Guthrie H D, Grimes R W, Cooper B S, Hammond J M
Germplasm and Gamete Physiology Laboratory, ARS, USDA, Beltsville, MD 20705, USA.
J Anim Sci. 1995 Sep;73(9):2834-44. doi: 10.2527/1995.7392834x.
The physiological regulation of follicular atresia was investigated during the early luteal phase after ovulation and during altrenogest-synchronized preovulatory maturation in pigs (gilts). Apoptosis in dispersed granulosa cells was determined by flow cytometry. Apoptotic (A0) cells contain low, subdiploid amounts of DNA fluorescence. Follicles were classified biochemically as atretic or nonatretic based on the percentage of A0 (% A0) cells, atretic with > or = 10%, and nonatretic with < 10% A0 granulosa cells. The % A0 granulosa cells/follicle ranged from .02 to 89. Follicles containing debris in their isolated granulosa cells were classified as morphologically atretic. The morphological and biochemical criteria of atresia were in agreement for 224 of 248 follicles. Internucleosomal DNA cleavage, the hallmark of apoptosis, was determined by autoradiographic analysis of [32P]3'-end labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [32P]3'-end labeled DNA fragments in the .18 to 20 kbp size range was correlated with the % A0 cells (R > .9, n = 22, P < .001). During altrenogest-synchronized preovulatory maturation, < 5% of large (> 6 mm in diameter) follicles were atretic. Among medium-sized follicles (3 to 6 mm) on d 1 and 3 of preovulatory maturation, only 17% were atretic, in contrast with d 5 when 87% were atretic. During the early luteal phase, atretic follicles/pig increased from 6% on d 5 to 50% on d 7 after estrus. Follicular fluid estradiol-17 beta concentration was greater (P < .001) in nonatretic than in atretic follicles on d 5 and 6 after estrus, but by d 7 estradiol-17 beta had decreased to a mean < 1 ng/mL in nonatretic and atretic follicles. The increase in apoptosis in granulosa cells and loss of estradiol-17 beta production in vivo indicated a high incidence of atresia among the first group of follicles grown after ovulation in pigs. These results indicate that apoptotic cell death was involved in degeneration of granulosa cells and atresia during two different stages of follicular development.
在猪（后备母猪）排卵后的黄体早期以及烯丙孕素同步化的排卵前成熟过程中，对卵泡闭锁的生理调节进行了研究。通过流式细胞术测定分散的颗粒细胞中的凋亡情况。凋亡（A0）细胞含有低含量的亚二倍体DNA荧光。根据A0（% A0）细胞的百分比，将卵泡生化分类为闭锁或未闭锁，A0颗粒细胞≥10%为闭锁卵泡，<10%为未闭锁卵泡。每个卵泡中A0颗粒细胞的%范围为0.02至89。分离的颗粒细胞中含有碎片的卵泡被分类为形态学上闭锁的卵泡。在248个卵泡中，有224个卵泡的闭锁形态学和生化标准一致。通过对颗粒细胞[32P]3'-末端标记DNA的放射自显影分析来确定凋亡的标志——核小体间DNA裂解。密度分析表明，大小在0.18至20 kbp范围内的[32P]3'-末端标记DNA片段的光密度与A0细胞的%相关（R>.9，n = 22，P<.001）。在烯丙孕素同步化的排卵前成熟过程中，直径大于6 mm的大卵泡中<5%为闭锁卵泡。在排卵前成熟第1天和第3天的中等大小卵泡（3至6 mm）中，只有17%为闭锁卵泡，而在第5天，这一比例为87%。在黄体早期，每头猪的闭锁卵泡从发情后第5天的6%增加到第7天的50%。发情后第5天和第6天，未闭锁卵泡中的卵泡液雌二醇-17β浓度高于闭锁卵泡（P<.001），但到第7天，未闭锁和闭锁卵泡中的雌二醇-17β平均浓度均降至<1 ng/mL。颗粒细胞凋亡增加以及体内雌二醇-17β分泌减少表明，猪排卵后生长的第一组卵泡中闭锁发生率很高。这些结果表明，凋亡性细胞死亡参与了卵泡发育两个不同阶段中颗粒细胞的退化和闭锁过程。
