Rosales-Torres A M, Avalos-Rodríguez A, Vergara-Onofre M, Hernández-Pérez O, Ballesteros L M, García-Macedo R, Ortíz-Navarrete V, Rosado A
Universidad Autónoma Metropolitana-Xochimilco, Depto de Producción Agrícola y Animal, Deleg Coyoacán, Mexico.
Mol Reprod Dev. 2000 Mar;55(3):270-81. doi: 10.1002/(SICI)1098-2795(200003)55:3<270::AID-MRD5>3.0.CO;2-H.
The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.
采用宏观、组织学和类固醇定量方法，评估了处于闭锁四个阶段的两种不同大小母羊窦状卵泡中凋亡和坏死的差异定量参与情况。使用膜联蛋白V结合和碘化丙啶（PI）摄取来检测健康活细胞（膜联蛋白V阴性/PI阴性）、早期凋亡细胞（膜联蛋白V+/PI-）以及坏死或晚期凋亡细胞（PI+）。此外，我们使用核小体间DNA片段化作为凋亡的定量估计。卵泡液和颗粒细胞中溶酶体酶的存在和分布用作坏死性细胞死亡的指标。DNA流式细胞术和凝胶电泳与闭锁进程呈正相关，小的闭锁卵泡比大于6mm的卵泡具有更高百分比的核小体间切割DNA。膜联蛋白/PI结合也表明，随着闭锁进程凋亡和坏死增加，在小卵泡中凋亡通常超过坏死。3 - 6mm卵泡的卵泡液中的酸性磷酸酶和氨基葡萄糖苷酶在健康卵泡和初始闭锁卵泡之间无显著变化，仅在晚期闭锁卵泡中活性有小幅但显著增加。相反，大于6mm卵泡中的溶酶体酶活性与闭锁阶段以及卵泡液中酸性磷酸酶和氨基葡萄糖苷酶的活性呈正相关。在颗粒细胞的游离或膜结合溶酶体酶活性中发现了类似的大小差异行为。在卵泡成熟的所有阶段均存在坏死，但主要是凋亡，这表明卵巢卵泡的生长和成熟涉及颗粒细胞的持续更新，由凋亡调节。调节这种平衡的机制可能参与卵巢卵泡最终的命运，即排卵或闭锁。
