Intercellular networks and luminal acidification in the male reproductive tract

精子在穿越附睾体时获得活动能力和受精能力。附睾腔是酸性的，碳酸氢盐浓度很低：这两个因素对精子在成熟和储存过程中保持静止都很重要。在之前的资助时期，我们表明腔酸化是由透明细胞通过质子泵V-ATPase实现的，并且它可以通过与周围上皮细胞的串扰来调节。我们现在计划使用一种系统生物学的方法来剖析主细胞和透明细胞之间的细胞间通信网络，该方法将信号通路中的分子事件与体内完整附睾中的细胞事件联系起来。我们认为，在性唤醒过程中，基底外侧旁分泌因子对主细胞的刺激导致随后通过分泌到附睾腔的三磷酸腺苷和碳酸氢盐激活透明细胞。这将使流明的pH值恢复到其静止的酸值。或者，这些因素中的一些可能会直接刺激透明细胞分泌质子。在目标1A中，将使用多光子活体显微镜(IVM)、质子分泌的功能离子通量分析和细胞内钙测量来检测基底外侧激素和激动剂刺激主细胞后的透明细胞激活。这些效应将与鲁米那、腺苷和碳酸氢钠引起的效应进行比较。目的1B将利用LC-MS/MS、RT-PCR、原位杂交、Western blotting和免疫荧光等方法，鉴定与腔介质(ATP、腺苷和碳酸氢盐)反应有关的心尖部嘌呤能受体(如A2B、P2X4)和碳酸氢盐转运体(如NBC3)。将分析它们在透明细胞激活中的作用，如目标1A所述。目的2将分别使用一种新的荧光素酶测定法和BCECF-葡聚糖来监测基底外侧刺激后通过IVM主细胞的腔内ATP和碳酸氢盐的分泌。将研究关键的顶端转运体和调节因子在三磷酸腺苷(如ClC-3，CFTR)和碳酸氢盐(如SLC26A6)分泌中的作用。在男性生育中起关键作用的cftr的作用将在永生化附睾细胞系中进一步剖析，并将确定囊性纤维化相关cftr突变的后果。目标3将探索激素和激动剂对透明细胞的直接激活，而不涉及主要细胞。细胞形态、V-ATPase顶端募集、V-ATPase依赖的质子分泌和细胞内钙离子的分泌将通过流式细胞仪从我们独特的转基因小鼠中分离出来。这些研究将为上皮细胞协同工作以建立和维持允许精子成熟和静止储存的酸性管腔环境的机制提供新的见解。总之，我们建议使用多学科方法来解决一个问题，这个问题对于我们理解睾丸后男性生育能力的调节是至关重要的。这里产生的数据将提供对男性生殖道的新见解，以及一般酸/碱运输上皮的生理学。

Spermatozoa acquire their motility and fertilization capacity as they traverse the epididymis. The lumen of the epididymis is acidic and has a low bicarbonate concentration: both factors are important for keeping sperm quiescent during their maturation and storage. In the previous funding periods, we showed that luminal acidification is achieved by clear cells, via the proton pumping V-ATPase, and that it can be regulated via crosstalk with surrounding epithelial cells. We now plan to dissect the intercellular communication networks between principal cells and clear cells using a systems biology approach that links molecular events in signaling pathways to cellular events in the intact epididymis in vivo. We propose that stimulation of principal cells by basolateral paracrine factors during sexual arousal leads to subsequent activation of clear cells via ATP and bicarbonate that are secreted into the epididymal lumen. This would allow luminal pH to be restored to its resting acidic value. Alternatively, some of these factors might directly stimulate clear cells to secrete protons. In Aim 1A, multiphoton intravital microscopy (IVM), functional ion flux assays of proton secretion and intracellular calcium measurements will be used to examine clear cell activation following stimulation of principal cells by basolateral hormones and agonists. These effects will be compared with those elicited by luminal ATP, adenosine and bicarbonate. Aim 1B will identify and characterize the apical purinergic receptors (e.g. A2B, P2X4), and bicarbonate transporters (e.g. NBC3) involved in the response to luminal mediators (ATP, adenosine and bicarbonate), using LC-MS/MS, RT-PCR, in situ hybridization, western blotting and immunofluorescence. Their role in clear cell activation will be analyzed, as described in Aim 1A. Aim 2 will monitor luminal ATP and bicarbonate secretion by principal cells by IVM following basolateral stimulation, using a novel luciferase assay and BCECF-dextran, respectively. The role of critical apical transporters and regulators involved in ATP (e.g. ClC-3, CFTR) and bicarbonate (e.g. SLC26A6) secretion will be examined. The role of CFTR, which plays a critical role in male fertility, will be further dissected in immortalized epididymal cell lines, and the consequences of cystic fibrosis-associated CFTR mutations will be determined. Aim 3 will explore the direct activation of clear cells by hormones and agonists, without principal cell involvement. Cell morphology, V-ATPase apical recruitment, V-ATPase-dependent proton secretion and intracellular calcium will be examined in EGFP-expressing clear cells isolated by FACS from our unique transgenic mice. These studies will provide new insights into the mechanisms by which epithelial cells work in a concerted manner to establish and maintain an acidic luminal environment that is permissive for sperm maturation and storage in a quiescent state. In summary, we propose to use a multidisciplinary approach to address a problem that is central to our understanding of post-testicular regulation of male fertility. Data generated here will provide novel insights into the male reproductive tract in particular, as well as the physiology of acid/base transporting epithelia in general.