APOPTOSIS IN BOVINE GRANULOSA-CELLS IN RELATION TO STEROID-SYNTHESIS,CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE RESPONSE TO FOLLICLE-STIMULATING-HORMONE AND LUTEINIZING-HORMONE, AND FOLLICULAR ATRESIA

Apoptosis is a process of selective cell deletion implicated as a mech anism underlying the process of ovarian follicular atresia The aims of this study were 1) to test the hypothesis that granulosa cell death d uring follicular (greater than or equal to 4.mm diameter) atresia in c ows occurs by apoptosis and 2) to define relationships between the occ urrence and degree of granulosa cell apoptosis, cAMP response to FSH o r LH, extant aromatase activity, and other previously established bioc hemical and morphometric indices of granulosa cell function and follic ular atresia in this species. Granulosa cells and follicular fluid fro m 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 cell s from each follicle was inferred from detection of internucleosomal D NA cleavage by 3'-end radiolabeling; it was quantitated both subjectiv ely from intensity of oligonucleosome labeling (apoptosis [AP] score = 0, 1, 2, or 3) and objectively by beta-counting of low molecular-weig ht gel fragments (labeling index; LI). Extant aromatase activity (ng e stradiol produced/10(6) cells/3 h) and cAMP response (pmol/10(6) cells ) to different doses of FSH or LH (1-10 000 ng/ml) was determined for granulosa cells from most healthy follicles (n = 39). Apoptosis was de tected in granulosa cells from all atretic follicles as well as from 7 6% of healthy follicles, from 80% (16 of 20) of follicles with follicu lar 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 inv ersely related to the number of granulosa cells recovered (as percenta ge maximum by follicle size). In healthy follicles, FSH stimulated cAM P synthesis in a dose-dependent manner in granulosa cells from all fol licles examined (greater than or equal to 4 mm), but only 36% of these had appreciable aromatase activity. The cAMP response to FSH (per cel l) increased with follicle size from 4-7 mm in diameter and remained h igh in granulosa cells from follicles greater than or equal to 8 mm wi th aromatase activity; in cells without aromatase activity, cAMP respo nse to FSH decreased with increasing follicle size greater than or equ al 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 w ith increasing follicle diameter greater than or equal to 8 mm, but to a greater degree in cells with aromatase activity than in cells witho ut Despite the high incidence of apoptosis in healthy follicles, LI wa s not related to aromatase activity, cAMP response to FSH or LH, or fo llicle size and did not influence relationships between these variable s. However, both AP score and LI were negatively related to follicular fluid estradiol and positively related to follicular fluid progestero ne levels. In conclusion, these results are consistent with the hypoth esis that death of granulosa cells during follicular atresia in cows o ccurs by apoptosis. However, the results also suggest that granulosa c ell apoptosis may occur in healthy follicles during the luteal phase o f the estrous cycle and/or very early in the process of atresia, befor e other morphological or biochemical changes are detected.