Loss Of Atg5 In Sertoli Cells Enhances The Susceptibility Of Cadmium-Impaired Testicular Spermatogenesis In Mice

Background:Cadmium（Cd）,an environmental contaminant,is a male reproductive toxicant and damages spermatogenesis and declines sperm quality.Sertoli cell autophagy is a necessary condition for maintaining self-function and germ cell development.Previous studies have found that the loss of autophagy in Sertoli cells aggravates cadmium-induced germ cell apoptosis.However,the role and mechanism of autophagy Atg5 knockout in cadmium-induced spermatogenesis remains unknown.Therefore,we constructed Sertoli cell-specific Atg5 knockout mice to explore the loss of autophagy on cadmium-impaired spermatogenesis.Objective:To investigate the effect and mechanism of Atg5 konckout in Sertoli cells on the susceptibility of Cd-impaired spermatogenesis.Methods:The study includes vivo animal experiments and vitro cell experiments.Vivo animal experiments consist of two components:Experiment one,Construct Atg5knockout mice.Amh-Cre+/Atg5^F/Wmale mice and Atg5^F/Ffemale mice were combined until appropriate age,and the genetypes of offspring were identified by mouse DNA genes.The male mice with Amh-Cre+/Atg5^F/Fgenetype were used to do experiments.Experiment two,Amh-Cre+/Atg5^F/Fmale mice and wild type male mice were divided into control groups and cadmium groups with type S according to body weight.All moues were exposed Cd Cl₂at 15 W by drinking water.After exposure for 5 W,the epididymis was taken for sperm quality evaluation,including sperm count,motility test and sperm malformation evaluation.Testes were taken to evaluate spermatogenic tubule staging.Germ cell development,meiosis and retinoic acid signals were detected by molecular experiments.Vitro cell experiments consist of two components.Experiment one,to explore the effect of cadmium on Sertoli cell protein level of WT1 and ALDH1A1,TM4 Sertoli cells were treated with Cd Cl₂（20μM）for 0,2,8 and 24 h to detect protein levels of WT1 and ALDH1A1.Experiment two,in order to further explore the effect of WT1 on cadmium-downregulated of ALDH1A1,TM4 cells were pretreated with p EX3-Wt1 overexpression plasmid for 24 h and then treated with Cd Cl₂（20μM）for 24 h to verify whether WT1 directly regulated ALDH1A1 expression.Results:In this study,Atg5 knockout of Sertoli cell was found to increase cadmium-induced vacuolation of spermatogenic tubules.Sertoli cell Atg5 deletion significantly aggravated cadmium-induced decreased sperm count,decreased sperm motility and increased sperm malformation rate in mice.Accordingly,Atg5^-/-combined with cadmium,the expression of MVH protein,a specific marker of mouse testicular germ cells,was further downregulated.The above results indicated that Atg5 knockout of Sertoli cells aggravated cadmium-damaged testicular germ cell development and spermatogenesis.In addition,this study found that Sertoli cell Atg5 knockout aggravated cadmium-decreased the number of testes spermatocytes.At the same time,Atg5^-/-combined with cadmium treatment group significantly reduced the levels of meiosis related proteins STRA8 and SYCP3.These results suggest that Sertoli cell Atg5knockout aggravates cadmium-inhibited testicular meiosis.Further studies showed that Atg5 knockout of Sertoli cells aggravated cadmium-down-regulated retinoic acid（RA）and retinoic acid receptor（RARα）levels in mouse testes.Accordingly,the expression of RARαprotein in mice was further downregulated after Atg5^-/-combined with cadmium.Moreover,Atg5 deletion of Sertoli cell significantly downregulated the protein expressions of ALDH1A1 and ALDH1A2 in cadmium-treated testes.These results suggest that Atg5 knockout in Sertoli cells aggravated cadmium-caused retinoic acid signaling impairment.We found by Ch EA3 database（https://maayanlab.cloud/chea3/）WT1 highest ranked in all prediction of transcription factors.The results also showed that Atg5 knockout significantly downregulated WT1 expression in cadmium-treated testes.In order to further verify whether WT1 directly regulates the expression of ALDH1A1,the p EX3-Wt1 plasmid was overexpressed in TM4 cells.The results showed that overexpression of Wt1 reversed the cadmium-induced downregulation of ALDH1A1 protein.In conclusion,Atg5 knockout of Sertoli cells aggravated cadmium-inhibited retinoic acid signaling in mouse testes by down-regulating WT1expression.Conclusions:Collectively,our data suggest that loss of Atg5 in Sertoli cells enhances the susceptibility of Cd-impaired testicular spermatogenesis via inhibiting WT1-mediated RA synthesis in mice.