Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells

The first objective of this study was to determine whether the death of bov ine granulosa cells (CC) isolated from small (less than or equal to 4 mm), medium (5-8 mm), and large (> 8 mm) follicles during follicular atresia occ urs by apoptosis. The second objective was to establish an in vitro model s ystem to elucidate the developmental (CC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric cr iteria as healthy or atretic. Apoptosis in GC from follicles of different s izes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 10(6) cells/mi in serum-free med ia at 39 degrees C to determine the effects of FSH and ICF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicle s. Apoptosis in CC was also detected in some healthy follicles. Degenerate CC displayed the morphological characteristics of apoptosis, including nucl ei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All CC classified as apoptot ic on the basis of their morphology contained fragmented DNA measured by th e terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TU NEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the CC layer, apoptotic cell de ath was more prevalent among antral CC than among mural CC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in CC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of CC from small follicles was higher tha n that from medium and large follicles. FSH attenuated apoptotic cell death in CC from medium follicles more effectively than in those from small foll icles. IGF-I also suppressed apoptosis in cultured CC from small follicles. In conclusion, this study showed that 1) CC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in CC and theca cells; however, apoptosis does not occur in cumulus cells even in at retic antral follicles; 3) CC from all small, medium, and large follicles u ndergo spontaneous onset of apoptosis when cultured under serum-free condit ions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.