Follicular atresia in pigs: measurement and physiology

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.