Effects of follicle-stimulating hormone on endothelin receptors in cultured rat ovarian granulosa cells

Two subtypes of the endothelin (ET) receptor (ETA and ETB) were studied in cultured ovarian granulosa cells. Immature 21-day-old female Wistar-Imamichi rats were implanted with diethylstilbestrol (DES) pellets for 5 days and granulosa cells were collected by repeated puncturing. Viable cells (2.5 or 5 x 10(5)) were cultured with 50-400 ng/ml of ovine NIH follicle-stimulating hormone (FSH) in the presence or absence of [I-125-Tyr(13)]ET-1 (50 pM) in 1 ml McCoy's 5a medium for 72 h. FSH gradually increased the [I-125-Tyr(13)]ET-1 binding to ganulosa cells, whereas FSH-untreated granulosa cells had no significant changes. The dose of 200 ng/ml of FSH was most effective for [I-125-Tyr(13)]ET-1 binding for 48-h culture, thereafter revealing a plateau. After 48 h of culture with 200 ng/ml of FSH, granulosa cells were further incubated with [I-125-Tyr(13)]ET-1 (10 pM-1 nM) and/or [I-125]IRL1620, the selective ETB receptor agonist (10 pM-1 nM) for 2 h for equilibrium study, and then the dissociation constant and the maximal binding capacity between receptors and ligands were determined by saturation curve and Scatchard plot analysis. ETA + ETB, ETB, and ETA (sites/cells) showed a 4.4-, 2.6-, and 7.5-fold increase, respectively. As for steroidogenesis, ET-1 (100 nM) or ET-3 (100 nM) suppressed FSH-induced progesterone and 17beta-estradiol production. These results indicate that FSH upregulates both ETA and ETB receptors in DES-treated immature rat granulosa cells, with no significant differences between ET-I and ET-3, and that ET-1 or ET-3 suppresses FSH-induced steroidogenesis. ETs may affect the granulosa cell function through the ETA and ETB receptors, and the increase in amount of ET binding does not reflect ET effects on granulosa cell function. The ET receptor plays an important role in the development of the ovary.