Effects Of Heat Stress On The Expression And Localization Of Heat Shock Proteins And Androgen Receptor In Boar Testes

Objective: Heat stress affected spermatogenesis and semen quality in mammals,but its molecular mechanism remains unclear.Heat stress increased germ cell apoptosis.However,a growing body of evidence demonstrated that germ cell apoptosis can be induced by heat stress and androgen deprivation.Androgen is mediated by androgen receptor(AR),and is essential for maintaining normal spermatogenesis.The expression of heat shock proteins(HSPs)was up-regulated in response to heat stress.Studies have shown that Hsp70 and Hsp90 are molecular chaperons of AR.Whether heat stress caused germ cell apoptosis by upregulating HSPs to regulate AR-mediated hormone signaling pathways,thereby,affecting spermatogenesis,this is still unclear.The purpose of this study was to explore the expression and localization of Hsp60,Hsp70,Hsp90 and androgen receptor(AR)in boar testes under heat stress conditions,and to study the molecular mechanism of spermatogenesis damaged by heat stress in boar testes.Methods: In this study,environmental heat stress and local testicular heating models were established in boars.Nine adult Landrace boars(14~18 months old)were randomly divided into three groups(n(28)3each).Environmental hyperthermia group(the spermatogenesis cycle in boars is about 41 d),three boars were subjected to a temperature of 37~40 ? for 3 h daily for 42 d;Local testicular heating group,the testes of three boars were exposed to 42 ? for 1 h with an electric blanket of controlled temperature.After the heat treatment,the boars were driven back to the room at 20~27 ?.Control group,three boars were housed at room temperature(20~27 ?).After 24 h of environmental heat stress and 6 h of local testicular heating,all boars were castrated and the testes of each were collected.All testes were respectively cut into small pieces,parts of them quickly frozen in liquid nitrogen to extract total RNA and total protein.The others were fixed in Bouin's solution to make paraffin sections.The expression and localization of Hsp60,Hsp70,Hsp90 and AR was detected by q RT-PCR,Western Blot and immunohistochemistry.Results:1.q RT-PCR results showed that environmental heat stress(37~40 ? 42 d)and local testicular heating(42 ? 1 h)up-regulated the expression of hsp60,hsp70,hsp90 and ar m RNA.2.Western Blot results showed that environmental heat stress(37~40 ? 42 d)and local testicularheating(42 ? 1 h)up-regulated the expression of Hsp60,Hsp70,Hsp90 and AR protein.3.Immunohistochemistry results showed that environmental heat stress(37~40 ? 42 d)and local testicular heating(42 ? 1 h)induced Hsp60 immunopositive staining from the cytoplasm of spermatocyte to the nucleus of spermatogonia,Hsp70 immunopositive staining from the cytoplasm of spermatogonia and Sertoli cell to the nucleus,and increased the expression of Hsp90 in spermatogonia and elongated spermatid and AR in Sertoli cell.Conclusion: Environmental heat stress(37~40 ? 42 d)and local testicular heating(42 ? 1 h)increased the expression of Hsp60,Hsp70 and Hsp90 in spermatogonia and Sertoli cell,which can protect spermatogonia and Sertoli cell from apoptosis under heat stress conditions,but can not prevent other germ cell apoptosis.This may be due to the increased expression of Hsp70 in Sertoli cell,which enhanced the inhibition on AR and reduced the sensitivity on androgen,thereby affecting spermatogenesis and semen quality.