Effect Of Heat Stress On The Expression And Localization Of Transforming Growth Factor-β3 And Blood-Testis Barrier Tight Junctions Molecules In Boar Testes

Objective:The high temperature in summer severely affected the reproduction rate of breeding boars,which was a generally serious problem in the production practice of domestic and foreign breeding boars farms.Under high temperature heat stress,the sperm deformity rate increased,the density decreased and the vitality decreased in boars.But the molecular mechanism has not yet well understood.Whether high temperature heat stress affects the expression and localization of the blood-testis barrier(BTB)regulatory protein transforming growth factor-β3(TGF-P3)and the BTB tight junctions(TJs)molecules Occludin and Claudin-11 in boar testes,disruptes the structure of the BTB,thus affects spermatogenesis and semen quality,there is no research report.The purposes of this study was to explore the expression and localization of the BTB regulatory protein TGF-β3 and the BTB TJs molecules Occludin and Claudin-11 in boar testes under heat stress,and then researched the molecular mechanism of heat-induced spermatogenesis damage in boar testes.Methods:The present study designed boar testes heat stress models.Adult boars(Landrace,14-18 months of age,n = 12)were used and randomly divided into four groups(n = 3 each),of which nine boars were treated with heat stress:Short time ambient heat stress group,three boars were subjected to ambient temperature 37-40 ℃ for 7 days,daily for 3 hours;Long time ambient heat stress group(A spermatogenesis cycle is 42 days),three boars were subjected to ambient temperature 37-40 ℃ for 42 days,daily for 3 hours;Local scrotal heating group,three boar were given local scrotal exposure to 42 ℃ for 1 h with a home-made electric blanket of controlled temperature.After heat treatment,the boars were driven back to normal temperature(20～27 ℃).The other three boars received no heat-treatment and were left at standard room temperature(20～27 0C)as control group.All boars were castrated and the testes were harvested.qRT-PCR,Western blotting and immunohistochemistry were used to explore the expression and localization of the BTB regulatory protein TGF-β3 the BTB TJs molecules Occludin and Claudin-11 in boar testesResult:1.The results of qRT-PCR showed that heat stress(Short time ambient heat stress,long time ambient heat stress and local scrotal heating)upregulated the relative expression levels of TGF-β3 mRNA,down-regulated the relative expression levels of the BTB TJs molecules Occludin and Claudin-11 mRNA in boar testes2.The results of Western blotting found that heat stress(Short time ambient heat stress,long time ambient heat stress and local scrotal heating)upregulated the relative expression levels of TGF-β3 protein,down-regulated the relative expression levels of the BTB TJs molecules Occludin and Claudin-11 proteins in boar testes3.Immunohistochemistry results found that TGF-β3 immunoreactivity staining was observed in the cytoplasm of all stages of germ cells and Sertoli cells in heat stress groups(Short time ambient heat stress group,long time ambient heat stress group and local scrotal heating group),and the depth and area of the positive staining in heat stress groups were higher than that of the control;The immunoreactivity staining of Occludin and Claudin-11 were limited to Sertoli cells and the expression of the obvious BTB strand was impaired in heat stress groups(Short time ambient heat stress group,long time ambient heat stress group and local scrotal heating group)compared with the control.Conclusion:Heat stress(Short time ambient heat stress,long time ambient heat stress and local scrotal heating)increased the expression of the BTB regulatory protein TGF-β3,down-regulated the expression of the BTB TJs molecules Occludin and Claudin-11.This indicates that heat stress might be disrupted the structure of the BTB and damaged spermatogenesis.