GONADOTROPIN-RELEASING-HORMONE DIRECTLY INDUCES APOPTOTIC CELL-DEATH IN THE RAT OVARY - BIOCHEMICAL AND IN-SITU DETECTION OF DEOXYRIBONUCLEIC-ACID FRAGMENTATION IN GRANULOSA-CELLS

The majority of ovarian follicles undergo atresia through a mechanism involving apoptotic cell death. Although GnRH and its agonists have be en shown to suppress ovarian growth and differentiation in hypophysect omized rats, studies on the induction of follicle atresia by GnRH are contradictory. In the present study, the direct effect of GnRH on the occurrence of apoptosis in the ovary was investigated in hypophysectom ized estrogen-treated immature rats. Starting 2 days after operation a nd estrogen capsule implantation, rats were treated with a GnRH agonis t (GnRHa; [desGly(10)D-Phe(6),Pro(9)-N-ethylamide] GnRH; 50 mu/injecti on, twice daily). Total ovarian DNA was isolated 48 h later, labeled a t the 3'-ends with [P-32]dideoxy ATP, and size-fractionated. Compared to that in control animals, treatment with GnRHa increased DNA fragmen tation in multiples of 180 basepairs, a hallmark of apoptosis, demonst rating that GnRH directly increases ovarian apoptotic cell demise. In contrast, FSH treatment (10 mu g/ injection, twice daily) decreased ap optotic DNA fragmentation, and the antiapoptotic effect of FSH was par tially blocked by concomitant treatment with GnRHa, The apoptosis-indu cing effect of GnRHa was time and dose dependent, with a significant i ncrease seen after 24 h of treatment and a maximal 5.5-fold increase w ith 10 mu g GnRHa/injection. Similar to studies using DNA isolated fro m whole ovaries, DNA obtained from isolated granulosa cells also showe d a time- and dose-dependent increase in DNA fragmentation after GnRHa treatment. The effect on DNA fragmentation by GnRHa was mediated by o varian GnRH receptors, because a potent GnRH receptor blocker, Azaline B, prevented GnRHa action. In addition, in situ end labeling of DNA u sing digoxigenin-dideoxy-UTP showed that DNA fragmentation was confine d to the granulosa cells of preantral and antral follicles. No GnRHa-i nduced apoptosis was detected in granulosa cells of primary follicles or in thecal and interstitial cells. These data suggest that GnRH dire ctly increases apoptotic cell death in the ovary, and the GnRH action is confined to the granulosa cells. These data provide a basis for fut ure studies on the mechanism of follicular atresia and the regulation of ovarian endonuclease by GnRH.