Studies on apoptosis in bovine follicles and embryos

The aims of this thesis were to further study the involvement of apoptosis in bovine ovarian follicles and embryos. In the first part of the thesis, studies in vitro and in vivo were carried out to investigate whether apoptosis is the underlying mechanism of follicular atresia and to characterize its regulation by hormones. Studies in vitro demonstrated that granulosa cells (GCs) in follicles undergoing atresia display both morphological and biochemical characteristics of apoptosis. Apoptosis was observed mainly in GCs and in scattered theca cells. Apoptosis was not evident in cumulus cells. These results suggest that apoptosis is the most common pathway of GC deletion which leads to the destruction of the GC layer and finally triggers follicular atresia. Apoptosis was also detected in GCs of some morphologically healthy follicles, indicating that apoptosis is detectable before other morphological and biochemical signs of degeneration appear. A system for culturing GCs in vitro was developed to study the hormonal regulation of apoptosis. Using this culture system, it was observed that the rate of DNA fragmentation in cultured GCs from small follicles was higher than that from medium and large follicles. Follicle stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) attenuated spontaneous apoptotic cell death in cultured GCs, suggesting that they are follicle survival factors.; An in vivo model in which the proestrus dominant follicle (DF) is maintained for 9 days was used to further study the precise regulation of the atresia of the nonovulatory DF induced by high concentration of progesterone (P4). With the time of P4 administration, DFs gradually underwent atresia and exhibited different levels of apoptosis in GCs, confirming our previous findings. P4 did not suppress apoptotic death in cultured GCs, suggesting P4-induced atresia of non-ovulatory bovine DFs is probably via regulation of luteinizing hormone (LH). In addition, it was demonstrated that follicular atresia was related to a shift in the ratio of death repressor (Bcl-2) to death inducer (Bax) protein expression.; Using an in vitro embryo production system, apoptosis was found to be related to bovine oocyte degeneration and embryo fragmentation, suggesting the existence of a natural preprogrammed cell death mechanism which can respond to external stimuli and/or internal defects in bovine oocytes and embryos. The ratio of Bcl-2 and Bax protein expression was demonstrated to be an important determinant of developmental competence for both oocytes and embryos.; This thesis provides important insight into the mechanisms of follicular atresia and early embryonic loss. Evaluation of the nature of these events would increase our understanding of the limitations with follicular dynamics and early embryonic development.