Research On Autophagy Induced By Environmental Estrogen Bisphenol A In Goat Sertoli Cells

Sertoli cells(SCs)are the only somatic cells in the seminiferous tubules of male testes,which can maintain and regulate the normal spermatogenesis process and play an important role in the development of spermatozoa.The structural and functional integrity of SCs is critical to the proliferation and maturation of spermatogenic cells,and its changes can lead to spermatogenic cell death.Therefore,SCs are widely believed to play a pivotal role in the maintenance of the testicular homeostasis.Autophagy is a biological process by which eukaryotic cells degrade their own subcellular structures through lysosomes,regulating cell growth,differentiation,and maintaining the homeostasis.This study aims to investigate the occurrence of autophagy and its possible regulatory mechanisms of SCs in the stress of bisphenol A(BPA),and to provide a new basis for studying the survival mechanism of SCs in oxidative stress,which further provide a theoretical reference to reveal the mechanism of spermatogenesis.Goat as an important economic animal,with economic value of meat,cashmere and milk other.Goat breeding industry is also one of most potential livestock industries in China.Therefore,in this study,goat SCs were used as the object.Firstly,the autophagy dual-fluorescence reporter reporter system of goat SCs was established,and the autophagy activity of goat SCs was also determined.Then,combining with autophay activator Rapamycin(Rap),the effects of BPA on the goat SCs were analyzed to investigate whether the goat SCs achieve self-protection mechanism through the autophagic approach under stress.Furthermore,the effects of autophagy key factor ULK1 on the biological characteristics and autophagy activity of goat SCs were studied by using overexpression and RNAi technique.The main experimental results are as follows:1.Goat SCs culture and autophagy dual-fluorescence reporter system construction.This study was designed to obtain goat SCs in vitro and to establish a dual fluorescence reporter system for detecting autophagic activity.The expression of specific gene and protein was detected by enzyme digestion and differential adherence method.The constructed double fluorescent reporter vector pmCherry-EGFP-LC3 was transfected into goat SCs,and the fluorescence was observed after starvation induction,autophagy gene LC3 expression was also detected by qRT-PCR and Western-blot.Then autophagy gene Beclinl and LC3 Ⅱ/LC3 Ⅰ were analyzed,and autophagosome of goat SCs was observed by transmission electron microscopy.Our results showed that the isolated and purified goat SCs attached in long column shape,and cell bodies were larger and had two or more protrusions on both sides.FASL,Innhibin,ABP marker gene and vimentin were strongly expressed.The expression of Beclinl and LC3 Ⅱ/LC3 Ⅰ increased significantly after starvation,and the cells had more initial autophagosome.Furthermore,the expression of Beclinl and LC3 Ⅱ/LC3 Ⅰ was also significantly increased after starvation,which showed that autophagy activity existed in goat SCs.In total,the double fluorescent autophagy reporter vector constructed in this study can provide an effective tool for further study of autophagy of goat SCs.2.Bisphenol A affects cell viability involved in oxidative damage and autophagy in goat SCs.This study aimed to probe the effects of BPA on induction of autophagy in goat SCs to various concentrations of BPA.Goat SCs were treated with different concentrations of BPA(100 μM,200 μM,300 μM,400 μM,500 μM,600 μM).The optimal oxidative damage concentration model was selected by detecting cell viability and ROS level.The expression of autophagy marker gene and apoptotic gene were measured by qRT-PCR and Western-blot.Autophagosome of goat SCs were also investigated under TEM.Autophagy activator rapamycin were further added to observe its role in goat SCs after BPA induction.Our results showed that exposure of SCs to 500 μBPA,decreased cell viability,triggered loss of mitochondrial membrane potential(△Ψm),increased ROS production,and induced apoptosis by increasing ratio of BCL2/BAX proteins,as well as autophagosome formation,BPA-induced autophagy was then further confirmed by autophagosome marker protein LC3 and Beclin1 expression.However,autophagosomes accumulated to their peak level under high BPA conditions in parallel with significantly up-regulated apoptosis.Furthermore,treatment with rapamycin alleviated a decrease in cell viability,intracellular ROS production and reduction of △Ψm,as well as decreased apoptosis.This study demonstrated that BPA had toxic effects on goat SCs by inducing autophagy and apoptosis and this process may involve ROS-dependent pathways.Modulation of autophagy induced by BPA may serve as a survival mechanism against cell apoptosis.3.Study on the role of ULK1 gene in autophagy induced by BPA in goat SCs.The aim of this study was to investigate the effect of autophagy initiation factor ULK1 on the autophagy of goat SCs and the biological properties of goat SCs.The overexpression vector of goat ULK1 gene was constructed and transfected into goat SCs.The expression level of specific marker gene,cell cycle distribution,autophagy and apoptosis-related genes were detected.The expression of ULK1 was further investigated by TEM,and the effect of ULK1 on the autophagy of goat SCs was investigated.The results showed that the expression levels of ABP,AMH,FASL,GATA4 and Inhibition were significantly decreased treated by ULK1 inference,and the expression levels of Beclin1,ATG5 and ATG7 genes were significantly decreased,and the protein expression levels of LC3 and Beclinl were significantly decreased(P<0.05).However,the marker gene,autophagy-related gene and protein expression level of goat SCs after overexpression of ULK1 showed a rising trend(P>0.05).Compared with BPA alone and overexpressing UKL1+BPA-induced treatment group,the SCs morphological structure of the goat SCs had a significant increase in mitochondrial cavity and a significant decrease in the number of autophagosomes.These results suggest that the ULK1 gene may influence the biological characteristics of goat SCs by regulating the autophagy of the cells.This study lays a foundation for further clarifying the important function of ULK1 gene of autophagy in goat SCs.