Kiss of the Mutant Mouse: How Genetically Altered Mice Advanced Our Understanding of Kisspeptin's Role in Reproductive Physiology

Abstract

The kisspeptin system has emerged as one of the most important circuits within the central network governing reproduction. Although kisspeptin physiology has been examined in many species, much of our understanding of this system has come from mice. Recently, the study of several innovative strains of genetically engineered mouse models has revealed intriguing and unexpected insights into the functions of kisspeptin signaling in the hypothalamus. Here, we review the advancements in our knowledge of the central kisspeptin system through the use of mutant mice.

The Kiss1 gene and its peptide product kisspeptin have been studied for their role in cancer metastasis since their discovery in 1996 (1). A few years later, the orphan receptor GPR54 was pinpointed as the cognate receptor for kisspeptin and was later given the generally accepted designation of Kiss1R (2, 3). (For clarity and ease of reading, we will use GPR54 to represent the kisspeptin receptor gene in this mini-review.) In 2003, the kisspeptin system emerged as a key player in reproductive physiology when three separate groups reported that a loss of kisspeptin signaling through GPR54 resulted in infertility and a perpetual prepubertal stasis in both mice and humans (4–6). Since then, the field of kisspeptinology has surged forward with studies in myriad species (7–12). Here we present a brief background on the organization and putative function of the kisspeptin system in mice followed by a more detailed description of how genetically engineered mouse models have contributed to our knowledge of kisspeptin physiology. For a broader overview, we refer the reader to more comprehensive reviews (13–16).

The Kiss1 gene and its receptor are localized in several tissues including ovary, testis, pancreas, pituitary, and brain (2, 17). Although there have been some examinations aside from cancer studies of the effects of kisspeptin signaling in the periphery, the physiology of the kisspeptin system is best defined in the hypothalamus. It is here that the kisspeptin system directly interacts with the small population of neurons that express GnRH (18–20). GnRH neurons represent a central locus that is essential to initiate and maintain the pulsatile release of the pituitary hormones (gonadotropins) that support gametogenesis and sex steroid production in the gonads (21–23). Nearly all GnRH neurons in the mouse express mRNA for GPR54, and exposure to kisspeptin stimulates a flood of GnRH output (19, 24, 25). The ability of femtomolar doses of centrally delivered kisspeptin to stimulate in vivo gonadotropin secretion elevates this neuropeptide to the status of most potent endogenous GnRH/gonadotropin activator yet discovered (24).

In the mouse hypothalamus, Kiss1 expression is primarily restricted to two sites: the arcuate nucleus (Arc) and the anteroventral periventricular region (AVPV) (24). This pattern seems to be set from early on, but expression in the AVPV is very low before puberty (19). In adult mice, it seems likely that kisspeptin neurons in both regions send stimulatory input to GnRH neurons; however, the two populations have contrasting characteristics. The most striking example of differences between kisspeptin neurons residing in different regions is their response to estradiol exposure. Nearly all Kiss1-expressing neurons are sensitive to regulation by the sex steroid estradiol, but expression of Kiss1 in neurons in the AVPV is stimulated by estradiol whereas Kiss1 expression in neurons of the Arc is suppressed (26–28). The differential regulation of Kiss1 mRNA suggests that the two populations serve different functions and may participate in both the positive and negative feedback effects of estradiol in the hypothalamus. Kiss1 neurons residing in the Arc have another characteristic that sets them apart from their counterparts in the AVPV: nearly all Kiss1 neurons in the Arc express the neuropeptides dynorphin and neurokinin B (28–31). All three neuropeptides are important players in the central regulation of reproduction, and this population of Arc neurons, the so-called KNDy cells, has become a subject of many recent investigations (32). Although many questions about the kisspeptin system remain to be addressed, many recent advances in knowledge and new enigmas have come from the study of genetically engineered mouse models. With this background in mind, we will discuss relevant physiological data generated from mouse strains produced and characterized by several different laboratories (Table 1).

Table 1.

List of engineered mouse strains

Strain designation	Originator, location, and year of first publication	Engineered mutation(s)	Descriptive publications
GPR54PTL	Paradigm Therapeutics Ltd.a (Cambridge, UK), 2003	IRES lacZ cassette replaces partial coding sequence of exons 1 and 2	6, 25, 59
GPR54SPR	Schering-Plough Research Institute (Kenilworth, NJ), 2003	IRES lacZ cassette replaces partial coding sequence of exon 2	5
GPR54HPC	Massachusetts General Hospital/Harvard Medical School (Boston, MA), 2007	Neomycin cassette replaces all coding sequence of exon 2	37, 39
GPR54Omer	Omeros Corp. (Seattle, WA), 2007	Retroviral insertion of nonsense codons in all reading frames; no loss of coding sequence	35, 36, 41
Kiss1PTL	Paradigm Therapeutics Ltd., 2007	IRES lacZ cassette replaces all coding sequence of exons 2 and 3	38
Kiss1HPC	Massachusetts General Hospital/Harvard Medical School, 2007	Neomycin cassette replaces all coding sequence of exon 2	37, 39
Kiss1-IRESCre	Center for Molecular Neurobiology (Hamburg, Germany), 2010	IRES followed by Cre sequence inserted after the Kiss1 gene	66, 69
Kiss1-CreUTSWJ2–4	University of Texas Southwestern Medical Center (Dallas, TX), 2011	BAC containing Kiss1 start sequence attached to Cre sequence inserted randomly into genome	63, 82
Kiss1-CreGFPUW	University of Washington (Seattle, WA), 2011	Cre-eGFP sequence inserted at the genomic Kiss1 translation start site	62
GPR54-IRESCre	Center for Molecular Neurobiology, 2011	IRES followed by Cre sequence inserted after the Kiss1R gene	69
GPR54-flox	Lexicon Pharmaceuticals (The Woodlands, TX), 2012	LoxP sites inserted to flank entire coding sequence of GPR54, also inserted LacZ sequence following final LoxP site	56

BAC, Bacterial artificial chromosome; eGFP, enhanced GFP.

^aParadigm is now Takeda Cambridge Ltd.

Global Loss of Function Models

The chronicle of kisspeptin as a regulator of fertility and puberty began with a defective gene. Two groups working independently in France and in the United States examined the genetic mutations of male and female family members suffering from idiopathic hypogonadotropic hypogonadism. Both groups determined that all the affected individuals were homozygous for deletions in GPR54, the gene coding for the kisspeptin receptor (4, 6). Observations from the human studies were reiterated in concurrent studies of genetically engineered mice bearing inactivating mutations to the GPR54 gene [GPR54 knockout (KO)] (5, 6). That is, humans lacking functional GPR54 and their murine counterparts fail to progress through puberty and, as adults, have low levels of sex hormones, underdeveloped gonads, and are infertile (4–6). The similarities in phenotype have also held true between several independently developed strains of GPR54 KO mice.

The GPR54 KO strain first described by Seminara et al. (6), in 2003 was designed by Paradigm Therapeutics (Cambridge, UK) (33) with a deletion of coding sequence in exons 1 and 2 from the GPR54 gene, leading to a complete loss of gene expression. These animals are characterized by small, underdeveloped gonads, low levels of circulating sex steroids, threadlike uteri and lack of estrous cycles in females, and no detectable mature oocytes or sperm (6). Also in 2003, Funes' group (5, 33) characterized a mouse strain generated by Schering-Plough Research Institute (Kenilworth, NJ) with an excision of a portion of exon 2 preventing expression of GPR54. This strain is likewise characterized by hypoplastic gonads and other reproductive organs in both males and females (5). Five years later, two more strains of GPR54 KO mice were characterized. One strain, developed by Omeros Corp. (Seattle, WA), Kauffman et al (35), and Steiner and co-workers (36) at the University of Washington, was generated by a gene trap method described by Gragerov et al (34) that prevents transcription of the GPR54 gene without removing any coding sequence. The other 2007 strain was generated by removal of the entire sequence of exon 2 by Seminara and associates (37) at Massachusetts General Hospital (affiliated with Harvard Medical School) located in Boston, Massachusetts. Although some variations have been uncovered among the four strains of GPR54 KO mice, all strains share the characteristics of apparently absent pubertal maturation and complete infertility (5, 6, 25, 35–37).

The year 2007 also saw the revelation of two strains of mice deficient in the GPR54 ligand, kisspeptin. The Boston group generated a strain with a targeted deletion of exon 1 in the Kiss1 gene whereas Paradigm Therapeutics developed a strain with deletions of exons 1 and 2 (37, 38). The phenotype of both strains is analogous to that of the GPR54 KOs. Loss of Kiss1 gene expression prevents pubertal maturation and renders the animals infertile with little to no effect on any other system that cannot be attributed to decreased levels of sex steroids (37, 38). Lapatto et al. (37) in Boston directly compared their new kisspeptin-deficient model to their receptor-deficient model and determined that loss of the Kiss1 gene produced a less severe reproductive phenotype. Male Kiss1 KO mice have small testes and low testosterone compared with wild-type mice, but these parameters are improved compared with GPR54 KO mice (37). Similarly, the female Kiss1 KOs have delayed vaginal opening but still achieve this milestone earlier than GPR54 KOs (37). It seems also that loss of kisspeptin expression may produce a more variable phenotype among individuals: some female Kiss1 KOs that had normal vaginal opening also had ovaries of normal weight compared with wild-type females (37). Although none of the tested Kiss1 KO animals proved to be fertile, it is not clear whether the animals with the most normal phenotypes were tested (37). The parallels between phenotypes of the receptor and ligand KO strains suggest that kisspeptin and GPR54 comprise a direct circuit within the hypothalamic network that coordinates reproduction. Coupled with the observation that kisspeptin fails to stimulate GnRH neurons or a rise in gonadotropins in mice lacking GPR54, these results suggest that kisspeptin can only exert its effects on GnRH neurons through this receptor (25, 35, 36).

Several lines of evidence derived from the classic loss-of-function mouse models provide a strong case that kisspeptin signaling through GPR54 is essential to drive the hypothalamic-pituitary-gonad (HPG) axis that governs reproduction. First, GnRH neurons themselves appear normal in the absence of GPR54, and exposure to exogenous GnRH stimulates a robust release of gonadotropins from the pituitary (6, 25, 36). Second, a series of experiments by Chan et al. (39) using the GPR54 KO and Kiss1 KO strains made in Boston suggest that, even in the absence of kisspeptin signaling, GnRH neurons do secrete minimal amounts of GnRH. They determined that, although vaginal opening was severely delayed in both strains of KO mice (more so for receptor deficiency than the ligand), some mice did eventually exhibit extended estrous cycles although no signs of ovulation were detected. This study and others have determined that some male Kiss1 KO and GPR54 KO mice are capable of generating sperm, and treatment with a GnRH inhibitor results in reduced weights of the already diminished testes and uteri of the KO mice (37–40). Finally, kisspeptin treatment in Kiss1 KO mice induces GnRH secretion that can lead to normalized gonadotropin levels (37, 38). Together, these observations suggest that the HPG axis downstream of GnRH neurons is intact and that a stimulatory input such as kisspeptin is required to drive the system. However, the moderation of phenotype in strains lacking Kiss1 expression compared with mice lacking the receptor points to the possibility that other peptides may interact with GPR54 or that the receptor may have some ligand-independent activity (37, 39).

The hypoplastic phenotype of the gonads and other sex organs of GPR54 KOs suggest that other sexually differentiated aspects may be affected by the absence of the receptor. Kauffman et al. (35) initiated a series of studies to determine whether kisspeptin signaling through GPR54 is necessary for the development of sex-specific characteristics of brain and behavior. They observed that male GPR54 KO mice have female-like histology in sexually dimorphic areas of the brain. Moreover, in the presence of a female in estrus, adult males lacking GPR54 do not exhibit normal olfactory preference nor do they express normal male-specific sex behaviors (35). Testosterone treatment restores all sex behaviors except for ejaculation in male GPR54 KOs but does not overcome the lack of olfactory preference (35). Likewise, adult female GPR54 KOs display normal lordosis behavior after priming with estradiol and progesterone (35). Further studies of neonates have indicated that GPR54 signaling is not required for the perinatal rise in testosterone but is necessary for the pubertal steroid surge in males (41). These studies argue that the kisspeptin system is a switch activated in puberty to impel the HPG axis and stimulate sex behaviors rather than a mechanism of sex differentiation during embryonic and neonatal development. However, the generation of olfactory preference and male-like development of the hypothalamus clearly rely upon the presence of GPR54. The mechanisms behind the sex-specific development of Kiss1 neurons are still under investigation (42).

The relationship between GPR54 signaling and sex-specific physiology of the female ovulatory cycle has also been explored. During the proestrous phase of the estrous cycle, rising levels of estradiol trigger an acute surge in circulating LH, the gonadotropin responsible for initiating ovulation (43–48). The AVPV region of the hypothalamus, a sexually dimorphic area that is larger and contains more cells in females than in males, is thought to play an important role in the generation of the preovulatory LH surge (49–51). Because the AVPV is a site of concentrated Kiss1 expression and that expression is enhanced by estradiol, it seems plausible that kisspeptin neurons in this region represent an integral part of the neural circuitry controlling ovulation (24, 26). Studies in rats support this idea: the majority of Kiss1-expressing neurons in the AVPV, but not the Arc, are transcriptionally active at the time of the LH surge, and infusion of kisspeptin antiserum into the preoptic area of the hypothalamus can severely diminish, if not entirely ablate, an estradiol-induced LH surge (52, 53). These intriguing results precipitated an examination of induced LH surges in female GPR54 KO mice. In 2007, Steiner and associates (36) in Seattle, Washington tested the ability of the Omeros GPR54 KOs to respond to positive feedback of a chronic infusion of estradiol. They observed that most of the GPR54 KO females challenged with estradiol experienced a robust increase in circulating LH and transcriptional activation of GnRH neurons. This surprising result indicated that, although important for the tonic stimulation of GnRH/LH secretion, kisspeptin signaling through GPR54 is not essential for the generation of an estradiol-induced LH surge.

This assertion, however, was challenged shortly thereafter. Herbison and associates (54) at the University of Otago in New Zealand performed similar experiments using the GPR54 KO mouse strain developed by Paradigm Therapeutics. In contrast to the Seattle study, this group observed that lack of kisspeptin signaling through GPR54 prevented any LH response to estradiol and progesterone priming. This result would argue that GPR54 signaling is essential to generate a sex steroid-induced LH surge. The reasons for such different results may lie in the different surge-induction protocols used by the two laboratories, the differences between GPR54 KO mouse strains, or another detail in the experimental designs, but this has yet to be reconciled. The authors from New Zealand posit that the Omeros KO strain may be subject to some residual expression of GPR54 that would allow kisspeptin signaling to stimulate GnRH neurons (54). However, this seems unlikely because kisspeptin treatment failed to stimulate either transcriptional activation in GnRH neurons or LH secretion in the KO mice (35, 36). Considering that other neurotransmitters are able to stimulate LH release in kisspeptin receptor-deficient mice, it seems plausible that, when sex steroid levels are normalized, glutamate or another signal is able to initiate the LH surge in the absence of kisspeptin signaling (55–58).

Reporters and Conditional Deletion and Reactivation Models

Another genetic strategy employed to study the kisspeptin system was the insertion of reporter and recombinase sequences to allow visual detection of target gene expression and to manipulate the expression of genes in Kiss1 neurons, respectively. The GPR54 KO developed by Paradigm Therapeutics and first characterized by Seminara et al. (6) in 2003 bears an internal ribosome entry site taking the place of coding sequence for the GPR54 gene followed by sequence coding for the LacZ gene. This design disrupts transcription of GPR54 while introducing expression of the LacZ protein product, β-galactosidase, controlled by the endogenous GPR54 promoter. The presence of β-galactosidase can be detected by histochemistry to reveal the location of cells that would normally express the kisspeptin receptor. Herbison and associates (59) used this technique to examine the putative expression of GPR54 through different stages of development. They observed that, in the hypothalamus, GPR54 expression is localized to GnRH neurons from birth through adulthood, supporting previous observations made by in situ hybridization (19, 59). Coupled with evidence from other studies indicating that kisspeptin production increases during puberty, these observations suggest that although the kisspeptin receptor is present in GnRH neurons of juveniles, there may be insufficient ligand to activate the receptor or the receptor itself may not yet be presented on the cell surface (19). This model also revealed the presence of GPR54-expressing cells in the thalamus, hippocampus, and in the hindbrain; however, the role of the receptor in these regions is not yet clear (59). The Paradigm GPR54 KO is not unique in its usefulness in exploring kisspeptin anatomy; several of the other previously described strains also bear reporter sequences regulated by their respective target genes (Table 1). However, at this time, Kiss1 and GPR54 expression patterns have yet to be examined in the other KO strains.

An additional approach to manipulating gene transcription and visualizing gene expression is use of the Cre-LoxP system. The Cre recombinase enzyme excises genomic DNA between two LoxP sequences inserted to flank a specific locus of the mouse genome (target gene) (60). Thus, a Cre-expressing animal can be bred with an animal bearing inserted LoxP sites that flank a gene of interest to produce a strain with a selective deletion of the target gene only in the Cre-expressing cells (60). Alternatively, the Cre-LoxP system can be used to activate transcription of a gene that was blocked by the insertion of a LoxP-flanked STOP sequence (61). For example, a mouse engineered to express Cre recombinase in Kiss1-expressing cells could be bred with a mouse bearing a Cre-inducible reporter gene, such as LacZ. Cre activity would remove the transcriptional blocker from the LacZ reporter gene only in Kiss1-expressing cells, allowing immunohistochemical visualization of kisspeptin cells. One important feature of this system is that once Cre is activated, the alterations it makes are irreversible. If the attached promoter causes Cre to be expressed in early development, the affected genes will remain silenced (or activated, depending on the LoxP site pattern). As with the global KO models, it must be taken into account that these very early changes in expression can lead to undefined compensatory changes in the developing mouse brain.

In 2012, Lexicon Pharmaceuticals (The Woodlands, TX) used the Cre-LoxP technique to create a new GPR54 KO: they crossed a strain bearing LoxP sites flanking the GPR54 gene (GPR54-flox) to a protamine-Cre mouse strain to remove the entire coding sequence of GPR54 from all cells (56). The Tena-Sempere (5, 6, 35, 56) laboratory in Cordoba, Spain has characterized the new strain and reported that the general phenotype is consistent with that of previously described global GPR54 KO mice, with, perhaps, an even more severe form of hypogonadotropic hypogonadism. The investigators used this model to test the effects of neurotransmitters with LH-stimulating properties on mice lacking the kisspeptin receptor. In agreement with previous studies, they demonstrated that glutamate receptor agonists, galanin-like peptide, and antagonists of the gonadotropin-inhibiting peptide RFRP3 all elicited an increase of circulating LH in GPR54 KO mice (51–54). It seems plausible, then, that with the proper steroidal milieu, other hypothalamic circuits could take over the GnRH-activating duties of kisspeptin. However, this group also observed that a neurokinin receptor agonist did not significantly alter LH in the absence of GPR54, suggesting that neurokinin's LH-inducing effect requires kisspeptin signaling (56).

Steiner and associates (62), in collaboration with the Palmiter laboratory at the University of Washington, developed a knock in mouse strain in which the coding sequence for both enhanced green fluorescent protein (GFP) and Cre recombinase enzyme are controlled by the endogenous Kiss1 promoter without altering endogenous Kiss1 expression. This strategy allows for both visualization of Kiss1-expressing cells via GFP visualization and manipulation of the expression of other genes via the Cre-LoxP system. Similar to the GPR54 KO-LacZ strain used by the Herbison group, the Kiss1-CreGFP strain features a reporter gene whose expression is controlled just as the endogenous target gene would be. Gottsch et al. (62) demonstrated this property of the model by ovariectomizing females and treating them with estradiol or vehicle. The GFP fluorescence patterns in these animals mirrored those of Kiss1 mRNA, suggesting that this model is useful not only for showing where Kiss1 is expressed, but also when (62). One limitation of this approach, however, is that the appropriate steroid conditions must be met to detect GFP in a specific location (e.g. estradiol must be low or absent for GFP to be visualized in the Arc of this model). In keeping with this requirement of the Kiss1-CreGFP strain, the investigators examined electrophysiological characteristics of GFP-expressing neurons in the Arc of ovariectomized females. They determined that this population of neurons displays several characteristics of pacemaker cells, including N-methyl-d-aspartate-induced burst firing activity (62). This observation suggests that kisspeptin neurons in the Arc (KNDy neurons) have a role in regulating the pulsatile activity of GnRH neurons, but this remains to be demonstrated.

The Cre-LoxP strategy was also employed by a collaboration of the Elias and Zigman laboratories at University of Texas Southwestern Medical Center (Dallas, TX) to generate a strain of mice expressing Cre under the control of the Kiss1 promoter. They first used a bacterial artificial chromosome to insert a transgene at a random location or locations within the genome (63). The transgene contained the coding sequence for Cre coupled to the promoter sequence for Kiss1 such that, in the transgenic mouse, any cell normally expressing the Kiss1 gene would also express Cre recombinase (63). This strain, known as Kiss1-Cre line J2–4, was then crossed to another mouse strain bearing a transgene coding for a Cre-inducible LacZ reporter gene (a LacZ sequence interrupted by a STOP sequence flanked by LoxP sites) (63). Using immunohistochemistry for β-galactosidase, Cravo and colleagues (24, 26, 63) confirmed earlier reports of Kiss1 expression patterns in the hypothalamus and colocalization with estrogen receptors in the female mouse. β-Galactosidase was also detected in the neocortex, a region where kisspeptin's presence had not yet been noted and where its function is still unknown (63). Further, Cravo and co-workers used the Kiss1-Cre crossed with a Cre-inducible GFP reporter gene to test whether Kiss1 neurons are sensitive to leptin signaling. Previous work in the male wild-type mouse had indicated that about 40% of Kiss1 neurons in the Arc express the functional leptin receptor gene Leprb, but this study in ovariectomized females revealed only a portion of those (∼15% of Kiss1 neurons in the Arc) show an intracellular response to leptin treatment (63, 64). Interestingly, kisspeptin neurons in the AVPV appear to be entirely insensitive to the direct effects of leptin as studies in both the Kiss1-Cre model and in wild-type mice indicate that leptin treatment fails to induce phosphorylation of signal transducer and activator of transcription 3 (a marker of leptin signaling) in this population (63, 65).

Challenging Dogmas with the Cre-LoxP System

Throughout the relatively short period of study for the kisspeptin system, two primary themes have emerged. First and foremost among these is that kisspeptin signaling through its receptor is necessary for normal progression through puberty and adult reproductive function. The hypogonadotropic hypogonadal phenotypes of a small army of independently developed kisspeptin- and GPR54-deficient mice suggest that this is correct (33, 56). Second is the theory that kisspeptin neurons mediate both the stimulatory and suppressive effects of sex steroids on GnRH neurons. The coexpression of estrogen and androgen receptors in kisspeptin neurons, the apparent site-specific regulation of Kiss1 mRNA expression by sex steroids, and the loss of tonic stimulatory drive for GnRH/LH secretion in the absence of kisspeptin signaling all support this theory (26, 27, 36).

The idea that kisspeptin signaling is essential for the regulation of GnRH by sex steroids was tested in 2011. Another Cre-expressing mouse strain was developed by Mayer et al. (66) in Hamburg, Germany and used in collaboration with the Levine laboratory at Northwestern University in Illinois. Their model features an internal ribosome entry site (IRES) and sequence encoding the Cre enzyme following the Kiss1 gene. This group was the first to use the Cre-Lox system to selectively manipulate endogenous genes in kisspeptin cells. They crossed their Kiss1-IRESCre mice with those bearing LoxP sites flanking a portion of the coding sequence for estrogen receptor α to generate mice lacking estrogen receptor α in kisspeptin neurons (66). These animals display precocious pubertal initiation demonstrated by early vaginal opening and circulating LH levels 3-fold higher than normal on d 15 after birth (66). However, after this early start, the mice fail to progress to normal estrous cyclicity and show no signs of ovulation as adults (66). These characteristics argue in favor of estrogen signaling in kisspeptin neurons having a dual effect on the central circuits regulating reproduction, first as a brake to restrain the timing of puberty and then as a stimulant to activate the HPG axis and support fertility in adulthood. Because very few Kiss1-expressing neurons were identified in the AVPV region of pubertal animals, it seems plausible that it is the Arc population that is responsible for restraining pubertal initiation (66). This idea is supported by previous observations in wild-type female mice that ovariectomy on d 14 (prepuberty) leads to an increase in both Kiss1 gene expression in the Arc and LH levels in circulation (67). Evidence that Kiss1 neurons in the AVPV are important for the awakening of the HPG during puberty in females has been presented in more recent studies (68). These studies uphold the theory that estrogen signaling in kisspeptin neurons is necessary for completion of pubertal maturation. It would be interesting to determine whether loss of estrogen signaling to Kiss1 neurons would prevent an estradiol-induced LH surge. The results of such an experiment may help resolve the issue as to whether kisspeptin (or another signal from Kiss1 neurons) is essential to generate the LH surge or if another population of estrogen-sensitive neurons could compensate for the loss.

Mayer and Boehm (69) followed this work by using the Kiss1-IRESCre to tackle the central theme of kisspeptin research. They crossed the Kiss1-IRESCre strain to mice bearing a transgene for diphtheria toxin A (DTA), which codes for a toxin deadly to any cell in which it is expressed. The DTA sequence is coupled to the promoter of a ubiquitously expressed gene, ROSA26, and contains a STOP sequence flanked by LoxP sites. Thus, in this strain, all cells that normally express the target gene Kiss1 at any time during development will also produce the toxin and be ablated (70). The resulting phenotype proved to be a head scratcher. Despite the previous evidence to the contrary, loss of nearly all Kiss1-expressing neurons early in development did not have any appreciable effect on the reproductive system save for a reduction in ovarian mass (69). The timing of vaginal opening of Kiss1-ablated females matched that of control females, and the mutant mice achieved regular, albeit extended, estrous cycles in adulthood (69). Moreover, all tested females became pregnant and delivered average-size litters (69). These observations challenge the original notion that kisspeptin signaling is essential to fully activate the HPG axis and drive the reproductive system. They also suggest that some element inherent in Kiss1 neurons may inhibit GnRH secretion and that kisspeptin signaling is necessary to overcome that suppression. It may be that in mice the imbalance between neuropeptides in KNDy neurons of the Arc caused by deletion of a single gene is more detrimental than removal of the entire population. Observations in sheep have hinted that this may be the case (71). Alternatively, the loss of an entire population of neurons may force the developing brain to compensate by forming connections that would not be made in a normal mouse hypothalamus. Yet another possible explanation for these results is that the tiny portion of surviving Kiss1 neurons is capable of generating the necessary kisspeptin signals to drive the HPG. The authors demonstrate by immunohistochemistry that fewer than 3% of kisspeptin neurons remain in toxin-treated mice, but it is unclear whether the investigators accounted for sex steroid status of the female mice used for the assays. It is possible that some surviving Kiss1 neurons escaped detection due to the effects of fluctuating estradiol on kisspeptin expression.

This work was accompanied by characterization of the complementary mouse mutant: the Cre-inducible DTA strain was crossed with a new strain of GPR54-IRESCre to generate mice with a loss of cells expressing the kisspeptin receptor (69). Females with this mutation are similar to females with ablated kisspeptin cells, i.e. almost entirely normal (69). Similar to the Kiss1-ablated mice, these animals have small ovaries and longer cycles, and they are fertile (69). The kisspeptin receptor is expressed in nearly all GnRH neurons, and ablation of all GPR54-expressing neurons leaves fewer than 10% of GnRH neurons surviving in the preoptic area, suggesting that only a miniscule number of GnRH neurons are truly required to support fertility (69).

In an important and elegant experiment, Mayer and Boehm (69) crossed their Kiss1- and GPR54-Cre expressing mice to a strain bearing a Cre-inducible diphtheria toxin receptor. The mice generated from these crosses would then be susceptible to ablation of Kiss1- or GPR54-expressing cells, respectively, simply by peripheral injection of diphtheria toxin. This technique has been previously employed to ablate other hypothalamic neuron populations, but it seems to be less thorough than DTA ablation, leaving up to 15% of target neurons behind after treatment (72–74). The investigators observed that acute loss of Kiss1-expressing neurons in adult females halted the estrous cycle and rendered the animals infertile (69). Females that were treated with diphtheria toxin on d 20, just before normal onset of puberty, also failed to achieve estrous cyclicity in adulthood (69). These results support the theory that embryonic loss of kisspeptin neurons triggers a compensatory rearrangement of hypothalamic circuitry to allow the HPG axis to function, and that this compensation occurs before puberty. The cessation of estrous cycles in toxin-treated mice also argues against the possibility that a very small population of kisspeptin neurons is sufficient to sustain fertility. In contrast, diphtheria toxin-induced ablation of GPR54-expressing cells in adult female mice did not alter estrous cycles, and half of the animals tested produced litters after being paired with males (69). Previous studies using in vivo tissue grafts have demonstrated that only a few GnRH neurons are required to drive gonadotropin secretion in mice lacking proper GnRH signaling; however, these studies of developmental and adult neuron ablation are the first studies to show that acute decimation of the GnRH neuronal population in mice creates only a minor hiccup in the function of the HPG axis (75, 76).

Finally, there is the pervading idea that kisspeptin neurons transmit metabolic information to GnRH neurons including signals from leptin. Leptin is a hormone made in adipose tissue, and the amount in circulation correlates to the presence of energy stores within the body (77, 78). Similar to loss of kisspeptin signaling, lack of this hormone shuts down the HPG axis (79, 80). Because GnRH neurons do not express the functional form of the leptin receptor, it was proposed that an upstream population of neurons may be mediators of leptin signals to GnRH neurons (81–83). Observations that conditions of low leptin levels suppressed Kiss1 expression and that a subset of Kiss1 neurons in the Arc express Leprb support a role for kisspeptin as a mediator of leptin's signals to GnRH neurons (64). To test this model, Elias and associates (84) crossed their Kiss1-Cre transgenic mice with a strain bearing LoxP sites flanking the Leprb gene to create a mouse lacking the leptin receptor in all Kiss1-expressing cells. They observed that these mice have no detectible abnormalities and progress through puberty to become fertile adults. Furthermore, the body weights of Kiss1-Cre/LepR-flox animals are similar to those of control animals (84). These studies demonstrate that leptin action in kisspeptin neurons is not required for normal pubertal development and fertility. In the same study, the investigators showed that a population of neurons (that do not express Kiss1) located in the ventral premammillary nucleus plays a critical role in leptin action in female pubertal development (84). However, developmental adaptations and redundancy may occur. It will be important to assess whether restoring leptin signaling selectively to kisspeptin neurons in an otherwise LepRb-deficient mouse is sufficient to mediate leptin's effect in the reproductive neuroendocrine axis.

Summary and Conclusions

The use of genetically altered mouse models has greatly expanded our knowledge of the kisspeptin system and its role in reproductive physiology. But these models have also exemplified the essential plasticity of neuronal circuitry in the developing brain. Studies in mutant mice first established dogmas of kisspeptinology and then challenged them with some surprising results. The vast majority of data so far support the notion that kisspeptin signaling through its cognate receptor GPR54 is required for pubertal maturation and fertility. In the complete absence of either Kiss1 or GPR54 expression, mice fail to become sexually mature and never achieve the ability to reproduce (5, 6, 25, 35–37, 39). And yet, the ablation of the entire Kiss1-expressing neuron population during early development seemingly has no adverse effect on puberty or fertility (69). Moreover, ablation of Kiss1 neurons in adulthood halts reproductive function whereas ablation of GPR54 neurons at the same period has little effect (69).

The role of kisspeptin signaling in the positive and negative feedback by estradiol was examined in female mice lacking kisspeptin receptors and in mice selectively lacking estrogen receptors in Kiss1-expressing neurons. The reproductive deficits in both types of mutants are consistent with an essential role for kisspeptin signaling in the development of ovarian cycles; however, it remains plausible that other circuits can compensate for the disruption of the kisspeptin system if the proper gonadal hormones are provided (36, 54, 66). Despite most expectations, Kiss1 neurons seem to have no significant role in directly relaying leptin signals to GnRH neurons (84). Thus, although the kisspeptin system is one important player in the neuroendocrine control of reproduction, it is subject to the influence and primacy of every other factor within the hypothalamus and beyond.

One important caveat to these conclusions is an oversight common to all the kisspeptin-related engineered mouse model studies. As mentioned before, early studies indicated that kisspeptin and its receptor can be found not only in the brain but also in peripheral tissues such as the ovary, uterus, testis, pituitary, and pancreas (5, 17). The role of the kisspeptin system in these tissues has received much less attention, but we can speculate that deletion of a gene or ablation of a cell population would affect these organs along with the brain. It would be worthwhile to characterize mouse strains with Kiss1 or GPR54 deletions in specific cell populations such as neurons or ovarian cells.