Molecular Chaperone Autophagy Mediated By Hsc70 Acetylation Regulates The Function Of Goat Ovarian Granulosa Cells

The low reproductive efficiency of sheep is related to follicular atresia caused by ovarian Granulosa cells（GC）apoptosis.Follicular atresia is a complex biological process regulated by a variety of physiological and environmental factors.The exact mechanism of regulating follicular atresia is still unclear.The effects of Chaperone-mediated autophagy（CMA）on follicular atresia and granulosa senescence have attracted increasing attention.The acetylation of Heat shock cognate protein 70（Hsc70）may be involved in chaperone autophagy,but the specific mechanism remains to be investigated.In this study,ovarian granulosa cells of Daizu Black sheep were selected as the research object.The effects of molecular chaperone autophagy mediated by Hsc70 acetylation on the function of ovarian granulosa cells were investigated by immunohistochemistry,immunofluorescence,q RT-PCR,Western Blot,adenovirus transfection,immunoprecipitation,mass spectrometry,flow cytometry and other methods.The following results are obtained:1.Effect of H₂O₂ on the molecular chaperone autophagy of ovarian granulosa cellsTotal protein and total RNA were extracted from the ovary of two and ten years of age,and the Chaperone-mediated autophagy（CMA）activity in ovary of different ages was detected by Western blot and q RT-PCR,respectively.Results showed that the expression level of Hsc70 m RNA in aging ovarian tissue significantly decreased（P<0.01）,the signature proteins Hsc70 and LAMP-2A significantly decreased（P<0.01）,and the substrate proteins GAPDH and MEF2D significantly increased（P<0.01）.cck-8 was used to detect ovarian granulosa cells treated with H₂O₂ at different concentrations and times.We found that 200μm/L H₂O₂treatment for 48 h had the best effect.Senescent cells were stained and senescent genes were detected later.Results showed that the number of senescent cells was the highest after treatment with 200μm/L concentration for 48 h,and the signposts of senescence genes P53 and P21 m RNA relative expression significantly increased（P<0.05）.After the senescence of granulosa cells induced by H₂O₂,CMA activity was detected.Results showed that the expression of the iconic proteins LAMP-2A（P<0.01）and Hsc70（P<0.05）significantly decreased during CMA in the treatment group,and the expression of the substrate proteins GAPDH（P<0.01）and MEF2D（P<0.05）significantly decreased.The CMA activity of ovarian granulosa cells was significantly inhibited by H₂O₂ treatment labeled with 200μm/L.2.Effect of Sirt2 on the acetylation of Hsc70Trichostatin A（TSA;HDAC family inhibitor）and nicotinamide（NAM;Sirtuin family inhibitor）were used to treat ovarian granulosa cells.NAM was found to promote the acetylation of Hsc70.Subsequently,granulosa cells were treated with the Sirt2inhibitor AKG2,and we found that the degree of acetylation of Hsc70 increased.This finding indicated that the acetylation modification of Hsc70 was affected by the regulation of Sirt2.3.Effect of Hsc70 deacetylation on chaperone autophagyHsc70 was acetylated at K512 as revealed by mass spectrometry.Hyperacetylation and deacetylation mutants were constructed and transfected into granulocyte cells.Immunoprecipitation results showed that acetylation increased when lysine was mutated to glutamine and decreased when lysine was mutated to arginine.Transfection of different mutants on CMA was observed,and Hsc70 wild-type was found to be significantly reduced in the Hsc70 deacetylation group compared with Hsc70 and LAMP2A（P<0.01）.The CMA substrate proteins GAPDH and MEF2D were significantly reduced in the Hsc70 hyperacetylation group（P<0.01）.In conclusion,Hsc70 protein mutated at the K512 site,and Hsc70 deacetylation significantly inhibited CMA.4.Effects of Hsc70 deacetylation on the proliferation,apoptosis,and senescence of ovarian granulosa cellsAfter transfection of Hsc70 and its mutants,the expression of proliferation-related protein PCNA was significantly decreased（P<0.01）,the S phase of granulosa cells was decreased,and the G2 phase was increased by deacetylation of Hsc70.Bcl-2/Bax in the Hsc70 deacetylation group significantly decreased（P<0.01）,and Bcl-2/Bax in the Hsc70hyperacetylation group did not significantly differ from that of wild-type Hsc70.Flow cytometry analysis showed that deacetylation of Hsc70 increased ovarian granulosa cell apoptosis.Hsc70 overexpression decreased cell senescence after hydrogen peroxide induced senescence.Hsc70 mutants increase senescence.These results indicated that Hsc70 deacetylation significantly inhibited the proliferation of ovarian granulosa cells and promoted senescence and apoptosis.Based on the above results:（1）CMA activity in aging ovarian tissue was lower than that in normal tissue;（2）granulosa cells treated with H₂O₂ can accelerate senescence and inhibit CMA process;（3）Hsc70 was acetylated at lysine site 512,and Sirt2 inhibited the acetylation of Hsc70.（4）Deacetylation of Hsc70 can inhibit the proliferation of ovarian granulosa cells and promote apoptosis and senescence.The results of this study provide a basis for the analysis of the mechanism of molecular chaperone autophagy mediated by Hsc70 acetylation modification on granulosa cell apoptosis.