GONADOTROPIN-RELEASING-HORMONE OVERCOMES FOLLICLE-STIMULATING HORMONES INHIBITION OF INSULIN-LIKE GROWTH FACTOR-5 SYNTHESIS AND PROMOTION OF ITS DEGRADATION IN RAT GRANULOSA-CELLS

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 we re cultured in serum-free medium for 72 h with GnRH-a and the conditio ned media were subjected to immunoblot analysis using rat IGFBP-5 spec ific antibody. GnRH-a caused a dose-dependent (ED(50) = 8.6 x 10(-11) M) accumulation of IGFBP-5, which migrated as 35 (non-glycosylated) an d 36 kDa (glycosylated) bands under reducing conditions. A maximally e ffective dose of GnRH-a (10(-9) M) caused a 4-fold increase in IGFBP-5 accumulation. In contrast, increasing doses of porcine follicle-stimu lating 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 ce lls 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) x 10( -10) M). The GnRH-a effects were completely blocked by co-incubation w ith GnRH-antagonist, IGFBP-5 mRNA accumulation levels were increased b y GnRH-a in a dose dependent manner. These results using cultured rat granulosa cells demonstrate that: (1) GnRH-a coordinately stimulates t he expression of IGFBP-5 protein and IGFBP-5 mRNA; (2) GnRH-a abolishe s 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 invo lved in the atretic process, by increasing IGFBP-5 production directly by itself and indirectly by altering the effects of FSH.