Role of BAX-mediated apoptosis in the sexual differentiation of hypothalamic Kiss1 neurons

The neuropeptide kisspeptin, encoded by the Kiss1 gene, is a crucial regulator of GnRH neurons, and hence, puberty and reproduction. In rodents, Kiss1 neurons are found in two regions of the hypothalamus, the arcuate nucleus and the anatomical continuum comprising the anteroventral periventricular (AVPV) and anterior periventricular (PeN) nuclei. Interestingly, the Kiss1 population in the AVPV/PeN is sexually dimorphic, with females having many more Kiss1 neurons than males. This Kiss1 sex difference has been proposed to underlie the ability of females but not males to produce an estrogen-induced GnRH surge. Although we have previously shown that sex steroids during the postnatal critical period guide the sexual differentiation of AVPV/PeN Kiss1 cells, the mechanism(s) by which sex steroids achieve this effect is unknown. Previous studies found that some sexually-dimorphic traits of the AVPV, such as total cell number, are dependent on BAX-mediated programmed cell death (apoptosis), whereas other sexually-dimorphic traits, such as tyrosine hydroxylase (TH) neuron number, are not. Here we used adult male and female BAX knockout (KO) mice and their wildtype (WT) littermates to determine if sexually dimorphic AVPV Kiss1 expression is dependent on BAX signaling. Because Kiss1 expression is significantly altered by adult sex steroid levels, all mice were gonadectomized in adulthood and treated with the same dose of estradiol before sacrifice. Brains were analyzed for Kiss1 and TH mRNA levels in the AVPV/PeN via in situ hybridization. We found that WT males had fewer Kiss1 neurons in both the AVPV and PeN than WT females, confirming the previously-identified sex difference. Interestingly, BAX KO males also had Kiss1 levels that were significantly lower than BAX KO females, indicating that sexual differentiation of the Kiss1 system was not altered in the absence of BAX-mediated apoptosis. Like Kiss1, TH expression was sexually dimorphic in the AVPV of both WT and BAX KO mice, being higher in females than males. However, unlike Kiss1, TH was not significantly different between sexes in the PeN, indicating that in this region, Kiss1 and TH are either two different populations or are under different transcriptional and/or developmental control. Our data suggest that while some sexually-dimorphic traits in the AVPV are induced by BAX-mediated apoptosis, both the Kiss1 and TH sex differences in this region are organized by BAX-independent processes.