Early onset of deoxyribonucleic acid fragmentation during atresia of preovulatory ovarian follicles in rats.
Early onset of deoxyribonucleic acid fragmentation during atresia of preovulatory ovarian follicles in rats.
复制标题
大鼠排卵前卵泡闭锁期间脱氧核糖核酸断裂的早期发生。
DOI：
10.1095/biolreprod55.5.1075
复制
发表时间：
1996
影响因子：
3.6
通讯作者：
Tsafriri,A
中科院分区：
文献类型：
--
作者：
Nahum,R;Beyth,Y;Chun,SY;Hsueh,AJ;Tsafriri,A
绝大多数哺乳动物卵泡的最终结局是闭锁死亡而不是排卵，影响了各种物种中70-99.9%的卵泡。最近的研究已经确定，卵泡闭锁变性是一个凋亡过程，预示着核酸内切酶降解的DNA在核小体间的网站，这产生DNA片段的大小倍数为185-200 bp的琼脂糖凝胶电泳后，被视为不同的梯形带。利用垂体切除术诱导排卵前卵泡闭锁的模型，并通过放射自显影法分析3'端标记的DNA片段，我们研究了闭锁变化的时间。DNA降解与闭锁的形态学体征、排卵能力和卵泡类固醇生成的变化有关。在发情前期当天早晨切除大鼠垂体，之后在不同时间收集最大卵泡。在5个卵泡的组中分析DNA片段化。垂体切除后4小时内低分子量DNA片段的增加可以忽略不计（101 ± 10%; 0 h时间= 100%），但在垂体切除术后8、12、24、48和72 h进展（分别为143 ± 20%、168 ± 27%、235 ± 29%、3299 ± 1075%和2249 ± 805%;p< 0.03，n = 5）。在48和72小时，DNA降解的程度高于在无血清培养基中培养24小时的卵泡中观察到的。同样地，用4 ′，6-二氨基-2-苯基吲哚盐酸盐对DNA进行染色，在垂体切除后8小时显示凋亡细胞核（p< 0.01），并且这种细胞核的百分比随后进行性增加。因此，DNA片段化的增加与先前研究中观察到的闭锁变化（6 h时排卵率降低，垂体切除术后6 h卵泡中孕酮蓄积自发增加，雄激素和雌激素减少）同时出现，并在24 h时可通过形态学变化检测到闭锁。在闭锁级联反应早期检测排卵前卵泡核小体间DNA降解，强调了细胞凋亡在卵泡闭锁中的中心作用。
Atretic demise rather than ovulation is the ultimate fate of the vast majority of ovarian follicles in mammals, affecting 70–99.9% of the follicles in various species. Recent studies have established that atretic degeneration of follicles is an apoptotic process, heralded by endonuclease degradation of DNA at internucleosomal sites, which generates DNA fragments in size multiples of 185–200 bp that are seen as distinct ladder bands after agarose gel electrophoresis. Using the well-characterized model of inducing atresia of preovulatory follicles in vivo by hypophysectomy and analyzing DNA fragmentation by autoradiography of size-fractionated DNA labeled at the 3' ends by [32p] dideoxy-ATP, we have examined the timing of atretic changes. DNA degradation was related to morphological signs of atresia, ovulability, and changes in follicular 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 follicles. The increase in DNA fragments of low molecular weight up to 4 h after hypophysectomy was negligible (101 ± 10%; 0 h time = 100%) but progressed 8, 12, 24, 48, and 72 h after hypophysectomy (143 ± 20%, 168 ± 27%, 235 ± 29%, 3299 ± 1075%, and 2249 ± 805%, respectively;p< 0.03, n = 5). At 48 and 72 h, the extent of DNA degradation 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-phenylindole hydrochloride revealed apoptotic nuclei at 8 h after hypophysectomy (p< 0.01), and the percentage of such nuclei progressively increased afterwards. Thus, the increase in DNA fragmentation appeared concomitantly with atretic changes observed in previous studies (a decrease in ovulability at 6 h, and a spontaneous increase in progesterone accumulation and decrease in androgen and estrogen in follicles explanted 6 h after hypophysectomy) and preceded atresia detectable by morphological changes at 24 h. Detection of internucleosomal DNA degradation in preovulatory follicles early in the atretic cascade underscores the central role of apoptosis in ovarian follicle atresia.