GnRH receptors in cultured rat granulosa cells: mediation of the inhibitory and stimulatory actions of GnRH.

The regulatory actions of FSH and the GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa) upon ovarian GnRH receptors were studied in granulosa cells obtained from ovaries of hypophysectomized diethylstilbestrol-treated rats. When granulosa cells were cultured for 48 h in the presence of FSH (5-250 ng/ml) the binding of 125I-GnRHa to granulosa cell receptors was increased in a dose-dependent manner, to a maximum of 3-4 fold above the control value. Addition of FSH (100 ng) also caused a dose-dependent increase of more than 100-fold in the accumulation of cAMP and progesterone in the culture medium. In freshly prepared cells, Scatchard analysis of GnRHa binding data revealed an equilibrium constant (Ka) of 1.1 x 10(10) M-1 and GnRH receptor concentration fo 401 fmoles/mg protein. Granulosa-cell GnRH receptors decreased during culture without FSH, but were maintained in the presence of 100 ng FSH at 580 femoles/mg protein, with Ka of 0.8 x 10(10) M-1. This action of FSH on GnRH receptors was significantly reduced by 10(-8) M GnRHa. Also, GnRHa concentrations of 10(-10) and 10(-8) M caused inhibition of FSH-induced cAMP and progesterone accumulation. In cells cultured with GnRHa alone, there was a slight enhancement of GnRH receptors by GnRHa concentrations up to 10(-8) M, and a decrease below control levels with higher amounts. Also, GnRHa concentrations from 10(-8) to 10(-5) M caused a 3-4-fold increase in cAMP accumulation in the absence of FSH. These results demonstrate that FSH maintains GnRH receptors in cultured granulosa cells, and that GnRHa attenuates this effect, as well as the other actions of FSH on granulosa cell maturation. It is also evident that GnRHa itself can slightly stimulate cAMP production and partially maintain GnRH receptors, but at high concentrations causes loss of the homologous receptor sites. These findings also emphasize the ability of GnRH agonists to exert both positive and negative direct effects on rat granulosa cell function.