Role of Fas/FasL on apoptosis of porcine follicular granulosa cells derived from isolated follicles during culture in vitro

Abstract

The whole antral follicles were isolated from porcine ovaries and classified as follows: healthy follicles (HF), early atretic follicles (EF) and progressed atretic follicles (PF). The isolated porcine follicles were used for routine histological section and HE staining after examination by eyesight. Morphological research shows that the accuracy rate of eyesight examination for HF is 92%. Healthy follicles were chosen for further experiment and divided into 3 groups: large follicles (greater than 5 mm in diameter), medium follicles (3-5 mm in diameter) and small follicles (less than 3 mm in diameter). All follicles were cultured for 8, 16 and 24 h, respectively and the apoptosis of of their granulosa cells were examined by Annexin V-FITC/PI double-labeling. It showed that the total apoptotic rate of granulosa cells derived from cultured follicles could reach over 70% at 8 h after culture and be 81.1% - 94.6% at 24 h after culture. Granulosa cells from groups were collected at 0, 8, 16, 24, 48 and 72 h after culture without serum and used for the examination of expression of FasL and Fas mRNA with real time PCR SYBRgreen method. The expression level of FasL mRNA of granulosa cells from different size of follicles increased with culture time and reached the highest level at 24 h after culture (P<0.05). Expression level of FasL mRNA of granulosa cells from small follicles was higher than those from large and medium follicles. There exists no difference for expression level of Fas mRNA of granulosa cells among groups before culture but significantly increased at 8 h after culture and reached the highest level at 48 h after culture. It showed in the present experiment that the follicular culture system without serum used could effectively induce the apoptosis of follicular granulosa cells. Cell apoptosis is the main cause of follicular atresia, the degree of which varied with the size of follicles. Small follicles seemed to be easier atretic than medium and large follicles.