Gonadotropin-releasing hormone overcomes follicle-stimulating hormone's inhibition of insulin-like growth factor-5 synthesis and promotion of its degradation in rat granulosa cells

Abstract

The effect of a gonadotropin-releasing hormone-agonist (GnRH-a) on the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5), a physiological marker for atresia, was investigated. Granulosa cells obtained from diethylstilbestrol (DES)-treated immature female rats were cultured in serum-free medium for 72 h with GnRH-a and the conditioned media were subjected to immunoblot analysis using rat IGFBP-5 specific antibody. GnRH-a caused a dose-dependent (ED 50 = 8.6 × 10 −11 M) accumulation of IGFBP-5, which migrated as 35 (non-glycosylated) and 36 kDa (glycosylated) bands under reducing conditions. A maximally effective dose of GnRH-a (10 −9 M) caused a 4-fold increase in IGFBP-5 accumulation. In contrast, increasing doses of porcine follicle-stimulating hormone (pFSH) caused a biphasic effect on IGFBP-5 accumulation. A low dose of pFSH (0.25 ng/ml) increased and higher doses of pFSH (22.5 ng/ml) decreased the 35 and 36 kDa IGFBP-5 bands. In the presence of high doses of pFSH (20.75 ng/ml), a 22 kDa band corresponding to a cleaved IGFBP-5 fragment appeared in the media. When the granulosa cells were cultured with a saturating dose of pFSH, co-addition of GnRH-a dose dependently inhibited the FSH effects (ED 50 = (2.3–3.7) × 10 −10 M). The GnRH-a effects were completely blocked by co-incubation with GnRH-antagonist. IGFBP-5 mRNA accumulation levels were increased by GnRH-a in a dose dependent manner. These results using cultured rat granulosa cells demonstrate that: (1) GnRH-a coordinately stimulates the expression of IGFBP-5 protein and IGFBP-5 mRNA; (2) GnRH-a abolishes the action of FSH to inhibit IGFBP-5 production and to induce IGFBP-5 protease activity; and (3) these GnRH-a effects are blocked by GnRH-antagonist. These results support the hypothesis that GnRH may be involved in the atretic process, by increasing IGFBP-5 production directly by itself and indirectly by altering the effects of FSH.