Intercellular networks and luminal acidification in the male reproductive tract

精子在穿过附睾时获得活动能力和受精能力。附睾腔呈酸性，碳酸氢盐浓度较低：这两个因素对于保持精子在成熟和储存期间保持静止都很重要。在之前的资助期间，我们表明管腔酸化是通过透明细胞，通过质子泵V-ATP酶实现的，并且它可以通过与周围上皮细胞的串扰进行调节。我们现在计划解剖主细胞和透明细胞之间的细胞间通讯网络，使用系统生物学的方法，在体内完整的附睾细胞事件的信号通路中的分子事件。我们提出，刺激主细胞的基底外侧旁分泌因子在性唤起导致随后激活透明细胞通过ATP和碳酸氢盐分泌到附睾腔。这将使管腔pH值恢复到其静息酸性值。或者，这些因子中的一些可能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测定和细胞内钙测量来检查基底外侧激素和激动剂刺激主细胞后的透明细胞活化。将这些效应与管腔ATP、腺苷和碳酸氢盐引起的效应进行比较。目的1B将使用LC-MS/MS、RT-PCR、原位杂交、蛋白质印迹和免疫荧光来鉴定和表征参与对管腔介质（ATP、腺苷和碳酸氢盐）的响应的顶端嘌呤能受体（例如A2 B、P2 X4）和碳酸氢盐转运体（例如NBC 3）。如目标1A所述，将分析它们在透明细胞活化中的作用。目的2将分别使用新的荧光素酶测定和BCECF-葡聚糖，通过基底外侧刺激后的IVM监测主细胞的管腔ATP和碳酸氢盐分泌。将检查参与ATP（例如ClC-3、CFTR）和碳酸氢盐（例如SLC 26 A6）分泌的关键顶端转运蛋白和调节剂的作用。CFTR在男性生育力中起着关键作用，其作用将在永生化附睾细胞系中进一步解剖，并将确定囊性纤维化相关CFTR突变的后果。目的3将探索透明细胞的直接激活激素和激动剂，没有主细胞参与。细胞形态，V-ATP酶顶端募集，V-ATP酶依赖性质子分泌和细胞内钙将在通过FACS从我们独特的转基因小鼠分离的表达EGFP的透明细胞中进行检查。这些研究将为上皮细胞以协调一致的方式工作以建立和维持允许精子在静止状态下成熟和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的方法来解决一个问题，这是我们的理解后睾丸调节男性生育能力的核心。这里产生的数据将提供新的见解，特别是男性生殖道，以及一般的生理学的酸/碱运输上皮细胞。

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.