Biology of Reproduction, Vol 51, 934-944, Copyright © 1994 by Society for the Study of Reproduction

ARTICLES

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

PD Jolly, DJ Tisdall, DA Heath, S Lun and KP McNatty
Wallaceville Animal Research Centre, Upper Hutt, New Zealand.

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 (> or = 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 (1-10,000 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 is a dose-dependent manner in granulosa cells from all follicles examined (> or = 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 > or = 8 mm with aromatase activity; in cells without aromatase activity, cAMP response to FSH decreased with increasing follicle size > or 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 > or = 8 mm, but to a greater degree in cells with aromatase activity than in cells without.(ABSTRACT TRUNCATED AT 400 WORDS)