β-防御素19/119 在精子趋化性中的作用及机制研究

Successful fertilization requires the sperm to travel along the female reproductive tract until they reach the egg. However, how these sperm are guided to the fertilization site remains poorly understood. It has long been observed that sperm show directional movement towards follicular fluid, a phenomenon known as sperm chemotaxis, indicating possible involvement of chemoattractants in guiding the sperm along their voyage in female reproductive tract. However, the molecular identity of these chemoattractants and their corresponding receptor(s) on sperm remain elusive. Our recent study has uncovered an important role of β-defensin 1 (DEFB1) in promoting sperm motility through its interaction with chemokine receptor 6 (CCR6) on sperm. Recent studies further found that CCR6 forms a complex with a sperm specific Ca2+ channel (CatSper) which has been implicated in sperm chemotaxis. Our preliminary data show that Defb19 (β-defensin 19) is ascending gradient expression from vagina towards the oviduct and robust expression in the ovary. Further studies found DEFB19/119 (DEFB19/DEFB119) recombination protein is chemotactically active to mouse sperm and human sperm. Most importantly, we found DEFB19/119 is interacting with CCR6 in mouse and human sperm. These observations prompt us to hypothesize that the binding of DEFB19/119 secreted by the cumulus-oocyte complex (COC), oviductal or uterine epithelia to a CCR6/CatSper complex on sperm cooperatively guide the migration of sperm through the female reproductive tract. To test this hypothesis, we propose to use latest method of chemotaxis analysis, in vivo animal models and human infertility specimen to prove DEFB19/119 mediated sperm chemotaxis. We also propose to use PLA staining, Calcium imaging and cAMP detection technology to confirm the mechanism of sperm chemotaxis. The proposed study may provide novel insight into our understanding on the mechanism(s) that guide sperm to the egg by DEFB19/119 secreted from the female reproductive tracts and may lead to the development of new conceptive, infertility diagnosis and treatment.

精子游向卵子是体内成功受精的前提，但精子是如何被引导游向受精发生地的问题尚不清晰。我们已报道，CCR6与CatSper结合形成复合体，而CatSper已知在精子趋化性中发挥作用。本项目前期发现：Defb19和其人类同源基因DEFB119在雌性生殖道中有表达，且初步证明DEFB19/119的重组蛋白可诱导小鼠和人类精子的趋化性。重要的是，研究发现DEFB19/119都可与CCR6相互作用。因此，我们推测：雌性生殖道中分泌的DEFB19/119与精子上CCR6-CatSper复合体的相互作用可能是精子趋向卵子的分子机制。本项目拟采用最新趋化性分析方法和动物模型及临床不孕症标本，进一步证明DEFB19/119介导精子趋化性的准确性及意义；采用蛋白邻位连接检测技术及检测钙离子的浓度和cAMP的生成，确定其诱导趋化性的机制。本项目将揭示新的精子趋向卵子的分子机制，并促进不育症的诊断和治疗手段的发展。

精子的趋化性是引导精子进入卵子的关键机制。然而，生理趋化剂的分子特性及其参与不孕症的过程仍然不清晰。本研究中，我们鉴定了人类DEFB119及其小鼠同源物DEFB19作为一种生理上的精子趋化剂。卵丘-卵母细胞复合体(COC)和女性生殖道上皮分泌的DEFB19/119蛋白触发激发了钙离子内流的信号，促进了获能精子中的趋化作用。在体内，我们通过输卵管结扎来阻止COC的趋化作用，减少了输卵管中的精子数量，而过表达DEFB19则恢复了到达输卵管中的精子数量。敲减或敲除内源性DEFB19显著减少了输卵管中的精子数量，并降低了生育能力。重要的是，我们观察到，在不孕症妇女中，DEFB119的水平与卵泡液的趋化效力之间存在显著的相关性。本研究揭示了DEFB19/119在精子趋化过程中的关键作用，并证实了DEFB119在特发性不育症中的作用及其在精子选择中的应用。