Sexual differentiation of galanin gene expression in gonadotropin-releasing hormone neurons

Abstract

In adult rats, only females are capable of generating an LH surge in response to high levels of ovarian steroids. This is because the ability to generate LH surges in adulthood in response to ovarian steroids is suppressed by high levels of testosterone during neonatal life. We have previously shown that galanin gene expression in GnRH neurons is induced by ovarian steroids in association with an LH surge, suggesting that galanin plays a role in the generation of the LH surge. If this is the case, then the induction of galanin gene expression in GnRH neurons in response to ovarian steroids might, like the LH surge itself, be sexually differentiated during the neonatal period. To test this hypothesis, we manipulated gonadal steroid levels in four groups of rat pups on the first day of life and looked at the ability of ovarian steroids to induce an LH surge and stimulate galanin gene expression in GnRH neurons of adult animals. The four groups consisted of female pups that were injected with either 10 micrograms of testosterone propionate (n = 6; testosterone-treated females) or vehicle (n = 6; control females) and male pups that were either castrated (n = 6; castrated males) or sham-castrated (n = 6; sham-castrated males). All animals were then gonadectomized at 59 or 60 days of age, except for the males that had been castrated neonatally. Seven weeks later, the animals were challenged with estradiol (E2) benzoate and progesterone (P) in a paradigm that reliably produces an LH surge. (The rats were injected sc with 15-30 micrograms of E2 on day 0 at 1030 h and 5 mg of P on day 2 at 1200 h.) The animals were then killed between 1800 h and 1840 h on day 2 at the time of the expected LH surge, and their brains were processed for double-label in situ hybridization to estimate cellular levels of galanin messenger RNA (mRNA) in GnRH neurons. We reconfirmed the classical observation that the ability to generate an LH surge in response to E2/P is suppressed by neonatal exposure to testosterone. Galanin mRNA levels in GnRH neurons showed a similar pattern: after E2/P priming, galanin mRNA levels in GnRH neurons were significantly higher in normal females and in males castrated neonatally than in normal males and in females treated with testosterone neonatally. These results demonstrate that the E2/P induction of galanin gene expression in GnRH neurons at the time of the LH surge is sexually differentiated during the neonatal period and suggest that the increase in galanin gene expression in GnRH neurons could be one of the mechanisms underlying the sex difference in gonadotropin release seen in rats.