Hsp90与共伴侣Cdc37通过Erk1/2及p38信号通路调控人精子获能的分子机制研究

Sperm capacitation is a prerequisite for acrosome reaction, sperm-egg fusion and fertilization under normal physiological conditions. Protein phosphorylation is an important signature for sperm capacitation. As a highly conserved molecular chaperone, heat shock protein 90 (Hsp90) plays important roles in maintenance of cell functions and regulation of signaling pathways. In the previous studies, we found that human sperm Hsp90 affects protein tyrosine phosphorylation and participates in progesterone-stimulated sperm capacitation. Further studies indicate that Hsp90 and its co-chaperone Cdc37 are expressed in the tail of human sperm. They can interact with each other and form protein complex. As Erk1/2 and p38 are also co-located in the tail of human sperm, and their Thr/Tyr phosphorylation are correlated with sperm capacitation. These results suggesting that the regulation of Hsp90 and Cdc37 in human sperm capacitation is associated with Erk1/2 and p38 signaling pathways. However the molecular mechanism is still unknown. Therefore, this study will focus on Hsp90 and Cdc37 to detect whether they can regulate Erk1/2 and p38 signaling pathways through Src; whether Hsp90 and Cdc37 can recruit protein kinases in Erk1/2 pathway and influence their phosphorylation level; as well as how Hsp90 and Cdc37 regulate sperm p38 pathway, through canonical p38 pathway or noncanonical p38 autophosphorylation pathway. The results of the study will reveal the mechanism of Hsp90 and Cdc37 in regulating human sperm capacitation. This will improve the regulation network of sperm capacitation and provide new theory for diagnosis and treatment of unexplained male infertility.

获能是正常生理条件下精子顶体反应及受精的必要前提，蛋白磷酸化水平是评价精子获能的重要指标。分子伴侣Hsp90有维持细胞功能、调节信号通路等重要功能。我们前期研究发现，Hsp90影响人精子酪氨酸磷酸化，参与调控精子获能；Hsp90与共伴侣Cdc37在人精子尾部表达，两者有相互作用，与人精子尾部的Erk1/2和p38蛋白定位十分一致，且该蛋白苏/酪氨酸磷酸化与获能密切相关，提示Hsp90和Cdc37参与调控精子Erk1/2及p38信号通路影响获能，但目前其分子机制不清楚。本项目拟围绕Hsp90与Cdc37开展四个层面研究：确定Hsp90与Cdc37是否通过调节Src影响上述信号通路？阐明其如何调节Erk1/2通路影响获能？明确它们怎样调控p38通路？是否存在p38自磷酸化途径？旨在揭示Hsp90与Cdc37参与调控精子获能进而影响受精的新机制，为填补精子网络调控理论及诊治男性不育提供科学依据。

热休克蛋白90（Heat shock protein 90, Hsp90）是一种高度保守的分子伴侣，具有维持细胞功能、调控细胞信号等重要的生物学功能。细胞分裂周期蛋白37（Cell division cycle protein 37, Cdc37）是Hsp90激酶特异性共伴侣。前期研究发现Hsp90和Cdc37均在人精子中表达，二者均定位于人精子头部和尾部区域，存在相互作用。Cdc37极有可能招募各种激酶，结合到分子伴侣Hsp90上，调控激酶稳定性及活性，进而调控人精子获能。在国家自然科学基金的资助下，我们在本项目中围绕Hsp90和Cdc37在人精子获能过程中调控MAPK信号通路开展研究。我们按照项目计划书设计的研究内容和技术路线，取得了以下一些新的重要研究结果：（1）发现Hsp90参与调控人精子获能后的超激活运动和顶体反应，影响获能及受精过程。（2）抑制Hsp90，可影响人精子中Hsp90-Cdc37复合物水平，进而影响Cdc37招募激酶到分子伴侣Hsp90上。（3）抑制Hsp90，影响Hsp90与Erk1/2的相互作用。Erk1/2从复合物中解离下来，随之降解，酶活性下降。（4）抑制Hsp90，影响Hsp90与p38的相互作用，p38从复合物中解离下来，发生磷酸化激活。Erk1/2磷酸化促进人精子超激活运动和顶体反应，而p38磷酸化抑制人精子运动，因此Hsp90通过维持Erk1/2磷酸化、抑制p38磷酸化而帮助人精子获能。这些研究结果为Hsp90和Cdc37在人精子获能过程中的作用机制提供新的见解，为深入揭示人类受精机制提供科学依据，为诊治不明原因性男性不育提供新的思路。