EARLY-ONSET OF DEOXYRIBONUCLEIC-ACID FRAGMENTATION DURING ATRESIA OF PREOVULATORY OVARIAN FOLLICLES IN RATS

Atretic demise rather than ovulation is the ultimate fate of the vast majority of ovarian follicles in mammals, affecting 70-99.9% of the fo llicles in various species. Recent studies have established that atret ic degeneration of follicles is an apoptotic process, heralded by endo nuclease degradation of DNA at internucleosomal sites, which generates DNA fragments in size multiples of 185-200 bp that are seen as distin ct ladder bands after agarose gel electrophoresis. Using the well-char acterized model of inducing atresia of preovulatory follicles in vivo by hypophysectomy and analyzing DNA fragmentation by autoradiography o f size-fractionated DNA labeled at the 3' ends by [P-32] dideoxy-ATP, we have examined the timing of atretic changes. DNA degradation was re lated to morphological signs of atresia, ovulability, and changes in f ollicular steroidogenesis. Rats were hypophysectomized on the morning of the day of proestrus, after which largest follicles were collected at various times. DNA fragmentation was analyzed in groups of five fol licles. The increase in DNA fragments of low molecular weight up to 4 h after hypophysectomy was negligible (101 +/- 10%; 0 h time = 100%) b ut progressed 8, 12, 24, 48, and 72 h after hypophysectomy (143 +/- 20 %, 168 +/- 27%, 235 +/- 29%, 3299 +/- 1075%, and 2249 +/- 805%, respec tively; p < 0.03, n = 5). At 48 and 72 h, the extent of DNA degradatio n was higher than that observed in follicles cultured in a serum-free medium for 24 h. Likewise, staining of DNA by 4',6-diamido-2-phenylind ole hydrochloride revealed apoptotic nuclei at 8 h after hypophysectom y (p < 0.01), and the percentage of such nuclei progressively increase d afterwards. Thus, the increase in DNA fragmentation appeared concomi tantly with atretic changes observed in previous studies (a decrease i n ovulability at 6 h, and a spontaneous increase in progesterone accum ulation and decrease in androgen and estrogen in follicles explanted 6 h after hypophysectomy) and preceded atresia detectable by morphologi cal changes at 24 h. Detection of internucleosomal DNA degradation in preovulatory follicles early in the atretic cascade underscores the ce ntral role of apoptosis in ovarian follicle atresia.