Alexander S. Kauffman

DISCOVERY OF POTENT KISSPEPTIN ANTAGONISTS DELINEATE PHYSIOLOGICAL MECHANISMS OF GONADOTROPIN REGULATION

Neurons that produce gonadotropin-releasing hormone (GnRH) are the final common pathway by which the brain regulates reproduction. GnRH neurons are regulated by an afferent network of kisspeptin-producing neurons. Kisspeptin binds to its cognate receptor on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion, thus gating down-stream events supporting reproduction. We have developed kisspeptin antagonists to facilitate the direct determination of the role of kisspeptin neurons in the neuroendocrine regulation of reproduction. In vitro and in vivo studies of analogues of kisspeptin-10 with amino substitutions have identified several potent and specific antagonists. A selected antagonist was shown to inhibit the firing of GnRH neurons in the brain of the mouse and to reduce pulsatile GnRH secretion in female pubertal monkeys; the later supporting a key role of kisspeptin in puberty onset. This analog also inhibited the kisspeptin-induced release of luteinizing hormone (LH) in rats and mice and blocked the postcastration rise in LH in sheep, rats, and mice, suggesting that kisspeptin neurons mediate the negative feedback effect of sex steroids on gonadotropin secretion in mammals. The development of kisspeptin antagonists provides a valuable tool for investigating the physiological and pathophysiological roles of kisspeptin in the regulation of reproduction and could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer.

Emerging ideas about kisspeptin– GPR54 signaling in the neuroendocrine regulation of reproduction

Neurons that produce gonadotropin-releasing hormone (GnRH) drive the reproductive axis, but the molecular and cellular mechanisms by which hormonal and environmental signals regulate GnRH secretion remain poorly understood. Kisspeptins are products of the Kiss1 gene, and the interaction of kisspeptin and its receptor GPR54 plays a crucial role in governing the onset of puberty and adult reproductive function. This review discusses the latest ideas about kisspeptin-GPR54 signaling in the neuroendocrine regulation of reproduction, with special emphasis on the role of Kiss1 and kisspeptin in the negative and positive feedback control of gonadotropin secretion by sex steroids, timing of puberty onset, sexual differentiation of the brain and photoperiodic regulation of seasonal reproduction.

The kisspeptin receptor GPR54 is required for sexual differentiation of the brain and behavior.

GPR54 is a G-protein-coupled receptor, which binds kisspeptins and is widely expressed throughout the brain. Kisspeptin–GPR54 signaling has been implicated in the regulation of pubertal and adulthood gonadotropin-releasing hormone (GnRH) secretion, and mutations or deletions of GPR54 cause hypogonadotropic hypogonadism in humans and mice. Other reproductive roles for kisspeptin–GPR54 signaling, including the regulation of developmental GnRH secretion or sexual behavior in adults, have not yet been explored. Using adult wild-type (WT) and GPR54 knock-out (KO) mice, we first tested whether kisspeptin–GPR54 signaling is necessary for male and female sexual behaviors. We found that hormone-replaced gonadectomized GPR54 KO males and females displayed appropriate gender-specific adult sexual behaviors. Next, we examined whether GPR54 signaling is required for proper display of olfactory-mediated partner preference behavior. Testosterone-treated WT males preferred stimulus females rather than males, whereas similarly treated WT females and GPR54 KO males showed no preference for either sex. Because olfactory preference is sexually dimorphic and organized during development by androgens, we assessed whether GPR54 signaling is essential for sexual differentiation of other sexually dimorphic traits. Interestingly, adult testosterone-treated GPR54 KO males displayed “female-like” numbers of tyrosine hydroxylase-immunoreactive and Kiss1 mRNA-containing neurons in the anteroventral periventricular nucleus and likewise possessed fewer motoneurons in the spino-bulbocavernosus nucleus than did WT males. Our findings indicate that kisspeptin–GPR54 signaling is not required for male or female copulatory behavior, provided there is appropriate adulthood hormone replacement. However, GPR54 is necessary for proper male-like development of several sexually dimorphic traits, likely by regulating GnRH-mediated androgen secretion during “critical windows” in perinatal development.

Circadian regulation of Kiss1 neurons: implications for timing the preovulatory gonadotropin-releasing hormone/luteinizing hormone surge.

The preovulatory GnRH/LH surge depends on the presence of estradiol (E(2)) and is gated by a circadian oscillator in the suprachiasmatic nucleus (SCN) that causes the surge to occur within a specific temporal window. Although the mechanisms by which the clock times the LH surge are unclear, evidence suggests that the SCN is linked to GnRH neurons through a multisynaptic pathway that includes neurons in the anteroventral periventricular nucleus (AVPV). Recently, Kiss1 neurons in the AVPV have been implicated in the surge mechanism, suggesting that they may integrate circadian and E(2) signals to generate the LH surge. We tested whether Kiss1 neurons display circadian patterns of regulation in synchrony with the temporal pattern of LH secretion. Mice housed in 14 h light, 10 h dark were ovariectomized, given E(2) capsules (or nothing), and transferred into constant darkness. Two days later, the mice were killed at various times of day and their LH and Kiss1 levels assessed. In E(2)-treated females, LH levels were low except during late subjective day (indicative of an LH surge). Similarly, AVPV Kiss1 expression and c-fos coexpression in Kiss1 neurons showed circadian patterns that peaked coincident with LH. These temporal changes in Kiss1 neurons occurred under steady-state E(2) and constant environmental conditions, suggesting that Kiss1 neurons are regulated by circadian signals. In the absence of E(2), animals displayed no circadian pattern in LH secretion or Kiss1 expression. Collectively, these findings suggest that the LH surge is controlled by AVPV Kiss1 neurons whose activity is gated by SCN signals in an E(2)-dependent manner.

Sex differences in the regulation of Kiss1/NKB neurons in juvenile mice: implications for the timing of puberty.

In mammals, puberty onset typically occurs earlier in females than in males, but the explanation for sexual differentiation in the tempo of pubertal development is unknown. Puberty in both sexes is a brain-dependent phenomenon and involves alterations in the sensitivity of neuronal circuits to gonadal steroid feedback as well as gonadal hormone-independent changes in neuronal circuitry. Kisspeptin, encoded by the Kiss1 gene, plays an essential but ill-defined role in pubertal maturation. Neurokinin B (NKB) is coexpressed with Kiss1 in the arcuate nucleus (ARC) and is also important for puberty. We tested whether sex differences in the timing of pubertal development are attributable to sexual differentiation of gonadal hormone-independent mechanisms regulating hypothalamic Kiss1/NKB gene expression. We found that, in juvenile females, gonadotropin secretion and expression of Kiss1 and NKB in the ARC increased immediately following ovariectomy, suggesting that prepubertal females have negligible gonadal hormone-independent restraint on their reproductive axis. In contrast, in similarly aged juvenile males, no changes occurred in LH levels or Kiss1 or NKB expression following castration, suggesting that gonadal hormone-independent mechanisms restrain kisspeptin/NKB-dependent activation of the male reproductive axis before puberty. Notably, adult mice of both sexes showed comparable rapid increases in Kiss1/NKB expression and LH secretion following gonadectomy, signifying that sex differences in the regulation of ARC Kiss1/NKB neurons are manifest only during peripubertal development. Our findings demonstrate that the mechanisms controlling pubertal activation of reproduction in mice are different between the sexes and suggest that gonadal hormone-independent central restraint on pubertal timing involves Kiss1/NKB neurons in the ARC.

RFamide-Related Peptide-3 Receptor Gene Expression in GnRH and Kisspeptin Neurons and GnRH-Dependent Mechanism of Action

RFamide-related peptide-3 (RFRP-3) is known to inhibit the activity of GnRH neurons. It is not yet clear whether its G protein-coupled receptors, GPR147 and GPR74, are present on GnRH neurons or on afferent inputs of the GnRH neuronal network or whether RFRP-3 can inhibit gonadotropin secretion independently of GnRH. We tested the following: 1) whether GnRH is essential for the effects of RFRP-3 on LH secretion; 2) whether RFRP-3 neurons project to GnRH and rostral periventricular kisspeptin neurons in mice, and 3) whether Gpr147 and Gpr74 are expressed by these neurons. Intravenous treatment with the GPR147 antagonist RF9 increased plasma LH concentration in castrated male rats but was unable to do so in the presence of the GnRH antagonist cetrorelix. Dual-label immunohistochemistry revealed that approximately 26% of GnRH neurons from male and diestrous female mice were apposed by RFRP-3 fibers, and 19% of kisspeptin neurons from proestrous female mice were apposed by RFRP-3 fibers. Using immunomagnetic purification of GnRH and kisspeptin cells, single-cell nested RT-PCR, and in situ hybridization, we showed that 33% of GnRH neurons and 9-16% of rostral periventricular kisspeptin neurons expressed Gpr147, whereas Gpr74 was not expressed in either population. These data reveal that RFRP-3 can act at two levels of the GnRH neuronal network (i.e. the GnRH neurons and the rostral periventricular kisspeptin neurons) to modulate reproduction but is unable to inhibit gonadotropin secretion independently of GnRH.

Regulation of Kiss1 Expression by Sex Steroids in the Amygdala of the Rat and Mouse

Kisspeptin (encoded by the Kiss1 gene) is an important regulator of reproduction. In rodents, Kiss1 is expressed in two hypothalamic regions, the arcuate nucleus and anteroventral periventricular/ periventricular continuum, where it is regulated by sex steroids. However, the distribution, regulation, and functional significance of neural kisspeptin outside of the hypothalamus have not been studied and are poorly understood. Here, we report the expression of Kiss1 in the amygdala, predominantly in the medial nucleus of the amygdala (MeA), a region implicated in social and emotional behaviors as well as various aspects of reproduction. In gonadally intact rats and mice, Kiss1-expressing neurons were identified in the MeA of both sexes, with higher Kiss1 expression levels in adult males than females in diestrus. In rats, Kiss1 expression in the MeA changed as a function of the estrous cycle, with highest levels at proestrus. Next, we tested whether Kiss1 in the MeA is regulated by the circulating sex steroid milieu. Kiss1 levels in the MeA were low in gonadectomized mice and rats of both sexes, and treatment with either testosterone or estradiol amplified Kiss1 expression in this region. Testosterones inductive effect on Kiss1 expression in the MeA likely occurs via estrogen receptor-dependent pathways, not through the androgen receptor, because dihydrotestosterone (a nonaromatizable androgen) did not affect MeA Kiss1 levels. Thus, in rodents, Kiss1 is expressed and regulated by sex steroids in the MeA of both sexes and may play a role in modulating reproduction or brain functions that extend beyond reproduction.

Development, Sex Steroid Regulation, and Phenotypic Characterization of RFamide-Related Peptide (Rfrp) Gene Expression and RFamide Receptors in the Mouse Hypothalamus

Arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3, encoded by the Rfrp gene) is the mammalian ortholog of gonadotropin-inhibiting hormone and can inhibit GnRH neuronal activity and LH release. However, the development and regulation of the RFRP-3 system in both sexes is poorly understood. Using in situ hybridization, we examined changes in Rfrp-expressing neurons in mice of both sexes during development and under different adulthood hormonal milieus. We found no sex differences in Rfrp expression or cell number in adult mice. Interestingly, we identified two interspersed subpopulations of Rfrp cells (high Rfrp-expressing, HE; low Rfrp-expressing, LE), which have unique developmental and steroidal regulation characteristics. The number of LE cells robustly decreases during postnatal development, whereas HE cell number increases significantly before puberty. Using Bax knockout mice, we determined that the dramatic developmental decrease in LE Rfrp cells is not due primarily to BAX-dependent apoptosis. In adults, we found that estradiol and testosterone moderately repress Rfrp expression in both HE and LE cells, whereas the nonaromatizable androgen dihydrotestosterone has no effect. Using double-label in situ hybridization, we determined that approximately 25% of Rfrp neurons coexpress estrogen receptor-α in each sex, whereas Rfrp cells do not readily express androgen receptor in either sex, regardless of hormonal milieu. Lastly, when we looked at RFRP-3 receptors, we detected some coexpression of Gpr147 but no coexpression of Gpr74 in GnRH neurons of both intact and gonadectomized males and females. Thus, RFRP-3 may exert its effects on reproduction either directly, via Gpr147 in a subset of GnRH neurons, and/or indirectly, via upstream regulators of GnRH.

BAX-Dependent and BAX-Independent Regulation of Kiss1 Neuron Development in Mice

The Kiss1 gene and its product kisspeptin are important regulators of reproduction. In rodents, Kiss1 is expressed in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV)/rostral periventricular (PeN) nuclei. In the AVPV/PeN, females have more Kiss1 and tyrosine hydroxylase (TH) neurons than males. We explored the ontogeny of the Kiss1 sex difference, and the role of cell death in establishing Kiss1 and TH cell number. We also determined whether Kiss1 cells in AVPV/PeN coexpress TH. AVPV/PeN Kiss1 neurons were first detected in both sexes on postnatal d 10, but the Kiss1 sex difference did not emerge until postnatal d 12. The role of BAX-mediated apoptosis in generating this sex difference was tested in adult Bax knockout (KO) and wild-type mice. Deletion of Bax did not diminish the sex difference in Kiss1 expression in the AVPV/PeN. TH expression was sexually dimorphic in the AVPV of both wild-type and Bax KO mice but, unlike Kiss1, was not sexually dimorphic in the PeN of either genotype. Double-label analysis determined that most Kiss1 neurons coexpress TH mRNA, but many TH neurons do not coexpress Kiss1, especially in the PeN. These findings suggest that several subpopulations of TH cells reside within the AVPV/PeN, only one of which coexpresses Kiss1. In the ARC, Kiss1 cell number was markedly increased in Bax KO mice of both sexes, indicating that although BAX-dependent apoptosis does not generate the sex difference in either Kiss1 or TH expression in AVPV/PeN, BAX does importantly regulate Kiss1 cell number in the ARC.

Sexually Dimorphic Testosterone Secretion in Prenatal and Neonatal Mice Is Independent of Kisspeptin-Kiss1r and GnRH Signaling

Kisspeptin, encoded by the Kiss1 gene, stimulates GnRH secretion and is therefore critical for sex steroid secretion at puberty and in adulthood. However, kisspeptins role in regulating sex steroid secretion earlier in development is unexplored. In rodents, testosterone (T) levels are higher in prenatal and newborn males than females. We determined whether kisspeptin-Kiss1r and GnRH signaling plays a role in sexually dimorphic perinatal T secretion in mice. Our results demonstrate that 1) T levels in newborn males are elevated at 4 h but not 20 h after birth, but hypothalamic Kiss1 and neurokinin B (NKB) levels in males are not different between these time points (and both are lower than in females); 2) serum T levels in newborn Kiss1r knockout (KO) males are higher than in newborn females and similar to wild-type (WT) males; 3) perinatal hypothalamic progesterone receptor (Pgr) expression, which is dependent on circulating levels of gonadally produced T, is significantly higher in prenatal and newborn Kiss1r KO and WT males than similarly aged females; 4) multiple measures of testicular growth and function are not different between developing Kiss1r KO and WT mice until after postnatal d 5; and 5) GnRH neurons of newborn males do not exhibit high c-fos coexpression, and newborn hypogonadal (hpg) male mice (lacking GnRH) secrete elevated T, similar to newborn WT males. We conclude that, unlike in puberty and adulthood, elevated T secretion in prenatal and neonatal mice is independent of both kisspeptin and GnRH signaling, and the necessity of kisspeptin-Kiss1r signaling for testicular function is first apparent after d 5.