睾丸特异的AQP3剪切异构体在精子低渗适应中的功能及机理

In the journey from the male to female reproductive tract, mammalian sperm experience a physiological hypotonic stress. Adaptation to such hypotonic stress is necessary for sperm to exert normal function in female reproductive tract. We've previously revealed AQP3 as an essential membrane protein for sperm regulatory volume decrease (RVD) upon physiological hypotonicity. AQP3 localized in mouse and human sperm tail, AQP3 knockout sperm increased vulnerability to hypotonic stress with abnormal tail bending, resulting in 50% decrease of male fertility. We now further discovered that AQP3 knockout testis shows up-regulated testis-specific splice variants which could be translated into truncated proteins, suggesting that these AQP3 splice variants are functionally compensating for the loss of original AQP3 in the knockout testis. To demonstrate the func.tion of testis-specific AQP3 splice variants, in this project, we generated two genetically engineered mice strain: 1) a new AQP3 knockout strain deleting all aqp3 splice variants, 2) a transgenic mouse strain overexpressing an AQP3 splice variant (specifically found in previous aqp3 knockout testis) under the testis-specific promoter protamine1. By cross-breeding these two mice strain, we could further get mice that only express the AQP3 splice variant, as to exam its role in sperm osmoadaptation. Moreover, since the AQP3 splice variants sequances do not contain the traditional water permeable domains, therefore suggesting that their functions in sperm RVD is independent on water permeability. We hypothesized that original AQP3 and AQP3 splice variants exert their role by acting as mechanosensors in initiating RVD processes. We'll further test this hypothesis by using cell model systems in vitro.

哺乳动物精子从雄性进入雌性生殖道经历一个生理性低渗应激，适应这种低渗环境是精子在雌性生殖道内行使正常功能的必要前提。申请人在本领域首次发现水通道蛋白AQP3是参与精子体积调控，实现低渗适应的一个关键蛋白。AQP3定位于精子尾部，其敲除鼠精子对低渗环境耐受下降，导致雄鼠生育力下降50%。我们随后进一步发现睾丸中存在组织特异的AQP3蛋白剪切体，且在敲除鼠睾丸中呈代偿性上调，提示这些剪切体的存在可能是敲除鼠保留部分生育力的原因。本课题拟利用两种工具小鼠：1）精子中特异过表达AQP3剪切体的转基因小鼠；2）剔除所有AQP3剪切体的敲除鼠,并通过二者杂交获得只具有AQP3剪切体的小鼠用于在体功能研究，揭示其在精子低渗适应中的功能。此外，由于AQP3剪切体序列不含通水关键结构域，我们推测其调控精子低渗适应的分子机理独立于水通透性，可能作为膜感受器参与细胞体积调控；本课题将对此机理进行深入探讨。

哺乳动物精子从雄性进入雌性生殖道经历一个生理性低渗应激,适应这种低渗 环境是精子在雌性生殖道内行使正常功能的必要前提。申请人在本领域首次发现水通道蛋白 AQP3 是参与精子体积调控,实现低渗适应的一个关键蛋白。AQP3 定位于精子尾部,其敲除 鼠精子对低渗环境耐受下降,导致雄鼠生育力下降 50%。我们随后进一步发现睾丸中存在组 织特异的 AQP3 蛋白剪切体,且在敲除鼠睾丸中呈代偿性上调,提示这些剪切体的存在可能 是敲除鼠保留部分生育力的原因。本项目通过对AQP3+/+和AQP3-/-小鼠的睾丸进行深度测序,发现在AQP3-/-睾丸中出现了大 量新的剪切体形式,其中一些剪切体包含了intron部分，我们通过5’RACE技术对这些新型剪切体的全长进行鉴定，发现了之前从未报道过的睾丸特异性的AQP3剪切体形式。并且在AQP3-/-小鼠的睾丸中,由于exon2-3区段的敲除,导致敲 除鼠睾丸中其他剪切体形式出现大量上升。我们进一步发现睾丸中的AQP3剪切体形式在不同时期呈现动态变化，其中这些剪切体在AQP3-/-小鼠的睾丸中的表达时期明显提前，并且我们发现了一条AQP3剪切体能够编码一个短小的AQP3蛋白，提示其对AQP3-/-精子渗透压适应的功能有代偿作用，是AQP3-/-小鼠维持部分生育力的重要原因。