Intercellular networks and luminal acidification in the male reproductive tract

精子在穿过附睾时获得运动能力和受精能力。附睾的管腔是酸性的，碳酸氢盐浓度低：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。在之前的资助期内，我们证明了腔内酸化是由透明细胞通过质子泵送v - atp酶实现的，并且可以通过与周围上皮细胞的串扰进行调节。我们现在计划使用系统生物学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完整附睾体内的细胞事件联系起来。我们认为，在性兴奋期间，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管腔的ATP和碳酸氢盐激活透明细胞。这将使腔内pH值恢复到其静息酸性值。或者，其中一些因素可能直接刺激透明细胞分泌质子。在Aim 1A中，多光子活体显微镜（IVM）、质子分泌的功能离子通量测定和细胞内钙测量将用于检查基底侧激素和激动剂刺激主细胞后的透明细胞活化。这些效应将与腔内ATP、腺苷和碳酸氢盐引起的效应进行比较。Aim 1B将使用LC-MS/MS、RT-PCR、原位杂交、western blotting和免疫荧光鉴定和表征参与对腔介质（ATP、腺苷和碳酸氢盐）反应的根尖嘌呤能受体（如A2B、P2X4）和碳酸氢盐转运体（如NBC3）。如Aim 1A所述，我们将分析它们在透明细胞活化中的作用。Aim 2将分别使用一种新的荧光素酶测定和bcecf -葡聚糖，通过IVM监测基底外侧刺激后主细胞的腔内ATP和碳酸氢盐分泌。我们将探讨ATP（如ClC-3、CFTR）和碳酸氢盐（如SLC26A6）分泌过程中关键的根尖转运蛋白和调节因子的作用。CFTR在男性生育能力中起着至关重要的作用，将在永生化附睾细胞系中进一步剖析，并确定囊性纤维化相关CFTR突变的后果。目的3将探讨激素和激动剂对透明细胞的直接激活，而不涉及主细胞。在我们独特的转基因小鼠中，细胞形态学、V-ATPase顶端募集、V-ATPase依赖的质子分泌和细胞内钙将被检测。这些研究将为研究上皮细胞协同工作的机制提供新的见解，以建立和维持一个酸性腔内环境，使精子在静止状态下成熟和储存。总之，我们建议采用多学科的方法来解决一个问题，这是我们对睾丸后男性生育能力调节的理解的核心。这里产生的数据将提供新的见解，特别是男性生殖道，以及酸/碱运输上皮的生理一般。

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.