Establishment And Evaluation Of A Stable Steroidogenic Goat Follicle Granulosa Cell Line And Research On The Function Of AIF In Goat Follicle Granulosa Cells

In mammals, storage capacity of tens of thousands of primordial follicles is estimated at birth, but the minorities of them can be selected for ovulation. More than 99% of follicles will undergo atresia which is a common physiological phenomenon during the overall process of follicular growth and development. Granulosa cell apoptosis is considered the main reason causing ovarian follicular atresia, but the molecular mechanisms of granulosa cell apoptosis are not fully understood. Apoptosis inducing factor(AIF) mediates caspase-independent apoptosis and chromatin condensation and fragmentation, but its role in granulosa cell apoptosis during goat follicular atresia is not clearly described. Although we can use primary ovarian granulosa cells to study the mechanism of granulosa cells apoptosis, but the ability of primary granulosa cells to proliferate in vitro limits many studies. In this study, we immortalized granulosa cells by hTERT, hTERT- granulosa cells(hTERT-GGCs) were selected as a model to explore the relationship between AIF and granulosa cell apoptosis. The main results were as follows:(1)Goat granulosa cells(GGCs) were aspirated with a fine needle from medium-sized(4-6mm) healthy follicles. GGCs were transfected with pCI-neo-hTERT, complete culture medium which contained 400μg/mL G418 were used to select transfected cells. Positive colonies were propagated and cultured in the following studies, named hTERT-goat granulosa cells(hTERT-GGCs). The RT-PCR and immunofluorescence showed that the expression of hTERT mRNA and protein in hTERT-GGCs were positive. RT-PCR demonstrated that some enzymes involved in steroidogenesis such as StAR, CYP11A1, CYP19A1 and CYP17A1 were detected in the hTERT-GGCs. The other receptors such as LHR and ER were also expressed in GGCs and hTERT-GGCs. As for progesterone, there was not significant difference between the progesterone levels in the hTERT-GGCs and GGCs group. The cell viability of hTERT-GGCs were significant increased compared with the GGCs. The hTERT-GGCs show a relatively higher telomere length compared with GGCs and the normal goat chromosome number, don’t have oncogenicity.(2)Immunohistochemistry results indicated that AIF was strongly expressed in pyknoticcells and scattered granulosa cell layer in atretic follicles. The levels of AIF protein were significantly up-regulated with follicular atresia and follicular diameter. The recombinant vectors are constructed successfully. The results of RT-PCR and western blot showed that pCD513B-U6-AIF-shRNA-1 had the best down-regulated effect(more than 80%) of the AIF expression. Therefore, pCD513B-U6-AIF-shRNA-1 was used for this study. The flow cytometry result showed that down-regulation of AIF increased cell viability compared to the sh-Negetive and control groups when cells were treated with serum starvation for 48 h.In conclusion, we immortalized goat mural granulosa cells by hTERT, and down-regulation of AIF increased cell viability in hTERT-GGCs treated with serum starvation.