Influence Of Heat Stress On Expression And Localization Of FasL/Fas Signal And TGF-β1 In Boar Testis

[Purpose]Recently,the influence of mild heat stress on male reproductive is an important potential trouble in pig production practice.It is still unclear that the effect of heat stress on spermatogenesis molecular mechanism in mammals testis.The exogenous apoptosis molecule Fas L/Fas signaling and transforming growth factor TGF-β1 are main molecules regulating apoptosis,and are also candidates for immune immunity of testis.These molecules interact,which may lead to increased apoptosis and increased testicular immunosuppression relative to the pathogen.This study was conducted to determine whether heat stress leads to testicular immune immunity through regulating Fas L/Fas signal and TGF-β1 expression,leading to decreased spermatogenic ability and resistance to pathogens.[Methods]3 heat stress models were established in 9 mature boars at 14 months of age.9 boars were randomly divided into 3 groups: control group(Control),environmental heat stress group(Environmental Heat Stress,EHS),local heat stress group(Local Heat Stress,LHS).None experimental treatment in control group.Boars in control group were kept in captivity at room temperature(20-25 ℃),and supplied with water and feeding daily.Environmental heat stress group was kept in captivity at 37-40 ℃ 3 h for 7 days,then driven back to room temperature(20-25 ℃).The left side testis scrotum of boars was wrapped by home-made temperature controlled heating pad in local heat stress group(42 ℃ heating 1 h).Testicular tissues of boars in each group were quickly obtained after experimental treatments.Part of testicular tissues were cut into about 1 cm3 pieces.Then fixed in Bouin’s solution for paraffin section.Part of testicular tissues were cryopreserved in liquid nitrogen for extraction of total RNA and total protein.Relative expression levels of m RNA and protein of Fas,Fas L,Caspase-8,TGF-β1 were detected by q RT-PCR and Western blot.Location of Fas,Fas L,Caspase-8,TGF-β1 in germ cells was observed by immunohistochemistry.[Results]1.Fas,Fas L:In environmental heat stress group,relative expression levels of Fasm RNA and protein showed up-regulation.Anti-Fas antibody was mainly located in spermatogenic cells,and cellular immune positive staining is deeper than control group.Relative expressions of Fas Lm RNA and protein showed down-regulation.Anti-Fas L antibody was mainly located in sertoli cells,spermatocyte,spermatid.In local heat stress group,relative expressions of Fas and Fas Lm RNA and protein both showedup-regulation.Cellular immune positive staining is deeper than control group.The location of the cells remained no difference.2.Caspase-8:In environmental heat stress group,relative expression levels of Caspase-8m RNA and protein showed up-regulation.Anti-Caspase-8 antibody was mainly located in round spermatid and spermatocyte.In local heat stress group,relative expression levels of Caspase-8m RNA and protein were higher than environmental heat stress group.Anti-Caspase-8 antibody was mainly located in round spermatid and sertoli cell.3.TGF-β1:In environmental heat stress group,relative expression levels of TGF-β1m RNA and protein showed up-regulation.Anti-CTGF-β1 antibody was mainly located in sertoli cell,spermatocyte,elongated spermatid and Leydig cell.In local heat stress group,relative expression levels of TGF-β1m RNA and protein were higher than environmental heat stress group.Anti-TGF-β1 antibody was mainly located in round spermatid,sertoli cell and Leydig cell.[Conclusion]Spermatogenesis in pig testis was affected by mild heat stress.The up-regulated expression of Fas L/Fas and TGF-β1 indicated increased apoptosis and increased immunosuppression in the testis of heat stressed pigs,providing a new molecular mechanism for reducing the spermatogenic ability and enhancing the susceptibility to pathogens in boars after heat stress.