Effects Of Autophagy And Its Regulation On Cell Proliferation In Buffalo Follicular Granulose Cells

Follicular development of mammals is an extremely complex physiological processe which is regulated precisely. Granulosa cells as the main somatic cells in follicle play an important role during follicle development and oocyte maturation, which decide the fate of follicle. Autophagy as one facilitated factor for cell surviving is programmed cell death II, which could regulate cell death or survival, to some extent, even reverse cells'fate from apoptosis. Till now, there are a lot of reports about autophagy of granulose cells in rat etc, however, few studies have been reported about autophagy and its relationship with apoptosis in granulosa cells during buffalo follicular atresia. In this study, we first classified follicle by different standards, including by follicular diameters and atretic status, to explore the relationship between autophagy and apoptosis in granulosa cells, and the expression pattern of autophagy-related and apoptosis-related genes in buffalo follicular granulosa cells. Secondly, rapamycin, an inhibitor of mTOR pathway, was used to regulate autophagy to investigate its effect on autophagy, apoptosis, growth curve, cell cycle, expression of related genes in granulosa cells cultured in vitro. The study would be helpful to optimize the culture condition of buffalo granulose cells in vitro.1. The analysis of autophagy and apoptosis in granulosa cells on different stages of buffalo folliclular developmentAutophagy and apoptotic status of buffalo follicular granulosa cells with different diameters or atretic status were detected by GFP-LC3 transfection, Annexin V-FITC/PI staining. Two classified methods were used in the experiments. Compared with small follicle (SF) and medium follicle (MF), the autophagy rate and unliving cell rate of granulosa cells in large follicle (LF) were the highest, while the apoptosis rate was the lowest. Compared with healthy atretic follicle (HF) and early atretic follicle (EF), the autophagy rate and apoptosis rate of granulosa cells in progressed atretic follicle (PF) reached the maximum. The expression of autophagy-related and apoptosis-related genes in granulosa cells from buffalo follicles with different diameters or atretic status was detected by qRT-PCR. The results revealed that all of the related genes expressed in buffalo granulosa cells with different diameters or atretic status. The expression levels of autophagy-related genes, such as Atg3, Atg5, Atg7, Atgl2, BECN1 and LC3, was up-regulated with the increasing of follicular diameters, while the expression level of ULK1 was lower in LF granulosa cells. The expressions of Atg3, Atg5, Atg12, BECN1 and LC3 were increased along with the progress of follicular atresia, Atg7 and ULK1 expressed lower. LC3 protein in buffalo ovary was related with the development of follicle, which showed higher expression in mature follicle than in primordial follicle.2. The effect of regulating autophagy on proliferation of buffalo granulose cells cultured in vitroFirstly, cells cultured in vitro were detected by transmission electron microscope, autophagosome and autophagolysosome were found in the autophagy cells. Secondly, both of the autophagy rate and apoptosis rate of buffalo granulose cells cultured with rapamycin added increased compared with the cells cultured with DMEM(10% FBS) in vitro. Cell cycle was analyzed by flow cytometry, the ratio of cells in G0/G1 phase was increased, and the ratio of cells in S and G2/M phase was decreased when treated with rapamycin. The expression of autophagy-related genes, such as Atg3, Atg5, ULK1, and apoptosis-related genes, such as Bcl-2, Bax, P53, was analyzed with the treatment of rapamycin. We found that, in contrast with buffalo granulose cells cultured with DMEM(10% FBS) in vitro, Atg3, Atg5, ULK1 and other autophagy-related genes increased their expression when treated with 0.2nM rapamycin, without a dose-dependent manner. The expression of apoptosis promoting gene, such as Bax, Caspase3 and P53, was increased with the rapamycin concentration increased in buffalo granulose cells cultured in vitro. The expression of Bcl-2, the anti-apoptosis gene, was decreased with the rapamycin concentration increased in buffalo granulose cells cultured in vitro. And there was no significant change for Fas expression in buffalo granulose cells whether treated with rapamycin or not.The above results indicated that autophagy and apoptosis were detected in granular cells during buffalo follicle growth. With the increase of follicle diameter and the atresia status, autophagy and apoptosis rates of granular cells increased gradually. Autophagy can be induced by rapamycin via inhibiting mTOR pathway in buffalo granulosa cell cultured in vitro. Autophagy and apoptosis would act together to regulate the growth of granulose cells.