Apoptosis in bovine granulosa cells in relation to steroid synthesis, cyclic adenosine 3',55'-monophosphate response to follicle-stimulating hormone and luteinizing hormone, and follicular atresia

Apoptosis is a process of selective cell deletion implicated as a mechanism underlying the process of ovarian follicular atresia The aims of this study were 1) to test the hypothesis that granulosa cell death during follicular (greater than or equal to 4-mm diameter) atresia in cows occurs by apoptosis and 2) to define relationships between the occurrence and degree of granulosa cell apoptosis, cAMP response to FSH or LH, extant aromatase activity, and other previously established biochemical and morphometric indices of granulosa cell function and follicular atresia in this species. Granulosa cells and follicular fluid from individual follicles 4-18 mm in diameter were collected from luteal-phase cow ovaries. Follicles were classified by morphometric criteria as "healthy" (n = 45) or atretic (n = 34). Apoptosis in granulosa cells from each follicle was inferred from detection of internucleosomal DNA cleavage by 3'-end radiolabeling; it was quantitated both subjectively from intensity of oligonucleosome labeling (apoptosis [AP] score = 0, 1, 2, or 3) and objectively by beta-counting of low-molecular-weight gel fragments (labeling index; LI). Extant aromatase activity (ng estradiol produced/10(6) cells/3 h) and cAMP response (pmol/10(6) cells) to different doses of FSH or LH (1-10000 ng/ml) was determined for granulosa cells from most healthy follicles (n = 39). Apoptosis was detected in granulosa cells from all atretic follicles as well as from 76% of healthy follicles, from 80% (16 of 20) of follicles with follicular fluid estradiol to progesterone ratios > 1, and from 71% (10 of 14) of follicles with extant aromatase activity (> 2 ng/10(6) cells/3 h). For healthy and atretic follicles, degree of DNA fragmentation was inversely related to the number of granulosa cells recovered (as percentage maximum by follicle size). In healthy follicles, FSH stimulated cAMP synthesis in a dose-dependent manner in granulosa cells from all follicles examined (greater than or equal to 4 mm), but only 36% of these had appreciable aromatase activity. The cAMP response to FSH (per cell) increased with follicle size from 4-7 mm in diameter and remained high in granulosa cells from follicles greater than or equal to 8 mm with aromatase activity; in cells without aromatase activity, cAMP response to FSH decreased with increasing follicle size greater than or equal to 8 mm. The cAMP response to LH was generally low or undetectable in granulosa cells from 4-8-mm follicles; it then increased linearly with increasing follicle diameter greater than or equal to 8 mm, but to a greater degree in cells with aromatase activity than in cells without. Despite the high incidence of apoptosis in healthy follicles, LI was not related to aromatase activity, cAMP response to FSH or LH, or follicle size and did not influence relationships between these variables. However, both AP score and LI were negatively related to follicular fluid estradiol and positively related to follicular fluid progesterone levels. In conclusion, these results are consistent with the hypothesis that death of granulosa cells during follicular atresia in cows occurs by apoptosis. However, the results also suggest that granulosa cell apoptosis may occur in healthy follicles during the luteal phase of the estrous cycle and/or very early in the process of atresia, before other morphological or biochemical changes are detected.