Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences / Hangzhou Medical College, 310013, Hangzhou, Zhejiang, China.
Reprod Biol Endocrinol. 2021 Mar 4;19(1):39. doi: 10.1186/s12958-021-00723-2.
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. 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.
Human 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.
Human 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.
Taken 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.
热休克蛋白 90（Hsp90）是一种高度丰富的真核分子伴侣，在客户蛋白成熟、蛋白质折叠和降解以及信号转导中发挥重要作用。此前，我们发现 Hsp90 和其共伴侣细胞分裂周期蛋白 37（Cdc37）均在人精子中表达。已知 Hsp90 参与人类精子获能，但具体机制尚不清楚。由于 Cdc37 是 Hsp90 的激酶特异性共伴侣，Hsp90 可能通过其他激酶调节人类精子获能。据报道，两种主要的丝裂原活化蛋白激酶（MAPK），细胞外信号调节激酶 1/2（Erk1/2）和 p38，在人类精子中的表达位置与人精子中的 Hsp90 和 Cdc37 相同。已显示磷酸化的 Erk1/2 可促进精子超激活运动和顶体反应，而磷酸化的 p38 则抑制精子运动。因此，在这项研究中，我们探讨了 Hsp90 是否通过 Erk1/2 和 p38 MAPK 信号通路来调节人类精子获能。
在获能期间，用人精子特异性抑制剂 17-烯丙基-17-去甲氧基格尔德霉素（17-AAG）处理人精子。使用计算机辅助精子分析（CASA）检测精子运动和超激活。通过使用荧光素异硫氰酸酯结合的豌豆凝集素（PSA-FITC）染色分析精子顶体反应。使用共免疫沉淀（Co-IP）实验评估 Hsp90、Cdc37、Erk1/2 和 p38 之间的相互作用。使用 Western blotting 分析评估蛋白表达和磷酸化水平。
17-AAG 抑制人精子超激活和顶体反应，提示 Hsp90 参与人类精子获能。此外，Co-IP 实验显示 17-AAG 降低了 Hsp90 和 Cdc37 之间的相互作用，导致 Erk1/2 从 Hsp90-Cdc37 蛋白复合物中解离。Western blotting 分析显示 Erk1/2 及其磷酸化形式的水平随后降低。Hsp90-Cdc37 复合物的减少也影响了 Hsp90 和 p38 之间的相互作用。然而，p38 从 Hsp90 蛋白复合物中解离并通过自身磷酸化被激活。
总之，我们的研究结果表明 Hsp90 参与了人类精子的超激活和顶体反应。特别是，Hsp90 及其共伴侣 Cdc37 与 Erk1/2 和 p38 形成蛋白复合物，调节它们的激酶活性。这些结果表明，Hsp90 通过 Erk1/2 和 p38 MAPK 信号通路调节人类精子获能。
