The Role Of Oxidative Stress In Inducing Autophagy And Its Effects On Apoptosis In Porcine Ovarian Granulosa Cells

Autophagy is an important physiological process in eukaryotic cells and participates in cell metabolism.In this study,we investigate the interplay between autophagy and apoptosis of porcine ovarian granulose cells in the response to oxidative stress.Apoptosis was assessed by flow cytometry or detecting expression level of apoptotic genes by RT-PCR,and the CCK-8 assay was used to measure cell viability after 12 hours of exposure to H2O2 at different concentrations.The expression of Microtubule-associated protein 1 light chain 3(LC3,including LC3-I and LC3-Ⅱ)and autophagy adaptor protein p62/SQSTM1 was detected by Western blotting.The number of autophagosomes was demonstrated by transfecting cells with the GFP-LC3 plasmid.Moreover,after 4 hours of 3-methyladenine(3-MA)treatment and 12 hours of oxidative stimuli,granulosa cell viability was detected by CCK-8,and apoptosis rate was calculated by flow cytometry.Furthermore,we made a preliminary study on the molecular mechanism:western blot was performed to analyze the expression of phospho-mTOR and phospho-Foxo1;while immunofluorescence was applied to visualize the nuclear/cytoplasm distribution of Foxol in granulosa cells stimulated with oxidative stress.The results showed that a 12 hours H2O2 exposure decreased cell viability evidently and significantly increased mRNA level of apoptotic genes and the percentage of apoptotic cells.The relative expression of LC-II increased first and then decreased within 6 hours of oxidative stress stimuli while the expression tendency of P62 was on the contrary.The relative expression of LC-II increased first and then decreased within 6 hours of oxidative stress stimuli while the expression tendency of P62 was on the contrary.Similarly,GFP-LC3 transfection significantly promoted the formation of autophagic puncta,which reduced dramatically after 6 hours of H2O2 treatment.These data may indicate that autophagy in porcine ovarian granulosa cells was activated at the early stage of oxidative stress,but would be restrained under prolonged oxidative stress.Furthermore,apoptotic rates in cells treated with 3-MA for 4 hours before 12 hours of H2O2 exposure decreased markedly conpared to H2O2-only-treated cells,and cell viability increased significantly in the 3-MA treated group.In addition,early oxidative stress induced phosphorylation of mTOR and Foxo1;oxidative stress at late stage increased nuclear transportation of Foxo1.Our findings demonstrate that early stage of oxidative stress promotes autophay,and the inhibition of autophay may reduce apoptosis in the later stage.Induction of autophagy may be associated with phosphorylation of both Foxol and mTOR;while apoptosis in the later stage of oxidative stress may require nuclear translocation of Foxol.The findings from this series of study may provide theoretical support for improving animal fertility and provide new therapy for female reproductive disorders.