Abstract
BackgroundHeat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s). As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37. Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility. Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.MethodsHuman sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation. Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation. The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining. The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments. Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation.ResultsHuman sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation. In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex. Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased. Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38. Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation.ConclusionsTaken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction. In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.
Highlights
Heat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction
Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) form a protein complex with extracellular signalregulated kinase 1/2 (Erk1/2) and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 mitogen-activated protein kinases (MAPKs) signaling pathways
Since 17-AAG is a geldanamycin derivative with an allylamino group at position 17 of the scaffold structure and a similar biological action [38], 17-AAG may reduce Hsp90Cdc37 levels by decreasing Hsp90 expression in human sperm, explaining this discrepancy. These findings suggest that 17-AAG reduces levels of the Hsp90-Cdc37 protein complex during human sperm capacitation, thereby affecting kinases recruited to Hsp90-Cdc37 via Cdc37
Summary
Heat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. We found that both Hsp and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signalregulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp and Cdc. Spermatozoa that have just been ejaculated cannot fertilize oocytes and must undergo a series of biochemical and physiological events in the female reproductive tract to become fertilization competent. This process is known as sperm capacitation [1]. Capacitated sperm usually exhibit protein tyrosine phosphorylation, hyperactivated motility, and acrosome reaction initiation [2], which aids the fusion of sperm into an oocyte. The mechanisms that act downstream of PKA during human sperm capacitation remain unclear
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