TESTOSTERONE ACTS DIRECTLY AT THE PITUITARY TO REGULATE GONADOTROPIN-RELEASING HORMONE-INDUCED CALCIUM SIGNALS IN MALE-RAT GONADOTROPES

We have recently shown that castration alters GnRH-induced calcium (Ca 2+) signaling in the gonadotropes of male rats. Instead of generating spike-plateau Ca2+ responses to high concentrations of GnRH (100 nM), the majority of gonadotropes from castrated rats have oscillatory Ca2 responses, which are generally only seen with low concentrations of G nRH in the gonadotropes of intact rats. This change in the nature of G nRH-induced Ca2+ responses is prevented by in vivo testosterone treatm ent. The aims of the present study were, therefore, to determine if te stosterone acts directly at the pituitary or via the regulation of hyp othalamic GnRH secretion. Accordingly, castrated male rats were treate d with a GnRH antagonist to ablate the effects of increased GnRH secre tion at the pituitary gland. GnRH antagonist treatment (10 mu g/100 g BW, twice daily for 7 days from the time of castration) decreased the concentration of LH in the serum of castrated rats (0.4 +/- 0.1 ng/ml cs. 11.2 +/- 0.4 ng/ml in untreated castrated rats, mean +/- SEM) but had no effect on the proportion of gonadotropes having oscillatory Ca2 + responses to 100 nM GnRH when compared with untreated castrated rats (63% in antagonist-treated castrated rats vs. 70% in untreated castra ted rats). The GnRH antagonist treatment did not, however, interfere w ith the ability of in vivo testosterone treatment (100 mu g/100 g body weight/day) to decrease the proportion of gonadotropes having oscilla tory Ca2+ responses to 100 nM GnRH (26% in testosterone-treated rats v s. 25% in testosterone and antagonist-treated rats). These findings in dicate that testosterone acts directly at the pituitary, and not by al tered GnRH secretion, to modulate GnRH-induced Ca2+ signals. To confir m this suggestion, cultured gonadotropes of castrated male rats were t reated in vitro with 10 nM testosterone. Testosterone treatment for tw elve, but not 4 h, restored the proportion of gonadotropes having osci llatory Ca2+ responses to that seen in gonadotropes from intact rats. The in vitro affects of testosterone over 12 h were prevented by conco mitant treatment with the protein synthesis inhibitor cycloheximide (1 0 mu M), which, when given alone, had no effect on GnRH-induced Ca2+ s ignals in cells from castrate male rats. Taken together, these finding s suggest that testosterone has a direct genomic action at the pituita ry to regulate GnRH-induced Ca2+ signals, via a process that involves new protein synthesis.