Developmental GnRH signaling is not required for sexual differentiation of kisspeptin neurons but is needed for maximal Kiss1 gene expression in adult females.

Kisspeptin, encoded by Kiss1, stimulates reproduction. In rodents, one Kiss1 population resides in the hypothalamic anterior ventral periventricular nucleus and neighboring rostral periventricular nucleus (AVPV/PeN). AVPV/PeN Kiss1 neurons are sexually dimorphic (greater in females), yet the mechanisms regulating their development and sexual differentiation remain poorly understood. Neonatal estradiol (E2) normally defeminizes AVPV/PeN kisspeptin neurons, but emerging evidence suggests that developmental E2 may also influence feminization of kisspeptin, although exactly when in development this process occurs is unknown. In addition, the obligatory role of GnRH signaling in governing sexual differentiation of Kiss1 or other sexually dimorphic traits remains untested. Here, we assessed whether AVPV/PeN Kiss1 expression is permanently impaired in adult hpg (no GnRH or E2) or C57BL6 mice under different E2 removal or replacement paradigms. We determined that 1) despite lacking GnRH signaling in development, marked sexual differentiation of Kiss1 still occurs in hpg mice; 2) adult hpg females, who lack lifetime GnRH and E2 exposure, have reduced AVPV/PeN Kiss1 expression compared to wild-type females, even after chronic adulthood E2 treatment; 3) E2 exposure to hpg females during the pubertal period does not rescue their submaximal adult Kiss1 levels; and 4) in C57BL6 females, removal of ovarian E2 before the pubertal or juvenile periods does not impair feminization and maximal adult AVPV/PeN Kiss1 expression nor the ability to generate LH surges, indicating that puberty is not a critical period for Kiss1 development. Thus, sexual differentiation still occurs without GnRH, but GnRH or downstream E2 signaling is needed sometime before juvenile development for complete feminization and maximal Kiss1 expression in adult females.

Assessment of epigenetic contributions to sexually-dimorphic Kiss1 expression in the anteroventral periventricular nucleus of mice.

The Kiss1 gene, which encodes kisspeptin and is critical for reproduction, is sexually differentiated in the hypothalamic anteroventral periventricular (AVPV)/rostral periventricular (PeN) nuclei. Specifically, female rodents have higher AVPV/PeN Kiss1 expression than males, but how this Kiss1 sex difference is induced in early development is poorly understood. Here, we explored the contribution of epigenetic mechanisms to the establishment of the AVPV/PeN Kiss1 sex difference, focusing on histone deacetylation and DNA methylation. First, we utilized postnatal pharmacological blockade of histone deacetylation and analyzed Kiss1 expression in the AVPV/PeN. Postnatal disruption of histone deacetylase modestly increased AVPV Kiss1 cell number in both sexes but did not alter the Kiss1 sex difference. Next, we assessed whether the level of CpG methylation, which can influence transcription factor binding and gene expression, in the murine Kiss1 gene differs between males and females. We found significant sex differences in methylation at several CpG sites in the putative promoter and first intron of the Kiss1 gene in the AVPV/PeN, but not in the arcuate (which lacks adult Kiss1 sex differences), suggesting that differential methylation of the Kiss1 gene may influence sexually-dimorphic Kiss1 expression in the AVPV/PeN. Transgenic impairment of methyl CpG-binding protein-2 function did not eliminate the Kiss1 sex difference, indicating that other methylation factors are involved. Interestingly, CpG methylation in the AVPV/PeN was lower in males than females, suggesting that transcriptional repressors may contribute to the AVPV/PeN Kiss1 sex difference, a possibility supported by in silico identification of putative repressor binding sites near some of the sexually-dimorphic CpG.

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.

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. Testosterone's 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.

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.