附睾γ-谷氨酰循环与钙稳态相互作用对精子成熟微环境形成与调控的机制研究

The production of high quality sperm with normal structure and function is the benchmark of male fertility. This is achieved by normal spermatogenesis in the testis and functional maturation in the epididymis. Epididymis establishes a unique luminal microenvironment for the immature testicular sperm to undergo a dynamic but tightly-controlled sequential changes, to let them become functionally competent – the ability of fertilizing an oocyte. For instance, the epididymal fluid is acidic and progressively low in calcium concentration down the epididymal tubule. Our previous studies have demonstrated an intriguing intercellular communication network underlying the barrier function of epididymal epithelium. Previously, we have discovered that basal cells are involved, in a paracrine manner, in the regulation of fluid reabsorption via the solute-transporting principal cells, and to modulate the luminal acidification via the proton-pump V-ATPase expressing clear cells. Recently, we have also reported that the epithelial calcium channel TRPV6 can electrically couple with the calcium-dependent chloride channel TMEM16A in principal cells of specific regions of epididymis. This study is not only consistent with the putative role of TRPV6 in calcium homeostatic regulation and male fertility, but also introduces a new TRPV6-TMEM16A coupled chloride-driven secretion mechanism in the epididymis, which is a pH-sensitive calcium-dependent and electrically regulatory process. Another feature of epididymal microenvironment is the very high glutamate concentration in the epididymal luminal fluid, which is as high as in the pre-synaptic verve ending vesicles and over three-hundred times of that in the blood. The biology need of such high glutamate in the caput epididymides is unknown, and the interplay of calcium homeostasis and glutamate biology is completely unexplored in sperm physiology, in spite of that both physiological features (i.e. high glutamate and low calcium) are essential for epididymal function. Glutamate metabolism has been known to link to oxidative energy metabolism and calcium homeostasis as well as glutathione redox balance. Consistent with the important role of γ‐glutamyl cycle in life processes, the glutathione synthesis enzyme GCLC knockout mice are embryonic lethal, whereas the γ‐glutamyl transpeptidase GGT knockout mice are infertile. This suggests that γ‐glutamyl cycle is particularly important in the reproduction axis. The goal of study is aimed to elucidate the functional mechanism underlying the interplay of gamma-glutamyl cycle and calcium homeostasis in the epididymis. The ultimate goal is to elucidate the mechanisms involved in the normal physiology of epididymal luminal microenvironment formation for sperm maturation, and to provide sights for the better understanding of the pathophysiology behind the more and more concerned “idiopathic” male infertility cases in the clinics.

附睾上皮细胞通过相互作用建立适宜的附睾腔微环境，这对于精子成熟和男性生育能力至关重要。调查统计发现，在过去几十年，男性不育比例呈上升趋势，但人们对其潜在的致病因素却知之甚少。申请人之前的研究工作表明附睾上皮在屏障功能的基础上形成了错综复杂的细胞通讯网络，不同的细胞协同合作，为精子健康提供了适宜微环境。在这个通讯网络中，附睾钙稳态形成的调控网络以及对精子调控的分子机制尚不完全清楚，而且钙稳态和氧化还原平衡在细胞内存在着相互调控，但其具体机制并不清楚。已知附睾精子需要少量的活性氧激活一些生理反应, 但是过量则产生有害效应。其内在机制还没有被阐明。因此，本课题以氧化还原调节系统γ-谷氨酰循环和钙稳态的关键蛋白为切入点来阐明氧钙互作平衡在附睾微环境形成中的机制及其对精子成熟的影响，旨在建立γ-谷氨酰循环与钙稳态的互作在附睾微环境与精子成熟的理论模型。

附睾在精子遗传变异和男性生育中起着重要作用，越来越多的证据表明，附睾上皮细胞建立的管腔微环境对附睾的正常功能至关重要。附睾中的低Ca2+稳态和氧化还原平衡是确保精子成熟的关键因素。我们的研究旨在揭示适宜的附睾管腔微环境所需的因素及其调节机制，特别是与这些独特的微环境特征相关的因素。我们先前的研究表明，维生素D相关的TRPV6-TMEM16A通道耦合器只出现在附睾头部和尾部远端。在本研究中，我们还发现维生素K依赖性GGCX介导的基质Gla蛋白（MGP）羧化以空间互补的方式调节附睾中的Ca2+稳态。我们发现羧化MGP在促进Ca2+依赖性蛋白质聚集中起着重要作用。人类GGCX基因中的一个SNP位点也与弱精子症有关。在深入研究中，我们提供了进一步的证据来支持管腔基质Ca2+作为羧化MGP清除小鼠附睾细胞外微环境中代谢产物的辅因子的概念。MGP介导的与分泌型载脂蛋白APOJ的聚集通过改变外在Ca2+浓度而改变。整合生物信息学分析表明，上皮管腔侧受体和多种维生素（如维生素D和K）参与了附睾中MGP结合聚集体的吸收。有趣的是，维生素K循环依赖性GGCX-MGP低钙调节是一种氧化还原敏感途径，而谷氨酸代谢（即γ-谷氨酰循环）与钙稳态和氧化还原平衡有关。一直以来，附睾管腔液中的谷氨酸浓度都非常高，与突触前末端囊泡中的谷氨酸浓度一样高，是血液中谷氨酸浓度的三百多倍。因此，为了探讨γ-谷氨酰循环在氧化还原敏感性钙稳态中的作用，我们研究了γ-谷氨酰循环的关键酶，即GGT、GCLC和GCLM，以及附睾中关键的相关调节因子。我们的结果表明，在维持还原状态的附睾近端区域，谷氨酸的主要来源是GSH/GSSG氧化还原循环。这一观察结果与钙稳态和氧化还原平衡在附睾中有相互作用的观点是一致的。综上所述，钙依赖的MGP清除增加了通过多种维生素和氧化还原平衡调解途径调控附睾钙稳态的可能性，作为解决精子功能障碍和男性生殖缺陷的杠杆治疗策略。进一步的研究对揭示男性不育症特别是特发性男性不育症的发病机制具有重要意义。