Extrapituitary actions of gonadotropin-releasing hormone: stimulation of insulin-like growth factor-binding protein-4 and atresia.

To understand how the intrinsic GnRH system functions in the ovary, we tested the effects of GnRH agonist (GnRH-a) on insulin-like growth factor-binding protein-4 (IGFBP-4) production, a novel marker of atresia. We also tested the ability of GnRH-a to stimulate atresia in vivo. When rat granulosa cells were cultured in defined medium for 2 days (controls), relatively large amounts of the 24,000 relative molecular mass IGFBP-4 accumulated in the medium. FSH (100 ng/ml) inhibited control IGFBP-4 protein levels and stimulated IGFBP-4 protease activity. GnRH-a increased (up to 4-fold) IGFBP-4 accumulation in the medium (ED50 = 1 x 10(-10) M), and the effect was blocked by a GnRH antagonist. Neither GnRH-a nor its antagonist had a detectable effect on protease activity. In coincubation experiments, GnRH-a effectively inhibited (ED50 = 3 x 10(-11) M) the FSH responses, and the effect of GnRH-a was blocked by GnRH antagonist. A 6-day time-course experiment showed that IGFBP-4 accumulation in control cultures remained constant for 2 and 4 days, after which it was undetectable. FSH (100 ng/ml) produced no measurable IGFBP-4 over the 6-day time course. The levels of IGFBP-4 increased markedly during the first 2 days of GnRH-a treatment, but were not significantly different from control levels on days 4 and 6. Similar results were obtained when cells were treated with FSH plus GnRH-a. Treating immature hypophysectomized estrogen-primed rats with GnRH-a in vivo caused a rapid and dramatic decrease (average, 60%) in the mitotic index of the granulosa cells of all preantral follicles (healthy and atretic) and increased pyknosis. These results demonstrate that 1) GnRH-a stimulates the expression of IGFBP-4 protein in rat granulosa cells in vitro; 2) GnRH-a abolishes the ability of FSH to inhibit IGFBP-4 expression and induce IGFBP-4 protease activity; and 3) GnRH-a stimulates atresia in preantral follicles in vivo. These results support the hypothesis that autocrine/paracrine secretion of ovarian GnRH might cause atresia by mechanisms involving increased IGFBP-4 synthesis.