附睾基底细胞跨膜传感与钙稳态的功能对精子成熟和储存在男性生殖道的研究

Establishing a congenial environment for the proper functioning of epithelia is essential for health and yet a complicated process. One epithelium in which a favorable milieu is essential for function is found in the epididymis of the male reproductive tract - an indispensable organ for sperm to acquire their fertilization capacity and motility as well as protection during storage. One unique feature in the epididymal lumen is low calcium acidic environment, which is critical for sperm maturation and male fertility. Infertility is nowadays a common public health problem and affecting one in six of reproductive-aged couples across the world, and a progressive reduction in male fertility has been observed in men over the past few decades. However, our knowledge of the causes of male infertility is limited. Unraveling of the key factors and mechanisms involved in calcium metabolism in acidic environment in the epididymis has far-reaching implications for human reproduction that it has equal potential to provide insights into the causes of some unexplained male infertility cases, yet may also provide insight into the development of novel male contraceptive. Recently the applicant's paradigm-shifting discovery shows that the so-called basal cells in fact extend "antenna-like" body projections that cross the tight-junction barrier to reach the lumen. This previously unrecognized property of basal cells is observed in several tissues located in the male reproductive, gastrointestinal and upper respiratory tracts, indicating that it is a widespread phenomenon. This prompts the necessary of re-interpretation of our understanding of epithelial biology, apical sensing, and signal transduction in health and disease. In addition, it has been reported that calcium-regulation hormonal vitamin D receptor and epithelial calcium ion channel TRPV6 knock out mice showed severely impaired male fertility. The potential role of the luminal reaching basal cells in the calcium homeostasis in the epididymis will be therefore examined in this study.3D confocal immunofluorescence microscopy and western blotting will be used to detect the expression and cellular localization of the calcium metabolism regulatory proteins in the epididymis. Functional studies will also be performed by measuring calcium transport and ionic currents using electrophysiology methods. Transgenic mice with cell-specific fluorescent proteins and mice with genetic deletion of occludin protein will also be employed. These studies will help to elucidate key cellular mechanisms that regulate the spatial and temporal calcium metabolism in the epididymis.

男性生殖道的上皮细胞制造一个适宜的腔内环境确保精子质量和男性生育能力。在过去几十年男性生育能力正逐步下降, 但我们对其原因及知识是有限的。附睾内的低钙酸性环境对精子成熟、储存和保护是至必须的,但其中机制尚不完全清楚。申请人最近的重要突破性发现揭示了附睾基底细胞实际上会伸出"天线状" 的突触穿过紧结蛋白屏障接触到管腔的顶端表面并可以充当为管腔内激素的传感器和发射器控制酸平衡。这个"顶端向性"特征在男性生殖道、唾液腺和上呼吸道都观察到，表明这是一个广泛的现象，促使我们对上皮细胞生物学、管尖传感和信号转导在健康和疾病的认识有重新诠释的必要。最近的研究显示出调节钙稳态的维生素D受体和上皮钙离子通道TRPV6基因敲除小鼠的雄性生育力都表现出严重受损。本研究的目的是揭示维生素D内分泌激素系统在附睾钙稳态的关键作用，以及基底细胞管腔采样和跨膜传感的功能可能扮演的角色，从而构建一个附睾钙稳态理论

男性生殖道的上皮细胞制造了一个适宜的腔内环境，用以确保精子质量和男性生育能力。在过去的几十年里，男性生育能力逐步下降,但我们对其原因及机制的认识是有限的。其中，附睾管腔内的低钙酸性微环境对精子成熟、储存和保护是必须的，其机制目前尚不完全清楚。本研究围绕附睾上皮细胞互作功能调控附睾微环境这一大科学问题，以钙稳态为切入点，进而阐明附睾调控精子成熟以及生殖功能的分子机制。具体来讲，本项目从钙稳态和附睾屏障两个方面，分别研究了附睾上皮细胞中TRPV6对钙离子运输的电生理特性和紧密连接蛋白occludin对附睾微环境以及对精子成熟的调控。我们发现对细胞外酸钙敏感的上皮钙通道TRPV6与钙依赖性的阴离子通道TMEM16A有电偶联的相互作用，这个偶联体不仅在调控附睾钙稳态中起着重要作用，而且它们在调节附睾腔液分泌以及微环境形成与维持中也扮演着重要角色。另外，关于附睾屏障，已知紧密连接蛋白occludin缺失会导致雄性小鼠不育，但具体机制并未深入研究。在本项目中，通过研究occludin敲除小鼠模型，我们发现在年轻小鼠中，occludin缺失对精子发生并无影响。但附睾功能发生异常，继而导致精子功能障碍和雄性不育。进一步研究表明occludin缺失时，附睾体部出现胀大并伴随有炎症发生，附睾尾部精子的数量减少、形态异常且受精能力丧失。而造成这些异常的原因主要是附睾中亮细胞数量减少导致附睾腔微环境的破坏，比如附睾腔酸碱平衡失调。综上所述，我们从钙稳态和附睾屏障角度出发，通过研究TRPV6对钙离子运输的电生理特性以及occludin对附睾上皮细胞和附睾精子的调控，揭示了附睾腔微环境稳态形成与维持及其调控精子成熟的新机制。