Modulation of gonadotropin-releasing hormone-stimulated luteinizing hormone release in cultured male equine anterior pituitary cells by gonadal steroids

The objective of the present study was to determine whether the testicular steroids, i.e., testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and estrone sulfate (E1SO4), play a physiological role in regulating LH release in the male horse by direct actions at the anterior pituitary gland. Enzymatically dispersed anterior pituitary cells from stallions (n = 4) or geldings (n 3) were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse medium. To determine the effects of the steroids on the LH response to GnRH, the cells were incubated for 24 h in fresh media with or without 10(-10) M E2 or 10(-8) MT or DHT followed by a 4-h incubation +/- GnRH (10(-11) to 10(-7) M). Media and cells were analyzed for LH by RIA. In the stallion, GnRH increased LH release (p < 0.001) in a dose-dependent manner (ED50 GnRH = 4.5 X 10(-9) M), and this response was unaltered by T or DHT but greatly enhanced by E2 (p < 0.001). E2 lowered the ED50 for GnRH to 5 X 10(-10) M and increased the maximum LH response to GnRH by 350%. The LH release in response to a constant dose of 1 nM GnRH was unaltered by varying doses of T, DHT, or E1SO4 (10(-11) to 10(-7) M). In contrast, E2 increased GnRH-stimulated LH release maximally at 10(-10) M with an ED50 of 2.9 X 10(-11) M, whereas E1 was less effective with an EDmax of 10-8 M and an ED50 of 3 X 10(-9) M, respectively. In the gelding, the responsiveness to GnRH (i.e., ED50 = 2.8 +/- 0.8 X 10(-10) M) was enhanced (p < 0.01) compared to that in the stallion; however, none of the steroids, i.e., T, DHT, E2, or E1SO4, altered GnRH-stimulated LH release. In conclusion, in the male horse, the negative feedback of the testicular androgens on LH release appears to be due to an action on the hypothalamus by inhibition of GnRH secretion rather than a direct effect at the pituitary. Furthermore, it appears that E2 may be important for maintaining pituitary responsiveness to GnRH.