Hsp90 And Co-chaperon Cdc37 Regulate Human Sperm Capacitation Through Erk1/2 And P38 MAPK Signaling Pathway

Background: Heat shock protein 90(Hsp90)molecular chaperone is a key regulator for eukaryotic cells to maintain protein homeostasis under physiological and stress conditions,involving various cellular processes such as protein folding.We have found that Hsp90 and its co-chaperon cell division cyclin 37(Cdc37)are expressed in the neck and tail regions of human sperm.It is known that Hsp90 participates in human sperm capacitation through an unknown underlying mechanism.Since Cdc37 is the kinase-specific chaperone of Hsp90,Hsp90 may regulate human sperm capacitation through kinases.It has been found that two mitogen-activated protein kinases(MAPK)are expressed in human sperm,namely extracellular signal-regulated kinase 1/2(Erk1/2)and p38,and their expression regions in sperm are similar to Hsp90 and Cdc37.It has been reported that phosphorylated-Erk1/2(p-Erk1/2)can promote sperm hyperactive motility and acrosome reaction,while phosphorylated-p38(p-p38)inhibits sperm motility.Therefore,in this study,we explored how Hsp90 regulates human sperm capacitation through Erk1/2 and p38 MAPK signaling pathways.Purpose: Previously,we found that Hsp90 and its co-chaperon Cdc37 are involved in the regulation of human sperm capacitation,but the specific mechanism is still unclear.In this experiment,we propose that Hsp90 may regulate the new mechanism of human sperm capacitation through the Erk1/2 and p38 MAPK signaling pathways,which will help to understand the mechanism of sperm capacitation and provide a basis for the establishment of a molecular detection method with high sensitivity and strong specificity,and then provides new diagnostic ideas for unexplained male infertility.Method: The Hsp90 specific inhibitor 17-allylamino-17-demethoxygeldanamycin(17-AAG)was used to treat human sperm during sperm capacitation.After that,a computerassisted sperm analyzer(CASA)was used to detect sperm motility.The FITC-Pisum sativum agglutinin(PSA-FITC)was used to detect the sperm acrosome reaction.Coimmunoprecipitation(Co-IP)was used to evaluate the interaction between Hsp90 and Cdc37,Erk1/2 and p38.Western blot analysis was used to evaluate Erk1/2 and p38 protein expression and phosphorylation levels.Image J software was used to analyze the gray value of Western blot.The data were analyzed by SPSS software,and P<0.05 indicated significant difference.This study explores the molecular mechanism of Hsp90 and cochaperon Cdc37 regulating human sperm capacitation.Results: The analysis of sperm motility parameters and the analysis of acrosome reaction after capacitation showed that the hyperactivated motility and acrosome reaction of the sperm after 17-AAG treatment were inhibited,suggesting that Hsp90 was involved in human sperm capacitation.In addition,Co-IP experiments showed that 17-AAG reduced the interaction between Hsp90 and Cdc37,Erk1/2,and p38.Western blot analysis showed that the level of Erk1/2 protein expression and its phosphorylated form in sperm decreased after 17-AAG treatment,indicating that Erk1/2 became unstable and degraded after dissociation from the Hsp90-Cdc37 complex.Phosphorylation levels are also reduced.However,p38 was separated from the Hsp90 protein complex,its own protein expression level did not change,and the phosphorylation level increased,suggesting that it may be activated by autophosphorylation.Conclusion: The results show that Hsp90 interacts with Cdc37 during sperm capacitation,forms protein complexes with Erk1/2 and p38,maintains the stability of Erk1/2 protein,promotes its phosphorylation state,and maintains the dephosphorylation state of p38.Combined with literature reports,phosphorylated Erk1/2 can promote sperm hyperactive motility and acrosome reaction,while phosphorylated p38 inhibits sperm motility.These indicate that Hsp90 and its co-chaperon Cdc37 regulate human sperm capacitation through the Erk1/2 and p38 MAPK signaling pathways.