Preliminary Studying On Spermatogenesis Disorder Induced In Heat Shock Transcription Factor 5 By Regulating The Expression Of TLR2 In Leydig Cells

Background and ObjectiveInfertility is a major public health problem facing the whole of society today.Men account for a major factor in 50%of infertility cases,and declining sperm quality is an important cause of infertility that has attracted widespread attention.Spermatogenesis is a complex and orderly refinement process regulated by a variety of factors and is highly susceptible to temperature changes.The internal and external harmful factors can easily lead to the disorder of temperature regulation of mammalian testis,triggering heat stress and inducing autophagy,apoptosis and DNA damage in testicular cells,resulting in abnormal sperm production and ultimately infertility.Heat stress is mainly regulated by heat shock transcription factors（HSFs）.The HSFs in eukaryotes are HSF1,HSF2,HSF3,HSF4,HSF5,HSFX and HSFY;among them,HSF1 and HSF2 have been clearly reported to have important roles in spermatogenesis;however,HSF5,which is specifically expressed in the testis,has rarely been reported in spermatogenesis.Current literature suggests that male zebra fish with hsf5^-/-exhibit absolute sterility,while female zebra fish have unaffected fertility,but the exact mechanism is unknown.This suggests to us that HSF5 may play an important role in the process of spermatogenesis.In order to explore its function,our group has already conducted a study in mouse spermatocyte（GC2）.However,in addition to spermatogenic cells,Leydig cells and Sertoli cells are also essential in the maintenance of normal spermatogenesis,therefore,in this study,we propose to construct Hsf5 knockdown models in mouse Leydig cells（TM3）and mouse Sertoli cells（TM4）.The present study aims to explore the biological functions of HSF5 in testicular somatic cells at the RNA and protein levels,and to further investigate its role in male reproduction,with a view to providing new directions for the treatment of male infertility.Methods1.Hsf5 knockdown models of TM3 cells and TM4 cells were constructed by small interfering ribonucleic acid,and Real-time quantitative polymerase chain reaction verifies whether the knockdown model was successfully constructed;2.Based on the constructed knockdown model,RT-qPCR and Western blot were used to detect the expression of other factors and proteins of the heat shock family associated with spermatogenesis;3.TM3 and TM4 cells with knockdown of Hsf5 were subjected to RNA-seq;and the RNA-seq results of TM3 cells were validated using RT-qPCR,WB and Td T-mediated d UTP Nick-End Labeling assays.Results1.Successful construction of Hsf5 knockdown models for TM3 cells and TM4 cells.2.The changes of related heat shock families detected by RT-qPCR showed that:in the Hsf5 knockdown group:The m RNA relative expression levels of Hspa2,Hspa5,Hsp90ab1 in TM3 cells were significantly down-regulated（p﹤0.05）.The m RNA relative expression levels of Hsf2,Hspa2,Hsp90aa1,Hsp90ab1 and Hspd1 in TM4 cells were significantly down-regulated（p﹤0.05）.According to the results of RT-qPCR,the genes with significant changes（p﹤0.01）in the two kinds of cells were selected for WB.The results showed that the relative expressions of HSPA2 and HSPA5 proteins in the Hsf5 knockdown group in TM3 cells were significantly down-regulated（p﹤0.05）.In TM4 cells,the relative expression of HSPA2protein in the Hsf5 knockdown group was significantly down-regulated（p﹤0.05）.3.A total of 1170 genes were screened out by RNA-seq in TM3 cells（p﹤0.05）,of which375 genes were up-regulated and 795 genes were down-regulated.Combined GO enrichment analysis and KEGG enrichment analysis showed significant enrichment in pathways related to the immune system,such as antibacterial and viral related pathways（GO:0009617,GO:0051607）,humoral and innate immunity related pathways（GO:0045087,GO:0006955）,Herpes simplex virusⅠinfection（ko05168）,Toll-like receptor（TLR）signaling pathway（ko04620）and NOD-like receptor signaling pathway（ko04621）.In particular,TLR signaling pathway was enriched,and TLR2,TLR4 and TLR6 were significantly down-regulated in this pathway（p﹤0.05）,while Fas-associated protein with death domain（FADD）,an intermediate factor in TLR2-mediated apoptotic pathway,was also significantly down-regulated（p﹤0.05）.The transcription factor prediction database JASPAR was then used to predict HSF binding sites for TLR signaling pathway-related factors,and it was found that both TLR2 and TLR4promoter regions had HSF binding sites,while TLR6 did not,so TLR2 and TLR4 were selected for validation.The RT-qPCR results showed that the relative expression of Tlr2 and Tlr4 m RNA were significantly down-regulated after knocking down Hsf5（p﹤0.05）.WB results showed that the relative expression of TLR2 protein was also significantly down-regulated after knocking down Hsf5（p﹤0.05）;however,the expression of TLR4 protein did not change significantly（p﹥0.05）.The TUNEL assay showed that the apoptosis rate of the normal control group with zymosan was higher than that of the normal control group without zymosan（p﹤0.05）;in the normal group with zymosan and the Hsf5 knockdown group with zymosan,the apoptosis rate of the normal group was significantly higher than that of the knockdown group（p﹤0.05）.4.By RNA-seq,TM4 cells were screened for 217 genes（p﹤0.05）,of which 80 genes were up-regulated and 137 genes were down-regulated in expression.The results of the combined gene enrichment analysis showed that TM4 cells were significantly enriched in pathways related to phagocytes,such as phagocytes regulation（GO:0050764）,initial phagocytes（GO:0032009）,and FcγR-mediated phagocytes（ko04666）.ConclusionsIn summary,the RNA-seq results suggest that immune-related pathways in Leydig cells were significantly enriched following the down-regulation of Hsf5 expression,especially the TLR signaling pathway.Our validation results suggest that in Leydig cells,HSF5 may regulate apoptosis through TLR2,resulting in altered androgen secretion and ultimately impaired spermatogenesis.This study provides a theoretical basis for exploring the role of HSF5 in the male reproductive system,and also provides some basis and inspiration for the mechanism of heat stress in causing reduced male fertility.