic and limbic region nuclei, whereas Kiss1 neurons originating from the AVPV display a narrower pattern of projections that target a smaller number of medially located hypothalamic nuclei (510). Among those, AVPV Kiss1 neurons appear to heavily project to the ARC, where Gpr54 expression and kisspeptin actions in specific neuronal populations have been very recently documented in mice (131). These anatomical differences in terms of fiber projections strongly suggest important functional divergences between these two Kiss1 neuronal populations. Indeed, striking differences between ARC and AVPV Kiss1 neurons were already disclosed in 2005, when it was documented that the responses of these two neuronal populations to manipulations of sex steroid milieu were totally opposite (422, 423). More recently, further dissimilarities between the ARC and AVPV of Kiss1 neurons have surfaced, as only the ARC population has been shown to express Dyn, as well as NKB and its receptor (241). Such differences illustrate that, despite the common usage of kisspeptins as neurotransmitter, Kiss1 neurons at the ARC and AVPV are likely to play substantially different roles in the control of the HPG axis. Moreover, the wide patterns of fiber projection, recently documented in the mouse (510), support the contention that Kiss1 neuronal populations are likely involved in the control of additional, nonreproductive functions.
As complement to the above data on the characterization of the patterns of distribution of Kiss1m RNA and kisspeptin-IR in the brain, some studies have addressed the location of GPR54 in different areas of the central nervous system. Admittedly, however, such analyses have lagged behind, and our current knowledge of the actual distribution of GPR54-expressing neurons is still incomplete, probably due in part to the lack of reliable antisera against the receptor protein. Nevertheless, initial in situ hybridization analyses in the rat demonstrated expression of Gpr54 mRNA in the hypothalamus, and specifically in GnRH neurons (186). In good agreement, early studies also demonstrated the expression of GPR54 transcripts in laser-captured GnRH neurons in the cichlid fish (329). Altogether, these initial observations provided further support for the original hypothesis of the direct mode of action of kisspeptins on GnRH neurons. More recently, a transgenic Gpr54 LacZ knock-in strategy has been deployed to further document the patterns of brain expression of the kisspeptin receptor in mice (169). Studies using this mouse line, which expresses a reporter LacZ gene under the Gpr54 promoter, have been the first attempt to provide a reliable map of GPR54-positive neurons in different brain areas and confirmed the expression of this receptor in GnRH neurons at the rostral POA. Of note, no Gpr54 expression was apparently detected in the ARC or rostral periventricular nucleus in that study. Yet, this approach may suffer from limited sensitivity, which may have compromised the detection of Gpr54 expression at low levels and/or in discrete populations in the ARC, as recently suggested by functional analyses in the mouse (131). Notably, in the above study, the highest receptor density was detected at the dentate gyrus of the hippocampus and abundant expression was observed, in addition to the POA, at the septum, anteroventral nucleus of the thalamus, posterior hypothalamus, periaqueductal grey, supramammillary and pontine nuclei, and dorsal cochlear nucleus (169). Worthy to note, for most of those sites, the putative function of kisspeptin signaling remains totally unknown. Notwithstanding this, the hippocampus had been previously pointed out as a potential site of expression and action of kisspeptins, as Kiss1 mRNA levels at this site were shown to be regulated by seizure activity and glutamate signals (14), while Kp-10 was able to upregulate synaptic transmission in hippocampal slices (16).
V. KISS1 NEURONS AND BRAIN SEX DIFFERENTIATION
The patterns of pubertal activation of the HPG axis and its subsequent function in adulthood are sexually dimorphic. A substantial component of such sexual dimorphism, that manifests functionally at relatively late phases of postnatal maturation, is defined during early developmental stages, also referred as critical windows, where specific circuitries within the brain, especially in the hypothalamus, undergo different developmental programs between males and females (146). This process is mainly hormonally driven and closely linked to other sex-determination phenomena, such as gonadal differentiation (146).
In rodents, where the molecular basis of this phenomenon has been deeply scrutinized, it is known that the process of brain sex differentiation takes place during a critical developmental period spanning from late embryonic to early postnatal age (459). In this process, key neuronal networks at the hypothalamus become organized in a permanent manner differentially in males and females (146, 288). This allows the timely activation of the reproductive axis at puberty, and the manifestation of sexually differentiated behaviors and neuroendocrine secretory patterns later in life (146, 288). One of the neurohormonal traits that more clearly displays a sexual dimorphism is the cyclic secretory activity of the GnRH/gonadotropin system, which is based on the capacity of estrogens, selectively in the female, to evoke the preovulatory surge of gonadotropins from puberty onwards (88, 146). Of note, recent experimental evidence suggests that, in addition to earlier (perinatal) periods, puberty itself may represent a second critical window for neuroendocrine development, during which changes in sex steroid input might induce permanent functional alterations of different neurohormonal axes (e.g., the corticotropic/stress axis) later in life (118). Indeed, the timing of puberty itself is also different between sexes, with females entering puberty earlier than males (319, 320, 322).
A. Physiological Roles and Mechanisms of Sexual Differentiation of Kiss1 Neurons
As reviewed in previous sections, studies in rodents have set the contention that the hypothalamic Kiss1 system, and especially the neuronal population in the AVPV, is markedly sexually dimorphic at puberty and adulthood, with females having much greater numbers of Kiss1 neurons at this site (69, 207). These observations, together with the reported stimulatory effects of estrogen on Kiss1 mRNA expression and kisspeptin content in this region (422, 423), and the well-known role of the AVPV in rodents in the generation of the preovulatory surge of gonadotropins selectively in the female (88), strongly suggested that this population of Kiss1 neurons plays an important role in the process of brain sex differentiation. Experimental data accumulated over the last few years fully support such a possibility. Thus conclusive evidence has been presented that Kiss1 neurons at the AVPV are highly sensitive to the organizing effects of sex steroids during the neonatal period of sexual maturation of the brain in the rat. As a clear illustration, neonatal exposure to high doses of androgen in female rats dramatically decreased the expression of Kiss1 mRNA at the AVPV in adulthood so that neonatally androgenized females displayed Kiss1 mRNA levels similar to those of adult males but much lower than cyclic females (207). The functional relevance of such a masculinization of the pattern of Kiss1 expression of the AVPV is further stressed by the facts that 1) contrary to cyclic female rats, exposure to estradiol in adulthood failed to increase Kiss1 mRNA levels at the AVPV of neonatally androgenized females (207) (see also FIGURE 5), and 2) analogous protocols of neonatal manipulation of the sex steroid milieu blunted the ability of estrogen to elicit positive feedback and surgelike responses in terms of LH secretion (178).
In the same line, it has been recently shown that neonatal orchidectomy in rats, as a means to eliminate androgen actions during the critical period of brain sex differentiation, induces the feminization of Kiss1/kisspeptin expression at the AVPV (i.e., higher expression than in control adult males) (178). Neonatal orchidectomy also resulted in the acquisition of positive feedback and surgelike LH responses to ovulatory doses of estradiol (178). These findings might be suggestive of a default pathway for the feminization of the AVPV population of Kiss1 neurons. However, recent data from the hypogonadal hpg mouse, which is severely deficient in GnRH secretion and therefore has very low sex steroids levels, evidenced that hpg females displayed lower numbers of kisspeptin neurons in the AVPV than wild-type controls and did not show the normal stimulatory response to estrogen at this nucleus, thus suggesting the importance of the gonadal hormone milieu during development for the complete functional differentiation of this neuronal population also in the female mouse (140).
In addition to androgens, the roles of estrogen exposure during early critical periods on the shaping of the hypothalamic Kiss1 system, and its functional relevance in terms of sexual differentiation, have been also evaluated in rodents. Thus neonatal estrogenization of female rats decreased the number of Kiss1 neurons in the AVPV and disrupted the positive-feedback effects of estradiol in terms of LH secretion in adulthood (178). Similarly, it has been reported that neonatal exposures to synthetic estrogens, known to disturb proper activation and function of the gonadotropic axis (459), persistently suppressed the hypothalamic expression of Kiss1 gene at the expected time of puberty and adulthood in male and female rats (298, 306). In good agreement, studies in α-fetoprotein (AFP) KO mice, where the congenital lack of this scavenger protein of circulating estrogens results in excessive estrogenic input during early development, have documented that in the female such an excessive exposure to estrogen perturbs the capacity to generate preovulatory-like LH surges and to activate Kiss1 neurons at the AVPV in adulthood (143). Moreover, the total number of Kiss1 neurons at the AVPV is also decreased in AFP KO female mice (143). In the same vein, recent analyses in aromatase null mice, where transformation of androgen into estrogen is blocked, have shown that the pubertal expansion of the AVPV population of Kiss1 neurons in the female is prevented (65), and kisspeptin-IR in this region is severely reduced at adulthood (22), again suggesting an organizing role of early estrogen. Admittedly, the above KO models do not allow proper discrimination between early organizing versus later activational actions of sex steroids. Yet, when taken as a whole, the above findings strongly suggest that, at least in rodents, the mechanisms whereby the functional patterns of the GnRH/gonadotropic axis become sexually differentiated involve structural/organizing changes in the population of Kiss1 neurons at the AVPV, which appear to be driven by sex steroid inputs at critical periods of development.
Of important note, some of the above characteristics in terms of sexual differentiation of the hypothalamic (AVPV) Kiss1 system in rodents might not be shared by other mammalian species; a contention that needs to be stressed given the predominance of rodent studies in this and other areas of kisspeptin physiology, and the tenable differences in the process of sexual differentiation of the brain across mammals. For instance, in the sheep, Kiss1 neurons responsible for mediating the negative- and positive-feedback effects of sex steroids appear to reside mostly within different subareas of the ARC (116), although a recent report has suggested that a subset of Kiss1 neurons in the POA is also involved in the generation of the preovulatory surge in this species (424). Similarly, initial studies in primates, including humans, suggested that kisspeptin neurons at the hypothalamus are mostly confined to the ARC/infundibular region (362, 383, 408), where expression of Kiss1 gene is under the inhibitory regulation of androgens and estrogens (383, 408). These observations seemed in agreement with the contention that, in contrast to rodents, the mechanisms for the generation of positive feedback and preovulatory gonadotropin surges in primates appear to reside within the ARC/infundibular nucleus (222, 347). Likewise, classical studies suggested decades ago that the mechanisms of sexual differentiation of gonadotropin control are, at least partially, different between rodents and primates, as LH surges, and even cyclic ovarian function following ovary transplantation, could be induced in male monkeys, not subjected to hormonal manipulation at early stages of development, if provided with an appropriate ovarian (estrogen) signal as adults (204, 315). Nonetheless, as indicated in section IVB, a recent report identified a discernible population of kisspeptin neurons at the hypothalamic periventricular region in female humans (180). Moreover, robust sex differences were noticed in that study in terms of numbers of kisspeptin-positive neurons and fibers, which were consistently higher in women (180). Therefore, this population might be also subjected to sexual differentiation in humans.
Remarkably, the above sex differences (females>>males) were evident not only at the periventricular area but also at the infundibular region (180). Indeed, while initial RNA data suggested that the ARC population of Kiss1 neurons is not sexually dimorphic in the rat (207), more recent IHC and in situ hybridization studies in rodents and sheep strongly suggest that similar sex differences in the number of Kiss1 neurons exist at this nucleus (83, 444). In fact, a very recent report evidenced that Kiss1 mRNA levels in the ARC are higher in female rats already during the neonatal period (48). Of particular relevance, a recent study in the sheep suggested that prenatal exposure to testosterone does not alter kisspeptin-IR at the ARC in adult females, but significantly suppresses the expression of NKB (83). All in all, the mechanisms and specific contribution of ARC/infundibular versus periventricular Kiss1 neurons to the process of brain sexual differentiation in different mammalian species, including rodents and nonrodents, remain to be fully elucidated and merit further investigation.
B. Sex Differentiation of Kiss1 Neurons: Targets for Endocrine Disruption?
The above experimental evidence not only set the contention that Kiss1 neurons in specific hypothalamic nuclei are putative components of the mechanisms of brain sex differentiation, but also documented the sensitivity of the Kiss1 system to the organizing actions of endogenous, and eventually exogenous, sex steroids, a phenomenon which may pose pathophysiological implications in terms of endocrine disruption (ED) of the HPG axis (451). In this sense, during the last two decades, considerable attention has been paid to the potential adverse effects on reproductive health of human and wildlife species of inappropriate exposures, especially during critical periods of development, to numerous natural and synthetic compounds, with sex steroid-like bioactivities, of either estrogenic, androgenic, or anti-androgenic nature (211, 410, 459). While the clinical relevance of such exposures is yet to be fully defined (386), some of the epidemiological trends of deterioration of reproductive function, such as altered timing of puberty and certain forms of infertility, might potentially derive from primary alterations of the process of normal differentiation of hypothalamic Kiss1 neurons, which could be potential targets for the actions of compounds with sex steroid-like activities during critical developmental windows (451).
While conclusive demonstration of such phenomenon, especially in terms of human reproductive health, is still pending, initial experimental data, coming mostly from rodent studies, strongly suggest that this is a tenable possibility. Thus neonatal exposure to the synthetic estrogen, estradiol benzoate (EB), even at doses well below the conventional protocols of neonatal estrogenization (459), induced a persistent decrease in hypothalamic expression of Kiss1 gene at the time preceding puberty in male and female rats (306), as well as in adults males (298), a finding recently confirmed in aging Fisher rats after perinatal exposure to EB (147). In addition, neonatal exposures to EB and other synthetic estrogens, such as the selective ligand of estrogen receptor (ER)α, PPT, as well as the phytoestrogen genistein, evoked variable degrees of suppression of kisspeptin fiber density at the AVPV and/or ARC in pubertal and adult female rats (24, 253).
In the same line, gestational exposure of rats to various polychlorinated biphenyls (PCBs), compounds with reported estrogenic, anti-estrogenic, or anti-androgenic activities, altered sexual differentiation of female neuroendocrine systems in the offspring, including the AVPV, where a significant reduction in the number of kisspeptin fibers was detected in adulthood (104). Likewise, recent data obtained in sheep evidenced that in utero exposure to a complex cocktail of ED contained in sewage sludge, used as agricultural fertilizer in pastures and thus an optimal model for investigation of “real-life” mixtures of ED, induced a significant decrease in Kiss1 mRNA levels at the rostral, mid, and caudal regions of the hypothalamus of exposed fetuses (27), specifically when exposures took place during critical periods of maturation. Finally, neonatal exposure to the xeno-estrogen bisphenol A (BPA) in rats significantly reduced hypothalamic Kiss1 mRNA levels during the infantile period (47) and puberty (306), and decreased kisspeptin IR at the ARC (as estimated in terms of fiber density) in adulthood (332), especially in females. Furthermore, recent preliminary studies in mice suggested that perinatal exposure to BPA partially obliterates the sexual dimorphism of kisspeptin-IR content in the hypothalamus, mainly because of the increase of kisspeptin content at the ARC (327). These findings have been recently complemented by the observation that perinatal exposure of male rats to BPA increased the number of Kiss1 neurons in the AVPV (21).
The above data provide strong circumstantial evidence for the possibility that inappropriate exposures to sex-steroid acting compounds during early critical windows might have an organizing impact on the maturation of the hypothalamic Kiss1 system, with durable consequences that manifest later in life. In support of this possibility, functional studies conducted in some of the above models have conclusively shown that the decreased pubertal expression of Kiss1 gene following neonatal exposures to estrogenic compounds is associated with defective gonadotropin secretion, both in basal conditions and postgonadectomy, which can be rescued by administration of exogenous kisspeptin (306). In the same line, some of the above protocols of neonatal exposure to chemicals with estrogenic actions, associated with decreased kisspeptin fiber density at discrete hypothalamic nuclei in adult female rats, caused also reduced GnRH neuronal activation (24). Strikingly, one report has recently documented that perinatal exposure to environmental doses of BPA was sufficient to allow the manifestation of surgelike LH responses following estradiol priming in adult male rats (21). As a whole, these findings strongly suggest that by targeting the developing hypothalamic Kiss1 system, compounds with sex steroid-like activities might interfere with normal maturation and later function of the reproductive axis. While the evidence supporting this possibility is compelling, it must be emphasized, as general call of caution, that most of the work conducted so far in this area has addressed putative mechanisms of disruption rather than the consequences of real-life exposures to complex mixtures of ED, presumably at low doses (451). On the other hand, the possibility exists that, in addition to Kiss1 neurons, environmental compounds with sex steroid-like activities may target other elements of the reproductive brain as well, including GnRH neurons, which have been proposed as direct targets for different ED (103, 146). As a final note, it must be stressed that the disrupting actions of synthetic compounds with sex steroid activity on the Kiss1 system may not be restricted to early developmental periods and could manifest at later stages of postnatal maturation. In this sense, a recent study demonstrated that chronic exposure of female mice to anabolic androgenic steroids perturbs GnRH neuronal activity by suppressing kisspeptin excitatory inputs from the AVPV (334).
VI. KISS1 NEURONS AND THE CONTROL OF PUBERTY
As highlighted in previous sections, puberty is a crucial developmental period when reproductive capacity is achieved. Although notable differences exist in the process of pubertal activation of the HPG axis across mammalian species, a key event in the onset of puberty is the progressive increase of the neurosecretory activity of GnRH neurons, which in turn maximally stimulates gonadotropin secretion to allow complete gonadal maturation and function thereafter (320, 328, 492, 498). Such pubertal changes in the pattern of GnRH secretion are the result of concerted modifications in trans-synaptic and glial inputs to the GnRH neuronal network (319, 321), as well as plastic changes in GnRH neurons themselves (335). Among these maturational events, functional and structural changes in neuronal afferents to GnRH neurons appear crucial as ultimate triggers of puberty, likely through the combined increase in excitatory inputs and decrease in inhibitory signals projecting to GnRH neurons (319, 320, 322, 462). The nature of such excitatory and inhibitory neuronal transmitters has been partially elucidated in recent years. In this context, recent evidence has documented an important role of kisspeptins in the control of puberty in mammals.
A. Kisspeptins as Gatekeepers of Puberty Onset
The first indication for an essential role of kisspeptin signaling in the control of puberty onset came from the initial studies in humans and mice with genetic inactivation of GPR54, a condition which, as described earlier in this review, resulted in impaired puberty progression and sexual immaturity (95, 401). A roughly similar, albeit milder, phenotype was later described in mice engineered to lack a functional Kiss1 gene (93, 234). Altogether, these data demonstrated the indispensable role of kisspeptins in the proper timing of puberty, both in humans and rodents. The fact, however, that these observations were made in patients or animal models congenitally devoid of kisspeptins or GPR54 did not allow to conclusively demonstrate the actual involvement of kisspeptin signaling in the activational mechanisms whereby puberty is triggered, as the above phenotypes might stem from earlier organizing defects associated with the congenital inactivation of Kiss1 or GPR54. Therefore, mechanistic studies were needed to characterize the specific roles of kisspeptins in the control of puberty onset.
While these mechanistic studies have been instrumental in dissecting out the modes of action of kisspeptins, the recent generation of kisspeptin antagonists has made it possible to specifically address the actual physiological relevance of this system in the control of the timing of puberty, at least in rodents. Thus studies in pubertal female rats have demonstrated that central infusion of an effective kisspeptin antagonist, peptide 234, for a period of 7 days preceding puberty, results in delayed puberty onset, as estimated by a combination of consensus indexes, such the age of vaginal opening and uterus and ovarian weights (339) (FIGURE 6). In the same vein, infusion of this kisspeptin antagonist suppressed GnRH secretion in prepubertal and pubertal monkeys (156). Of note, terminal LH and FSH levels in rats infused with the antagonist were not significantly decreased, in spite of signs of impuberism following intracerebral administration of the antagonist (339). This might indicate that kisspeptin signaling is more directly related with the generation of key excitatory events, such as the prepubertal LH mini-surges that precede the first ovulation, than with the maintenance of basal, generally low, gonadotropin levels. In any event, these recent observations fully support the contention that kisspeptins play an important activational role in the onset of puberty.
Notwithstanding the solid genetic and pharmacological data supporting an important role of kisspeptin signaling in puberty onset, a recent study suggested that pubertal maturation in female mice can be attained even after congenital ablation of Kiss1- or Gpr54-expressing neurons (273). For obvious reasons, these findings have attracted enormous attention and have raised considerable debate in the area. A simplistic interpretation of these data would imply that kisspeptin signaling is dispensable for puberty onset in rodents. However, this contention does not match with the state of impuberism of humans and mice lacking functional Kiss1 or GPR54, neither does it fit with the antagonist data summarized above. Moreover, considering that GnRH neurons do express GPR54, it could be similarly concluded on the basis of this report that congenital ablation of GnRH neurons does not prevent puberty progression. Of note, neuronal ablation in the above models might have not been complete. In addition, in that study, timed elimination of Kiss1-expressing neurons from postnatal day 20 did alter normal female maturation and prevented the attainment of reproductive capacity (273). All in all, the above set of data may be indicative of the existence of robust compensatory mechanisms, brought about by the congenital elimination of key neuronal components of the reproductive brain, that may apparently enable pubertal maturation only with a marginal subset of Kiss1 or GnRH neurons (273). The latter is in keeping with previous literature suggesting that reproductive capacity can be achieved even after a severe congenital reduction of the number of GnRH neurons (172). Yet, the fact that inducible ablation of these Kiss1- or Gpr54-expressing neurons at later, prepubertal stages, when plastic compensatory changes are less likely to occur, did prevent normal pubertal maturation clearly evidences the relevant role of kisspeptin signaling in puberty onset.
B. Mechanisms for Activation of the Hypothalamic Kiss1 System at Puberty
In a first attempt to ascertain whether the Kiss1 system plays a role in puberty onset, expression analyses of the mRNA levels of both Kiss1 and Gpr54 were monitored in rat and monkey hypothalamic samples at different stages of postnatal maturation. Studies in male and female rats documented that, despite detectable expression across postnatal development, a clear-cut increase in Kiss1 and Gpr54 mRNA levels takes place at the hypothalamus coinciding with the onset of puberty (298). This phenomenon was later confirmed in non-human primates, where Kiss1 and Gpr54 mRNA expression was shown to increase approximately threefold at the mediobasal hypothalamus during the transition from the juvenile to the mid-pubertal stage of the female (404). Similarly, hypothalamic Kiss1 mRNA levels raised in male monkeys along puberty (404). While these studies were the first to suggest the heightening of kisspeptin signaling during puberty, their lack of anatomic resolution, in the case of rat studies, and the absence of peptide data fueled subsequent analyses aiming to provide a more complete understanding of the developmental changes undergone by the hypothalamic Kiss1 system along puberty. Nonetheless, the above initial studies in the monkey already suggested that the pubertal increase in Kiss1 expression takes place in the ARC, as indirectly evidenced by in situ hybridization of brain sections from adult female monkeys showing that Kiss1-expressing cells are mostly circumscribed to the medial ARC in this species (404).
In spite of this initial evidence in the monkey, it is emphasized here that studies addressing the roles of kisspeptin signaling in puberty have been mostly conducted in rodents, with the potential bias of species differences, which are well-known to exist for the overall neuronedocrine mechanisms of puberty onset (322, 344, 345). Keeping this limitation in mind, it is worth to stress that a number of studies in rodents have documented an increase in the expression of Kiss1 mRNA and/or the number of kisspeptin-IR neurons in the AVPV during pubertal maturation. Thus initial studies already demonstrated a dramatic upsurge in Kiss1 mRNA levels and kisspeptin-IR at the AVPV across the pubertal transition in female mice, as detected by in situ hybridization and IHC (69, 161), findings that have been confirmed recently (140). A similar phenomenon has been reported in male mice, although the absolute number of kisspeptin-IR neurons at the AVPV was found to be much lower (>10-fold) than in females (69). Similar developmental trends in the number of Kiss1 mRNA-expressing neurons have been described in male and female rats (182). Likewise, an elevation in kisspeptin-IR at the AVPV has been demonstrated in pubertal female rats (441). Such an increase in the number of AVPV Kiss1 neurons during puberty seems to be dependent on the stimulatory actions of estrogen at this site, as it is blunted in conditions of low or null estrogenic input before puberty, such as gonadectomy, absence of aromatase activity or in hpg mice (65, 140) (see below). The functional relevance of such elevation of kisspeptin tone in the AVPV for the timing of puberty in rodents is further supported by the significant increase during the pubertal transition of the number of kisspeptin fiber projections originating from the AVPV and of their appositions with GnRH neurons, which show a sharp rise between the early juvenile and peripubertal periods in mice (65). Admittedly, the correlate, if any, of the above developmental changes for the timing of puberty in nonrodent species remains unexplored.
Although the changes in Kiss1/kisspeptin expression in the AVPV in rats and mice are now well documented, it remains somewhat contentious whether a similar increase takes place in the ARC during rodent puberty. Nonetheless, studies in male and female rats, using in situ hybridization or qRT-PCR of specific hypothalamic regions, have demonstrated an elevation of Kiss1 mRNA levels at the ARC during the pubertal transition in this species (441, 444), yet the magnitude of such an increase might be considered modest compared with the reported elevation in the AVPV, especially in the female (444). Furthermore, kisspeptin-IR has been shown to significantly increase in the ARC of male and female rats along puberty (28, 441). In contrast, a recent study in mice was unable to demonstrate an elevation of Kiss1 mRNA levels at this nucleus, although the use of qRT-PCR on dissected hypothalamic fragments might have obscured the detection of modest changes (140). The functional relevance of the putative increase of kisspeptin tone in the ARC during male and female puberty in rodents awaits further investigation. As indicated above, however, the situation in primates is likely different, as consistent increases in Kiss1 expression have been reported in the MBH, presumably in the ARC, in male and female monkeys during the pubertal transition, therefore suggesting that the pubertal activation of ARC Kiss1 neurons may play a relevant role in primates (404).
The molecular mechanisms for the increase of hypothalamic Kiss1 expression at puberty have begun to be explored only recently. By a combination of molecular analyses, Mueller et al. (291) have provided compelling evidence for the ability of a number of transcription factors, such as TTF1, CUX1, YY1, and EAP1, which are likely involved in the onset of puberty, to bind to the human KISS1 promoter and to regulate KISS1 transcriptional activity (291). Thus, whereas EAP1 and CUX1-p110 were repressive, TTF1 and CUX1-p200 enhanced transcription of KISS1 gene (291). The physiological relevance of these observations is reinforced by the fact that all four transcription factors display nuclear expression in Kiss1 neurons of the MBH of the rat (291). Yet, the functional implications of such coexpression is not always clear; for instance, the ability of EAP1 to inhibit KISS1 gene expression does not apparently match with the reported increase of this factor at the time of puberty (166). Further analyses are needed to decipher the networks of trans-activators and repressors of Kiss1 gene, and their mechanistic implications in the physiological control of pubertal timing.
In addition to changes in the ligand, a number of studies have attempted to evaluate potential modifications in GPR54 signaling, as mechanistically relevant for the role of kisspeptin in the timing of puberty (for a recent example, see Ref. 156). Besides the initial expression data suggesting a net increase in Gpr54 mRNA expression at the hypothalamus during rat and monkey puberty (298, 404), the combination of in situ hybridization and electrophysiological recordings in female mice allowed to propose that a marked increase in the number of kisspeptin-responsive GnRH neurons takes place during puberty. Thus, while only 27% of GnRH neurons were activated by kisspeptin in juvenile mice, >90% of GnRH neurons were depolarized in adult animals in response to kisspeptin (161). Notably, such a phenomenon was not primarily caused by an increase in the mean expression levels of Gpr54 mRNA per cell but apparently by an enhancement in the efficiency of this receptor to couple with its effector signaling systems during the pubertal transition (161). In addition, it has been recently reported in mice that the percentage of GnRH neurons expressing Gpr54 increases gradually between the infantile and the (pre)pubertal period (169). In good agreement, the sensitivity to the stimulatory effects of kisspeptin, in terms of LH responses in vivo, augments significantly during pubertal maturation, in both rats and mice (57, 161). Of interest, studies in female rats have also documented that pubertal animals are less prone than adult cyclic females to desensitization of the stimulatory effects of Kp-10 in terms of LH responses (381). Such resistance to desensitization might help to explain the apparent state of full activation of the HPG axis despite the increase of kisspeptin inputs during the onset of puberty.
The functional relevance of the above changes, which collectively suggest the heightening of the kisspeptinergic tone/effects during puberty, is further supported by the observation that in the monkey there is a detectable increase in the pulsatile release of kisspeptin-54 at the median eminence during puberty, which is highly coincident with the elevation of GnRH pulse frequency at this stage (210). However, whether the median eminence is the primary site for the actions of kisspeptins at puberty remains to be solved. Yet a recent study demonstrated that direct infusion of Kp-10 into the MBH and median eminence dose-dependently stimulated GnRH secretion in pubertal monkeys (156). Further support for a functional role of kisspeptins in puberty onset comes from pharmacological studies in rats, where central repeated administration of Kp-10 to immature females, without any other neuroendocrine manipulation, was able to induce precocious vaginal opening, as external sign of puberty, and early activation of the HPG axis, as evidenced by potent gonadotropic and estrogenic responses (302) (see FIGURE 6). Similarly, repetitive administration of Kp-10 to monkeys at the end of the juvenile phase of primate development elicited a precocious and sustained train of GnRH discharges, analogous to that found during puberty (349). These observations suggest that proper activation of GPR54 during certain developmental windows is apparently sufficient to trigger the neuroendocrine events leading to the onset of puberty.
Notably, ontogenic analyses of GnRH and LH responses to exogenous Kp-10 in male and female rats conclusively demonstrated that, albeit with lower sensitivity, the gonadotropic system is fully capable to robustly respond to kisspeptin even at early (infantile) stages of postnatal maturation (57). Of note, important differences exist between rodents and primates in the secretory profiles of gonadotropins during infantile/juvenile stages, with primates having a sustained quiescent period characterized by very low levels of gonadotropins (372). Thus it is not possible to infer, on the basis of rat data (57), whether the gonadotropic axis of monkeys and humans may potently respond to kisspeptin stimulation during the infantile period. Yet, a clinical report has documented a case of compound heterozygote mutation of GPR54 in a boy associated with cryptorchidism and micro-penis (402), which suggests stimulatory effects of kisspeptins on GnRH/gonadotropin secretion already during the infantile period.
All in all, integration of the available experimental evidence, coming mostly from rodent studies, makes it tempting to propose a multifaceted mechanism whereby the hypothalamic Kiss1 system participates in the control of puberty onset. This is schematically presented in FIGURE 7 and is likely to include, at least, four major, related components: 1) an elevation in the endogenous kisspeptin tone, which seems to be sufficient per se to drive the GnRH/gonadotropin axis to a state of full activation; 2) an increase in the sensitivity to the stimulatory effects of kisspeptin in terms of GnRH/LH responses; 3) an enhancement of GPR54 signaling efficiency, which is apparently coupled to a state of resistance to desensitization to kisspeptin stimulation; and 4) an increase in the number of kisspeptin neurons, at the AVPV and/or the ARC depending on the species, as well as of their projections to GnRH neurons, originating mainly from the AVPV in rodents. As stressed elsewhere in this section, not all the above phenomena may be equally important in all mammalian species, and further studies are needed to fully characterize the activational roles of Kiss1 neurons in the regulation of pubertal timing in higher primates.
C. Are Kisspeptins the Trigger of Puberty?
The observations summarized in previous sections conclusively demonstrate the essential roles of kisspeptins and GPR54 in the central control of puberty. However, they do not provide definitive evidence for the hierarchical position of Kiss1 neurons within the networks governing the pubertal activation of GnRH neurons and fall short in elucidating the putative upstream regulators of the developmental changes that the Kiss1 system undergoes during puberty. Similarly, the above studies do not address the potential interactions of kisspeptins with other well-known neuropeptide regulators of puberty. Indeed, while some knowledge has been gained in these areas, most of the above issues remain ill defined to date. One of the major pending questions is whether kisspeptins are the long-sought trigger of puberty.
Studies on the factors regulating the pubertal increase of Kiss1/kisspeptin expression can help to provide a better understanding of the actual roles of this system in the control of puberty onset. However, while the potential roles of different neuropeptides and hormones in the regulation of kisspeptin signaling have been explored to some detail in adult animal models, specific analyses on the signals driving the pubertal changes of the Kiss1 system have been mostly restricted to evaluation of the roles of sex steroids, although the influence of metabolic signals has been partially explored also. Again, for obvious experimental limitations, analyses in this area are dominated by rodent studies, which may hamper direct extrapolation of these findings to other mammalian species. Nonetheless, reports from mice have demonstrated that the marked increase in kisspeptin neurons at the AVPV during puberty is highly dependent on the input of gonadal steroids, and specifically estrogen, as revealed by studies on the effects of ovariectomy, with or without estradiol replacement, in prepubertal mice, as well as in aromatase KO and hpg mice (65, 140). This situation is reminiscent of the positive feedback of estrogen on Kiss1/kisspeptin expression at this nucleus in adulthood. From a developmental standpoint, these observations would imply that an initial activation of ovarian function, as main source of endogenous estrogen, must take place in a kisspeptin-independent manner, therefore suggesting that kisspeptins are not the primus switch of puberty, at least in female rodents. In contrast, the kisspeptin system might operate as an estrogen-dependent amplifier of GnRH neurosecretory activity at puberty, which is, nonetheless, essential for normal puberty to occur (65).
In addition, these observations raise the intriguing possibility that the time-window preceding puberty might be considered as a secondary critical period, when changes in the sex steroid milieu may have durable consequences in terms of timing of puberty and, eventually, reproductive function later in life. This possibility, however, remains speculative, since testing of the effects of adult exposures to estrogen in terms of Kiss1/ kisspeptin expression in the AVPV of rodents subjected to prepubertal gonadectomy has not been conducted so far. Similarly, the actual levels of estrogen, and hence, of ovarian activation, required for the subsequent pubertal activation of the kisspeptin system, require further analysis. Finally, considering that the pubertal activation of GnRH/gonadotropin secretion in primates is largely gonadal independent (346, 372), it is arguable that a similar phenomenon of ovarian-induced activation of Kiss1 neurons may not operate in humans.
Another issue that merits investigation is to what extent sex steroids may participate in the regulation of the ARC population of Kiss1 neurons at puberty. This might be of special relevance in the male, where the subset of Kiss1 neurons of the AVPV is underdeveloped. Although more studies are needed in this front, a recent report by Mayer et al. (271), using genetically engineered female mice selectively lacking ERα in all Kiss1 cells, demonstrated that congenital elimination of estrogen actions in the ARC caused a precocious activation of Kiss1 neurons at the nucleus, due to the elimination of estrogen-mediated negative feedback. Such premature activation of ARC Kiss neurons was associated with phenotypic signs of earlier puberty, such as precocious vaginal opening. However, pubertal progression was arrested in this model, and first ovulation failed to occur, probably due to defective maturation of the Kiss1 neuronal population at the AVPV (271). These data, which need to be expanded as to control for potential developmental/compensatory events, are nonetheless indicative of the fine balance between ARC and AVPV populations of Kiss1 neurons in defining the timing of puberty, at least in rodents.
In addition to the above effects, age- or sex-dependent differences in Kiss1 responses to sex steroids might be also important in the timing of puberty. For instance, expression analyses have demonstrated that there is a significant reduction in the sensitivity to the inhibitory effect of estrogen on Kiss1 mRNA levels in the ARC during the course of puberty in rats, a developmental change which might help to release the HPG axis from the gonadal restrain during the pubertal transition (441). In addition, comparative analyses of Kiss1 mRNA changes in response to gonadectomy in male and female mice at the end of the infantile period have demonstrated that, whereas a rapid elevation in Kiss1 expression in the ARC is detected in females, male mice display a much delayed increase of Kiss1 mRNA levels. These observations suggest the existence of gonadal-independent factors restraining the pubertal activation of the Kiss1 system specifically in the male (208). While such a potential sex dimorphism has been argued to explain why females enter into puberty earlier than males (206), it must be stressed that the above phenomenon may not be present in other mammals, therefore bringing a call of caution on its overall relevance in the generation of sex-dependent differences in the timing of puberty.
VII. KISS1 NEURONS AND THE REGULATION OF GnRH NEURONS AND GONADOTROPIN SECRETION
Upon disclosure of the potential roles of the Kiss1 system in the control of the HPG axis, one of the first goals of the subsequent physiological studies was to address whether kisspeptins are able to modulate adult gonadotropin secretion, as surrogate marker of GnRH neuronal activation (374, 448). The initial demonstration of the extraordinarily potent secretagogue effects of kisspeptins on LH and FSH secretion in different species fuelled specific mechanistic studies on the major sites and modes of actions of kisspeptins on the central networks governing the HPG axis. As illustrated in the following sections, extensive efforts in this area have allowed, in a relatively short period of time, not only to characterize the physiological and pharmacological effects of kisspeptins on gonadotropin secretion in different species, but also to gain substantial insights into the mechanisms of action of kisspeptins on GnRH neurons and their potential interaction with other major central regulators of the HPG axis.
A. Kisspeptin Effects on Gonadotropin Secretion: Physiological and Pharmacological Characterization
The ability of kisspeptins to potently elicit gonadotropin secretion was documented shortly after the first reports on the reproductive consequences of GPR54 inactivation in humans and mice in late 2003. Indeed, already by the end of 2004, at least four groups had independently reported the potent stimulatory effect of Kp-10 and Kp-54 on LH secretion in the rat and mouse (149, 268, 298, 464). Those initial studies were soon confirmed and extended to other relevant mammalian species, such as the sheep, monkey, and human (101, 279, 280, 404). To date, the stimulatory effects of different molecular forms of kisspeptins on gonadotropin secretion have been documented in numerous mammals, including, in addition to those mentioned above, goats, pigs, and cows (121, 164, 242). As a whole, the pharmacological studies conducted so far strongly suggest that kisspeptins are able to elicit LH and FSH secretion both in males and females (101, 102, 299, 300), an effect that is already detected at early stages of postnatal development, including the infantile and/or juvenile periods in the rat, mouse, and monkey, although in the latter, only the juvenile phase has been tested (57, 161, 349). In addition, the stimulatory effects of kisspeptins have been detected in different physiological states of the gonadotropic axis, such as various phases of the ovarian cycle, pregnancy, lactation, and ageing (311, 380, 505). Furthermore, the fact that kisspeptins appear astonishingly potent via different routes of administration, both centrally [intracerebroventricular (icv) and intrahypothalamic] and systemically [intravenous (iv), intraperitoneal (ip), and subcutaneous (sc)] (268, 298, 300, 333, 336, 476), further emphasizes the robustness and potential applied interest of the effects of kisspeptins on gonadotropin secretion, both in terms of translational medicine and animal production.
As delineated by rodent, sheep, and primate studies, there are several remarkable features in the gonadotropic effects of kisspeptins, including its protracted duration and sensitivity. As a clear example, studies in rats have documented a greater than 3-h elevation of circulating LH levels following icv injection of 0.1–1 nmol doses of Kp-10 and Kp-52 (300, 336). Similarly, peripheral administration of Kp-10 and Kp-54 has been shown to elicit durable, although less protracted, LH responses in rats, sheep, monkeys, and humans (49, 101, 102, 300, 404, 476), with longer secretory responses being observed after iv administration than after other routes of peripheral injection (300, 476). Systematic comparison of the releasing effects of shorter (Kp-10) and longer (Kp-52, Kp-54) forms of kisspeptins in vivo has demonstrated greater LH secretory responses to the full-length peptides, probably reflecting a higher half-life and resistance to degradation (281, 336, 476). The threshold doses for the stimulatory effects of kisspeptins are very low, as already demonstrated by initial studies where doses of Kp-10 between 100 fmol-1 pmol (central administration), and ∼0.5 μg/kg (peripheral administration), were sufficient to evoke robust LH secretory discharges in the rat and monkey (149, 300, 349, 476). Similarly, doses of Kp-54 in the femtomolar range elicited detectable LH responses in mice (149). This combination of high sensitivity and duration in terms of gonadotropin responses defines kisspeptins as outstanding activators of the HPG axis. Indeed, the integral analysis of the published data on the LH-releasing effects of kisspeptins and other neuropeptide regulators of gonadotropin secretion evidences that, except for GnRH itself, kisspeptins are likely the most potent stimulators of LH secretion identified so far in mammals (385, 448).
While the studies conducted to date have conclusively demonstrated the ability of kisspeptins to stimulate the secretion of both gonadotropins, detailed pharmacological analyses carried out mainly in rodents have also documented differences in the patterns of LH and FSH responses to kisspeptin stimulation, even though some species differences may exist. Thus studies in the male rat have demonstrated that, compared with the rapid (within 5 to 15 min) and robust (up to 10-fold increase over basal levels) responses in terms of LH release, FSH secretion following icv injection of Kp-10 was somewhat delayed (from 30 min onwards) and of lower relative magnitude (∼2-fold increase) (299). Moreover, FSH responsiveness to Kp-10 in vivo seems to be ∼200-fold less sensitive than LH, with an EC50 of ∼2 and 400 pmol for LH and FSH, respectively (299). Although less detailed than in rodents, analogous dose-response analyses in humans also suggest that relative LH responses to kisspeptin are more robust (e.g., in terms of fold increase and threshold doses) than FSH ones (101, 102, 139). Some of these differential features of LH and FSH responses to kisspeptins, as delineated by rodent studies, are illustrated in FIGURE 8.
Several mechanisms might theoretically account for the above phenomenon, including 1) differences in the pattern of secretion between both gonadotropins, which is in general more constitutive for FSH than for LH (274); 2) differences in the eventual effects of kisspeptins on the patterns of GnRH release (as examples, see Refs. 82, 139), as the predominant activation of a profile of high-frequency GnRH pulses may preferentially induce LH secretion; and/or 3) differences in the regulatory actions of peripheral factors, mainly gonadal peptides, such as inhibins, that selectively regulate FSH secretion (42, 94). In any event, besides their physiological interest, the above observations may have pharmacological implications, regarding the design of protocols of selective activation of LH secretion. Of additional interest, studies in rodents suggest that the patterns of FSH responses to Kp-10 diverge between male and female rats so that male responses are delayed and more durable, whereas female responses are rapid and of shorter duration. Such a sex dimorphism seems to depend on differences in the gonadal steroid milieu (see FIGURE 9).
As good complement of the pharmacological studies summarized above, the recent development of peptide antagonists has allowed to initiate the characterization of the physiological roles of endogenous kisspeptins in the control of gonadotropin secretion. Extensive biological tests in diverse animal species, including the monkey, sheep, mouse, and rat, by a combination of in vitro and in vivo approaches and assessment of different end-points, from electrophysiological recordings to various hormonal targets, have documented the suitability of these compounds to suppress kisspeptin signaling in vivo (384). Somewhat unexpectedly, however, results from these studies have jointly suggested that, even though blockade of kisspeptin effects has an inhibitory impact on GnRH neuronal activation and GnRH pulsatility, as well as on different activational events of the gonadotropic axis, such as the response to gonadectomy and the preovulatory surge of gonadotropins, the different protocols of kisspeptin antagonism do not evoke a consistent decrease in the basal levels of circulating gonadotropins in the various species tested (384). Admittedly, the possibility of incomplete receptor blockade cannot be ruled out, especially since some degree of variability in terms of antagonism effectiveness has been detected for the different compounds among various laboratories. Yet, this is unlikely to be the sole explanation for this phenomenon given its repeated observation in numerous species and under different experimental conditions. Thus the above findings might be indicative of a genuine physiological role of kisspeptins, as preferentially involved in the generation of specific stimulatory phenomena of the HPG axis but not in the maintenance of basal gonadotropin levels. This possibility is further supported by the detection of residual GnRH pulsatility and gonadotropic activity in models of inactivation of GPR54 and Kiss1 (81, 460).
Finally, another aspect of the pharmacology of kisspeptins that has received quite some attention is the possibility of desensitization of gonadotropin responses following repeated or continuous exposure. Initial studies demonstrated that iv administration of intermittent boluses of Kp-10 to juvenile monkeys (1 every hour for 48 h), male rats (1 every 75 min for 5 h), or ewes (1 every hour for 6 h) evoked a sustained discharge of conserved LH pulses (49, 349, 476), suggesting that, under optimal conditions, kisspeptins may be used for the durable activation of the gonadotropic axis. Notwithstanding these observations, protocols of continuous infusion, initially reported in juvenile and adult monkeys, evidenced that, after an early phase of robust and sustained hypersecretion, LH responses faded away despite persistent administration of kisspeptins (365, 399). An analogous phenomenon has been also demonstrated in rats. Thus chronic sc administration of Kp-54 to adult male rats induced a transient LH releasing effect, which was no longer detectable on the second day of infusion (463). Similarly, in adult female rats, the stimulatory effects of icv infusion of Kp-10 disappeared after 48 h of the beginning of treatments (381).
Mechanistic studies in monkeys suggested this is a classical desensitization phenomenon at the level of Gpr54, since LH responses to Kp-10 were lost, but those to the glutamate agonist NMDA and GnRH were preserved, during the desensitized state (i.e., on day 4 after beginning of infusions) (399). Similarly, electrophysiological studies in mice evidenced desensitization of Gpr54 upon kisspeptin stimulation of GnRH neurons (110, 250). Hormonal studies in female rats, however, have pointed out that the process whereby gonadotropin responses to kisspeptins are desensitized in vivo might involve additional components, such as changes in the patterns of GnRH secretion in response to sustained kisspeptin stimulation, since the time course for desensitization was significantly different between LH and FSH (LH faster than FSH), and depended on the prevailing functional state of the HPG axis (381). Thus conditions such as the developmental stage and the metabolic status had a clear-cut impact, as desensitization was significantly attenuated in pubertal animals and during fasting (381). Similarly, the dose and duration of kisspeptin administration are determinant for the manifestation of desensitization events, as revealed by very recent studies involving Kp-10 infusion in men (139). In any event, it is important to stress that, while the loss of gonadotropin responses to continuous kisspeptin administration has been well characterized in various species, to our knowledge none of the protocols of desensitization reported to date has been shown to cause a sustained suppression of basal gonadotropin secretion. Pending further pharmacological characterization, this phenomenon is apparently in contrast to the well-known effects of chronic administration of GnRH agonists (78, 248), and may be indicative of the specific features of kisspeptins in the differential control of basal versus stimulated gonadotropin secretion.
B. Kisspeptin Actions on GnRH Neurons
The initial demonstration of the potent gonadotropin releasing effects of kisspeptins prompted mechanistic studies addressing their major site(s) of action. As primary suspect, it was hypothesized that the actions of kisspeptins are conducted via activation of GnRH neurons at the hypothalamus (see FIGURE 4). A wealth of experimental data, obtained mainly in rodents, sheep, and primates, have firmly supported this contention. Thus the effects of kisspeptins in terms of gonadotropin secretion are completely eliminated by pretreatment with GnRH antagonists, as demonstrated in rats, mice, and monkeys (149, 268, 299, 300, 404). In addition, as summarized in section IVB, GnRH neurons do express the Gpr54 gene (161, 169, 186). In good agreement, kisspeptins are able to induce the expression of c-fos, as a marker of early cellular activation, in GnRH neurons in rodents (186, 268). Likewise, it has been demonstrated that kisspeptins can evoke very potent depolarization responses in GnRH neurons, as measured by voltage recordings in hypothalamic slices from GnRH-GFP mice (110, 161, 250, 337, 514), and induce the release of GnRH both ex vivo (by rat hypothalamic explants) (57, 58, 464) and in vivo (to the cerebrospinal fluid in the sheep) (280). Of note, dose-response analyses in GnRH neurons and hypothalamic explants, using depolarization or GnRH secretion as end-points, have documented similar profiles for the stimulatory effects of kisspeptins, with EC50 in the low nanomolar range (58, 110). Finally, further circumstantial evidence for a direct effect of kisspeptins on GnRH neurons came from studies using cell lines parentally related with GnRH neurons, such as GT1–7, where expression of Gpr54 gene has been documented, and kisspeptins have been shown to activate intracellular responses, as well as GnRH gene expression and secretion (189, 316, 354). The relevance of the latter model, however, is tempered by the apparent variability in GT1–7 responses to kisspeptins among different research groups, some of which have been unable to demonstrate any consistent stimulatory effects (310).
The molecular and electrophysiological mechanisms whereby kisspeptins activate GnRH neurons and, hence, induce GnRH secretion have been also exposed. As indicated in section III, studies using hypothalamic explants and isolated GnRH neurons have documented the ability of kisspeptins to stimulate GnRH secretion and to induce strikingly potent, durable depolarizing responses in these neurons, which are associated with the activation of a PLC-Ca2+ pathway. Such depolarization is brought about mainly by the concerted closure of inwardly rectifying K+ channels and the opening of Na+-dependent, nonselective cationic channels, which mainly include canonical transient receptor potential channels (TRPC), some of whose subunits have been shown to be expressed in kisspeptin-responsive GnRH neurons (250, 514). The unusually prolonged depolarizing response of GnRH neurons to kisspeptins appears to depend on mechanisms intrinsic to this neuronal population. On the other hand, electrophysiological recordings allowed the identification of two distinct GnRH neuronal populations in the septal area of mice: one which is highly sensitive to the stimulatory effects of kisspeptins and another that is insensitive to kisspeptin, but responsive to the activation of metabotropic glutamate receptors (110). The physiological role of the latter in mediating potential kisspeptin-independent, hypophysiotropic effects of GnRH remains unclear and has begun to be investigated only recently (134).
Considerable efforts have been devoted recently to elucidate the precise neuronal pathways whereby Kiss1 neurons impinge on and activate GnRH neurons. Taking into account the neuroanatomical features of Kiss1 neurons and their projections known to date, as well as the well-established distribution of GnRH neurons, it has been proposed that the major direct kisspeptin afferents received by GnRH neurons originate from the AVPV, in rodents, or the POA, in sheep (20, 66). In contrast, direct projections of ARC Kiss1 neurons onto GnRH neurons had remained elusive, although a very recent report has documented that ∼20% of ARC Kiss1 neurons project to the rostral POA in mice, and hence may directly innervate GnRH neurons, which are predominantly located at this site (510). In fact, even before such anatomical evidence was obtained, it had been assumed that ARC neurons participate in the regulation of GnRH secretion either by interneuronal pathways, of as yet unknown nature, or by direct actions at the level of GnRH nerve terminals located at the median eminence (91). In fact, pharmacological tests showing rapid LH responses to kisspeptins after their peripheral administration are compatible with this latter site of action (476). Moreover, direct effects of kisspeptins on GnRH nerve terminals have been documented by the use of mediobasal hypothalamic explants in mice (92). Of note, the overall number of kisspeptin appositions received by GnRH neurons, regardless of their location, seems to be rather modest. This feature is suggestive of predominant interneuronal or even nonsynaptic forms of communication between Kiss1 and GnRH neurons (269). Further mapping of the actual distribution of GPR54 within the anatomy of GnRH neurons will aid to solve the above uncertainties on the preferential sites of action of kisspeptins.
Another issue that has attracted considerable attention is whether kisspeptin neurons might be a relevant component of the so-called GnRH pulse generator, a functional entity described in section I responsible for driving the pulsatile release, in terms of frequency and amplitude, of GnRH. This possibility is supported not only by the pharmacological profile of kisspeptins, as extraordinarily potent activators of GnRH/gonadotropin release, but also by recent anatomical and functional data in monkeys and rodents. On the former, Kiss1 neurons have been shown to be predominantly sited in an area corresponding to the posterior two-thirds of the ARC within the MBH, which was demonstrated by classical lesion studies to be the putative location for the GnRH pulse generator in primates (362). In addition, studies using a selective antagonist evidenced a sustained suppression of GnRH neuronal activation and secretory function following blockade of kisspeptin signaling in mice and monkeys (384). Moreover, selective infusion of this antagonist into the ARC significantly suppressed GnRH pulsatility in female rats, further supporting the potential involvement of Kiss1 neurons in the control of the pulsatile GnRH release (246). As a note of caution, one report using high doses of Kp-10, administered iv, could not identify any discernible effect of kisspeptins on the frequency of GnRH pulse generator, as indirectly measured by recording of multiunit electrical activity volleys in the ARC (219). Anyhow, recent studies in goats and mice have further documented the potential role of ARC Kiss1 neurons, which coexpress also NKB and Dyn, in the precise control of the pulsatile release of GnRH (304, 491).
C. Hierarchy and Interactions of Kiss1 Neurons With Other Afferent Regulatory Pathways
The recognition of the dominant roles of kisspeptins in the control of the gonadotropic axis has made it mandatory to revisit the proposed mechanisms and neuronal pathways whereby other central and peripheral signals regulate the GnRH/gonadotropin system. Efforts in this front will allow defining the hierarchy and eventual interplay of kisspeptins and other relevant input signals controlling GnRH neurons. Indeed, this particular area of kisspeptin physiology has gained considerable attention recently due to the identification of NKB and Dyn as potential partners and coreleased transmitters of Kiss1 neurons, a phenomenon that, due to its potential relevance and growing interest, will be specifically covered in the following section. In turn, we will concentrate here on the potential relationships of kisspeptins and other critical regulators of the HPG axis, such as glutamate, GABA, and the RF-amide gonadotropin-inhibitory peptides RFRPs.
Initial attempts to elucidate the hierarchy and potential interactions between kisspeptinergic and glutamatergic neurotransmission in the control of gonadotropin secretion involved the testing of the gonadotropin-releasing effects of kisspeptins following blockade of ionotropic glutamate receptors, of the NMDA and non-NMDA types, as well-known elements in gonadotropic control. Those studies evidenced that gonadotropin responses to high doses of Kp-10 were preserved despite effective inhibition of glutamate receptors (299, 300). These observations suggested that either these two pathways are independent, as supported by the presence of GPR54 and ionotropic glutamate receptors in GnRH neurons, or that kisspeptins are downstream of glutamate afferents to GnRH neurons. In keeping with the latter, NMDA has been recently shown to induce bursting activity in Kiss1 neurons, as revealed in Kiss1-CreGFP knock-in mice (151). Of note, those initial pharmacological tests involved the use of high doses of Kp-10 (1 nmol icv), which might have obscured more subtle regulatory phenomena. Indeed, we have recently obtained evidence that blockade of NMDA and non-NMDA receptors partially blunts LH responses to lower, submaximal (50 pmol icv) doses of Kp-10 in the rat (Pinilla and Tena-Sempere, unpublished data), an observation that suggests a potential interplay between these two critical regulators of GnRH secretion.
In agreement with the latter, electrophysiological studies have demonstrated that blockade of fast synaptic transmission, as a way to eliminate ionotropic glutamatergic (and GABAergic) inputs resulted in a decrease of GnRH neuronal responses to kisspeptin (337). Collectively considered, these observations suggest that at least part of kisspeptin effects on GnRH neurons might be indirectly mediated via activation of glutamate (and/or GABA) afferents to GnRH neurons. In favor of this possibility, recent work has documented that, in addition to direct postsynaptic effects on GnRH neurons, Kp-10 is able to enhance glutamatergic and GABAergic transmission to these neurons, acting in a presynaptic manner (338). It must be stressed, however, that since direct, independent effects of kisspeptins on GnRH neurons are well documented, it remains possible that blockade of one pathway (e.g., glutamate) may impair GnRH responsiveness to the other. Indeed, the relative importance of indirect versus direct effects of kisspeptin in the physiological control GnRH neurons is not sufficiently clarified and requires further analyses.
In this line, recent studies using Gpr54 KO mice have suggested that at least part of the excitatory effect of glutamate on GnRH neurons, acting via NMDA receptors presumably at GnRH cell bodies, is independent of kisspeptin signaling, whereas its stimulatory actions at GnRH nerve terminals require the presence of functional Gpr54 (90). Whether this implies that glutamate activates kisspeptins afferents to the median eminence or that preserved kisspeptin signaling is permissive for direct glutamate actions at this levels is yet to be elucidated. As an additional note, a recent study identified in the mouse a subpopulation of septal GnRH neurons that on the basis of electrophysiological recordings appears to be insensitive to kisspeptins but highly sensitive to agonists of type I metabotropic glutamate receptors (110). Taken together, the above observations suggest that whereas ionotropic glutamate and kisspeptin pathways may display some degree of bidirectional interactions in the control of GnRH secretion, metabotropic glutamate pathways may operate on kisspeptin-independent circuits, as recently confirmed in Gpr54 KO mice in vivo. It must be stressed, though, that the functional features of the subset of GnRH neurons in the septum might differ from those of the POA, as evidenced by comparative analyses of kisspeptin responses during puberty (110, 161).
Evidence for the interaction between GABA and kisspeptin neurotransmission in the control of GnRH neurons has been also presented. The nature of this interaction, however, is confounded by the fact that, depending on the experimental conditions and receptors activated, GABA can be excitatory or inhibitory for GnRH neurons (88, 168, 170, 321). In this sense, recent studies have described that activation of GABA-B receptors hyperpolarizes GnRH neurons and that this effect can be blocked by submaximal doses of Kp-10, as potential mechanism for part of the stimulatory actions of kisspeptins (513). Conversely, several studies have documented the ability of GABA to excite GnRH neurons under specific conditions, with a detectable increase in GABA transmission at key physiological states, such as the preovulatory surge, and discernible modulatory roles of sex steroids and metabolic cues upon GABA inputs to GnRH neurons (88). This putative excitatory action is compatible with the reported increase induced by kisspeptin on the frequency and amplitude of GABAergic postsynaptic currents in GnRH neurons (338). While the mechanisms subserving this apparent switch between stimulatory or inhibitory actions of kisspeptins on GABA transmission are unknown, the above data support the possibility of dynamic interactions between Kiss1 and GABA pathways in the control of GnRH secretion.
In addition to classical neurotransmitters, such as glutamate and GABA, considerable attention has been focused recently in elucidating the eventual interactions between kisspeptins and RFRPs, as mammalian orthologs of the GnIH cloned in avian species, whose function as putative gonadotropin-inhibitory signal in mammals has been documented now by a number of independent studies in different species (63, 340, 419, 479). While systematic review of these studies is beyond the scope of this review, they collectively point out that RFRP is an inhibitory signal for the gonadotropic axis, acting probably at both central (likely on GnRH neurons) and pituitary levels. The structural and functional commonalities between kisspeptins and RFRP, as both belong to the superfamily of RF-amide peptides and are involved in the control of the HPG axis, albeit with opposite actions, made it tempting to propose that it is the dynamic balance and interplay between these two sets of factors that drives the function of the reproductive system (231). Admittedly, however, the integral role of this tandem in the central control of reproduction has not been fully substantiated in mammals, especially because the magnitude and physiological relevance of RFRP effects on the gonadotropic axis appear to be much more modest than those of kisspeptins. Nonetheless, the available evidence is compatible with an anatomical and functional interplay between the above inhibitory and stimulatory pathways in the control of some aspects of reproduction in mammals.
Morphological evidence and expression data support the putative interplay between Kiss1 and RFRP systems in different species. In this sense, studies in the sheep have documented an inverse correlation between the expression levels of Kiss1 and RFRP mRNAs in the ARC and DMN, respectively, during the breeding season, when the number of appositions to GnRH neurons also changes inversely: increased number for Kiss1 neurons, decreased for RFRP neurons (421). These observations may suggest that, in this seasonal breeder, kisspeptins and RFRP reciprocally interact to regulate GnRH neuronal activity. Such interplay may involve not only direct, opposite actions at the level of GnRH neurons, but might also include reciprocal regulatory effects in terms of expression or biological actions. Thus studies in rats have recently documented prominent expression of the canonical receptor for RFRP, GPR147, in the AVPV, which might be compatible with a modulatory role of RFRP on Kiss1 neurons in this area (357), yet this possibility remains to be proven. In addition, functional electrophysiological analyses have documented the ability of RFRP-3 to inhibit the excitatory actions of kisspeptins on POMC neurons (131). Finally, while the differences in the magnitude of absolute LH responses to kisspeptins and RFRPs have precluded direct analysis of their coadministration in vivo, combined injection of Kp-10 and the antagonist of RFRP receptor, RF9, modestly increased the responses to kisspeptin alone (341). Altogether, the above data strongly suggest the possibility of interactions between kisspeptins and RFRP in the control of the HPG axis.
In spite of the above evidence, the physiological relevance of such interplay is toned down by a number of observations. First, several reports have failed to document reciprocal changes between Kiss1 and RFRP expression; for instance, during the late follicular phase in the monkey or the pubertal period in the rat, both transcripts are upregulated at the hypothalamus (357, 429). Second, while the involvement of kisspeptins in key reproductive phenomena, such as puberty onset or the preovulatory surge of gonadotropins, is well documented, the roles of RFRP pathways in those events appear to be much more modest, if any (63, 419). Third, differences seem to exist in the major sites of actions of kisspeptins and RFRP, as the latter appears to operate, at least partially, via inhibition of GnRH actions directly at the gonadotrope level (64, 340). Finally, it remains possible that, in contrast to the well-conserved functions of kisspeptins, the role of RFRPs in the control of reproduction in mammals may vary across species, with a more prominent role in seasonal breeders (63, 419). In this context, it is interesting to note that GnIH was first identified and seems to play a prominent regulatory role in birds, where the kisspeptin system has been apparently eliminated during evolution (479). This may suggest that coevolution of both systems is under opposite forces, with a reciprocal equilibrium between the dominant roles of kisspeptins or GnIH/RFRP, depending on the species and its reproductive strategy.
VIII. NEUROKININ B AND DYNORPHIN AS COTRANSMITTERS OF ARC KISS1 NEURONS
Among the array of different neuropeptides that potentially interplay with kisspeptins in the control of the HPG axis, NKB has been, by far, the one that has attracted most of the attention recently. NKB belongs to the family of tachykinins, which includes also neurokinin A (NKA), substance P, and hemokinin-1, as well as neuropeptides K and γ (7). Three tachykinin receptors have been identified so far: NK1R, NK2R, and NK3R; NKB preferentially activates NK3R, which is considered the canonical NKB receptor (367). The gene encoding NKB is named TAC3 in humans and Tac2 in rodents, whereas the NK3R is encoded by the TACR3/Tacr3 gene (367). Although the roles of NKB and other tachykinins in the regulation of gonadotropin secretion had been previously addressed in various species (200, 367, 389), interest on the participation of NKB and NK3R in the control of the HPG axis has grown considerably recently, due mainly to two almost coincident findings: 1) the initial demonstration in the sheep that the population of Kiss1 neurons located at the ARC actually coexpress NKB, as well as the endogenous opioid peptide, Dyn, as reported in late 2007 (145); and 2) the observation published in late 2008 that inactivating mutations in TAC3 and TACR3 in humans are associated with HH (475), a phenotype similar to that previously reported for GPR54 mutations. These observations collectively pointed out the important physiological role of ARC neurons expressing kisspeptins and NKB, as well as Dyn, in the control of mammalian reproduction, and boosted a number of studies on the anatomical distribution, regulation, and functional relevance of this neuronal population.
A. Neuroanatomical Evidence
As indicated above, the first anatomical evidence for the coexpression of the three neuropeptides was first published in 2007, when Goodman et al. (145) documented that the previously characterized population of Kiss1 neurons of the ARC in the sheep contained also NKB and Dyn. Such coexpression appears to be a specific feature of this set of ARC neurons, which were subsequently proposed to be named KNDy neurons to recognize their ability to produce the three neuropeptides (241). Thereafter, the presence of such KNDy neurons has been documented by a combination of in situ hybridization and IHC techniques in the mouse, goat, and monkey, although in the latter only NKB coexpression was analyzed (304, 305, 363, 491). Likewise, the presence of NKB has been recently demonstrated in as much as 77% of all kisspeptins neurons of the infundibular nucleus in humans (180). Notably, not only the cell bodies but also fiber projections from KNDy neurons coexpress these peptides, as demonstrated in the sheep, where triple-labeling analyses have identified the joint presence of kisspeptin-, NKB- and Dyn-IR, as well as in monkeys and humans (180, 241, 363).
Detailed neuroanatomical studies have initiated the mapping of KNDy fiber distribution and NK3R expression within the mammalian brain. Regarding fiber projections, IHC analyses in the sheep have documented the presence of KNDy fibers projecting to GnRH cells bodies in the POA (241), although the global existence of ARC projections to the POA has also been questioned in this species (19). In addition, there is evidence that KNDy neurons project to the median eminence in the sheep and monkey, where they appear to establish some contacts with GnRH nerve terminals (241, 363). The situation in rodents is less clear; an initial IHC study published in 2005, hence, before the recognition of the existence of KNDy neurons, documented the presence of abundant NKB fibers in the median eminence in rats, where they appeared to make contact with GnRH nerve terminals (229). However, subsequent studies of coexpression of Kiss1 and NKB have been based on in situ hybridization analyses and, therefore, did not allow evaluation of fiber projections.
Concerning the receptor, initial IHC analyses in the rat reported the presence of NK3R-positive GnRH axons at the median eminence, therefore providing the basis for potential direct actions of NKB on GnRH nerve terminals at this site (229). In line with these findings, Tacr3 mRNA has been detected in mouse GnRH neurons (466). Previous IHC analyses, however, showed that only a small fraction of GnRH somata expressed this receptor in the rat (229). More recently, IHC analyses in the sheep have demonstrated the existence of numerous NK3R-IR neurons and fibers in the POA and various hypothalamic regions including the ARC (9). Importantly, dual-labeling studies concluded that NKB neurons in the ARC also expressed NK3R in this species (9). In good agreement, expression of Tacr3 mRNA has been demonstrated in ARC KNDy neurons in the mouse (304, 305). However, IHC analyses in the sheep showed no detectable coexpression of GnRH and NK3R, neither at the level of cell bodies (POA) nor at the median eminence (9). The latter is in contrast to the data previously obtained in rats and might reflect either species (sheep vs. rat) or methodological differences between studies. Yet, recent in situ hybridization studies in the mouse have failed to demonstrate detectable expression of Tarc3 in GnRH neurons, which did not respond to NKB agonist either (305). From a mechanistic perspective, it will be critical to solve whether or not there is meaningful expression of NK3R in rodent GnRH neurons.
In addition, neuroanatomical analyses have disclosed two additional interesting features of the KNDy neuronal population in the ARC that are likely to have important functional implications. First, KNDy neurons appear to be profusely interconnected, as indirectly suggested by earlier studies in the sheep and rat where expression of NKB or Dyn was taken as marker of this population (43, 230, 241). Second, studies in the sheep have indicated that virtually all KNDy neurons express ERα, as well as progesterone (PR) and androgen (AR) receptors, therefore suggesting an integral role of this neuronal population, and hence of NKB, in the feedback control of gonadotropins (241). Similarly, studies in the human, monkey, and rodent have documented the expression of ERα in neurons expressing kisspeptin, NKB, and/or Dyn (43, 366, 367). Finally, it is important to stress that, while the coexpression of the three neuropeptides is considered an important functional signature of this set of neurons, it is possible that this population expresses another neuropeptides and transmitters (241), which may cooperate in the regulation of GnRH neurons or another function(s). Likewise, it is highly plausible that NKB neurons, not expressing Kiss1/kisspeptins, exist in the ARC, at least of rodents (Bentsen and Mikkelsen, personal communication).
B. Functional Evidence
Although NKB had been previously involved in the control of the HPG axis in mammals (367), its physiological importance did not gain general recognition until the association between inactivating mutations in TAC3/TACR3 and human cases of HH was first reported (475). The later observations drew attention to previous functional studies, addressing the roles of NKB in the control of gonadotropin secretion, and prompted new analyses to document the effects of NKB in the regulation of GnRH neurons and its potential interplay with kisspeptins and other cotransmitters, such as Dyn.
The analysis of the available data on the effects of NKB, or the agonist of NK3R, senktide, on LH secretion yields contradictory observations, with either null, inhibitory, or stimulatory actions being reported. Thus senktide administration to ovariectomized (OVX) mice or rats, without hormonal replacement or very low (or ineffective) estrogen substitution, resulted in inhibition of LH secretion (217, 304, 389), an effect that seems counterintuitive with the reported state of hypogonadotropism in humans with genetic inactivation of the NKB system. In addition, NKB was ineffective in altering LH secretion in adult male mice (73), although a recent report documented robust gonadotropin responses following icv administration of the NK3R agonist senktide in male mice (305). In line with the later study, compelling evidence has been presented for the potent stimulatory effects of NKB and/or senktide on LH secretion in the adult ewe and juvenile monkey (31, 363), with the stimulatory effects in the ewe being detected during the follicular phase of the cycle. In the same vein, we have recently observed robust LH releasing responses to icv injection of senktide in cyclic female rats and OVX rats with physiological supplementation of estradiol (301). The effects of senktide in the monkey were abrogated by pretreatment with a GnRH antagonist (363).
Altogether, the above findings demonstrate that, under appropriate physiological conditions, NKB signaling can exert potent stimulatory effects on GnRH secretion, thereby activating LH secretion. However, these observations also illustrate that the stimulatory actions of NKB are less universal than those of kisspeptins and might depend on several physiological parameters, such as the sex steroid milieu and early brain differentiation, endogenous GnRH secretory activity, and developmental stage. There might exist also important species differences, as indirectly supported by initial reports showing that, in contrast to humans, mice with inactivating mutations of Tacr3 gene appeared to have preserved fertility (415). However, different reproductive deficits have been very recently disclosed by detailed analyses in male and female mice from a novel Tacr3 KO line (508). In any event, such phenotype, ranging from preserved reproduction to subfertilty in Tacr3 null rodents, is in contrast to the consistent reproductive deficits found in humans and mice with inactivating mutations of GPR54. Finally, part of the above discrepancies may stem from the use of NKB, which binds also with lower affinity to other tachykinin receptors, or senktide, which is a selective NK3R agonist (363; see also Refs. 70, 305). In this sense, considering that the reproductive NKB pathways have been suggested to preferentially operate via NK3R, the use of senktide would provide a higher degree of specificity in physiological hormonal studies.
The additional presence of Dyn in KNDy neurons has prompted also functional analyses of the gonadotropic effects of this neuropeptide. Dyn is a member of the family of endogenous opioid peptides encoded by the prodynorphin gene that acts via the kappa opioid receptor (KOR) (509). However, in contrast to NKB, whose role in the control of gonadotropin secretion had been only partially characterized previously, solid evidence had set the contention that Dyn, as is the case for other endogenous opioids, is an inhibitor of gonadotropin secretion (144, 509). In good agreement, Dyn has been recently shown to inhibit LH secretion in mice and to suppress multiunit activity (MUA) volleys, as surrogate markers of GnRH pulses, and pulsatile LH secretion in goats (304, 491). In the same line, we have obtained evidence in the cyclic female and the young adult male rat that Dyn moderately inhibits basal LH secretion and LH responses to Kp-10 (Pinilla and Tena-Sempere, unpublished data).
From a physiological perspective, the question arising from the previous observations is whether and how the three neuropeptides, kisspeptin, NKB, and Dyn, as produced by a single neuronal population, engage into the mechanisms responsible for the control of pulsatile GnRH secretion. Recent studies in mice and goats strongly suggest that NKB and Dyn may operate, likely in a yin-yang manner, as positive and negative modifiers of the pulsatile release of kisspeptins by KNDy neurons at the ARC (304, 491). According to this model, which is supported by the anatomical demonstration of dense interconnections among KNDy neurons and the recent hormonal data on the effects of senktide on LH secretion in the sheep, goat, monkey, and rat (see above), NKB and Dyn would act autosynaptically on ARC KNDy neurons, which in turn would project to and activate GnRH nerve terminals at the median eminence, using kisspeptin as output neuropeptide effector. Further support to this autoregulatory mode of action comes from the finding that senktide, as agonist of NKB, is capable to activate Kiss1-expressing neurons in the ARC of female rats (301, 305), whereas its ability to stimulate LH secretion was totally prevented in Gpr54 KO mice (134). In the same vein, recent evidence suggests that the stimulatory actions of NKB on GnRH secretion take place upstream of Gpr54 also in the monkey (364). This hypothetical model, which is tentatively depicted in FIGURE 10, integrates a wide diversity of functional and anatomical observations, yet whether it applies to different mammalian species and both sexes awaits further confirmation. Likewise, it remains to be solved whether the actions of NKB and Dyn are conducted autosynaptically (i.e., on the very same neuron releasing the neuropeptides) or upon adjacent and/or contralateral KNDy neurons in the ARC, as this neuronal population is profusely interconnected (367).
IX. KISS1 NEURONS AS CONDUITS FOR THE NEGATIVE-FEEDBACK CONTROL OF GONADOTROPIN SECRETION
In parallel to the analyses of the gonadotropin-releasing effects of kisspeptins in adult animals, initial studies already addressed whether the expression and function of the hypothalamic Kiss1 system may be under the control of sex steroids, as major regulators of the gonadotropic axis. These studies, which are summarized in the next two sections, not only set the contention that Kiss1/kisspeptin expression in the adult hypothalamus is sensitive to sex steroids, but allowed also to surface important divergences in terms of regulation and physiological roles of the different sets of kisspeptin neurons (see FIGURE 11). The molecular basis for the sex steroid control of Kiss1 expression, the putative roles of sex steroids as modulators of kisspeptin responsiveness, and the mechanisms for the generation of the preovulatory surge are also discussed below.
A. Anatomical and Expression Data
The first evidence for the potential regulation of the hypothalamic Kiss1 system by sex steroids came from rodent studies in models of gonadectomy (GNX), with or without sex steroid replacement. Thus elimination of gonadal factors in adult male and female rats evoked a robust increase in Kiss1 mRNA levels in whole hypothalamic fragments, as detected by semiQ RT-PCR (298). These responses in terms of mRNA expression closely paralleled the expected changes in circulating gonadotropin levels, therefore suggesting a possible causative association (298). Protocols of sex steroid replacement further confirmed the functional relevance of these changes, as testosterone supplementation to GNX males and estradiol treatment in GNX females reversed both the hormonal (LH and FSH) and gene expression (Kiss1) responses to gonadectomy (298). These data were rapidly refined by in situ hybridization analyses, which confirmed the elevation of Kiss1 mRNA levels following GNX in male rats, as well as in male and female mice (186, 422, 423). Importantly, these studies were able to map the above changes to the ARC, where Kiss1 mRNA levels were consistently increased after GNX and suppressed following sex steroid replacement (422, 423).
These observations in rodents have been later confirmed and extended in a variety of mammalian species, including the sheep, pig, monkey, and human, where expression of Kiss1 in the ARC or equivalent infundibular nucleus has been shown to increase in conditions of low/absent sex steroid input from the gonads, such as GNX or menopause, with the latter in primates only (215, 383, 420, 468). Collectively considered, these findings point out that the subset of Kiss1 neurons in the ARC/MBH is an important node for the integration of sex steroid signals responsible for negative feedback, and for their transmission to GnRH neurons. Of note, limited attention has been paid so far to evaluate potential changes in GPR54 expression in models of GNX, with or without hormonal replacement, although the available evidence suggests a modest regulation (298). This supports the contention that sex steroid control of the Kiss1 system at the transcriptional level is mainly conducted via modulation of the ligand.
The roles of the different sex steroids in the regulation of Kiss1 expression at the ARC, and the receptors putatively involved in such control, have been also studied, mostly in rodents. As a whole, these analyses have documented that estrogen and testosterone are able to downregulate Kiss1 mRNA expression at the hypothalamus, and more specifically in the ARC, of male and female rats and mice (298, 305, 422, 423). Similarly, the ability of testosterone and/or estradiol to suppress Kiss1 mRNA levels at the ARC has been reported in the sheep and monkey (408, 420). Intriguingly, the effects of progesterone, a key sex steroid signal for feedback regulation in the female, have been scarcely addressed to date. Yet, studies in the sheep demonstrated a partial inhibition of Kiss1 mRNA levels after progesterone treatment (420), whereas in the rat no effect was detected (380). As a note of caution, the latter studies in the rat were based on the use of RT-PCR on whole hypothalamic fragments, which might have oversighted subtle changes in Kiss1 expression levels.
With regard to the receptors involved, studies using selective ER ligands in the rat, as well as ERα and ERβ KO mice, conclusively demonstrated that the negative effects of estrogen on Kiss1 expression are mediated via ERα (298, 422, 423). In good agreement, the use of KO models of selective elimination of ERα in Kiss1 cells has demonstrated that the lack of estrogen action induces a robust elevation of Kiss1 expression in the MBH, which was associated to a marked increase in serum LH levels, therefore supporting the physiological relevance of the ARC ERα-Kiss1 pathway in the negative-feedback control of gonadotropins (271). As indicated in previous sections, a remarkable feature of Kiss1 neurons is that virtually all of them express ERα, while only a modest fraction expresses ERβ (422, 423). This is of great physiological relevance, especially considering the dominant roles of ERα in the negative-feedback control of gonadotropins but the conspicuous lack of this receptor in GnRH neurons (167, 171). Thus it is tenable to propose that Kiss1 neurons are conduits for, at least part of, the effects of estrogen on GnRH secretion.
Concerning the effects of androgens, comparative analyses of the actions of testosterone, estradiol and the nonaromatizable androgen DHT suggested that the androgenic control of Kiss1 expression in the ARC is mediated via ER, following aromatization of testosterone, and the AR (423), as confirmed also by the use of mouse models with a hypomorphic allele to the AR or null mutations of this gene (305, 423). In support of the negative regulatory actions of sex steroids (mainly estrogen) on Kiss1 gene expression, studies using a human neuroblast cell line reported the ability of estradiol to suppress its mRNA levels, even though DHT was stimulatory in this setting (286). In addition, the molecular mechanisms putatively involved in the inhibitory effects of estrogen on Kiss1 gene expression have been partially exposed recently using the MDA-MB-231 breast cancer cell line, where ERα-dependent suppression of Kiss1 expression was shown to be conducted via a nonclassical mechanism, independent of estrogen response elements (ERE) or Sp1 interactions with the promoter region (181). Evidence of such a non-ERE mediated mechanism for the negative-feedback control of Kiss1 has been recently obtained also in mouse models (see below).
Identification of the coexpression of NKB and Dyn in ARC Kiss1 neurons prompted further analyses of the dynamics of these three factors following manipulations of the sex steroid milieu. Previous literature and recent data jointly point out that expression of NKB and Kiss1 genes undergoes similar changes following GNX and/or sex steroid replacement, as documented in the mouse, sheep, monkey, and human (304, 305, 367). In contrast, expression of Dyn, which is known to conduct inhibitory actions of gonadotropin secretion, has been shown to decrease following sex steroid withdrawal in the sheep and primates (127, 367), whereas in rodents estrogen seems to suppress Dyn mRNA levels at the ARC (150, 304, 305). These observations reinforce the proposal of a functionally distinct population of KNDy neurons that appear to play a dominant role in the negative-feedback control of gonadotropins in mammals. Of note, a recent study addressing the time course of the changes in expression of KNDy genes following menopause or GNX in the female monkey suggested that, while long-term suppression of sex steroid levels are associated with marked increases in Kiss1 and NKB mRNAs, more short-term changes in sex steroids were apparently without any discernible effect (114). This intriguing possibility requires further confirmation, as previous analyses had suggested a faster pattern of ARC responses following GNX (422, 423). Admittedly, however, while most of the studies produced to date have focused on setting the principle that ARC Kiss1 (and associated NKB and Dyn) expression is under the control of sex steroids, little efforts have been made so far to conduct a detailed analysis of the temporal correlation between changes in hypothalamic (ARC) levels of Kiss1 mRNA and circulating gonadotropins. Similarly, while many studies have assayed changes in mRNA expression, there is very limited information on the actual fluctuation of kisspeptin levels at the ARC in response to GNX and sex steroid replacement. In this context, a very recent study has documented parallel changes (suppression) of Kiss1 mRNA and kisspeptin-IR in the ARC of female guinea pigs under negative-feedback actions of estradiol (34).
B. Functional Data
In addition to the expression data summarized above, compelling functional evidence indicates that the hypothalamic Kiss1 system is an important component of the pathways for the transmission of the negative-feedback effects of sex steroids. Importantly, while expression analyses in models of manipulation of sex steroid levels had already suggested this possibility, more direct demonstration of such a role has come from studies involving the use of Gpr54 and Kiss1 KO mice, conditional ERα KOs, and in vivo administration of kisspeptin antagonists, as made available very recently. Notwithstanding this supportive evidence, it is clear that targets other than Kiss1 neurons participate also in the negative-feedback control of gonadotropin secretion by sex steroids. As a clear example, recent data from pituitary-specific ERα KO mice have corroborated a wealth of previous experimental evidence suggesting the contribution of direct pituitary effects of estradiol in the negative-feedback regulation of gonadotropins (414), a phenomenon that had been well documented in the monkey (296, 348) and was confirmed very recently also in humans (407).
Studies in Gpr54 and Kiss1 null mice have demonstrated that this system is essential for the compensatory rise of circulating gonadotropins following GNX in males and females (81, 112, 134). Thus female Gpr54 and Kiss1 KO mice did not display appreciable changes in LH and FSH concentrations after GNX (81, 112). Similarly, LH concentrations remained unchanged after GNX in male Gpr54 and Kiss1 KOs (112, 134). Intriguingly, two independent studies have reported a modest but detectable elevation of FSH levels following GNX in male Gpr54 and Kiss1 KOs (81, 134). The possibility exists, however, that such elevation may stem from changes in the input of non-steroid gonadal factors, such as inhibins, which are known to selectively suppress FSH release. It can be argued that, since gonads from the above KOs are severely hypofunctional, the acute effects of GNX may not be detectable given the removal of very low amounts of sex steroids. This, however, does not seem to be the case, since GNX Gpr54 KO mice subjected to supplementation with supraphysiological doses of testosterone failed to display an increase in serum LH levels following withdrawal of the sex-steroid replacement (134). This would imply that disruption of negative-feedback responses in this model is primarily due to the lack of kisspeptin signaling.
In the same line, recent in vivo studies have provided further insight into the mechanisms whereby estrogen participates in the negative-feedback control of ARC Kiss1 gene expression and, hence, gonadotropin secretion. Thus, with the use of an elegant mouse model engineered to lack classical ERα signaling through direct biding to DNA at consensus ERE sequences, it has been recently documented that the inhibitory effects of estrogen on Kiss1 mRNA levels at the ARC are conserved even in the absence of proper signaling via ERE-mediated pathways, therefore suggesting that a component of such a negative action is conducted through a nonclassical ERα signaling mechanism (150). This observation fits well with previous findings in the same model suggesting that negative feedback relies mainly on ERα-mediated, ERE-independent mechanisms (141). Of note, the above study also demonstrated that estrogen-induced suppression of Dyn mRNA at the ARC does require classical ER-DNA interactions. This would suggest divergences in the molecular mechanisms whereby estrogen conveys its inhibitory actions on the expression of two different neuropeptide systems within the KNDy neurons.
Finally, further support for the relevance of the Kiss1 system in the negative-feedback control of gonadotropins has been recently provided by in vivo studies, in rats and mice, documenting the effects of kisspeptin antagonism on gonadotropin levels in models of GNX. Despite some degree of variability in the effectiveness of the antagonists across studies, these analyses have demonstrated that the post-GNX rise of gonadotropins is prevented in mice and partially suppressed in rats after blockade of Gpr54 (384). While differences in the doses and protocols of administration of the antagonist in these two species may partially account for the divergence in the magnitude of gonadotropin suppression, these results prove the principle that acute blockade of kisspeptin signaling reduces the hypersecretory responses of gonadotropins following removal of negative-feedback signals from the gonads.
X. KISS1 NEURONS AS CONDUITS FOR THE POSITIVE FEEDBACK OF ESTROGEN
Expression and functional studies in the context of sex steroid regulation of Kiss1 expression not only allowed to delineate a tenable pathway for conveying the negative-feedback effects of gonadal steroids onto GnRH neurons, via Kiss1 neurons in the ARC, but disclosed also the existence of a second population of Kiss1 neurons, displaying opposite responses to sex steroids, which seems ideally suited for mediating the positive-feedback effects of estrogen in the generation of the preovulatory surge of gonadotropins. Admittedly, however, this population has been well characterized only in rodents, whereas the anatomical and functional features of its putative correlate in other mammalian species remain to date partially unfolded.
A. Anatomical and Expression Data
Coincident with the identification of the ARC population of Kiss1 neurons described above, Smith and colleagues (422, 423) reported the presence of another area of abundant expression of Kiss1 mRNA located at the AVPV of the mouse hypothalamus. Analysis of the dynamic changes of Kiss1 expression at this nucleus following GNX and sex steroid replacement demonstrated that, contrary to the ARC, neurons at the AVPV displayed decreased Kiss1 mRNA levels after elimination of gonadal steroids and increased expression following sex steroid replacement. This pattern of responses was initially detected in both male and female mice; however, responses in the female were much more prominent (422, 423). Subsequent in situ hybridization and IHC studies in rats and mice fully confirmed these original observations (2, 66). Moreover, expression analyses in cyclic and estradiol-primed OVX female rats demonstrated that there is an increase in Kiss1 mRNA expression at the AVPV preceding the spontaneous (preovulatory) as well as estrogen-induced LH surges, a period when Kiss1 neurons in this area become activated, as reflected by increased expression of c-fos (2, 426). Collectively considered, the above observations have consolidated the notion that Kiss1 neurons in the AVPV are the target for the stimulatory effects of estrogen and important conduits for its action in terms of generation of the preovulatory surge of gonadotropins in spontaneous ovulatory rodents. Of note, Kiss1 neurons in the medial POA (equivalent to the AVPV) are also upregulated by estradiol and become activated following a mating stimulus in the reflex ovulator, the musk shrew (185). To our knowledge, the specific roles of progesterone in the control of the AVPV population of Kiss1 neurons remain mostly unexplored, although expression of PR has been documented in a subset (up to 60%) of this population (67).
In keeping with a prominent role of AVPV Kiss1 neurons in the generation of GnRH/gonadotropin surges in rodents, recent studies have documented that Kiss1 mRNA levels and Kiss1 neuronal activation at this nucleus display a marked circadian pattern in female rats, mice, and hamsters (382, 499, 504), which appears to be dependent, to a great extent, on estrogen input and is fully compatible with the timed generation of gonadotropin surges. Molecular analyses suggested that such rhythmic changes in Kiss1 transcription are due to the dynamic fluctuation of the expression of the transcriptional factor, D-site binding protein (DBP), in AVPV Kiss1 neurons (504). Likewise, circadian inputs to this neuronal population come from the SCN, via vasopressinergic (AVP) pathways, as evidenced by the presence of AVP neuronal projections to Kiss1 neurons in the mouse and the ability of AVP to enhance Kiss1 neuronal activity at the AVPV in hamsters (484, 499).
As indicated in section IVB, the existence and functional roles of populations of Kiss1 neurons equivalent to the rodent AVPV in other mammalian species have remained contentious, although the anatomical data made recently available strongly suggest the presence of a subset of Kiss1 neurons in rostral periventricular/preoptic areas in the sheep, pig, and human. However, the magnitude of this population, compared with that in the ARC/infundibular region, appears to be smaller than in rodents, although some variability among species has been also detected (68). Furthermore, expression analyses on the effects of sex steroids on the expression of Kiss1 gene at such rostral sites in these species remain less well characterized. In any event, studies in pigs have demonstrated that estradiol upregulates the expression of Kiss1 mRNA at the periventricular nucleus (468), whereas in the sheep, a rise in Kiss1 mRNA has been detected at the neuronal population located at the POA during the late follicular phase, i.e., coinciding with the endogenous peak of estradiol preceding the gonadotropin surge (424). The situation in the sheep appears to be especially complex, as in situ hybridization studies have documented that a subset of Kiss1 neurons in the ARC, mainly sited in its caudal portion, are also activated by estrogen, as evidenced by c-fos labeling after exogenous administration of estradiol and the increase of Kiss1 mRNA levels in this area during the late follicular phase (116, 424). These observations suggest that the Kiss1 circuits involved in positive feedback in the sheep may include those originating from the caudal ARC and the POA populations (424). In fact, a very recent study revealed a substantial activation of Kiss1 neurons in the POA coinciding with the preovulatory surge in the ewe, suggesting the functional relevance of such population in this process in ovine species (177). To our knowledge, the putative role, if any, of the rostral population of Kiss1 neurons in primates in terms of positive feedback control of gonadotropins has not been evaluated to date. In fact, compelling evidence obtained in humans strongly suggests that, in contrast to rodents, the preovulatory midcycle surge of gonadotropin does not need an increase in the pulsatile secretion of GnRH to occur but rather relies on the augmentation of GnRH signaling at pituitary (3, 160). In this context, the actual contribution and physiological relevance of central Kiss1 pathways in the surge-generation system in primates needs further investigation.
B. Functional Data
Functional evidence for the crucial roles of AVPV Kiss1 neurons in mediating positive feedback and, thereby, contributing to the generation of the preovulatory surges has come from functional genomic and pharmacological studies, conducted mainly in rodents. Analyses of the ability of estrogen to activate GnRH neurons and to evoke LH secretory peaks have been conducted by several groups in female Gpr54 and Kiss1 null mice. Of note, while these studies have not disputed the potential role of AVPV Kiss1 neurons in mediating positive feedback, some discrepancies on whether this function may be dispensable, at least under certain developmental conditions, have been detected between reports. Thus, while Dungan et al. (112) observed that estrogen is capable to induce some degree of activation of GnRH neurons and to induce attenuated LH surges in Gpr54 KO mice, Clarkson et al. (67) failed to detect any activation, as measured in terms of c-Fos IR, in GnRH neurons, nor did they observe discernible LH secretory responses in Kiss1 or Gpr54 null mice following conventional protocols of sex steroid priming. The basis for such discrepancy remains obscure, but the wealth of expression and functional data obtained in congenital Kiss1 and Gpr54 KO mice models strongly support an important role of AVPV Kiss1 neurons in the generation of the preovulatory surge of gonadotropins. In good agreement, recent pharmacological studies have demonstrated that central infusion of a kisspeptin antagonist to cyclic female rats prevented the occurrence of the preovulatory surges of LH and FSH (339), data that confirmed earlier observations from immunoneutralization experiments, where infusion of a specific antibody against Kp-54 into the POA blocked the proestrus LH surge and disrupted estrous cyclicity (216). Similarly, a recent study has demonstrated that treatment with a kisspeptin antagonist significantly attenuated estradiol-induced LH surges in the ewe (425).
In addition to the above evidence, functional genomic studies have allowed also to dissect out the molecular mechanisms for the positive feedback of estrogen on AVPV Kiss1 expression in vivo. Thus analyses in the mouse line described in the previous section, which is devoid of classical ERα signaling via ERE but retains nonclassical ERα actions, have demonstrated that the stimulatory effects of estrogen on Kiss1 mRNA are totally dependent on ERα-ERE mechanisms, suggesting a classical genomic mode of action (150). Again, this observation fits well with previous data in the same model that suggested that the positive-feedback effect of estradiol in terms LH secretion requires an intact ERE-dependent pathway (141).
Finally, pending further electrophysiological studies in some of the transgenic models tagging Kiss1 neurons in vivo generated recently (77, 151, 271), the identification of the subset of Kiss1 neurons in the AVPV has allowed to initiate the electrophysiological characterization of this population. Thus a recent report documented the firing patterns of neurons at the AVPV in adult female mice. Postrecording IHC demonstrated that Kiss1 neurons at this nucleus, which are <20% of total neurons studied, display predominant bursting or tonic firing patterns, which tend to increase on the day preceding ovulation (109). This study reported also that all AVPV neurons responded to glutamate (depolarization) and GABA (cessation of firing), whereas they showed almost null responses to kisspeptin itself (109). While the phenotype of these neurons is heterogeneous, as they include both Kiss1 and non-Kiss1 neurons, the above electrophysiological data may help to delineate potential coregulators of this estrogen-sensitive population of Kiss1 neurons.
C. Estrogen as a Putative Modifier of Kisspeptin Responsiveness: Roles in the Preovulatory Surge
Although the above rodent data clearly demonstrated that estradiol is a major transcriptional regulator of the Kiss1 system, with the ability to activate Kiss1 gene expression in the AVPV during the period preceding ovulation, the mechanisms whereby estrogen contributes to the full expression of the preovulatory surge of gonadotropins are certainly more complex and multifaceted, and likely involve actions at different sites of the neuroendocrine network driving LH and FSH secretion. As a good example, estrogen, mainly via ERα, is known to play a crucial role in the generation of GnRH self-priming, acting at the pituitary level (393, 458). Concerning its effect on kisspeptin signaling, compelling evidence suggests that, in addition to the transcriptional effects summarized above, estrogen and other sex steroids, such as progesterone, carry out an important function in shaping GnRH/gonadotropin responsiveness to kisspeptin, a phenomenon that may also contribute to the mechanisms for positive feedback and generation of the preovulatory surge.
Evidence in support of a role of sex steroids in modulating kisspeptin responsiveness came first from pharmacological studies, testing the gonadotropin releasing effects of kisspeptins in cyclic female and OVX rats, with or without sex steroid replacement (380). These analyses demonstrated that, although Kp-10 induced potent LH secretory peaks in all phases of the estrous cycle, maximal responses were detected during the proestrus-to-estrus transition, i.e., at the peri-ovulatory period (380). Similar studies in women showed that LH responses to Kp-54 and Kp-10 were greater during the preovulatory phase (102, 191), whereas in the sheep, during the breeding season, LH secretion in response to Kp-10 was enhanced at the late-follicular period of the estrous cycle (428). In addition, analyses in OVX rats documented that elimination of ovarian steroids did not eliminate the capacity of Kp-10 to further increase the stimulated gonadotropin secretion, but maximal LH and FSH responses were only detected following estradiol and progesterone supplementation (380). Such a phenomenon may stem, at least partially, from the ability of estrogen to induce the rhythmic expression of Gpr54 in GnRH neurons, as evidenced recently by studies in GT1–7 cells (474). Given the mandatory role of both elevated estrogen levels and activated PR for the generation of the preovulatory surge of gonadotropins (243, 378, 379), it can be argued that one of the mechanisms whereby the effects of sex steroids are brought about is via the induction of a state of maximal responsiveness to kisspeptins, whose tone is supposed to increase also at the period preceding ovulation (2, 426). Admittedly, however, it remains possible that, at least partially, the above effects may stem from changes in pituitary responsiveness to GnRH (13).
Further pharmacological dissection of the roles of sex steroids in modulating gonadotropin responsiveness to kisspeptin in the female was provided by studies testing the effects of selective ER and PR agonists and antagonists in rats (378, 379). Results from these experiments demonstrated that selective blockade of ERα not only blunted the endogenous preovulatory surge of LH, but induced also a dramatic suppression of LH responses to exogenous Kp-10, when administered during the preovulatory phase. In contrast, while selective blockade of ERβ failed to cause major changes in the preovulatory rise of LH, it did augment the magnitude of acute responses to Kp-10, therefore suggesting a yin-yang balance between ERα (potently stimulatory) and ERβ (modestly inhibitory) signaling in shaping the LH responses to kisspeptins during the preovulatory period (379). Of note, none of the above differences was detected after GnRH stimulation, suggesting that the impact of ERα or ERβ blockade takes place upstream from the pituitary. In good agreement with these ER antagonist data, supplementation of OVX rats with a selective ERα agonist and progesterone was maximally effective in inducing robust LH secretory responses to Kp-10 (379). Roughly similar phenomena have been described for FSH responses to kisspeptin in the female rat following selective activation or inactivation of ERs and PR (378). Taken as a whole, the above results strongly suggest that estrogen, acting via ERα, in conjunction with progesterone, stimulates GnRH/gonadotropin responsiveness to kisspeptins, as a tenable mechanism to complement its transcriptional effects on Kiss1 gene expression and, therefore, to induce a complete preovulatory surge of gonadotropins. The observation that ERβ signaling might operate as a negative modifier of GnRH/LH responses to kisspeptins is suggestive of a potential mechanism whereby estrogen contributes to self-restraining its positive-feedback effects, thereby allowing a proper preovulatory surge to occur.
XI. KISS1 NEURONS AND THE METABOLIC REGULATION OF PUBERTY AND REPRODUCTION
Among the multiple regulators of the HPG axis, it is well established that the amount of energy reserves and the metabolic state of the organism play a crucial role in modulating the timing of puberty and reproductive capacity later in life (123, 174, 452). Such a dynamic interplay manifests not only in physiological conditions but also over a wide range of metabolic disturbances, from energy insufficiency to severe energy excess, in which alterations of reproductive function are frequently observed (61). Thus, as reproductive function, especially in the female, is highly energy-demanding but dispensable for the survival of the individual, sophisticated mechanisms have been selected during evolution to allow specific inhibition of the HPG axis in conditions of sustained energy deficit (50). In addition, other forms of metabolic stress, such as morbid obesity, are also frequently coupled to reproductive disorders, ranging from altered pubertal timing to hypogonadism and subfertility in adulthood (4, 33, 252, 358). Although awareness of this connection existed for ages on the basis of intuitive knowledge, the neurohormonal mechanisms for such a nexus between energy homeostasis and reproduction have begun to be elucidated only recently, by the identification of multiple interactions between numerous neuroendocrine pathways which jointly participate in the physiological control of energy balance and reproductive maturation and function (123, 452). Compelling evidence, summarized in the following sections, strongly suggests that Kiss1 neurons are key components of the neuroendocrine pathways whereby energy homeostasis and reproduction are functionally coupled.
A. Metabolic Regulation of Kiss1 System: Expression Changes and Functional Consequences
The prominent roles of kisspeptins in the control of the HPG axis made it reasonable to propose that the Kiss1 system could play also a central function in bridging energy homeostasis and reproduction. Experimental studies directed to test this hypothesis assumed that if such a role is physiologically relevant 1) it would be possible to identify alterations in the expression and/or function of this system in conditions of metabolic distress; and 2) exogenous administration of kisspeptins may ameliorate some of the reproductive phenotypes associated with such conditions (449). Overall, the experimental data gathered so far confirm the validity of these assumptions. In addition, initial studies aimed also at identifying whether kisspeptins might have on their own any detectable effect in terms of food intake or body weight control, as it had been reported previously for other neuroendocrine signals, such as leptin, with important roles in the metabolic control of reproduction. Those initial analyses, however, failed to demonstrate changes in the pattern of food intake or the expression of appetite-regulating neuropeptide genes at the hypothalamus, such as neuropeptide Y (NPY), proopiomelanocortin (POMC), Agouti relate peptide (AgRP), and cocaine- and amphetamine-related transcript (CART), following icv injection of kisspeptins to rats (58, 464). These observations suggested that the Kiss1 system is specifically engaged into the networks controlling reproduction, but apparently not food intake. This contention has been recently challenged by electrophysiological studies showing that Kp-10 is able to activate POMC neurons in the ARC, which produce the anorexigenic neuropeptide αMSH, while it inhibits NPY neuronal firing, NPY being a potent orexigen (131). It remains to be solved how such robust effects in terms of neuronal firing ex vivo apparently fail to correlate with corresponding anorectic responses in vivo, as documented by the earlier studies mentioned above. Of note, however, it was very recently reported that central kisspeptin injections were able to suppress food intake by increasing meal intervals in mice (435). Again, the basis for the discrepancy with the initial rat studies is yet to be unfolded.
In the last few years, efforts have been made to evaluate whether the expression of the hypothalamic Kiss1 system is under the regulation of metabolic cues. Admittedly, most of the analyses conducted so far on this topic have focused on the potential alterations of Kiss1 expression, and/or kisspeptin function, in conditions of negative energy balance associated with different degrees of hypogonadotropic hypogonadism. As a clear example, analyses in pubertal male and female rats subjected to up to 72-h fasting demonstrated a drop of Kiss1 mRNA levels at the hypothalamus, which was associated with a concomitant decrease in circulating LH levels (58). Similar inhibitory responses to fasting have been observed also in adult female rats (40, 270) and adult male mice, where time course analyses of the effects of fasting on hypothalamic expression of Kiss1 gene demonstrated a rapid decrease in its mRNA levels, with a detectable reduction as soon as 12 h after beginning of food deprivation (257). In the same vein, other models of metabolic stress coupled to conditions of negative energy balance and suppressed gonadotropic function have been shown to display analogous inhibitory changes in hypothalamic Kiss1 expression. For instance, in a model of uncontrolled type 1 diabetes induced by streptozotocin (STZ) injections, a marked suppression of Kiss1 mRNA was observed in the hypothalamus, with a close parallelism between defective gonadotropin concentrations and reduced hypothalamic Kiss1 mRNA levels, both at basal conditions and after GNX (59, 60). Altogether, these observations set the concept that conditions of persistent energy deficit, coupled to suppression of gonadotropic function, are also linked to a reduction of Kiss1 expression in the hypothalamus, which predicts a lowering of the kisspeptin tone with potential mechanistic implications (see below).
Most of the above studies, however, did not provide a precise neuroanatomical location for the changes of Kiss1 expression in conditions of metabolic stress. This is of potential importance given the different functional features of the ARC and rostral/periventricular populations of Kiss1 neurons (317, 374). To what extent these two populations are affected by metabolic stress is yet under active investigation. Initial studies in GNX leptin-deficient (ob/ob) male mice demonstrated that Kiss1 mRNA levels are significantly suppressed at the ARC in this model of congenital absence of leptin (418). In good agreement, studies in gonadal-intact female rats subjected to chronic calorie restriction around puberty (20% for 7 days) documented a decrease of Kiss1 mRNA expression in the ARC (376). On the other hand, short-term fasting in adult OVX female rats with estrogen replacement has been reported to reduce Kiss1 mRNA levels at the AVPV (198). Finally, a recent study demonstrated that dietary restriction induces a marked suppression of Kiss1 mRNA levels at the ARC and POA in the sheep (20). As a whole, these findings suggest that both ARC and AVPV Kiss1 neurons may be targets for metabolic regulation. Yet, the relative sensitivity and functional relevance, as well as the age and sex dependency, for such a metabolic regulation of ARC and/or AVPV Kiss1 neurons need further investigation. Similarly, further studies are needed to evaluate to what extent the above changes in mRNA levels translate into changes of kisspeptin content at these hypothalamic sites. Yet, a recent report has documented that 48-h fasting in pubertal female rats induces a significant suppression of kisspeptin-IR and the number of kisspeptin-positive neurons in the ARC (52). While these initial observations need replication in other species and conditions of metabolic stress, they further support the contention that states of persistent energy insufficiency are coupled to a decrease in kisspeptin content/tone at the hypothalamus.
Whereas the above data prove the principle that the hypothalamic Kiss1 system is sensitive to changes in the body energy status and may be suppressed by conditions of persistent negative energy balance, it remains to be defined whether other forms of metabolic stress associated with excess of energy stores, such as morbid obesity, might have an impact on the Kiss1 system as well. Such a possibility is of translational relevance, given the epidemic dimension of obesity worldwide and its potential reproductive comorbidities (4, 33, 252, 358). Indeed, although studies in this front remain scarce, evidence from animal models suggests changes in the expression and/or function of the hypothalamic Kiss1 system in conditions of obesity. Thus, while initial expression analyses using whole hypothalamic fragments from diet-induced obese mice failed to demonstrate significant changes in relative Kiss1 mRNA levels, further neuroendocrine characterization of this model revealed that these obese animals also displayed dramatically suppressed levels of circulating testosterone (257). Therefore, the expression levels of Kiss1 mRNA in this condition can be considered inappropriately low, given the lack of negative-feedback restraint. This might be suggestive of a state of subtle functional impairment of the hypothalamic Kiss1 system in obesity. In the same line, a very recent study has demonstrated that persistent obesity in DBA/2J mice, which are prone to fertility problems due to diet-induced obesity, evoked a marked suppression of Kiss1 mRNA levels in the ARC and AVPV, as well as in the number of kisspeptin-IR neurons in the AVPV (355). On the other hand, shorter protocols of exposure to a high-fat diet in male rats have been shown to increase Kiss1 mRNA levels at the hypothalamus (40). Likewise, our recent data suggest that early-onset obesity in female rats causes elevation of Kiss1 mRNA levels in the hypothalamus and a trend for increased numbers of kisspeptin-IR fibers at the AVPV at the time of puberty (55). Altogether, the above findings document the potential sensitivity of the hypothalamic Kiss1 system to conditions of obesity, a contention that needs to be investigated further. Notably, it has been recently proposed that the hypothalamic Kiss1 system might be altered in human obesity and type 2 diabetes (138), yet this appealing hypothesis still awaits experimental validation.
In addition to the expression data summarized above, functional analyses have provided further support for the relevance of the metabolic regulation of Kiss1 system in terms of control of the HPG axis. This is especially relevant considering that expression data on their own do not necessarily provide a conclusive demonstration for a causative relationship between inhibition of Kiss1 system and failure of the gonadotropic axis in conditions of metabolic stress. Yet, the rescue of gonadotropic function by kisspeptin administration in those conditions, where the endogenous kisspeptin tone is presumably suppressed, would support the importance of this system in transmitting nutritional and metabolic cues to the centers governing the reproductive axis. Indeed, this is apparently the case in different experimental models of metabolic stress and chronic administration of kisspeptins. Thus repeated icv injections of Kp-10 to female rats submitted to chronic subnutrition during the peripubertal period, where 30% reduction in daily calorie intake prevented puberty onset and suppressed circulating LH levels, was sufficient to rescue vaginal opening and to induce potent gonadotropic and estrogenic responses (58). Likewise, repeated injections of kisspeptin to hypogonadotropic diabetic male rats ameliorated testicular and prostate weights, and normalized circulating LH and testosterone levels (59). In fact, central injection of Kp-10 to uncontrolled diabetic male and female rats induced robust gonadotropic responses, and in males, also androgenic responses, despite the lowering of the prevailing gonadotropin/sex steroid levels due to the diabetic state (59, 60). In both models of metabolic distress (i.e., subnutrition and uncontrolled diabetes), rescue of the reproductive parameters was achieved in spite of the lack of direct effects of chronic kisspeptin treatment on body weights or any detectable improvement of other metabolic indexes.
In the same vein, pharmacological analyses of acute gonadotropin responses to kisspeptins in conditions of food deprivation or metabolic stress have further documented the impact of energy imbalance on the function of hypothalamic Kiss1 system. Thus gonadotropin secretion in response to a single bolus of Kp-10 in male and female rats subjected to acute fasting, as to induce a state of negative energy balance, was not only preserved, but even enhanced in fasted animals, despite the lowering of the prevailing levels of gonadotropins associated with food deprivation (58, 476). In addition, conditions of persistent energy deficit appear to modify the patterns of gonadotropin desensitization to continuous exposure to kisspeptins, as circulating LH levels remained elevated for longer time periods after infusion of Kp-10 in female rats submitted to chronic subnutrition (381). Altogether, the above evidence suggests a state of hyperresponsiveness to kisspeptin in situations of energy insufficiency. A similar state of enhanced responses to kisspeptins has been reported in terms of GnRH secretion by hypothalamic explants from fasted rats (58). A potential explanation for these observations is that in conditions of negative energy balance, the decrease of the endogenous kisspeptin tone brings about a compensatory state of augmented responsiveness to the neuropeptide. The molecular mechanism for such a state might involve an increase in the expression of Gpr54, as 72-h fasting in pubertal rats elicited an increase in its hypothalamic mRNA levels (58). Of note, however, shorter protocols of fasting in adult mice have been shown to reduce Gpr54 mRNA expression (257), observations which might be suggestive of complex dynamics in the changes of kisspeptin release and receptor sensitivity during the course of conditions of persistent negative energy balance. As a further illustration of such a complexity, it has been reported that fasting decreases net responsiveness of the HPG axis to kisspeptin stimulation in the monkey (488). In any event, while several factors, including differences in terms of species (rat vs. mouse and monkey), age (pubertal vs. adult), and duration of fasting, may account for some the above discrepancies, it seems clear that metabolic stress does not only alter Kiss1/kisspeptin expression at the hypothalamus, but very likely causes also functional alterations in terms of GnRH/gonadotropin responsiveness to the stimulatory effects of kisspeptins in different species.
B. Metabolic Regulators of Kiss1 Neurons: Leptin
Demonstration of the impact of different conditions of metabolic distress on Kiss1/kisspeptin expression and function at the hypothalamus raised the immediate question of which signals are ultimately responsible for such control. Given its paramount importance as integral metabolic regulator of different neuroendocrine axes, including the HPG system, leptin was considered as the first suspect. Indeed, as briefly mentioned elsewhere in the review, leptin is universally recognized as a major signal of energy abundance, originating from the adipose tissue and secreted in proportion of body fat stores, which stimulates/facilitates the activation of GnRH neurons (50, 123, 174, 452). However, despite the general recognition that leptin acts centrally, mainly at the hypothalamus, to promote or permit puberty onset and to maintain fertility, the apparent lack of functional leptin receptors in GnRH neurons, as solidly confirmed recently (356), suggested the existence of afferent pathways, sensitive to the effects of leptin, that would convey its action onto GnRH neurons. Experimental evidence strongly suggests that Kiss1 neurons are a component of the afferent hypothalamic network whereby leptin indirectly modulates GnRH function. Yet, the data supporting this contention come from rather extreme in vivo models of leptin deficiency and in vitro experiments using immortalized cell lines, and further neuroanatomical and molecular characterization of the networks whereby leptin effects are transmitted onto Kiss1 neurons, and hence to GnRH neurons, is needed.
Direct actions of leptin on Kiss1 neurons were first proposed in 2006 by studies in ob/ob male mice that documented that 1) the congenital lack of leptin was associated with a significant suppression of Kiss1 mRNA levels in the ARC, 2) such a low expression was partially rescued by central administration of leptin, and 3) a significant proportion (>40%) of Kiss1 neurons in the ARC expressed the mRNA encoding Ob-Rb, i.e., the functional leptin receptor (418). Of note, gonadectomized animals, which are expected to display an increase of Kiss1 expression in the ARC, were used in that initial study, as a way to control for the potential confounding factor of fluctuations in endogenous T levels (418). The fact that ARC Kiss1 neurons express the leptin receptor gene has been recently confirmed by RT-PCR in a transgenic mouse line that allows GFP tagging, and subsequent isolation, of Kiss1 neurons in vivo (77). However, these analyses revealed also that only a 15% fraction of Kiss1 neurons in the ARC displays conventional pSTAT3 responses to leptin administration, whereas Kiss1 neurons in the AVPV do not apparently express leptin receptor gene (77).
As further support for the impact of leptin on the hypothalamic Kiss1 system, it was independently reported, also in 2006, that in uncontrolled diabetic male rats, which suffer severe hypoinsulinemia and hypoleptinemia, central infusion of leptin, but not of insulin, normalized the defective hypothalamic expression of Kiss1 gene (59). Notably, such a protocol of icv infusion of leptin ameliorated various reproductive parameters, including LH and T secretion, in diabetic animals, despite persistent metabolic alterations (59). Taken as a whole, data from the above in vivo models of central administration of leptin demonstrated that hypothalamic expression of Kiss1 is under the positive control of this adipocyte hormone, with presumable direct actions of leptin on Kiss1 neurons, at least in the ARC. Also in keeping with such direct actions, leptin has been shown to induce the depolarization of ARC Kiss1 neurons via activation of TRPC channels in the guinea pig (353), a species where Kiss1 neurons in the ARC have been shown to express functional leptin receptors (353). Likewise, leptin has been reported to increase Kiss1 mRNA levels in the murine hypothalamic cell line, N6 (257), as well as in primary cultures of human fetal GnRH-secreting neuroblasts (286). Also recently, studies in the sheep have documented the expression of leptin receptor gene in the ARC and POA populations of Kiss1 neurons, and leptin has been shown to increase Kiss1 mRNA levels at those nuclei (20). Altogether, these data provide evidence for a tenable neuroendocrine leptin-kisspeptin-GnRH pathway, whereby sufficient levels of leptin would allow proper maturation and function of GnRH neurons and, hence, of the HPG axis.
Despite the solid experimental evidence summarized above, the possibility that leptin effects on Kiss1 neurons are solely conducted via direct actions has been very recently challenged by two independent studies, which jointly suggest that the mode of action of leptin on Kiss1 neurons may be, preferentially, indirect. Thus, using mice congenitally devoid of leptin receptors in Kiss1-expressing cells, Donato et al. (106) recently reported that puberty onset can proceed in the absence of leptin signaling in Kiss1 neurons. As a note of caution, however, in that study the specificity of selective elimination of leptin receptors only in postnatal Kiss1 neurons may be compromised by the wider expression patterns of Kiss1 gene within the brain and other peripheral tissues at early stages of maturation. In addition, considering that in the above model, leptin receptors were congenitally absent in Kiss1 cells, it is tenable that developmental compensatory mechanisms might have occurred. Notwithstanding these possibilities, a very recent IHC study failed to detect the functional isoform of leptin receptor in GnRH or Kiss1 neurons, except for a modest subset of ARC Kiss1 cells. In contrast, leptin receptor-expressing cells were clearly found in the ventral premammillary nucleus (PMV) and POA (254). Of note, the PMV have been recently suggested as an important center for conveying the permissive effects of leptin onto GnRH neurons (106). Intriguingly, this study also identified uncharacterized neuronal populations, sited in the ARC and AVPV, which expressed leptin receptors and were located in close vicinity to Kiss1 neurons (254). Therefore, it is plausible that an important component of the documented effects of leptin on Kiss1/kisspeptin expression in the hypothalamus is transmitted via intermediate pathways (254). Yet, further analyses are needed to solve the apparent discrepancy between RNA and protein data in terms of leptin receptor expression in Kiss1 neurons, with detectable expression of leptin receptor mRNA but negligible levels of its protein. These discrepant results may derive from methodological limitations, such as differences in the threshold of detection for the different techniques, and/or the expression of alternative isoforms of leptin receptors, whose functional relevance in the hypothalamus has been previously documented in other settings (455).
Regardless of its predominant mode of action, the ability of leptin to modulate the hypothalamic Kiss1 system poses not only obvious physiological interest, but might have also potential pathophysiological implications in diverse reproductive disorders, linked to dramatic changes of leptin levels, such as strenuous conditions observed in anorexia nervosa, extreme physical exercise, or low weight (493, 494), where kisspeptin analogs may have also potential therapeutic implications, considering the preserved, if not augmented, responses to kisspeptins in such situations of metabolic stress, at least in rodents (58, 476). In this sense, previous reports have documented the ability of leptin to rescue ovarian cyclicity in women with hypothalamic amenorrhea linked to conditions of energy insufficiency (87, 494). In this context, it is predictable, although yet to be fully proven, that exogenous kisspeptin might conduct similar positive effects in affected humans, in keeping with recent data obtained in women with hypothalamic amenorrhea (192), and previous results in rodent models of subnutrition in puberty (see above).
In addition, the ability of leptin to modulate the hypothalamic Kiss1 system is likely not restricted to the adult age, but rather Kiss1/kisspeptin expression appears to be sensitive to earlier metabolic insults. Admittedly, this issue remains scarcely studied. Yet, our initial evidence in models of postnatal under- and overnutrition in female rats suggests that leptin may be also an important regulator of Kiss1/kisspeptin expression at puberty. Thus, in rats with restricted feeding during lactation, which were leaner and displayed delayed vaginal opening, there was a close correlation between low circulating leptin levels at puberty and reduced hypothalamic expression of Kiss1 mRNA and kisspeptin-positive neurons (55). Conversely, heavier female rats due to postnatal overnutrition, which showed earlier entry into puberty, presented elevated serum leptin concentrations and Kiss1 mRNA levels at the hypothalamus (55). Whether these alterations stem from nutritional/leptin changes at early postnatal periods and/or puberty is yet to be defined. A very recent study suggested that the sensitivity of the hypothalamic Kiss1 system to inhibition by acute fasting develops during the juvenile period (187). Yet, protocols of persistent undernutrition, rather than acute fasting, are probably needed to exclude the possibility of early organizing effects of the nutritional status on the development of Kiss1 neurons.
In spite of the above expression/functional evidence suggesting the involvement of Kiss1 neurons in mediating, at least part of, leptin effects on the centers governing the HPG axis, these data do not preclude the possibility of additional, Kiss1-independent pathway(s) transmitting the reproductive effects of leptin. As mentioned earlier in this section, a clear example is the neuronal circuitry involving the PMV, which has been recently shown to be the target of leptin and, despite the lack of Kiss1 neurons at this site, an essential center for conveying its effects onto the reproductive axis (107). Whether this circuit and those involving Kiss1 neurons in other hypothalamic nuclei are related or independent is yet to be elucidated. To add further complexity to this phenomenon, a recent paper by True et al. (477) suggested that leptin, within the physiological range, is not the critical signal for recovery of gonadotropin secretion and hypothalamic Kiss1 expression during the exit of conditions of negative energy balance. In that study, restoration of apparent physiological levels of leptin in female rats submitted to persistent caloric restriction or acute fasting could not normalize LH concentrations or Kiss1 mRNA expression, thus suggesting the contribution of additional metabolic modulators. That study, however, did not exclude the possibility that threshold leptin levels are permissive for proper LH concentrations and Kiss1 mRNA expression. Moreover, the fact that high doses of leptin could unambiguously modulate LH and Kiss1 levels in different models of metabolic stress (see above) strongly suggests that, indeed, leptin may be a major regulator of kisspeptin expression and, hence, of gonadotropic function under specific metabolic conditions.
C. Other Metabolic Regulators of Kiss1 Neurons
Despite the predominant attention drawn by leptin as a major signal for the metabolic control of the hypothalamic Kiss1 system, it was reasonable to predict that other neuroendocrine factors, involved in the joint control of energy balance and reproduction, may participate also in the modulation of Kiss1 neurons. Indeed, this possibility has been substantiated by different experimental observations in recent years. Of interest, the regulatory actions of such factors in rodents have been shown to involve the modulation not only of Kiss1 expression at the hypothalamus but also of the patterns of GnRH responses to kisspeptin. As further illustration of the complexity of these interactions, a bidirectional communication between kisspeptins and key metabolic neuropeptides, such as NPY and α-MSH, has been recently described in the sheep, whose physiological relevance in terms of body weight control in mammals is yet to be defined. Some of the putative metabolic regulators of the Kiss1 system, other than leptin, are itemized below.
This is a gut-derived hormone secreted by specific endocrine (X/A) cells of the gastric mucosa, which operates as a circulating orexigenic molecule, the only known peripheral hormone with such a function (447). Indeed, on the basis of its biological profile, ghrelin has been regarded as a functional antagonist of leptin (447). Notably, ghrelin levels have been shown to increase before meals, and its circulating concentrations appear to be inversely correlated with the body mass index; thus ghrelin has been considered as an index of energy insufficiency (447, 483). In keeping with such a role as signal of energy deficit, compelling evidence has been presented that ghrelin operates as a negative modifier of puberty onset and/or gonadotropin secretion in a variety of species, including rodents, sheep, monkeys, and humans, acting presumably at central levels (124, 265, 447). Very recently, ghrelin has been shown to inhibit hypothalamic Kiss1 mRNA expression in female rats (128), a phenomenon which may contribute to the suppression of the HPG axis induced by conditions of elevated levels of ghrelin, such as fasting. Anyhow, the physiological relevance of ghrelin in the metabolic control of the Kiss1 system is yet to be fully elucidated.
This potent orexigenic neuropeptide, which is prominently produced by a population of neurons in the ARC that coexpress AgRP, has been well characterized as a hypothalamic effector for part of leptin actions on food intake (289, 396). However, contradictory data on the actions of NPY in the control of the HPG axis have been reported, as it appears to play either stimulatory or inhibitory actions depending on the stage of maturation, sex steroid milieu, and receptor subtype activated (199, 351). Expression analyses in hypothalamic samples from NPY null mice demonstrated significantly lower levels of Kiss1 mRNA in the absence of NPY signaling in vivo (257). Moreover, the lack of NPY prevented the suppression of Kiss1 mRNA expression in the hypothalamus induced by fasting (257). In keeping with these in vivo data, studies using the hypothalamic cell line N6, which expresses the Kiss1 gene, demonstrated that NPY can enhance Kiss1 mRNA expression in vitro (257). Of note, leptin has been demonstrated to suppress the expression of NPY at specific neuronal populations in the ARC (395), yet both leptin and NPY appear to be positive regulators of Kiss1 expression in the hypothalamus. Thus it is reasonable to hypothesize that NPY is not the mediator for the stimulatory effects of leptin on Kiss1 gene expression, but rather represents an independent central activator of the Kiss1 system, in keeping with its reported stimulatory effects on the HPG axis under specific conditions. Recent evidence suggests a higher degree of complexity in the interactions between NPY and Kiss1 neurons, as studies in the sheep have documented reciprocal innervations between these two populations (20). In addition, it has been shown that kisspeptin increased NPY gene expression in the ARC (20). In good agreement, studies using the murine NPY-secreting cell mHypoE-38 demonstrated that Kp-10 is able to enhance NPY mRNA levels and NPY secretion by this hypothalamic cell line, through a mechanism involving ERK1/2 and p38 MAPK (213). Altogether, the above data strongly suggest the existence of hypothalamic networks whereby kisspeptins and NPY regulate each other as potential mechanisms for the integral control of energy balance and reproduction. The nature of such reciprocal interactions, however, may vary depending on the species and/or physiological states, as it has been recently published that Kp-10 can inhibit NPY-expressing neurons in the ARC of mice, through an indirect mechanism involving the enhancement of GABA afferents (131). Anyhow, the physiological relevance of such apparent bidirectional interplay between kisspeptin and NPY signaling in the hypothalamus needs to be further characterized.
These neuropeptide products of the POMC gene are well-recognized anorectic signals produced by a subset of ARC neurons, whose functional balance with NPY neurons in the same nucleus represents a first-order relay for the transmission of the appetite-controlling actions of long-distance signals, such as leptin and ghrelin (289, 396, 516). The ability of melanocortins to modulate the function of the HPG axis had been long recognized, albeit only fragmentarily addressed, but their putative roles as metabolic regulators of reproduction has gained momentum recently due to observations suggesting that they may mediate the permissive effects of leptin on the reproductive axis (18, 19). In this context, it has been demonstrated that POMC and Kiss1 neurons display mutual contacts in the ovine brain and that the melanocortin agonist MTII not only elicits LH secretory pulses in the sheep but enhances also Kiss1 mRNA levels in the POA, whereas it decreases them in the ARC (19). In addition, a fraction of Kiss1 neurons in AVPV in mice have been shown to express the melanocortin receptor MC4R (77). As was described for NPY, the interactions between POMC and Kiss1 systems appear to be bidirectional, as Kp-10 was reported to inhibit POMC gene expression at the ARC in the sheep (20), while in the mouse kisspeptin has been demonstrated to directly activate ARC POMC neurons (131). Such a combination of excitatory and inhibitory actions illustrates the complexity of the interplay between these two pathways. Furthermore, the ability of NPY projections to suppress kisspeptin excitation of POMC neurons in mice documents a putative multilateral mode of interaction between kisspeptin networks and key orexigenic and anorexigenic signals (131), whose physiological importance in terms of metabolic control of fertility awaits further investigation.
4. Insulin-like growth factor I and insulin
Compelling evidence coming from functional genomic studies demonstrated a decade ago the critical role of insulin signaling in the brain for the metabolic control of the HPG axis, as brain-specific inactivation of insulin receptor resulted in metabolic alterations and infertility (41). In the same line, insulin has been proposed as major direct regulator of GnRH function (351). In this context, the observations of suppressed hypothalamic expression of Kiss1 mRNA in models of uncontrolled type 1 diabetes prompted specific analyses of the role of insulin in the regulation of the Kiss1 system. Yet, despite its important metabolic/reproductive roles, insulin does not appear to significantly contribute to the regulation of Kiss1 expression, at least in rodents, as evidenced by in vivo studies, where diabetic male rats infused with insulin directly into the brain failed to display any significant change in Kiss1 mRNA levels at the hypothalamus. In the same line, in vitro studies showed that Kiss1 mRNA levels remain unaltered in hypothalamic N6 cells stimulated with insulin (59, 257). Conversely, insulin-like growth factor I (IGF-I) has been reported to enhance Kiss1 mRNA levels at the AVPV in prepubertal female rats (176), thus suggesting a putative role in the control of Kiss1 expression at this site. Yet, it must be stressed also that blockade of IGF-I receptor did not alter Kiss1 mRNA levels in the periventricular hypothalamic area in adult female rats (465).
To our knowledge, the putative roles of other metabolic signals in the control of Kiss1 expression have not been evaluated in detail, except for the orexigenic neuropeptide melanin-concentrating hormone (MCH), which is prominently expressed in the lateral hypothalamus and has been very recently shown to suppress kisspeptin-induced stimulation of GnRH neurons (501). Yet, the ability of MCH to modulate other aspects of the system, such as Kiss1 expression, remains to date unexplored. On the other hand, the interplay between the Kiss1 and GnIH/RFRP system (reviewed in sect. VIIC) may involve also the ability of RFRP to attenuate the excitatory actions of kisspeptin on POMC neurons, a phenomenon with potential implications in terms of metabolic control of the gonadotropic axis (131). Finally, it is worth noting that, despite the known inhibitory impact of lipo- and glucodeprivation on gonadotropin secretion and on the neuroendocrine centers governing reproductive function (129, 387), the possibility that nutritional cues may directly regulate Kiss1 neurons has not been addressed to date.
D. Molecular Mechanisms for the Metabolic Regulation of Kisspeptin Signaling
In addition to the identification of the putative hormone and neuropeptide signals responsible for the metabolic control of the hypothalamic Kiss1 system, efforts have been focused recently in trying to unveil the molecular mechanisms underlying this phenomenon. Admittedly, while there have been some developments in the area, our understanding of the pathways whereby leptin and other metabolic factors conduct their regulatory roles on Kiss1 expression, and eventually kisspeptin functions, is still very limited, and in some cases controversial. In any event, compelling evidence published in recent years suggests that both Crtc1 and mTOR signaling in the brain are potential components of the pathways whereby leptin directly or indirectly modulates Kiss1 gene expression at the hypothalamus.
Crtc1, which stands for cAMP responsive element-binding protein-1 (Creb1)-regulated transcription coactivator-1 (also termed TORC1, for transducer of regulated Creb), is a cytoplasmic coactivator that becomes activated and shuttles to the nucleus upon dephosphorylation in response to cAMP and calcium signals to modulate the expression of target genes. Conversely, phosphorylation of Crtc1, via salt-inducible kinase (SIK), sequesters it in the cytoplasm, thus preventing its transcription modulatory activity (323, 442, 443). Genetically modified mouse models have been generated to dissect out the physiological relevance of Crtc1 pathways in the control of different metabolic routes. In this context, it has been reported that Crtc1 KO mice display not only obese and hyperphagic phenotypes, but are also infertile (8). The underlying mechanisms for such a combined alteration of energy homeostasis and reproduction seem to involve the disturbance, due to the lack of Crtc1, of the ability of leptin to stimulate the expression of Kiss1 and CART genes; the latter is an anorexigenic factor whose absence may explain the obesity phenotype in Crtc1 null mice. Regarding the control of Kiss1 transcriptional activity, leptin has been shown to induce the dephosphorylation and activation of Crtc1, which in turn stimulates the recruitment of Crtc1 to Kiss1 gene promoter. In addition, dephosphorylation of Crtc1 enhanced Kiss1 gene expression in GT1–7 cells, and Crtc1 overexpression increased Kiss1 promoter activity (8). Of important note, however, a very recent report has been unable to replicate the consequences of functional inactivation of Crtc1 on mouse fertility (39). Although the reasons for such discrepancy are yet to be solved, this discordant observation brings a call of caution on the actual physiological/indispensable role of Crtc1 in mediating leptin effects on the HPG axis in different mammalian species.
Another mechanism for the metabolic regulation of the Kiss1 system may involve intracellular energy sensors acting at certain brain (hypothalamic) circuits. Several systems have been identified during the last years as key elements for sensing the cellular energy status, thus allowing the coupling of energy availability of any cell and its functional activity (45, 84, 263, 295, 394, 478, 502). In addition to such a ubiquitous role, some of these energy sensors have been shown recently to operate also in discrete neuronal populations within the hypothalamus as gauge systems playing a physiological role in the control of energy homeostasis at the whole body level. A good example for such a factor is the mammalian target of rapamycin (mTOR) and its downstream effectors. Thus mTOR, and specifically the rapamycin-sensitive mTORC1 pathway, is an essential component of the energy-sensing mechanisms of the cell, whereby external nutrient/hormonal signals and cellular functions are coupled (84, 263, 394, 478, 502). In addition, mTOR signaling at certain hypothalamic nuclei is involved in the central control of food intake and energy homeostasis (75, 500). Thus central activation of mTOR was reported to suppress feeding, and mTORC1 signaling in the ARC was proposed as the key component for the mechanism whereby leptin conveys its anorectic effects (75). However, the roles of hypothalamic mTOR signaling in the control of energy homeostasis are probably more complex and involve nuclei other than the ARC (486). In fact, recent experimental evidence suggests that, rather than promoting a uniform regulation of hypothalamic mTOR activity, nutritional and metabolic signals modulate the mTORC1 pathway within the medial basal hypothalamus in a nucleus- and cell-specific manner (486). In any event, the apparent connection between leptin, hypothalamic mTORC1 signaling, and energy homeostasis made it tempting to hypothesize that this molecular pathway might also participate in mediating some of the effects of leptin in the central control of the HPG axis.
This possibility has been recently substantiated by a combination of pharmacological and expression analyses in pubertal female rats (376). These studies have documented that while activation of central mTOR signaling by icv administration of l-Leu induced acute LH secretory responses and partially rescued the state of suppressed gonadotropin levels induced by subnutrition, its blockade by repeated rapamycin injections disrupted the normal timing of puberty in female rats, as demonstrated by delayed vaginal opening, decreased ovarian and uterus weight, impaired ovarian follicular development, and suppressed ovulation. Of note, inhibition of central mTOR by rapamycin reversibly reduced circulating LH levels but did not alter LH responses to key activators of the gonadotropic axis, such as kisspeptins, therefore suggesting that mTOR signaling is mandatory for proper function of the afferent network governing GnRH/gonadotropin secretion. Interestingly, the permissive actions of leptin on puberty onset were abolished by the concomitant inhibition of mTOR signaling, as evidenced by the blocking effects of rapamycin on the capacity of leptin to increase the rates of vaginal opening and ovulation in pubertal female rats subjected to chronic food restriction (376). As a potential mechanism for the inhibitory effects of suppressed mTOR signaling on the reproductive axis, persistent blockade of central mTOR was shown to cause a significant suppression of Kiss1 mRNA levels in the ARC and, to a lesser extent, the AVPV. Thus, although additional functions of mTOR (e.g., at the level of GnRH neurons) cannot be excluded, the available data support the contention that central mTOR signaling may play an important role in transmitting the positive effects of leptin on the reproductive axis, and that this action is, at least partially, conducted via modulation of Kiss1 expression at key hypothalamic nuclei. Yet, the neuroanatomical substrate for such a leptin-mTOR-kisspeptin pathway, e.g., if it is composed of one or multiple neuronal elements, is yet to be defined (377).
Other brain fuel-sensing mechanisms might be also involved in the central control of the HPG axis. One possible candidate for such regulation is AMP-activated protein kinase (AMPK), another member of the metabolite-sensing protein kinase family (45, 295). AMPK is a heterotrimeric protein formed by a catalytic α subunit and the regulatory β and γ subunits. AMPK senses changes in the AMP/ATP ratio by competitive binding with both molecules. In conditions of energy insufficiency, ATP is consumed and excess AMP accumulates in the cell, promoting AMPK activation, which in turn phosphorylates and inactivates diverse ATP-consuming metabolic cascades. As described for the mTOR pathway, brain AMPK signaling has been proposed as a pivotal regulator of energy balance and food intake, and activation of AMPK stimulates appetite (74, 196). In good agreement, leptin has been shown to inhibit hypothalamic AMPK activity, whereas the orexigenic hormone ghrelin stimulates it (10). Given the well-known interplay between AMPK and mTOR, e.g., AMPK inactivates mTOR in different cell systems (184, 478), these two metabolic cell sensors have been proposed to reciprocally cooperate in the central control of energy homeostasis (75). Whether such an interaction applies also to the metabolic regulation of the HPG axis is unknown. Indirect evidence, however, supports such a possibility, as AMPK activation inhibited GnRH secretion in murine GT1–7 cells and moderately altered estrous cyclicity in adult rats (76, 495). Such inhibitory actions of AMPK pathways are very much in line with its proposed role as sensor of energy insufficiency and functional antagonist of mTOR. In this context, the possibility that AMPK may participate in the metabolic control of Kiss1 expression merits specific investigation.
XII. KISS1 NEURONS IN OTHER PHYSIOLOGICAL AND PATHOLOGICAL STATES OF THE HPG AXIS
As summarized in previous sections, there is compelling evidence supporting the involvement of kisspeptin signaling in the control of a variety of essential aspects of the maturation and function of the HPG axis. These likely include the control of gonadotropin secretion at key functional states of the gonadotropic system, such as pregnancy and lactation in the female, as well as ageing. Similarly, emerging evidence suggests a central role of Kiss1 neurons in mediating the inhibitory effects of stress conditions and acute inflammation on the HPG axis. Experimental data supporting such roles are briefly summarized below.
A. Pregnancy and Lactation
As indicated elsewhere in this review, the functionality of the HPG axis in the adult female undergoes dramatic physiological changes throughout the different stages of the ovarian cycle, as well as during pregnancy and lactation. While the involvement of the hypothalamic Kiss1 system in the control of gonadotropin secretion during the cycle is now well defined, its putative roles in the control of the gonadotropic axis during pregnancy and lactation have received so far much limited attention. Analysis of the eventual impact of kisspeptins on gonadotropin secretion in pregnancy seems a sensitive issue, given the fact that circulating kisspeptin dramatically increases (up 7,000-fold) during human pregnancy (179). Yet, despite the proven stimulatory effects of systemic administration of kisspeptins on LH and FSH secretion in different species, including humans, gonadotropin levels are partially suppressed, rather than increased, during gestation (249, 287).
The low levels of gonadotropins in the face of very high concentrations of circulating kisspeptins during human pregnancy may be the result of the inhibitory influence of markedly elevated levels of estradiol and progesterone, as well as prolactin, all of which can operate as potent suppressors of gonadotropin secretion. Yet, to our knowledge, the influence of sex steroids and prolactin upon gonadotropin responsiveness to kisspeptins in humans has not been explored to date. In addition, desensitization to the gonadotropin stimulatory effects of kisspeptins, in the face of the persistent elevation of their circulating levels, is also likely to take place in human pregnancy. Given the obvious experimental limitations in humans, pharmacological studies to test the latter possibility have been conducted in rats at mid and late pregnancy. Those analyses demonstrated that, despite variable degrees of suppression of their basal level, gonadotropin responses to exogenous Kp-10 were fully preserved, if not enhanced in terms of relative LH increases, during gestation (380). Furthermore, the sensitivity to low doses of Kp-10 was augmented, and hypothalamic expression of Gpr54 mRNA was not apparently altered, in rat pregnancy (380). While these findings would argue against the possibility of robust desensitization of Gpr54 following continuous gestational exposure to persistently elevated levels of kisspeptins in rodents, it must be stressed that the rat may not be an optimal model for such comparative analyses since, in contrast to humans, it has not been possible to document the existence of a sustained increase of circulating levels of kisspeptins during rat pregnancy due to the lack of reliable rodent assays. In fact, the expression levels of Kiss1 transcript in rodent placenta seem to be lower than in humans (461). In addition, an increase in hypothalamic expression of Kiss1 mRNA has been reported in the pregnant rat, whose physiological relevance, in face of decreased basal LH levels, is obscured by the fact that the anatomical location of such changes has not been provided (380). All in all, the experimental data available fall short in explaining the basis for the state of low circulating gonadotropins in the face of dramatically elevated kisspeptin levels in pregnant women, and set the scene for additional studies addressing the potential desensitization of the hypothalamic effects of kisspeptins selectively in human gestation.
In addition, the eventual contribution of changes in the hypothalamic Kiss1 system to the state of hypogonadotropism and anovulation associated with lactation has been also studied. The available evidence, coming from rodent models, points out that lactation has a discernible inhibitory impact on the expression and function of this system. Thus expression analyses of Kiss1 by qRT-PCR and in situ hybridization evidenced significant suppression of its mRNA levels at the ARC during lactation (505). Similarly, the number of kisspeptin-IR neurons was lower in lactating dams (503). Of note, these analyses were conducted in OVX lactating rats as a means to avoid the potential confounding factor of changes in endogenous estrogen levels. Yet, previous analyses in gonadal-intact lactating rats had failed to show any change in Kiss1 mRNA levels, although the lack of anatomical resolution of the latter studies may have hampered detection of subtle changes (380). Also of interest, a concomitant suppression of NKB mRNA expression in the ARC has been documented in lactating rats (503), while the levels of Kiss1 mRNA are also suppressed in the AVPV during lactation. Curiously enough, kisspeptin content in this nucleus appears to be augmented in lactating dams, eventually suggesting a suppression of peptide release (431), that needs to be confirmed.
In addition to these alterations in terms of expression, rodent studies have disclosed a state of reduced responsiveness to kisspeptins during lactation, as documented in gonadal-intact lactating dams. Thus low doses of Kp-10 that were fully effective in cyclic female rats were shown to be totally ineffective in terms of induction of LH release in lactating mothers (380). Accordingly, it is tempting to propose that, among other adaptative responses, lactation induces a state of reduced Kiss1/kisspeptin expression and decreased responsiveness to the gonadotropin releasing actions of kisspeptins, which is likely involved in the generation of the characteristic inhibition of the HPG axis observed during this period. The mechanisms for the above changes in the Kiss1 system are yet to be disclosed, but apparently the decrease in leptin and insulin levels associated with lactation does not have a major causative role, at least in rodents (503).
Changes of the hypothalamic Kiss1 system have been also studied in the context of reproductive senescence. Of note, studies in rodents and humans on this particular topic are likely to show important divergences, due to the intrinsic differences in the process of ageing of the HPG axis in primates and nonprimates. Studies in rodents, where ageing is associated with a primary decrease of GnRH neurosecretory activity and defective generation of the preovulatory surge in the female, have demonstrated that in middle-aged female rats there is a detectable decrease in Kiss1/kisspeptin levels at the AVPV associated with attenuated preovulatory peaks of LH, whose magnitude could be restored by central infusion of kisspeptin into the mPOA (235, 311). This stimulatory effect of kisspeptin was associated with increased glutamate and decreased GABA release in the POA, whereas blockade of glutamate NMDA receptors prevented it (311). These observations suggest that such an effect is conducted, at least partially, via modulation of glutamate and GABA neurotransmission, as supported also by electrophysiological studies in young female mice (337, 338). Altogether, the available evidence indicates that reproductive senescence in female rodents, and eventually in other nonmenopausal species, involves a primary failure of the Kiss1 circuits involved in the generation of the preovulatory surge, therefore causing alterations in ovulation.
In contrast to the situation in rodents, in female humans and other primates, ovarian failure is a primary event in reproductive senescence, thus leading to the withdrawal of negative sex steroid feedback and the postmenopausal rise of gonadotropins. In these conditions, it has been well characterized that an increase in Kiss1 mRNA levels takes place at the infundibular region of postmenopausal women and monkeys, as a reflection of the role of this neuronal population in the negative-feedback control of gonadotropins (114, 215, 383). Whether some degree of failure of Kiss1 neurons takes place at more advanced stages of ageing in human females, and to what extent a similar phenomenon may apply to the male (in primate and/or nonprimate species), is yet to be investigated.
C. Stress and Inflammation
Significant functional changes of the HPG axis are also observed in conditions of acute stress and immune/inflammatory challenge, which are frequently associated with transient suppression of gonadotropin secretion. In this context, it has been evaluated whether the impact of such conditions is conveyed through alterations of the hypothalamic Kiss1 system. Studies using administration of the bacterial lipopolysaccharide S (LPS), as model of acute inflammation linked to alteration of gonadotropic function, have documented a significant decrease in kisspeptin-IR levels in the ARC of adult male rats (53). Likewise, LPS administration to female rats induced a significant decrease of Kiss1 mRNA in the ARC and mPOA areas, which was associated with a transient decrease in pulsatile LH secretion (188, 218). Of note, studies in male rats documented that changes in kisspeptin-IR were not merely due to the anorectic responses coupled to LPS injection, thus suggesting the participation of specific hormonal or immune effectors (53). In the same vein, while rat studies have shown that LH responses to kisspeptins are detectable in LPS-treated males, the absolute magnitude of such responses was decreased under such stress conditions (53). This is in contrast to the trend to increased LH responses to kisspeptin observed in situations of food restriction, thus suggesting the specific impact of inflammation-associated changes, independent of the state of reduced food intake.
Other forms of stress applied in adulthood also have a discernible effect on the elements of the Kiss1 system. Thus restraint stress and insulin-induced hypoglycemia, which caused perturbations of the pulsatile release of LH in female rats, induced also changes in Kiss1 (and/or Gpr54) mRNA levels in the ARC and mPOA, the latter encompassing the AVPV as a major source of Kiss1 expression (218). Actually, LPS treatment in adult female rats induced also a decrease in Gpr54 expression at both sites. Likewise, administration of corticotropin-releasing hormone (CRH) or corticosterone, as to mimic the increase of both factors in stress conditions, caused similar reductions in Kiss1 mRNA levels in the ARC and mPOA (218). Therefore, it is plausible that different forms of stress may impact the expression of the elements of Kiss1 system, at least partially, via changes in CRH/glucocorticoid levels. On a related front, it has been demonstrated in rodents that either chronic or binge types of alcohol intake, which can be considered an external stressor with deleterious effects on GnRH/LH secretion, have an inhibitory impact on the basal expression of Kiss1 gene in the ARC and AVPV (repeated administration) (433), or on its response to IGF-I (binge exposure) (175). Finally, transient exposure to stress stimuli, such as LPS administration, during early stages of reproductive maturation, as the neonatal period, disturbs the timing of puberty and decreases Kiss1 mRNA levels in the mPOA in pubertal rats (224). These observations, together with those of models of early-onset obesity also in female rats (54), would suggest that the developing Kiss1 system is sensitive to the organizing effects not only of sex steroids, but also of different metabolic and immune stressors, which may cooperate in shaping the maturation of this hypothalamic system, thereby contributing to define the normal, or eventual pathological, course of puberty onset and reproductive function later in life.
XIII. KISS1 NEURONS AND THE SEASONAL CONTROL OF REPRODUCTION
As reviewed in previous sections, compelling evidence has accumulated in recent years to demonstrate the regulation of the hypothalamic Kiss1 system by a number of key modulators of the HPG axis, from sex steroids to nutritional and metabolic signals. In addition to those, reproductive function in many vertebrate species is exquisitely sensitive to environmental cues signaling the season and day length to the centers governing reproductive function (142). Experimental data obtained in such seasonal breeders, mostly hamsters, sheep, and some fish species, have now set the contention that the hypothalamic Kiss1 system is sensitive to such environmental signals, thus defining a putative pathway whereby these cues engage in the networks governing GnRH secretion. Despite the obvious commonalties, these studies have allowed to identify also interesting differences among species in terms of neuroanatomical organization, regulation, and functional relevance of such a seasonal control of the Kiss1 system. While this section concentrates on data coming from studies in mammals, specific comments on seasonal regulation of the Kiss system in nonmammalian vertebrates can be found in section XV of this review.
A. Neuroanatomical and Expression Data
The first evidence for the putative role of Kiss1 neurons as mediators of the photoperiodic control of the reproductive axis came from studies in male Syrian hamsters, where exposure to short days (SD), which in this species drives an inhibitory signal to the HPG system, resulted in a marked suppression of Kiss1 mRNA levels in the ARC (370). Interestingly, those male Syrian hamsters that were refractory to the photoinhibitory effects of SD did not display decreased Kiss1 expression at this nucleus (370). Similarly, studies in female Siberian hamsters evidenced striking differences in the profiles of expression of Kiss1/kisspeptin at the hypothalamus between photo-sensitive and photo-refractory animals (155, 266). Yet, these studies surfaced also intriguing species differences, since Siberian hamsters responded to SD with reduced Kiss1/kisspeptin expression in the AVPV but increased levels in the ARC (155, 266). The possibility that such discrepancy might solely reflect sex differences is unlikely, since later analyses documented the ability of SD to elevate kisspeptin-IR at the ARC of male Siberian hamsters (152), while consistent inhibitory responses to SD, in terms of Kiss1 mRNA expression, have been demonstrated in the ARC and AVPV of male and female Syrian hamsters (12). The basis for this apparent divergence in the pattern of response of the ARC Kiss1 neuronal population to similar photoperiodic stimuli between these two closely-related species remains so far unsolved.
In addition to data from hamsters, evidence from studies in the sheep, another model of seasonal breeding of obvious physiological and applied interest, has confirmed the role of the hypothalamic Kiss1 system in the photoperiodic control of reproduction. Of note, in this species, the reproductive axis is activated by SD, while long days (LD) induce anestrus. During such anestrous season, Kiss1 mRNA levels at the ARC were significantly suppressed (420). Importantly, such suppression was detected in OVX ewes, used as a model to avoid the potential confounding factor of seasonal variations of sex steroids. In the same line, Kiss1 mRNA levels and kisspeptin-IR at the ARC of OVX ewes supplemented with estradiol were consistently higher during the breeding season (421). This suggests that the above photoperiodic changes are caused by modifications of photic afferents rather than being secondary to alterations of sex steroid milieu. In good agreement, exposure to SD in male Siberian hamsters caused a marked suppression of kisspeptin-IR in the AVPV irrespective of gonadal status (152). Likewise, inhibition of Kiss1 mRNA expression in the ARC by SD in male and female Syrian hamsters was not caused either by the changes in circulating sex steroid levels, but was rather primarily derived from an hypothalamic action (12). Yet, some degree of interaction/cooperation between sex steroid background and photoperiod in terms of control of Kiss1 mRNA expression was detected in both species (12, 152). For instance, the suppression of Kiss1 mRNA at the AVPV in female Syrian hamsters is likely secondary to the observed changes (inhibition) of ovarian sex steroid secretion in SD. In addition, the suppressive effects of estradiol on ARC Kiss1/kisspeptin expression were greater during the nonbreeding season. Altogether, the above data suggest that gonadal factors and photoperiodic cues interactively participate in the precise control of the Kiss1 system in seasonal breeders (12, 152).
Finally, expression/anatomical analyses have helped also to initiate the study of other putative regulatory signals involved in the seasonal control of Kiss1 and reproduction. For obvious reasons, the pineal-derived factor melatonin has received substantial attention in this context, as a major inhibitory signal for the photic control of the HPG axis (369). Studies in the Syrian hamster, involving pinealectomy and melatonin treatment, have suggested that this transmitter plays an important role in the control of the hypothalamic Kiss1 system (370), although the effects of melatonin appear to depend on the breeding season, the sex steroid milieu, and/or the hypothalamic nucleus (12). Thus melatonin administration to hamsters under LD conditions, whose endogenous melatonin levels are expectedly low, resulted in suppression of Kiss1 mRNA at both ARC and AVPV, although the AVPV suppression only manifested in the presence of testicular hormones (12). Conversely, pinealectomy of SD hamsters, whose endogenous melatonin levels are expectedly high due to photo-inhibitory conditions, increased Kiss1 expression, but only in the ARC (12). The above data further stress the importance of the interaction of central signals, such as melatonin, and peripheral sex steroids in the precise regulation of Kiss1 expression in the hypothalamus by environmental seasonal cues.
B. Functional Data
Additional support for the physiological relevance of the Kiss1 system in the photoperiodic control of reproduction came for functional studies addressing the effects of kisspeptin administration in animal models with photo-inhibition of the HPG axis. While some conflicting results have been reported, the overall view is that kisspeptin administration in conditions of photoperiodic suppression of reproduction can rescue gonadotropin function, as evaluated in terms of acute gonadotropin responses, trophic testicular effects and induction of ovulation. Thus acute kisspeptin injection induced robust LH responses in Siberian hamsters exposed to SD (155), while chronic central administration of Kp-10 to SD Syrian hamsters reversed testicular atrophy despite persistent photo-inhibitory conditions (370). A similar rescue of testicular activity has been recently reported in photo-inhibited Syrian hamsters by protocols of twice daily ip injections of Kp-54 (11). In the same vein, kisspeptin infusion in sheep during the anestrous season has been shown to induce ovulation in >80% of treated animals, in contrast with the low ovulatory rates (<20%) in nontreated individuals (49). Taken as a whole, these observations suggest that restoration of defective kisspeptin tone would be sufficient to reactivate the HPG axis during the nonbreeding period, which indirectly reinforces the physiological relevance of seasonal regulation of the hypothalamic Kiss1 system. Admittedly, however, some reports have questioned the ability of kisspeptin infusions or repeated injections to reverse or prevent testicular regression in SD Siberian hamsters (153). Besides potential species differences, disparities in the protocols and doses of administration might help to explain these contradictory findings. As an additional source of variability, responses to kisspeptins have been recently reported to vary as a function of sex and reproductive condition in Siberian hamsters (154).
XIV. PUTATIVE EXTRAHYPOTHALAMIC ACTIONS OF KISSPEPTINS IN THE CONTROL OF REPRODUCTION
While the wealth of experimental data gathered to date conclusively point to a primary site of action of kisspeptins at the hypothalamus, compelling, as yet fragmentary, evidence obtained in different species suggests additional regulatory effects of kisspeptins at other levels of the reproductive system. Admittedly, such peripheral actions might be considered modulatory, as responsible for the fine tuning of specific tissue functions, in clear contrast to the indispensable roles of kisspeptin signaling in the brain for proper puberty onset and fertility (374, 448). Yet, the subordinated nature of the extrahypothalamic effects of kisspeptins should not preclude the analysis of those functions, which may become relevant in specific physiological or pathophysiological circumstances.
The possibility of kisspeptin actions directly at the pituitary level to control gonadotropin secretion was first proposed by studies on the characterization of the pharmacological effects of Kp-10 on LH and FSH release in the rat. Those analyses revealed stimulation of pituitary LH secretion in vitro by Kp-10 in the nanomolar range, whereas FSH release was only marginally increased by Kp-10 when coincubated in the presence of GnRH (299, 300). Of note, in contrast to in vivo data, the magnitude of LH responses to Kp-10 in vitro was somewhat modest and significantly lower than that of LH responses to GnRH (300). Thereafter, the ability of kisspeptin to elicit LH release acting directly at the pituitary has been further documented in rodents, where Ca2+ responses have been identified in gonadotropes following direct kisspeptin stimulation of dispersed rat pituitary cells (158), as well as in bovine (119, 437), ovine (427), and porcine (437) species, even at the nanomolar range in some cases. Similarly, Kp-10 was capable to elicit LH secretion directly from female baboon pituitaries (256). Furthermore, the ability of Kp-10 to elicit LH secretion directly at the pituitary level has been recently documented also in nonmammalian vertebrates, such as goldfish (507).
In addition, expression studies in the rat have suggested that both Kiss1 and Gpr54 mRNAs, as well as the corresponding peptides, are expressed at the pituitary (158, 371), with detection of kisspeptin- and Gpr54-IR in pituitary gonadotropes (371). Moreover, these rat studies suggested that expression of Kiss1 and Gpr54 genes is hormonally regulated: estrogen, acting via ERα, enhances Kiss1 but reduces Gpr54 mRNA levels, whereas GnRH selectively enhances Gpr54 expression at the pituitary (371). In the same line, the presence of kisspeptin-positive cells in the intermediate and anterior lobe of monkey pituitary has been recently documented, thus adding further morphological support to the possibility that kisspeptins may act directly at the level of the pituitary (361). Yet, no evidence for colocalization of kisspeptins in gonadotropes was found in that study (361). In addition, in the sheep, kisspeptins have been detected in hypophysial portal blood (427), whereas release of Kp-54 at the level of the stalk-median eminence, coincident with GnRH pulses and with the potential to reach the portal system, has been reported in the pubertal monkey (210). Collectively considered, the above data strongly suggest that kisspeptins may conduct specific stimulatory actions directly at the pituitary to stimulate gonadotropin secretion, mainly LH.
In spite of the above evidence, it is important to stress that a few other studies have been unable to detect any direct action of kisspeptins in the rat pituitary (268, 464). Of note, the latter studies explored the effects of kisspeptins on adult pituitary tissue, whereas direct stimulatory effects have been described in pituitaries from pubertal rats (158, 300), a feature which may account, at least partially, for such discrepant results. In addition, while detectable levels of kisspeptins have been reported in the hypophysial portal blood in the sheep, their concentrations do not display detectable fluctuations during key reproductive states, such as the preovulatory surge (427). Overall, whereas these results do not invalidate the possibility of direct pituitary actions of kisspeptins in the control of the gonadotropic axis, they bring a call of caution on their potential physiological relevance, which nevertheless warrants further investigation.
As was the case for the pituitary, initial expression analyses already revealed the presence of Kiss1 and/or GPR54 mRNAs in the testis and the ovary of humans and rodents. The possibility, however, of direct gonadal actions of kisspeptins remains scarcely studied to date. Notwithstanding this, evidence has been presented for the expression of Kiss1 gene and/or protein in the rat, monkey, and human ovary. In addition, expression of GPR54, at the mRNA and protein level, has been also documented in some of these species. Concerning the testis, only limited evidence for the presence and function of the elements of the Kiss1 system in male gonads has been presented to date. Further comments on the gonadal expression of Kiss and GPR54 genes in nonmammalian vertebrates can be found in section XV of this review.
Regarding the ovary, studies in the rat were the first to document the expression of Kiss1/kisspeptin and Gpr54 in the female gonad at different stages of postnatal maturation, across the cycle and in response to hormonal manipulations (56). Of interest, those studies demonstrated that, while Gpr54 mRNA levels remained rather low and stable across the ovarian cycle, ovarian Kiss1 expression increased during the pubertal transition and peaked at the afternoon of proestrus, i.e., preceding ovulation (56). In good agreement, more recent studies in the Siberian hamster demonstrated enhanced kisspeptin-IR during the ovulatory transition, i.e., proestrus and estrus (405). Ovarian expression of Kiss1 appears to be under the regulation of pituitary gonadotropins, since protocols of gonadotropin priming were able to enhance Kiss1 mRNA levels in the ovary of immature rats, while prevention of the preovulatory surge of gonadotropins blocked the rise of ovarian Kiss1 expression (56). In the same line, photo-inhibition of the gonadotropic axis in hamsters by SD evoked a significant reduction of ovarian Kiss1 mRNA levels (405).
In addition, recent analyses in a model of ovulatory dysfunction induced by inhibition of prostaglandin synthesis demonstrated a marked suppression of ovarian Kiss1 mRNA levels during the ovulatory period coincident with altered ovulatory dynamics. Moreover, gonadotropin-induced expression of Kiss1 was blocked by inhibition of prostaglandin synthesis (135). Collectively considered, the above observations are suggestive of a putative role of locally produced kisspeptins in the control of ovulation, whose nature and physiological relevance is yet to be defined. On the other hand, the demonstration of prominent kisspeptin-IR at various ovarian compartments such as the theca layer of growing follicles, the corpus luteum, and the interstitial gland, may indicate additional ovarian functions of locally-born kisspeptins (56, 135). Admittedly, the fact that humans and rodents with impaired GPR54 signaling can ovulate, if properly primed with gonadotropins, argues against an indispensable role of local kisspeptins in the control of ovulation (325, 401). Yet, to our knowledge, detailed analyses of the ovulatory responses, or any other ovarian functions, in individuals with null mutations of GPR54 following gonadotropin priming have not been reported to date. All in all, the above evidence, together with the highly conserved patterns of expression of Kiss1/kisspeptin between rodent, monkey, and human ovaries (135), point out the possibility of direct ovarian actions of kisspeptins that, even if subordinated to the major central roles of the Kiss1 system, merit further investigation. Similarly, the possibility that ovarian-derived kisspeptins may have an impact on gonadotropin secretion has been recently suggested on the basis of hormonal analyses in HH patients primed with exogenous gonadotropins (23), yet the nature and relevance of such a regulatory loop need to be characterized further.
Regarding the testis, there is much paucity of data on the potential local expression or actions of kisspeptins, apart from initial reports on the expression of KISS1 and, faintly, GPR54 in human testis (318). While, to our knowledge, detailed expression analyses have not been published yet, our unpublished studies evidence that the Kiss1 gene is expressed in the testis of various mammalian species, including the rat, mouse, and monkey, in addition to the human, with detectable expression of Kiss1 mRNA in the rat testis across postnatal maturation. Likewise, kisspeptin-IR has been detected at the rat testicular tissue, mainly in interstitial Leydig cells (Garcia-Galiano, Gaytan, and Tena-Sempere, unpublished data). In the same line, the presence of kisspeptin-IR has been very recently documented in human spermatozoa (342). From a functional standpoint, preliminary analyses suggest that, while Kp-10 per se does not alter basal testosterone secretion by rat testicular explants, it potentiates testosterone responses to gonadotropic stimulation ex vivo (Garcia-Galiano et al., unpublished data). These observations are in agreement with previous functional analyses of the consequences of kisspeptin infusion on testosterone secretion in male monkeys, where despite evidence for desensitization of gonadotropin responses to continuous administration of high doses of Kp-10, testosterone levels were consistently higher than LH concentrations, therefore suggesting potential stimulatory effects of kisspeptins directly at the testicular level (365). In a related front, Kp-13 has been recently shown to evoke Ca2+ responses and changes of motility in human sperm (342). Again, the mechanisms, sites of action and eventual physiological relevance of these phenomena need to be clarified.
C. Expression and Actions at the Placenta
Although the central neuroendocrine roles of kisspeptins have been the major focus of research in this field, the peptide was first isolated and structurally identified by mass spectrometry from human placenta (228). In fact, demonstration of abundant KISS1 gene expression in the human placenta, together with the recognition of the anti-metastatic activity of kisspeptins (228, 318), attracted speculation about their potential role in regulating the invasion of placental trophoblast cells into the endometrium. There are striking similarities between the biology, behavior, and regulatory mechanisms of invasive placental cells and cancer cells. For example, matrix metalloproteases (MMPs) have been implicated in tumor metastasis (506, 512) and trophoblast invasion (71), likely due to their ability to affect cellular interactions with the extracellular matrix, and hence cell division, migration, and morphogenesis (290). In fact, kisspeptins have been shown to inhibit MMP activity. In addition, angiogenesis is also a prerequisite for both placental formation and cancer spread (293). Interestingly, kisspeptins and GPR54 are expressed in human blood vessels and induce vasoconstriction (275), which suggests a potential role in angiogenesis. In fact, recent studies on human placental blood vessels and umbilical vein endothelial cells (HUVEC) demonstrated expression of GPR54 and documented the ability of Kp-10 to inhibit angiogenesis and to suppress proliferation and migration of HUVEC, without affecting their viability or apoptosis (360).
Kisspeptins and GPR54 appear to be appropriately located at the feto-maternal interface. KISS1 gene and kisspeptins are highly expressed in the syncytiotrophoblast (ST) in normal human placenta (30), with increased KISS1 mRNA levels in early and molar placentas (190). Of note, the outer ST lies adjacent to blood vessels, facilitating passage of kisspeptins into the maternal bloodstream. In turn, GPR54 is localized in the villous and invasive extravillous human cytotrophoblasts (30, 190), thus providing the basis for putative autocrine and paracrine regulation of invasion by trophoblast cells. In addition, it has been reported that KISS1 and GPR54 mRNA levels are higher in first trimester than in term placentas (30). This contrasts with the profile of changes of circulating Kp-54 that rises as pregnancy progresses: ∼1,000-fold increase in the first trimester, and up to ∼7,000-fold in the third trimester (179). Of interest, there appear to be parallels in both the spatial and temporal expression of Kiss1 and GPR54 genes at the placenta between women and the laboratory rat. Thus Kiss1 mRNA is expressed in rodent trophoblast giant cells, responsible for early invasion of the spiral arteries and replacement of the endovasculature, which are equivalent to the human extravillous trophoblast (461). Yet, the marked increase in circulating levels of Kp-54 in human pregnancy has not been confirmed so far in rodents.
The highest expression of Kiss1 and GPR54 in trophoblast cells during the first trimester in humans and at embryonic day 12.5 in rats coincides with the peak of trophoblast invasion which is critical for pregnancy. In addition, the fact that Kp-10 inhibits the migration and invasion of trophoblast cells provides further support to the notion that kisspeptins may play a role in restraining trophoblast invasion and regulating implantation (30, 173). Circulating concentrations of kisspeptins were found lower at early stages of pregnancy (between 8–14 wk) in women who had small-for-gestational-age neonates (417). Likewise, kisspeptin levels were lower in women with gestational bleeding (209), and decreased plasma kisspeptin concentrations have been reported in the second trimester of pregnancy in women who subsequently developed preeclampsia and/or intrauterine growth retardation (17, 251). At first consideration, these observations do not appear to resonate with a role of kisspeptin in invasion. However, the lower kisspeptin levels detected in women prone to develop pathologies later in pregnancy may simply reflect the dysfunctional state of the placenta. In addition to these alterations in circulating concentrations, the levels of KISS1 mRNA and kisspeptin peptide content in term trophoblasts from preeclamptic patients appeared to be elevated compared with those of control pregnancies (352), whereas decreased kisspeptin expression has been reported in the first trimester trophoblast on women with recurrent pregnancy loss (330). In addition, expression analyses in rat oviduct indirectly suggested a possible role for kisspeptins in preventing ectopic pregnancy (136). Admittedly, however, there are a number of potential confounding factors in this ensemble of studies. These include the possible lack of relationship between tissue mRNA and peptide content, circulating peptide levels, and site of action, which may be disconnected in the above biological system. Another important call of caution comes from the uncertainty about the specificity of immunoassays employed for measuring circulating kisspeptins, and the fact that some of the studies were retrospective, with handling and storage conditions being important preanalytical factors for successful kisspeptin measurement in biological fluids (359). As an additional note of caution, the data on circulating and tissue levels of KISS1/kisspeptins in relation to gestational pathologies are only correlative and do not elucidate whether the changes observed are cause or effect.
More direct proof for the putative roles of kisspeptin signaling in placental physiology may come from studies in mice and humans with null mutations of GPR54 or Kiss1 genes. While due to their HH, untreated patients with GPR54 mutations are unable to spontaneously conceive, it has been reported that few infertile women due to GPR54 inactivation have given birth to healthy infants after induction of ovulation (325). This would preclude an indispensable role of kisspeptin signaling for completion of human pregnancy. It must be stressed, however, that the features of placental formation and invasion in these patients have not been thoroughly evaluated, so it remains possible that the lack of GPR54 might have induced subtle alterations of placental morphology and/or function that, despite not preventing gestation, could have an impact in the organ physiology. To our knowledge, similar studies in Gpr54 KO mice, involving induction of ovulation and insemination, have not been reported to date. These might help to further elucidate the actual roles and relevance of kisspeptin signaling in placental function. In any event, it is also worth noting that since kisspeptins are proposed to have a restraining influence on excessive or premature implantation, the phenotype of Kiss1/GPR54 deficiency may not be very obvious. A clearer pathological phenotype might be revealed by overexpression of kisspeptins, a model which has yet to be developed. In sum, while there seems to be convincing evidence for the expression of KISS1/kisspeptins and GPR54 in normal human placenta, and for its eventual alteration in several gestational pathologies, a major role for kisspeptin signaling in pregnancy has not been yet fully established and warrants further investigation.
XV. COMPARATIVE ENDOCRINOLOGY OF KISS/KISSPEPTINS AND GPR54
The features of the Kiss1 system summarized in previous sections are defined mostly on the basis of studies in mammalian species, mainly rodents, sheep, and primates, including humans. Indeed, most of the initial efforts to unveil the physiological roles of kisspeptins in the control of reproduction were focused in mammals, while similar studies in nonmammalian species lagged behind. However, significant achievements have been made in the last few years concerning the characterization of the genomic organization, functional roles and regulatory mechanisms of the elements of the Kiss1 system in nonmammals, including fish, reptiles, and amphibians (5, 479, 481). These findings have been enormously instrumental to confirm the crucial and conserved roles of kisspeptins and GPR54 in the integral control of reproduction and have allowed gaining an accurate view of the molecular evolution and diversity of the elements of this ligand-receptor system. In this section, we will comprehensively review our current knowledge on the genomic structure and multiplicity of Kiss/kisspeptins and GPR54 in different animal phyla and will highlight the major functional features of this neuroendocrine system in nonmammalian (mostly fish) vertebrates, as emerging from comparative endocrinology studies.
A. Molecular Evolution of Kiss1 and GPR54
While the initial report on the structure of the KISS1 gene dates back to 1998, our understanding on the genomic organization of Kiss1 (and the paralogous Kiss2) has significantly enlarged only recently, mostly in the context of studies addressing the molecular evolution of the genes encoding kisspeptins (5, 456, 479, 481). These studies have surfaced the diversity of Kiss/kisspeptins in different species, thus challenging the original dogma coming from mammalian studies on the absolute selectivity of the ligand-receptor tandem, each encoded by a single gene in any given species. In fact, it is now well documented that, at least in fish and amphibian genomes, two or even three different Kiss genes exist, thus adding complexity to the evolutionary history and functionality of this system.
The original report of the human KISS1 gene suggested that it is composed of four exons and three introns, with the first and second exons being nontranslated and the last two exons encoding the 145-amino acid precursor peptide (496). Yet, some controversy has persisted on the actual genomic structure of Kiss1 in mammals, as the putative structure of the human gene has been more recently challenged by an alternative proposal for genomic organization consisting of three exons; the first nontranslated exon encompassing exons 1 and 2 of the original study (255). In addition, the mouse Kiss1 gene has been proposed to contain only two translated exons, which correspond to the last two exons of the human gene (481). In all mammalian models reported to date, the minimal active sequence encoding Kp-10 is contained in the last exon.
Despite extensive efforts, molecular cloning of the Kiss1 gene in nonmammals was not achieved until 2008, i.e., 10 years after the first study on the human, when its structure was first disclosed in several teleost species, including zebrafish and medaka (32, 202, 482). Such a delay was caused by the overall low degree of sequence homology among Kiss1 cDNAs from mammalian and nonmammalian species. Anyhow, these initial studies evidenced that the region encoding the functional kisspeptin decapeptide was notably conserved, thus highlighting not only the functional relevance of this core amino acid sequence for reproductive control, but also the strategy for the systematic cloning of Kiss1 in different phyla. Indeed, in the last 3 years, sequences of the Kiss1 gene have been reported in a number of additional fish and other nonmammalian species, as extensively reviewed recently elsewhere (5, 479, 481). Interestingly, partially discordant genomic structures have been also proposed for Kiss1 in fish; for instance, in zebrafish, the Kiss1 gene has been reported to be composed by either two or three exons (32, 221, 482), with the first exon being not translated in the latter model.
Remarkably, in the course of the above investigations, it was recognized also that a second form of Kiss gene, termed Kiss2, exists in the genome of several fish species, as initially reported in medaka, zebrafish, and sea bass (122, 221). The genomic structure of such Kiss2 gene has been unanimously recognized as to be composed by two exons and one intron. The exciting observation of the presence of a second Kiss gene in nonmammalian genomes was later confirmed in other fish species, as well as in certain reptiles, amphibians, and even mammalian monotremes (5, 481). Furthermore, in the amphibian, Xenopus tropicalis, a third Kiss isoform, termed Kiss1b, has been reported (240). Altogether, the above findings on the variable existence of Kiss paralogs documented the previously unsuspected molecular diversity of the genes encoding kisspeptins, and allowed to draw the evolutionary history of this family of ligands.
From an evolutionary perspective, chromosome synteny analyses have set the consensus that, due to whole-genome duplication events, two paralogs of the Kiss gene, putatively termed Kiss1 and Kiss2, existed already in the genome of ancient vertebrate ancestors (5, 481). Divergent evolution between the tetrapod lineages resulted in the retention of Kiss1 gene in placental and marsupial mammals, but in its loss in avian and reptiles (5, 481). As a brief note, there seems to exist a Kiss1-like sequence in the genome of some reptiles, but due to the presence of mutations in conserved cleavage sites, this is considered to be a pseudogene not encoding any functional protein (481). In clear contrast, the Kiss2 gene was selectively lost in placental and marsupial mammals, whereas it was retained in mammalian monotremes, such as platypus, and reptiles, such as lizards (122, 481). Of note, birds apparently lost also the Kiss2 gene from their genome during evolution, thus resulting in the lack of both Kiss1 and Kiss2 genes in avian species (122, 481). In contrast, a third isoform, termed Kiss1b, has been described in amphibians, probably as a paralog of the conserved Kiss1 gene (240). Concerning the teleost lineage, several fish species have retained both Kiss1 and Kiss2 genes, although selective disappearance of the Kiss1 gene has taken place in certain species. Thus, as shown in FIGURE 12, the differential evolution has defined different sets of Kiss genes in different groups of species, with placental mammals having only one Kiss1 gene, many fish species and mammalian monotremes having two Kiss genes (namely, Kiss1 and Kiss2), some fish and reptile species having only one Kiss2 gene, amphibians having up to three Kiss genes (Kiss1a, Kiss1b, and Kiss2), and birds having neither Kiss1 nor Kiss2 genes. Note that for nomenclature of nonmammalian Kiss genes, we have partially adopted the proposal of Azakome et al. (5), but for the sake of homogeneity, we have used the terms Kiss1 and Kiss2 to name these paralogs.
With regard to the amino acid sequences of the encoded kisspeptins, comparison among species and phyla has been systematically conducted for the COOH-terminal decapeptides derived from both Kiss1 and Kiss2 genes. Such analyses have demonstrated a remarkable degree of conservation for each subgroup of kisspeptins, and even between the decapeptides encoded by Kiss1 and Kiss2 genes (5, 221). As shown in FIGURE 12, all Kiss1-derived peptides have a conserved core decapeptide sequence with variability only at residue 3. In addition, while most Kiss1-encoded kisspeptins possess a Tyr (Y) residue at position 10 (COOH terminus), the human and one of the amphibian Kp-10 have a Phe (F) residue. In the same vein, the core decapeptide encoded by Kiss2 is fully conserved except for some variability at residue 3. In addition, comparison between Kiss1 and Kiss2 decapeptides identified three sites of complete divergence: residues at positions 1 (Y in Kiss1 vs. F in Kiss2), 5 (S in Kiss1 vs. P in Kiss2), and 10 (Y in most of Kiss1 vs. F in Kiss2) of the core sequence. On this basis, it has been proposed that Kiss1-derived kisspeptins conform the so-called Y-Y form (with a subgroup of the Y-F form in humans and amphibians), while Kiss2-encoded kisspeptins are of the F-F form (5, 221).
Thorough sequence analyses of the regions upstream the core Kiss1 and Kiss2 decapeptides in nonmammals suggested that functional kisspeptins of longer amino acid length may exist in fish and amphibians. Thus the precursors encoded by fish Kiss1 gene and the amphibian Kiss1b gene possess a conserved dibasic cleavage site 5 amino acids upstream the initiation of the canonical Kp-10, suggesting that the final product could be a pentadecapeptide with NH2-terminal pyroglutamylation (240, 481). In addition, the precursor derived from the Kiss1a gene in Xenopus tropicalis may give rise to a tetradecapeptide (240, 481). Finally, the Kiss2 gene is likely to generate a functionally active dodecapeptide, as initially suggested by the presence of a basic amino acid (Arg) three residues upstream the canonical Kp-10 sequence and later confirmed by immunoaffinity purification (240).
In parallel with the disclosure of the molecular diversity of Kiss genes, the canonical receptor GPR54 was also shown to be putatively encoded by multiple genes in different animal phyla. Indeed, up to four different GPR54 paralogs likely existed in ancient vertebrate ancestors, due to two successive rounds of whole genome duplication (5, 276). In this context, it is notable that all GPR54 genes cloned so far display a similar overall structure, composed of 5 exons and 4 introns, and a higher degree of sequence homology than that showed by Kiss genes across phyla (481). In terms of evolution, the recent proposal of Um et al. (481) based on sequence homology and synteny analyses suggests that a first duplication event gave rise to two major isoforms of GPR54, termed GPR54–1 and -2. Subsequently, additional duplication, thereby generating up to four paralogs, and selective evolution in the different tetrapod and teleost lineages resulted in the presence of a variable number of GPR54 gene isoforms in modern animal species (481) (see FIGURE 12). Thus, while placental mammals have only one GPR54–1 gene, tentatively named as GPR54–1a, most fish species, including medaka, goldfish, and zebrafish, as well as mammalian monotremes, such as platypus, possess two GPR54 genes (namely, GPR54–1 and GPR54–2). Of important note, synteny analyses revealed that the GPR54–1 gene in fish has a substantially different genomic location compared with placental mammals, suggesting it to be a different paralog, named GPR54–1b (481). Intriguingly, the amphibian genome seems to have two conserved GPR54-1 genes, of the 1a (i.e., similar to mammalian) and 1b (i.e., similar to fish) types, as well as one GPR54–2 gene, similar to that found in fish species. In addition, some fish and reptile species possess only one GPR54–2 gene, although apparently arisen through different evolutionary routes, as revealed by synteny analyses showing that the neighboring chromosomal regions of reptile and fish GPR54–2 genes are totally different. Thus two isoforms of GPR54–2 seem to exist as well, one in reptiles and monotremes, and the other in fish and amphibians (481). Finally, as was the case for the ligands, avian species have apparently lost also all the genes encoding GPR54, thereby suggesting an active evolutionary pressure to eliminate all the elements of this system from their genomes (5, 481). Note that while we have systematically used the nomenclature proposed by Um et al. (481) for nonmammalian GPR54 genes, correlation with an alternative terminology, recently proposed by Azakome et al. (5), has been provided in FIGURE 12, whenever possible.
B. Functional Roles of Kisspeptins in the Control of Reproduction in Nonmammalian Vertebrates
In addition to the considerable efforts directed towards the elucidation of the molecular structure and evolution of Kiss and GPR54 genes in nonmammals, our understanding of the potential physiological roles of kisspeptins in the control of the reproductive axis in those species has significantly increased recently, as a result of the combination of neuroanatomical expression and pharmacological studies. Notably, the recognition of substantial differences for the organization and function of this system across nonmammalian vertebrates, together with the identification of at least two forms of Kiss/kisspeptins and GPR54 in nonmammals, endowed those studies with an additional level of complexity, which brings a call of caution when generalizing observations from one species to another.
While the potent gonadotropin releasing effects of kisspeptins in mammals was documented shortly after the disclosure of their reproductive roles, similar analyses in fish were not published until rather recently, due to the delay in the cloning and subsequent characterization of fish Kiss/kisspeptins. In fact, it was not until 2009 when at least two independent studies reported the ability of both Kiss1- and Kiss2-encoded Kp-10 to activate the gonadotropic axis in zebrafish and sea bass, using LH/FSH beta mRNA or gonadotropin levels as end-points (122, 221). These studies also pointed out that in those species the Kiss2 decapeptide was apparently more effective in terms of activation of pituitary gonadotropin gene expression and secretion (122, 221), thus suggesting that the Kiss2 system might have taken a dominant role in the control of the gonadotropic axis in fish. This contention was later supported by the observation that some fish species have selectively lost the Kiss1 gene during evolution, whereas specific loss of Kiss2 has not apparently occurred in any of the fish species studied to date (5, 481). Of note, however, subsequent functional studies in goldfish evidenced that, in that particular species, the Kiss1-encoded decapeptide is a more potent gonadotropin secretagogue (245), therefore suggesting important species differences regarding which Kiss system is actually the dominant in the control of the HPG axis in fish. This notion has been recently reinforced by expression analyses in medaka, which support the view that the Kiss1 system, not the Kiss2, is the one that plays a leading role in the regulation of hypophysiotropic GnRH1 neurons and thereby gonadal function in this fish species (284) (see below).
In addition to in vivo studies, molecular cloning of the different kisspeptins and their receptors allowed detailed in vitro analyses of the functional characteristics of these ligand-receptor pairs. In a series of elegant studies, Lee et al. (240) documented that nonmammalian GPR54–1 preferentially binds to Kiss1-derived tetradeca- and pentadecapeptides, which displayed also higher affinity at the GPR54–1 level than the corresponding decapeptide forms (240). In contrast, nonmammalian GPR54–2 did not clearly discriminate between Kiss1- and Kiss2-encoded peptides (240). However, the Kiss2 dodecapeptide was more effective than the corresponding decapeptide in terms of GPR54–2 activation (240). It is interesting to note that biochemical characterization of zebrafish GPR54–1 and -2 demonstrated that while GPR54–1 operates mainly via the PKC pathway, as described for mammalian kisspeptin receptors, GPR54–2 seems to act via both PKC and PKA cascades (32). Altogether, the above differences in terms of receptor binding and signaling may explain, at least partially, some of the differential responses obtained in the various in vivo tests reported so far. In addition, it is important to stress that such pharmacological studies in vivo have been based on the use of Kiss1 and Kiss2 decapeptides (122, 221, 245), whereas longer (15, 14, and 12 amino acid) kisspeptins have been shown to display greater biological activity in vitro (240). All in all, further characterization of the gonadotropic effects of the native Kiss1 and Kiss2 peptides in a wider number of fish, and other nonmammalian species is needed to fully delineate their differential roles in the control of the HPG axis.
In addition to functional tests, neuroanatomical and expression analyses have been initiated, mainly in fish species, to better characterize the physiological roles of kisspeptins in nonmammals. Since cloning of GPR54 sequences preceded that of Kiss genes, the first expression analyses, using RT-PCR approaches, aimed to define the patterns of tissue distribution of GPR54 transcripts and their eventual regulation during key reproductive states. These initial studies, some of which were published before the recognition of the different receptor isoforms, demonstrated the expression of GPR54 in the brain of different fish species, such as tilapia, grey mullet, cobia, fathead minnow, grass puffer, halibut, sole, and zebrafish (32, 125, 264, 276, 277, 285, 314, 329, 406). Moreover, by comparison with the expression profiles of the different GnRH transcripts and the stages of maturation postfertilization, indirect evidence was accumulated for the potential involvement of kisspeptin signaling in the control of puberty onset in fish. For instance, studies in male and female fathead minnows demonstrated that a peak in brain expression levels of GPR54–2 mRNA occurs before the onset of puberty, as documented on the basis of histological analysis of gonadal maturation (125). Those studies, however, were devoid of precise anatomical resolution, which hampered the mechanistic interpretation of the expression data. Nonetheless, it is worthy to recall that the first evidence ever reported on the putative presence of kisspeptin receptors in GnRH neurons, and of its developmental regulation, was obtained in tilapia, by the use of RT-PCR analysis of laser-captured GnRH neurons, thus suggesting the possibility of direct kisspeptin effects on these cells (329).
As for the receptor, expression of Kiss genes in fish brain has been documented by the use of RT-PCR analyses in different species, including medaka, zebrafish, goldfish, and sea bass (122, 221, 398, 406, 507). In addition to those, neuroanatomical in situ hybridization studies addressing the specific distribution of Kiss gene expression in different brain areas have been also published. The latter not only allowed to initiate the mapping of Kiss neurons in fish, but have disclosed also substantial species differences in terms of distribution of Kiss mRNA subtypes. Thus detailed studies in medaka have documented the hypothalamic expression of Kiss1 mRNA at the nucleus ventralis tuberis (NVT) and the nucleus posterioris periventricularis (NPPv), as well as its weaker presence at other extrahypothalamic areas, such as the habenula (202). In contrast, studies in zebrafish suggested that Kiss1 gene is not significantly expressed at the hypothalamus but highly expressed in the habenula (221). With regard to the expression of Kiss2 gene, prominent hypothalamic expression of this transcript has been detected at the nucleus recessus lateralis (NRL) in medaka (284). Analogous studies in zebrafish have demonstrated the expression of Kiss2 mRNA in the dorsal and ventral zones of the periventricular hypothalamus (221).
The lack of specific antisera selectively detecting Kiss1 versus Kiss2 peptides had prevented the detailed mapping of kisspeptin neuronal cell bodies and projections in the fish brain until recently, when antibodies against the COOH terminus of zebrafish prepro-Kiss1 and -Kiss2 peptides were first generated (403). Histochemical studies using these antisera have allowed the confirmation of the patterns of brain distribution of Kiss1 and Kiss2 mRNAs, initially revealed by in situ hybridization in zebrafish, with prominent location of Kiss1 neurons in the habenular nucleus, and the abundant presence of Kiss2 cells in the ventral and dorsal hypothalamus (403). Importantly, these studies have permitted also the mapping of kisspeptin fibers in this species, showing that Kiss1 neurons display a restricted pattern of projections, targeting the interpeduncular and raphe nuclei, whereas Kiss2 neurons project to a wider number of areas, including the preoptic area and the ventral and caudal hypothalamus (403). Notably, Kiss2 neurons have been shown to make close appositions with hypophysiotropic GnRH3 neurons in zebrafish. In the same study, in situ hybridization analyses of the brain distribution of GPR54–1 and -2 mRNAs were first applied to this fish species. These analyses revealed a perfect match between Kiss2-neuron projections and GPR54 expression in different brain regions, including those harboring GnRH neurons (403). While these recent data nicely document the presence of two independent kisspeptin systems in the zebrafish brain, further comparative studies mapping Kiss- and GPR54-expressing neurons are needed to define the anatomical features of the different Kiss1 and Kiss2 systems in fish and other nonmammalian species.
In any event, the neuroanatomical data on mRNA distribution of Kiss1 and Kiss2 obtained to date, together with the recent peptide mapping summarized above (403), support the important functional role of kisspeptins in the control of key aspects of gonadotropin function in fish. As a clear example, in situ hybridization studies in medaka have documented that Kiss1 neurons at the NVT, but not at NPPv, are under the control of sex steroids and environmental (photic) cues, as illustrated by the effects of OVX, with or without estrogen replacement, and changes in light cycle upon Kiss1 mRNA expression at this nucleus (202). On the other hand, Kiss2 neurons at the NRL do not seem to be sensitive to changes in the sex steroid milieu or day length in this species (284). Interestingly, in the NVT, OVX decreased Kiss1 expression while estrogen replacement stimulated the levels of this transcript in medaka (202). Likewise, estrogen treatment enhanced Kiss2 mRNA levels in the dorsal and caudal hypothalamus of juvenile zebrafish (403). This is reminiscent of what is observed in the AVPV of female rodents and suggests the potential involvement of NVT Kiss1 neurons in medaka, and hypothalamic Kiss2 neurons in zebrafish, in the positive-feedback control of gonadotropin secretion. To our knowledge, the possibility that, as it is the case for ARC/infundibular neurons in mammals, other Kiss1 or Kiss2 neurons may participate in mediating the negative-feedback effects of sex steroids in fish has not been documented to date. In addition, long-day cycles, which are associated with increased gonadal maturation, evoked a rise of Kiss1 mRNA levels at the NVT in medaka, suggesting a functional association between changes in endogenous kisspeptin tone and the environmental control of breeding in this species (202).
Finally, expression analyses of Kiss and GPR54 genes in different fish species are also helping to highlight the potential repertoire of sites of action of this system in the integral control of the reproductive axis and, eventually, to delineate additional nonreproductive functions. For instance, prominent expression of Kiss1 gene at the habenula, as documented in medaka and zebrafish, strongly suggests a functional role at this site, even though no direct projections from this region to the preoptic area, where hypophysiotropic GnRH neurons are located, have been demonstrated so far (221). Nonetheless, the habenula is one of the most conserved structures in the vertebrate brain, which is located in the close vicinity of, and receives afferents from, the pineal gland. Therefore, the habenular Kiss1 system may participate in the control of reproductive, and eventually nonreproductive, functions by environmental photic cues and biological rhythms (221). In addition, as is the case in different mammalian species, expression of Kiss1 and GPR54 genes has been recently reported in goldfish pituitary, where homologous Kp-10 was capable to directly elicit LH, GH, and prolactin responses (507). Likewise, Kiss2 and GPR54 mRNAs have been detected in the pituitary of pufferfish (406). Of note, prominent expression of GPR54 was recently reported also in the pituitary of the European eel, where kisspeptin has been shown to conduct paradoxical direct inhibitory effects on LHβ expression (331). In addition, a number of studies have documented the expression, and eventual developmental regulation, of GPR54 and/or Kiss mRNAs in male and female gonads from different fish species (122, 221, 245, 276, 277, 314, 398). Again, there is a clear parallelism with the situation reported in some mammals. However, the functional relevance of such gonadal expression of the elements of the Kiss system in fish gonads remains to be elucidated, although dramatic changes in the gonadal levels of Kiss1 mRNA have been reported during spermiation and late vitellogenesis in chub mackerel (398). In any event, the above expression data strongly suggest that, in addition to prominent effects at the hypothalamus, central extrahypothalamic (e.g., habenula) as well as peripheral (pituitary, gonadal) actions of kisspeptins might contribute to the fine-tuning of the reproductive axis in fish. Similarly, the expression Kiss and/or GPR54 genes in numerous peripheral tissues in fish might evidence additional nonreproductive actions of kisspeptins at those sites, in keeping with fragmentary evidence obtained in mammals.
XVI. NONREPRODUCTIVE FUNCTIONS OF KISSPEPTINS: FACTS AND HYPOTHESES
Although this review is specifically devoted to cover the reproductive aspects of kisspeptin physiology, it is important to mention that evidence from different species suggests potential additional, nonreproductive functions of kisspeptins. In most cases, the physiological relevance of such nonreproductive roles of kisspeptins is yet to be conclusively demonstrated. Anyhow, a brief critical summary of the available data in this area is provided herein, to endow the reader with a wider perspective of the whole spectrum of putative biological actions of kisspeptins. This exercise would help also to stress the paramount relevance of the reproductive functions of kisspeptins over their potential nonreproductive roles, some of which will be itemized below.
For obvious reasons, the initial studies on KISS1 and kisspeptins were carried out in the context of human cancer, an area where the putative metastasis-suppressor actions of kisspeptins drew considerable attention initially (237–239). The pioneering observations that KISS1 expression was associated with suppressed metastasis of human melanomas and breast carcinomas, as well as the demonstration that the KISS1-encoded peptide, Kp-54, inhibited dissemination of melanoma cells, paved the way for a considerable number of studies, aiming to assess the patterns of KISS1 gene expression and/or the biological actions of kisspeptins in additional types of tumors and cell lines. Collectively considered, these studies documented that Kp-54 and other kisspeptins are provided with antimetastatic activities in several malignancies, such as papillary thyroid carcinoma, breast cancer, melanoma, and pancreatic and ovarian carcinoma (193, 238, 239, 267, 318). In addition, loss of KISS1 gene expression was identified as a bad prognosis factor for tumor progression and metastasis in esophageal squamous cell carcinoma, pancreatic cancer, gastric carcinoma, bladder cancer, and non-small-cell lung cancer, among others (100, 183, 294, 388, 515). Nonetheless, despite molecular and clinical evidence for a potential role of KISS1 in metastasis regulation (163), conflictive data on the function of KISS1 as universal metastasis suppressor gene have been reported, while its usefulness as a prognosis factor has been also discussed in some forms of cancer (203, 205). For instance, high expression levels of KISS1 and GPR54 have been associated with poor prognosis in estrogen-sensitive forms of breast carcinomas (262). Intriguingly also, there is a conspicuous lack of reports on the metastatic potential of tumors in GPR54 or Kiss1 null models or patients. All in all, the relevance and eventual therapeutic implications of this system in the control of cancer progression warrant further clarification.
B. Central Nervous System
Besides their role in the control of cancer spread, initial analyses on kisspeptins included also the study of the patterns of expression of Kiss1 and GPR54 in various tissues. Despite the lack of precise anatomical resolution, those studies already evidenced prominent expression of both genes in different areas of the central nervous systems, a contention which was later backed up by IHC analyses. As a whole, these initial observations were taken as suggestive of a role of kisspeptins as transmitters for different brain functions that, on the sole basis of distribution/expression data, were proposed to include nociception and visceral regulation (38, 111). These putative functions of kisspeptins, however, have not received too much attention, except for their potential role as mediator of synaptic transmission in areas, such as the hippocampus, where abundant expression of GPR54 has been also documented. Indeed, as indicated in section IVB, expression of Kiss1 mRNA at the hippocampus had been shown to be regulated by glutamate inputs and seizure activity (14, 15). In addition, Kiss1 expression at the hippocampus of male rats increases after gonadectomy (14). Moreover, kisspeptin stimulation of hippocampal slices has been demonstrated to enhance synaptic transmission at this area (16). Despite this suggestive evidence and the potential roles of kisspeptins in other neuronal systems, their eventual implication in relevant brain functions, such as neurogenesis or neuroprotection, has not been addressed to date. Likewise, the involvement of kisspeptins in visceral regulation, other than via neurohormonal mechanisms, and nociception remains to be thoroughly investigated. Yet, a very recent report has provided initial functional evidence for a role of kisspeptin signaling in the control of pain sensitivity in mice (432).
C. Other Neuroendocrine Axes
Initial structural analyses of the kisspeptin precursor already suggested that it encodes a secreted factor(s), as evidenced by the presence of a signal peptide and conserved cleavage sites. These structural features prompted specific neuroendocrine analyses that documented the ability of kisspeptins to stimulate oxytocin release (228), a phenomenon that has been very recently confirmed by the demonstration that peripheral administration of Kp-10 increases the electrical activity of oxytocin neurons (392). The disclosure of the reproductive effects of kisspeptins renewed the interest on their neuroendocrine actions, but these have been mostly explored in the context of the control of gonadotropin secretion. In any event, evidence is mounting that kisspeptins might participate in the control of the secretion of other pituitary hormones, such as growth hormone (GH) and prolactin (PRL). On the former, a limited number of studies have documented the ability of Kp-10 to stimulate GH secretion by rat and bovine anterior pituitary cells in vitro (120, 158, 195), and to elicit GH secretion after systemic administration in cows (194, 497). In the same line, our recent evidence demonstrates the ability of Kp-10 to directly stimulate pituitary GH secretion in the monkey (256). Such direct actions of kisspeptins on the somatotrope axis are indirectly supported by the demonstration that Gpr54 mRNA is expressed within the sheep pituitary in somatotrope-enriched cell fractions, among other cell populations (427). Admittedly, however, other studies have failed to demonstrate the capacity of kisspeptins to alter GH secretion when administered in vivo to goats, calves, gilts, and monkeys (121, 164, 242, 361). Therefore, the physiological role, if any, of the Kiss1 system, either at central or pituitary levels, in the control of the GH axis remains contentious. Similarly, fragmentary evidence has suggested that kisspeptin may regulate the secretion of PRL when administered in vivo, although inhibitory and stimulatory effects have been reported in rats (298, 438). In addition, direct stimulatory actions of kisspeptins on PRL secretion by bovine pituitaries in vitro have been also reported (119). Yet, other studies have not documented effects of kisspeptins on PRL release in other species (164), thus casting some doubt on their conserved role in PRL regulation. Conversely, recent reports in rodents have demonstrated expression of PRL receptors or cellular responses to PRL in AVPV and/or ARC Kiss1 neurons in rodents and sheep (227, 244, 416), therefore suggesting the involvement of PRL in the regulation of central kisspeptin pathways, a phenomenon of potential relevance during lactation. To our knowledge, the putative roles of kisspeptins in the control of other pituitary axes remain scarcely studied to date. In this context, a very recent report failed to demonstrate consistent effects of kisspeptins on the activity of the hypothalamic-pituitary-adrenal axis in male rats, even though modest changes in the central expression of key stress-related genes, such as CRH and AVP, were found following kisspeptin administration (368).
D. Metabolic Tissues
The possibility of the expression and/or actions of Kiss1/kisspeptins in some metabolic peripheral tissues has been also addressed, although the available data in this front remain scarce. As a clear example, expression of Kiss1 and GPR54 genes has been demonstrated in human and rodent pancreatic islets, with discernible peptide-IR being detected in α- and β-cells but not in the exocrine pancreas (165). However, the functional relevance of such a local kisspeptin signaling system needs further clarification, since in vitro studies, using isolated human and mouse islets, as well as perfused rat pancreas, have documented either stimulatory or inhibitory effects of kisspeptins on glucose-induced insulin secretion (37, 165, 412, 485), disparities which might be related to the experimental conditions, such as glucose concentrations, tissue or cell preparations, and species differences. In fact, recent systematic analyses of the effects of Kp-10 and Kp-13 on insulin secretion by perifused islets from human, pig, rat, and mouse species under fixed glucose conditions (20 nM) have documented consistent stimulatory responses (36). In the same line, systemic administration of Kp-10 has been reported to increase insulin levels in conscious rats (37), and heightened glucose-induced insulin secretion in monkeys (490). Yet, preliminary evidence argues against a detectable impact of systemic administration of Kp-10 on circulating glucose and insulin levels in humans (137).
In addition, expression studies in rats demonstrated the expression of Kiss1 mRNA in the white adipose tissue (WAT), whose levels fluctuated depending on sex steroid milieu and nutritional conditions. For instance, sex steroids and fasting increased Kiss1 expression, whereas a high-fat diet reduced it (40). Despite the lack of demonstration of kisspeptin (peptide) expression in the WAT so far, such a dynamic regulation is suggestive of a potential modulatory role of adipocyte-born kisspeptins, a possibility that has been recently supported by the observations that circulating kisspeptin levels are increased in obese girls (343), and that kisspeptins can stimulate the secretion of certain adipokines, such as adiponectin, but not others, such as leptin and resistin (489). Notwithstanding this fragmentary evidence, the roles of peripheral kisspeptins in the control of metabolism, and glucose or fat homeostasis, remain dubious and need further clarification.
E. Other (Miscellaneous) Actions
Finally, mention will be made in this section of the possibility, documented so far by an as yet limited number of studies, that kisspeptins may have an impact on cardiovascular function and fluid homeostasis. Thus a report by Mead et al. in 2007 (275) described the expression of kisspeptin and GPR54 in smooth muscle cells from aorta, coronary arteries, and umbilical vein, with discernible kisspeptin binding and vasoconstrictor actions on these vessels in humans. In addition, abundant presence of kisspeptins and their receptor was demonstrated at atherosclerotic plaques of the coronary artery (275). While this study raised considerable attention, these initial observations were not followed up by additional studies on the cardiovascular roles of kisspeptins in other species or pathophysiological conditions until recently, when the ability of Kp-10 to induce edema formation and to evoke constriction of the peripheral microvasculature has been reported in mice (391). In addition, Kp-10 has been shown to conduct anti-angiogenic actions on endothelial cells in vitro and to inhibit placental vessel formation ex vivo (360). Furthermore, evidence for the expression of GPR54 in human and rodent myocytes and intramyocardial blood vessels has been presented very recently, and direct inotropic actions of kisspeptins have been documented in the human, rat, and mouse (261). These observations pave the way for additional studies on the physiological relevance and pathological implications of kisspeptin signaling in cardiovascular function. As a note of caution, however, it is stressed here that the impact of kisspeptins on vascular dynamics has been recently challenged by studies in men that showed no effects of Kp-10 on blood pressure even at doses two orders of magnitude greater than those required to elicit LH release (137).
Also recently, evidence has been presented that Kiss1/kisspeptins and Gpr54 are expressed in different kidney compartments in rats, and their levels are variably altered in conditions of chronic renal impairment, with an intriguing dissociation being detected between changes of mRNA and peptide contents in different models of kidney failure (409). While the functional relevance of such expression is unclear, recent data from Gpr54 null mice suggest that congenital lack of kisspeptin signaling results in retarded embryonic kidney branching morphogenesis and glomerular development, with Gpr54 KOs having higher risk of displaying low glomerular numbers in adulthood (511). In a potentially related front (i.e., fluid homeostasis), administration of Kp-10 to rats has been recently shown to inhibit diuresis and natriuresis via a central action through elevation of vasopressin levels. In addition, kisspeptin-IR and GPR54 expression has been documented in human adrenal gland (297, 440), where Kp-54 has been shown to stimulate aldosterone secretion (297). The potential contribution of the above features to alterations in cardiovascular or fluid homeostasis in conditions when kisspeptins levels are expected to increase, such as pregnancy, has been suggested, but their actual relevance is yet to be defined.
XVII. CLINICAL ASPECTS OF KISSPEPTINS IN REPRODUCTIVE HEALTH
As illustrated in previous sections, most of the studies on the reproductive roles of kisspeptins have focused on the elucidation of diverse physiological aspects, concerning the expression, regulation, mode of action, and biological effects of these peptides and their receptor in the HPG axis. Notwithstanding, the available evidence suggests that, in addition to their paramount physiological relevance, kisspeptins may pose interest in terms of applied medicine, as a target for the development of improved therapeutic alternatives for the manipulation of the reproductive system. The clinical dimension of kisspeptins and GPR54 is further emphasized by their original recognition as potential disease-causing factors in humans. Comments on the above issues, of potential clinical relevance, are included in this section.
A. GPR54 and KISS1 gene mutations in reproductive disorders
As indicated in section IIB, identification of the reproductive roles of KISS1/kisspeptins and GPR54 took place in the course of genetic studies in patients with iHH, therefore providing clear evidence for the potential relevance of inactivating mutations of these genes in the generation of reproductive disorders. Admittedly, the overall frequency of mutations in GPR54 (or KISS1) gene is strikingly low even for a rare disease as iHH (62). Initial reports of genetic inactivation of GPR54 in humans identified a 155-nt deletion, eliminating the splicing acceptor site between intron 4 and exon 5, a homozygous L148S mutation, and the compound R331X and X399R mutations of GPR54 in individuals with phenotypic HH (95, 401). These initial clinical reports have been later confirmed by identification of independent missense mutations of GPR54 in additional iHH patients, including C223R and R297L substitutions (compound heterozygote) (402), an insertion at 1001_1002C (with the subsequent change in the open-reading frame) (233), a L102P mutation (460), and a combined insertion/deletion mutation at the 3′ splicing acceptor site of exon 2 (446), as well as additional rare variants of GPR54 in patients with late-onset HH (62). More recently, a new homozygous, loss-of-function mutation involving a p. F272S substitution in the GPR54 peptide sequence has been described in male and female individuals with severe forms of familial HH of early onset (313). A schematic presentation of the major known inactivating mutations of GPR54 gene is depicted in FIGURE 13.
Since functional analyses of some of the above mutations have been conducted in heterologous cell systems, these studies have allowed also to gain some insight on the structure-function relationship of GPR54. Of note, a considerable degree of variability in the impact of the above mutations on receptor function has been documented, ranging from severe impairment of signaling (e.g., L148S) to mild or no impact on function (e.g., ins/del mutation at intron 2). Also of interest, neuroendocrine studies of individuals affected by the same mutation have revealed quite significant differences also in terms of hormonal alterations; this has been documented, for instance, for the L102P mutation that, despite the dramatic suppression of receptor function in vitro, resulted in an array of clinical manifestations ranging from mild to severe gonadotropin deficiency (460). This observation illustrates the importance of the potential interactions between genetic variability at GPR54 and other loci in defining the clinical phenotype associated with suppressed kisspeptin signaling.
To our knowledge, no single report has been produced to date on homozygous inactivating mutations affecting the KISS1 gene, but only polymorphisms, in HH patients (411). (see note added in proof). Yet, a recent study has documented up to 10 different heterozygous rare sequence variants that, in some cases, were associated with analytical evidence of GnRH deficiency in humans (80). In addition, two recent studies have documented the first suspected activating mutations of KISS1 and GPR54 in humans. Two different missense mutations, leading to P74S and H90D substitutions, have been identified in the KISS1 gene in a cohort of children with central precocious puberty (411). Genetic and biochemical analyses of these mutants revealed that none of them affected the region encoding the active peptide core of kisspeptins, neither did they alter IP production by the corresponding peptides in cell assays (411). Yet, the clinical phenotype of these mutations was rather robust (e.g., the P74S mutation was identified in homozygous state in a boy with onset of puberty at 1 yr of age), and serum incubations from controls and affected individuals suggested a greater stability of Kp-54 caused by the P74S mutation. Whether this is the sole cause for such a dramatic advancement of puberty is yet to be solved. On the other hand, additional studies in patients with precocious puberty of central origin allowed also the identification of the first case of an activating mutation of GPR54 (see also FIGURE 13), an autosomal dominant R386P substitution which did not result in constitutive activity of the receptor but rather prolonged its pattern of IP response upon stimulation, therefore suggesting decreased desensitization (445). These observations have been recently refined by the demonstration that the mutant R386P GPR54 displays a reduced rate of proteasome degradation, thus resulting in enhanced recycling to the membrane and receptor numbers of surface receptors (29). As for the ligand, there appears to exist a certain degree of disparity between the severity of the clinical phenotypes and the biochemical features of the R386P mutated receptor, which may be suggestive of additional contributing factors, interacting with the reported variations in the prepro-kisspeptin and/or GPR54 sequences, for the generation of the state of precocious puberty of affected humans.
In addition to missense mutations, the eventual contribution of polymorphic nucleotide variations in KISS1 and GPR54 gene sequences in the definition of key reproductive traits, such as the age of puberty, has been evaluated. To date, three studies have been produced linking certain polymorphisms in GPR54 (one report) and KISS1 (two independent reports) with precocious puberty in Asian girls, although in general the strength of the associations was very modest. Nonetheless, one of the variations of the KISS1 sequence was independently identified in two different populations and was shown to cause an amino acid substitution (P110T) of as yet unclear etiopathological relevance. To our knowledge, whether polymorphic variations of the KISS1 and GPR54 genes might be linked to other reproductive traits or disorders in humans has not been evaluated to date. Yet, recent studies have revealed the potential association between several polymorphisms in the coding sequences of Gpr54 and/or Kiss1 genes and the prolificacy/litter size in sheep and goats (46, 89). The eventual correlate of these preliminary findings in terms of human reproductive health remains unknown.
B. Kisspeptins as Pharmacological Targets: Development of Agonists and Antagonists
The development of superactive agonists and antagonists of different neuroendocrine peptides, such as GnRH, has provided powerful tools to interrogate the physiological control of various endocrine systems, and to identify potential therapeutic agents for their intervention in a diverse range of pathologies, from prostatic cancer to pituitary tumors. In principle, development of agonists and antagonists of GPR54 holds the potential for equivalent utility and therapeutic advances.
Two approaches may be implemented in developing neuroendocrine peptide analogs. The first is the empirical and/or rational modification of the native peptide structure to improve agonist activities or generate antagonist properties. In general, these studies aim to increase receptor binding affinity and/or to improve in vivo half-life and reduce metabolic clearance. The former is accomplished through stabilizing the peptide in a high-affinity binding conformation and by generating additional peptide interactions with the receptor. The latter is achieved through amino acid substitutions that decrease susceptibility to proteolytic degradation (e.g., d-amino acids, blocked termini) or enhance binding to plasma proteins (e.g., hydropholic side chains such as tryptophan). The peptide analog approach has the advantage that small changes are made to a natural peptide that has been selected during evolution to specifically target its cognate receptor. Thus off-target side effects are far less frequent than for entirely chemical small molecules. The disadvantage of peptide analogs is the need to administer them parenterally. However, this is largely compensated by slow release depot preparations that may be effective even for months after a single injection.
The second approach for analog development is the classical pharmaceutical industry high-throughput screening of small-molecule libraries for binding and/or intracellular signaling at target GPCRs, such as GPR54, expressed in appropriate cell lines. Hit molecules are then clinically refined to provide the appropriate pharmacokinetic and dynamic profile. These molecules have the advantage of oral bioavailability but the disadvantage of unexpected off-target effects. These two approaches have been used to develop kisspeptin analogs targeting GPR54 with different success, and will be briefly described below.
1. Agonists of GPR54
There is substantial literature in patents and journals on the development of kisspeptin agonists, whose detailed articulation exceeds the scope of this review. Of note, the primary motivation behind this major pharmaceutical drive was the development of agonists with antimetastatic properties, as originally proposed for KISS1-derived peptides, including Kp-54. In contrast, little attention has been paid so far to the development of agonists to superstimulate the HPG axis. Initiatives directed at obtaining peptide agonists made use of two kernel pieces of information, namely, that the minimal structure required for agonist activity is Kp-10, and that the evolutionarily conserved COOH-terminal RF-amide moiety of this large superfamily of peptides is essential for receptor engagement and is part of a binding pharmacophore Phe6, Arg9, and Phe10-NH2 (70, 324, 470).
Different small peptidergic agonists of GPR54, generated as pentapeptide derivatives of the COOH-terminal region of Kp-10, have been produced and characterized in vitro (312, 469–472). These studies documented the ability of such pentapeptide products to activate GPR54 with different biopotency, thus reinforcing the functional relevance of this terminal region in receptor activation. Of note, derivatives containing alkene- or fluoroalkene-dipeptide isosteres displayed similar bioactivity as the parent decapeptide, whereas other pentapeptide products were of significantly lower activity (469). In addition, small kisspeptin variants with nonhydrolyzable Gly-Leu dipeptide isosteres have been generated as a means to avoid degradation by MMP-9, with the resulting GPR54 agonists being fully active but significantly resistant to protease-mediated cleavage (473). Remarkably, while the above agonists have been thoroughly tested in vitro, their capacity to modulate gonadotropin secretion was in most cases not evaluated. Yet, our preliminary tests evidenced that some of the above agonists, such as compound C-34, are rather weak activators of gonadotropin secretion in vivo (Pinilla and Tena-Sempere, unpublished data). These findings stress the importance of biological testing of the above analogs in relevant animal models, as their functional features in terms of receptor activation in vitro may not translate into equally potent gonadotropic responses in vivo.
With the use of analogous peptidergic strategies, different kisspeptin decapeptide analogs with variable biopotency have been recently generated. Thus systematic Ala substitutions of rat Kp-10 sequence, coupled to detailed structural and functional analyses, revealed that the variant [Ala6]-Kp10 possesses higher EC50 in vitro and weak, but detectable, agonistic activity in terms of LH secretion in vivo, without discernible antagonistic actions (157). More recently, a similar approach, involving not only Ala scanning but also multiple enantiomer exchanges, has allowed identification of a decapeptide variant, [dY1]-Kp10, with lower activity in vitro but enhanced biopotency in terms of induction of LH and testosterone secretion in vivo (79). The above studies illustrate the possibility to generate agonists of GPR54 with specific features in terms of amplitude and duration of gonadotropic responses versus the native kisspeptins (e.g., low-potency, long-acting analogs), which may pose potential therapeutic advantages in certain clinical settings.
In addition to the above peptidergic approach, large-scale screening of small-molecule libraries has been also implemented to identify agonists of GPR54 with potential to generate orally-active analogs, able to cross the blood-brain barrier (232). While a preliminary report using this approach documented the identification of one hit with dose-dependent GPR54 agonistic activity (197), the EC50 of such compound appeared to be very high and, to our knowledge, its ability to stimulate the gonadotropic axis in vivo has not been reported to date.
2. Antagonists of GPR54
Since endogenous kisspeptins are essential signals for normal reproduction, administration of agonists might be especially revealing in physiological or pathophysiological conditions where the kisspeptin tone is predicted to be low, such as prepubertally, during seasonal anestrus, in leptin deficiency or uncontrolled diabetes. In contrast, antagonist administration may be more instrumental for dissecting out the actual roles of kisspeptins not only in normal physiological processes, such as sexual differentiation, puberty onset, ovulation, and feedback control by gonadal steroids, but also in pathological conditions of increased gonadotropin secretion, such as precocious puberty or polycystic ovarian syndrome. In addition, kisspeptin antagonists have potential interventive applications in a wide spectrum of sex steroid hormone-dependent diseases such as prostatic and ovarian cancers, endometriosis, and uterine fibroids. However, despite such potential interest, generation of GPR54 antagonists has lagged behind the development of agonists. Yet, there has been significant progress in this front recently.
With the use of a peptidergic approach, the first series of GPR54 antagonists was published in 2009 (384). These were generated by systematic modifications of the core decapeptide sequence of Kp-10, involving NH2-terminal truncations and specific residue substitutions, which are schematically depicted in FIGURE 14. From the initial series of >100 compounds synthesized using this strategy, a limited subset of peptide derivatives with promising antagonistic activities in vitro were identified (384). Among those, the peptide antagonist 234 ([D-A]NWNGFG[D-W]RF-NH2) was shown to have a high binding affinity and potent inhibitory effects upon Kp-10 stimulated IP production in a heterologous cell reporter system. As described in previous sections, the antagonistic properties of the 234 compound have been documented in a variety of species, including monkey, sheep, rat, and mouse, using different experimental approaches, therefore defining this peptide analog as the first GPR54 antagonist with the ability to interfere kisspeptin signaling in vivo (339, 384). Of note, derivatives of compound 234, engineered to enhance its ability to cross the blood-brain barrier by means of insertion of a penetratin motif, have been also generated and validated in vivo, thus paving the way for future therapeutic applications after systemic administration (339). Notwithstanding, while the antagonist 234 was initially reported to have no intrinsic agonist activity, more extensive analyses have subsequently revealed a weak agonistic action of ∼10% of maximum at micromolar concentrations (283). In addition, some degree of variability in the efficiency of compound 234 derivatives in terms of blockade of kisspeptin effects has been detected among different batches and experimental conditions. These observations, and the need to improve the physicochemical properties of the first-generation antagonists, have fueled further efforts in the synthesis of refined peptidergic variants of Kp-10 with antagonistic activity, based on the knowledge generated from studies on the structure-activity relationships of the initial compounds. Preliminary analyses in this front demonstrate that some of these analogs have improved physicochemical properties and antagonistic activities in vitro and are currently being thoroughly validated using different in vivo models (Millar, unpublished data).
Finally, small-molecule synthesis and screening strategies have been also applied recently to the generation of antagonists of GPR54 (225, 226, 232). While, to our knowledge, high-throughput screening of libraries of small molecules with potential for oral activity and/or enhanced permeability at the blood-brain barrier have not resulted so far in the identification of active antagonists in vivo (232), synthesis of a series of 2-acylamino-4,6-diphenylpyridines has recently allowed the generation of small-molecule antagonists of GPR54 (225), some of which have been reported to have in vivo efficacy, in terms of suppression of the post-GNX rise of LH levels, and high brain penetration (226). Whether these compounds will be the basis for the production of orally-active GPR54 antagonists and, if so, whether they will represent a therapeutic advantage over more classical peptidergic antagonists remain to be determined.
C. Therapeutic Opportunities of Kisspeptins and Their Analogs
On the basis of the physiological and pharmacological data reviewed in previous sections, kisspeptin analogs may find utility in the treatment of some forms of reproductive dysfunction, in both males and females. In addition, different sex hormone-dependent diseases might benefit from the therapeutic manipulation of kisspeptin signaling. With regard to kisspeptin agonists, these may be used in the pharmacological activation of the HPG axis in conditions of reproductive insufficiency of central origin, provided that the GnRH neuronal system is intact. In addition, they may also be diagnostic agents for determining the neuroendocrine site of dysfunction of the HPG axis, as well as the gonadotropic potential of the patient. The proven desensitization of gonadotropin responses after chronic or continuous exposure to kisspeptins makes mandatory the optimization of protocols of intermittent administration of the analogs (349, 476), or the development of compounds with low-potency and long-lasting stimulatory properties. Efforts in this sense have been recently published (79), yet it remains to be defined whether such longer acting kisspeptin analogs induce also gonadotropin desensitization, as described for the native peptides. In addition, very recent studies in men suggest that protocols of continuous infusion of Kp-10, at relatively low doses (4 μg·kg−1·h−1), can evoke persistent stimulatory responses, in terms of LH and testosterone secretion, even 22.5 h after beginning of the infusion protocol. These findings nicely illustrate the need for optimal selection of the features (dose, pattern, and duration) of administration of kisspeptins to avoid tachyphylaxis of the gonadotropic system (139). In any case, demonstration of the ability of different forms of kisspeptins, such as Kp-54 and Kp-10, to potently stimulate gonadotropin secretion in humans after their systemic administration provides the basis for the development of optimal protocols of activation of the HPG axis by the use of kisspeptin analogs with agonistic activity (82, 101, 102, 137, 139, 192).
In principle, kisspeptin analogs might have potential therapeutic applications similar to those of GnRH analogs, which have found extensive pharmacological use in various hormone-dependent diseases and in vitro fertilization (IVF). However, as illustrated in FIGURE 15, important differences appear to exist in terms of gonadotropic responses to acute and continuous infusion of GnRH and kisspeptin analogs. Thus GnRH agonists are mainly used because of their ability to induce robust gonadotrope desensitization, while antagonists prevent receptor activation by endogenous GnRH. Both treatments result in the marked suppression of gonadotropin secretion with the consequent reduction in circulating gonadal steroid hormones, which are lowered to GNX levels. Such a dramatic reduction results frequently in important side effects, such as hot flushes, reduced lean body mass, decreased libido, and bone loss.
In clear contrast, kisspeptin antagonists have been shown to reduce LH pulse frequency and amplitude but do not appear to affect basal LH secretion (384). These features suggest that such antagonists may find specific clinical utility in conditions where reduced gonadotropic drive is wanted but maximal suppression of sex steroids is contra-indicated. In principle, pathologies such as benign prostatic hyperplasia, endometriosis, and uterine fibroids, where partial lowering of gonadal steroids could improve the conditions without the side effects of complete suppression of sex steroid hormones, might benefit from treatment with kisspeptin antagonists. Similarly, the ability of kisspeptin antagonists to inhibit the ovulatory LH surge without lowering basal LH secretion represents an attractive possibility as female contraceptive (339), in which follicular development and estrogen production would continue but ovulation may be prevented. These features are in contrast to the current hormonal contraceptive methods in which supraphysiological levels of sex steroids are required to inhibit gonadotropins and ovulation. Likewise, kisspeptin antagonists may also find application in IVF, to prevent premature luteinization while maintaining basal LH, which might be of value in some women during hormone-induced superovulation (108, 113). In addition, there may also be an advantage in partial suppression of gonadotropins by kisspeptin antagonists during IVF, as opposed to current protocols of complete suppression with GnRH analogs, since the doses of gonadotropin replacement may be considerably lower. Finally, the fact that continuous administration of kisspeptins leads to desensitization of their gonadotropin responses without a detectable lowering of basal gonadotropin levels strongly suggests that, if any, protocols of desensitization using kisspeptin agonists may have clinical utilities similar to those described above for kisspeptin antagonists.
XVIII. OPEN QUESTIONS AND FUTURE DIRECTIONS IN KISSPEPTIN RESEARCH
As illustrated in previous sections, kisspeptin physiology has been a fertile area of research in recent years, with numerous achievements and important contributions to our global knowledge of how reproductive function is controlled in mammals and other vertebrates. On the basis of this impressive progress, we believe it is not bombastic to state that the identification and subsequent characterization of kisspeptins is a major breakthrough in contemporary neuroendocrinology, probably equivalent to the isolation of GnRH (in 1970s) or the identification of leptin (in 1990s), just to mention two paradigmatic examples. It is important to stress, though, that as is the case in other dynamic areas of biosciences, our knowledge of the functions of kisspeptins in health and disease is still rather incomplete, and additional, exciting developments are expected in this field. While such a contention has been emphasized in the different sections of this review, in this last section we intend to identify especially challenging or contentious areas of kisspeptin physiology, which we predict will attract considerable attention and research efforts in the coming years. These are not only related to important conceptual aspects of the function of kisspeptins and their regulation, but also to the development of improved tools for their analysis or the identification of potential therapeutic applications of kisspeptin analogs. An itemized and condensed presentation of such a forecast in kisspeptin research can be found also in TABLE 2.
A. Neuroanatomy of Kiss1 Neurons and Their Projections in the Brain
While the mapping of Kiss1 (and Kiss2) neurons in the brain of different vertebrates has substantially advanced recently, some features of this neuronal system require further elucidation. In mammals, the anatomical and functional characteristics of the rostral population of Kiss1 neurons, equivalent to the rodent AVPV, in larger species, including primates, need to be clarified. In addition, it will be critical to expand recent reports in the mouse (510), tracing the fiber projections of ARC/infundibular Kiss1 neurons, to elucidate their repertoire of biological functions and how this population engages into the neural pathways governing GnRH secretion in different mammals. Similarly, the potential interneuronal or nonsynaptic communication between Kiss1 and GnRH neurons warrants further investigation. Finally, neuroanatomical mapping of Kiss neurons in nonmammals is so far restricted to a few model species, such as medaka and zebrafish (284, 403), and needs to be extended to additional nonmammalian species.
B. Distribution and Molecular Regulation of GPR54 in the Brain
Despite recent progress in this area (169), our knowledge of the anatomical distribution of GPR54 within the brain remains scarce and needs substantial improvement in rodent and nonrodent species. In addition, there is still limited information on the array of genes and cellular networks that are activated upon kisspeptin stimulation in GnRH neurons, and how these cellular responses lead to specific patterns of GnRH release. Likewise, little is known about the factors that regulate GPR54 expression in GnRH neurons and other brain areas, where GPR54 signaling has been proposed to play a role but whose nature and molecular substrate remain largely unexplored. Finally, additional studies are warranted on the putative roles of GPR54 signaling in peripheral tissues, to delineate the whole repertoire of reproductive and nonreproductive actions of kisspeptins.
C. Roles and Mode of Action of KNDy Neurons in the Central Control of GnRH Secretion
While evidence is rapidly accumulating to demonstrate the existence of a discernible ARC population of KNDy neurons that operates as nodal center for the regulation of pulsatile GnRH secretion (304, 491), several aspects of such a network require further elucidation. For instance, the actual effects and mode of action of NKB and Dyn on KNDy neurons remain partially unfolded. In addition, better characterization of the distribution of the elements of the KNDy pathway would be desirable, e.g., whether or not KNB may act directly on GnRH nerve terminals. Likewise, our knowledge of the pattern of fiber projections from ARC KNDy neurons onto GnRH neuronal cell bodies and nerve terminals remains limited. Efforts in this front will help to substantiate the neuroendocrine pathways whereby NKB, Dyn, and kisspeptins engage into the networks driving GnRH release.
D. Interactions and Hierarchy of Kiss1 Neurons and Other Central Regulators of the HPG Axis
Our knowledge of the potential interactions of kisspeptins with other central transmitters involved in the regulation of GnRH remains incomplete, and additional studies are needed to dissect out the eventual interplay between kisspeptins and key components of the reproductive brain, such as glutamate, GABA, and RFRPs, among others. These studies will help to elucidate the relative importance of direct versus indirect actions of kisspeptins on GnRH neurons. The proven expression of GPR54 in GnRH neurons supports the importance of direct kisspeptin afferents in the control of GnRH secretion, a contention reinforced by preliminary, as yet unpublished, data showing that selective rescue of Gpr54 expression in GnRH neurons in Gpr54 null mice appears sufficient to attain puberty and reproductive capacity (220). Notwithstanding this evidence, solid electrophysiological and pharmacological data suggest the existence of additional, indirect pathways for the transmission of kisspeptin effects, whose nature is presently under active investigation.
E. Roles of Kisspeptins in the Timing of Puberty: Trigger, Amplifier, or Dispensable Regulator?
Compelling evidence strongly suggests the involvement of kisspeptins in the timing of puberty in mammalian and nonmammalian vertebrates. Yet, this consensus has been challenged by recent observations showing that mice with congenital ablation of Kiss1- and Gpr54-expressing neurons can apparently undergo reproductive maturation (273). While some possible reasons for these perplexing observations have been discussed in detail in section VI, the actual basis for kisspeptin-independent pubertal activation in such extreme conditions warrants further analysis. Moreover, with the assumption of a relevant role of kisspeptin signaling in the physiological control of puberty, the appealing proposal that, rather than the trigger, Kiss1 neurons at the AVPV would operate as amplifiers of the neurosecretory activity of GnRH neurons at the time of puberty has been only been documented in the mouse and requires further confirmation regarding critical aspects, such as: 1) which is the amount of estrogen input, and therefore of ovarian activation, that is needed to allow the pubertal expansion of Kiss1 neurons to proceed in rodents; and 2) whether this mechanism may operate also in other mammalian species including primates, where pubertal activation of gonadotropin secretion is well known to take place also in the absence of ovarian sex steroids (85, 86). In addition, the mechanisms responsible for the pubertal activation of the HPG axis in male puberty and the eventual role of the ARC/infundibular population of Kiss1 neurons in this process remain to be fully solved.
F. Roles and Mode of Action of Leptin in the Control of Kiss1 Neurons: Direct or Indirect Effects?
While a wealth of data have documented the capacity of Kiss1 neurons to sense the metabolic state of the organism, and solid experimental evidence has demonstrated the impact of changing levels of leptin on Kiss1/kisspeptin expression in the hypothalamus, strong evidence is mounting that kisspeptin-independent pathways may play also an important role in mediating at least part of leptin effects on GnRH neurons (106, 107). In addition, recent data have been presented supporting that, at least part of, leptin effects on Kiss1 neurons may be indirectly mediated (254). Elucidation of the relative importance of direct versus indirect effects of leptin on Kiss1 neurons, as well as of Kiss1-independent pathways, in different physiological and pathophysiological metabolic settings warrants further investigation.
G. Synaptic and Transcriptional Interactions of Kisspeptins and Metabolic Regulators: A Role in Food Intake?
Very recent findings have suggested the potential bidirectional interplay between kisspeptins and several neuropeptide regulators of energy homeostasis and feeding, such as NPY and melanocortins (20, 131, 213). While this possibility seems to be at odds with initial reports on the lack of direct effects of kisspeptins on food intake and body weight in rodents (58, 464), the recent combination of expression and electrophysiological data makes it tempting to revisit the putative roles and mechanisms of action, if any, of kisspeptins in the direct control of feeding and energy homeostasis.
H. New Physiological Models for Kisspeptin Research
The rapid accumulation of new information in the kisspeptin field makes it reasonable to predict the development of more sophisticated animal, and eventual cellular, models that will allow gaining a deeper insight into the major physiological roles and regulatory mechanisms of kisspeptins. A clear example in this front is functional genomic analyses by the use of genetically modified (GM) mouse lines, of which a new generation is just emerging (1, 77, 106, 115, 151, 271–273). In fact, until very recently, the only GM animal models in this area were congenital KO mice of Kiss1 or Gpr54 (72), which nonetheless have been instrumental for elucidation of different aspects of kisspeptin signaling, and have permitted the identification of interesting differences regarding the impact of selective elimination of either kisspeptins or Gpr54 on various reproductive parameters (72, 278). However, in the last year, new GM models for kisspeptin research, including the first Kiss1-Cre/GFP mouse lines, have been reported. These lines will enable the direct functional assessment of the electrophysiological properties of Kiss1 neurons in living tissue sections. In addition, these new GM models have allowed the initiation of studies involving selective ablation of specific signals/factors, such as ERα and leptin receptors, in Kiss1 cells. It is anticipated that the use of these and related models will revolutionize the field, by allowing selective manipulation of key additional pathways (of which NKB, NK3R, Dyn, AR and PR are just but few options) in Kiss1 cells. In addition, the possibility to identify and profile the various populations of Kiss1 neurons will permit to gain further knowledge about their differential features, which in turn may pave the way for more specific ablation strategies.
I. New Analytical Tools for Kisspeptin Research
Along with the generation of novel experimental models, it is expected that further improvements will be achieved in the methodological front. For instance, further progress is expected regarding optimization of protocols and reagents for IHC analysis of kisspeptins and GPR54 in different mammalian and nonmammalian species; on the latter, the first antibodies against Kiss1 and Kiss2 peptides have been reported only few months ago. Likewise, the lack of widely accessible immunoassays for measuring the actual levels of kisspeptins in different biological samples has hampered our understanding of key aspects of kisspeptin physiology and pathophysiology. For instance, the reported rise of circulating Kp-54 levels during human pregnancy has not been confirmed in rodents or other mammals due to the lack of specific assays. Similarly, absolute quantification of changes of kisspeptin content in the brain in various physiological or experimental conditions, such as pubertal development or gonadectomy, has not been possible so far. Thus development of universal assays for detection of kisspeptins in different biological samples is eagerly awaited.
J. Therapeutic Opportunities of Kisspeptins
The wealth of physiological and pharmacological studies reviewed herein have set the scene for the potential application of kisspeptin analogs in clinical settings, where these compounds might pose therapeutic advantages over established protocols of hormonal manipulation of the HPG axis based on the use of GnRH analogs. This area of kisspeptin research, however, is still at its infancy and is likely to concentrate further efforts in the near future. Based on their pharmacological profiles, kisspeptin analogs might be not only useful therapeutic options but eventually also diagnostic tools, e.g., for the development of tests to evaluate gonadotropic potential in conditions of suppressed HPG function such as metabolic disorders. On the other hand, the biological properties of kisspeptins in other species of obvious economical interest in terms of production are likely to make kisspeptin analogs tenable tools for the manipulation of the HPG axis in such species (such as ovine, bovine, and fish), in a variety of conditions, ranging from prevention of early puberty to enhancement of reproductive capacity. In this context, the rational design of kisspeptin-based protocols of reproductive control in animal production is likely to concentrate substantial attention in the near future.
In sum, we have witnessed in the last few years the emergence of kisspeptins as master regulators of the HPG axis and, hence, important players in the processes whereby the individual undergoes normal sexual differentiation of the brain and puberty onset, and attains reproductive capacity. In the course of investigations of the physiological roles of this system, we have become aware of the functional diversity of kisspeptins, which appear to be controlled by sophisticated regulatory mechanisms and are involved in most, if not all, possible critical aspects of the central control of reproduction. While this review article has intended to provide an exhaustive and comprehensive overview of our knowledge of kisspeptin physiology, as emerging from an ever-growing number of studies in different species, we have also aimed to pinpoint controversial and unsolved issues, as a means to set the scene for future challenges in this field. We hope that, by doing so, we will contribute to foster active discussion and to fuel renewed research efforts among scientists working in the field of kisspeptin physiology, an area that we believe is critical for our understanding of reproductive biology and promises to be a continuous source of scientific excitement in the near future.
The work from the authors' laboratory summarized in this article was supported by Grants BFU 2005-07446, BFU 2008-00984, and BFU 2011-25021 (Ministerio de Ciencia e Innovación, Spain; covered in part by funds from FEDER, EU); Project PI042082 (Ministerio de Sanidad, Spain); Project P08-CVI-03788 (Junta de Andalucía, Spain); and EU Research Contract DEER FP7-ENV-2007-1. CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Sanidad, Spain).
No conflicts of interest, financial or otherwise, are declared by the authors.
We are indebted to the members of the research team at the Physiology Section of the University of Cordoba, who actively participated in the generation of experimental data discussed herein during the last seven years. M. Tena-Sempere is especially grateful to Drs. V. M. Navarro, J. Roa, F. Gaytan, and J. M. Castellano for their substantial contribution to some of the studies reviewed in this article. The substantial contribution of Dr. Navarro and Dr. R. A. Steiner in completion of studies about the roles of NKB signaling in the control of gonadotropin secretion in the female rat, which have been revised here, is also acknowledged.
Address for reprint requests and other correspondence: M. Tena-Sempere, Dept. of Cell Biology, Physiology, and Immunology, Faculty of Medicine, University of Córdoba, Avda. Menéndez Pidal s/n, E14004 Córdoba, Spain (e-mail:).
NOTE ADDED IN PROOF
During the very last stage of proofreading this manuscript, the first inactivating mutation of KISS1 gene in humans has been reported (475a).