Direct effects of free and conjugated steroids on GnRH stimulated LH release in cultured equine anterior pituitary cells.

Enzymatically dispersed anterior pituitary cells from donor mares were cultured for 48 h in alpha-modified Eagles' medium containing 10% steroid-free horse serum. The cells were then incubated for 24 h in fresh medium oestrogen followed by a 4-h incubation with or without GnRH. Media and cell extracts were analyzed for LH by radioimmunoassay. In the first series of experiments, pituitary cells from Day-3 dioestrous mares were preincubated with ethanol (control) or different concentrations of E2 (10(-11) to 10(-7) M) for 24 h prior to a 4-h incubation without (basal) or with 1.0 nM GnRH. E2 increased (P less than 0.001) GnRH-stimulated LH release with a maximal response obtained at 10(-10) M E2 with an ED50 of 3 x 10(-11) M E2. Also E2 enhanced greatly the responsiveness of the pituitary to varying concentrations of GnRH by decreasing the minimum effective dose and ED50 by 93% and 78%, respectively, and by increasing the maximum response to GnRH by 250%. E2 had no effect on basal LH secretion. In a second experiment, the effects of E2, E1 and E1S on basal and GnRH stimulated LH release were evaluated. Although E1 increased GnRH-stimulated LH release, the maximally effective dose of E1 to enhance LH release and the ED50 for E1 were 100 times greater than those found for E2. E1S had no oestrogenic activity even at concentrations as high as 100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)