Mechanisms Of Heat Exposure Promoting Apoptosis And Pyroptosis Of Mouse Sertoli Cell

Reproductive sensitivity to increases in temperature that organisms often experience in the natural environment is well known in mammals,where adaptations that allow testicular cooling of 2 to 8℃ below core body temperature are essential to allow normal male fertility.Testicular heat exposure caused by occupational factors,lifestyle,and clinical diseases can lead to different degrees of reproductive problems.Objective: To reveal the important role in the transcriptional regulation of testicular heat exposure and the specific mechanisms that promote apoptosis and pyroptosis in mouse sertoli cells.Methods: Testicular tissues were collected from scrotal heat exposure group （43℃）and control group（33℃） respectively to isolate RNAs and used for whole transcriptome sequencing.The sertoli cell TM4 heat excitation model was also constructed,and the effects of heat exposure on sertoli cells were investigated by Western blot,immunofluorescence,TUNEL,and projection electron microscopy experiments.Results: Using differential expression analysis,2721 genes were differentially expressed in testicular tissue of mice after heat exposure.The differentially expressed genes were enriched by Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis in the meiotic cell cycle（GO:0051321）,cytoplasm（GO:0005737）,membrane rafts（GO:0045121）,MAPK signaling（mmu04010）,Hippo signaling pathway（mmu04390）and Apoptosis（mmu04210）with potentially important functions.This study provides data for high-throughput sequencing of testicular heat exposure models and lays the foundation for further studies of male reproductive disorders associated with elevated testicular temperature.In addition,we found in vitro experiments that many pyroptosis vesicles were found after heat stimulation of sertoli cells TM4.Pyroptosis is a programmed cell death mediated by gasdermin,which is manifested by cell distension until the cell membrane ruptures,leading to the release of cell contents and thus activating a strong inflammatory response.We examined the microstructure of TM4 cells after heat stress by transmission electron microscope（TEM）,the protein associated with pyroptosis by western blot,and the extent of DNA damage in TM4 cells after heat stress by in situ end-transferase labeling（TUNEL）.The results showed that the TM4 cell membrane rupture,mitochondrial breakage damage and autophagic vesicle production were observed after heat stress,and the N-terminal of GSDME shear was increased and the TUNEL result was positive.In conclusion,thermal excitation induced apoptosis and pyroptosis in TM4 cells.Conclusion: In summary,high-throughput sequencing results showed differentially expressed m RNAs after testicular heat exposure,and enrichment analysis revealed that Hippo pathway and apoptosis-related pathways were significantly enriched.As well as a brief exploration of the mechanism of heat-excited TM4 cell-induced pyroptosis effect was performed.These findings provide a basis for further studies of male reproductive disorders associated with elevated testicular temperature and provide new entry points for the design of novel contraceptives.