The Primary Study On Effects Of Decline In Chaperone-Mediated Autophagy Activity On Sertoli Cells Response To Stress

When the cells are in stress and intracellular pressure increases,will activate cell autophagy.Autophagy can be divided into Macroautophagy,Microautophagy and Chaperone-mediated autophagy(CMA).When cellular stress,Macroautophagy/Microautophagy quick start function,but of short duration(?10h),and CMA late start,but the long duration,and three kinds of autophagy interact complement.The activity of CMA depends mainly on the amount of LAMP-2A(Lysosomal-associated membrane protein 2A)in the lysosomal membrane,and the decrease of LAMP-2A can cause the function of CMA dysfunction and "functional aging cells".These cells under the effect of various stress factors,easily lead to intracellular error protein aggregation,metabolic disorders and eventually enter apoptosis.The majority of cells will maintain the basic CMA activity,in response to cell damage,maintain intracellular protein quality and so play an important role.Our results also showed that LAMP-2 and LC3 proteins were detected in the brain,ovary,testis,liver,spleen,kidney and lung tissues of mice,indicating that the above tissues had certain CMA and Macro/Microautophagic activity;consistent with the reported results.However.we immunohistochemistry and immunofluorescence of mouse testes showed that LAMP-2 is abundantly expressed in Leydig cells(Lc)and Sertoli cells(Sc)and was expressed in Spermatogonia(Sg);but not expressed in Spermatocytes(Sp),Round sperm cells(Rs)and Elongated sperm cells(Es),indicating that spermatocytes do not have CMA activity.Associated with:mouse testicular heat shock or experimental cryptorchidism,Leydig cells,Sertoli cells,Spermatogonia and Spermatocytes,Round sperm cells,Elongated sperm cells in almost the same temperature environment,Respectively.resulting in survival and apoptosis of the two different results,suggesting that Sertoli cells and other cells with CMA fi,notion may play an important role in resisting adverse effects of stress on cells,avoiding cells into the apoptotic path.In this regard,we use TM4 cells with a reduction in CMA function to studied in the case of reduction in CMA function,Sertoli cells in response to heat shock and other stress conditions,and to explore the role of CMA functions in Sertoli cells and Sertoli cells which loss CMA function Whether product similar cytological performance to spermatocytes.From the perspective of autophagy to explain the principles of Sertoli cells against heat shock.The main results showed that:1.The use of Anisomycin to inhibit the CMA of TM4 cells,when the Anisomycin concentration greater than 0.04ptM,compared with the non-heat shock group,the survival rate of heat shock cells significantly reduced.The preliminary description shows that the lack of CMA function reduces the resistance of TM4 cells to heat shock.2.Lentiviral infection knockdown Lamp-2 gene expression decreased TM4 cells CMA activity,cultured under normal circumstances,the short time on the Macro/Microautophagic activity of the compensatory increase is limited,but the cells in stress,CMA inhibition will lead to a significant increase in Macro/Microautophagic activity compensatory,especially in high-intensity stress such as heat shock,cell autophagy is also stronger;indicating that Sertoli cells and other Somatic cells are similar-there is a complete autophagy protection system in the cell,Macro/Microautophagic and CMA there is complementary and compensatory protection.3.After Anisomycin inhibits CMA function or knockdown LAMP-2 reduces CMA activity,the cell grows state deteriorated after heat shock treatment.Meanwhile,with the prolongation of heat shock time,the apoptosis rate is getting higher and higher.It is suggested that the maintenance and increase of CMA activity play an important positive role in Sertoli cells to resist heat shock damage and avoid cell entering the apoptosis and so on.4.After the decrease of CMA activity,the activity of cell proliferation was decreased under the action of stress factors such as starvation and heat shock.It is suggested that the decrease of CMA activity not only increases the level of cell apoptosis under stress,while also reduces the cell proliferation ability.Based on the above results,we atnalyzed the TM4 cells which knockdown LAMP-2 or treatment with Anisomycin to inhibited CMA function,just like the liver cells which Lamp-2 gene knockout,spermatocytes which loss of CMA activity because of not express LAMP-2 and HSC-70t(Heat shock cognate protein70t).become a "functional aging cells",due to CMA function decreased or lost and resistance to heat shock and other stress factors in the decline,and ultimately to initiate apoptotic pathway.