Luminal Acidification in the Male Reproductive Tract

男性生殖道管腔酸化

• 批准号: 7106508
• 负责人: SYLVIE BRETON
• 金额: $ 32.89万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-03 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106508
• 关键词: acid base balance; cell differentiation; cell population study; epididymis; fertility; flow cytometry; fluorescence microscopy; gene expression; genetic promoter element; genetic regulation; genetically modified animals; green fluorescent proteins; laboratory mouse; laser capture microdissection; male reproductive system; phenotype; polymerase chain reaction; reproductive development; sex differentiation; spermatogenesis; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): Millions of couples in the United States are affected by infertility. In a large fraction of these couples, the infertility originates from a male defect that is associated not only with oligozoospermia but also with asthenozoospermia. Spermatozoa acquire their motility and capacity to fertilize as they traverse the epididymis, but our knowledge of the contribution of post-testicular mechanisms to these critical sperm functions is poor. The lumen of the epididymis and vas deferens (VD) is maintained at an acidic pH and has a low bicarbonate concentration: both are important for sperm maturation and for the maintenance of sperm in a quiescent state during their storage period. Thus, defective epididymal acidification might impair male reproductive capacity. Luminal acidification involves a concerted interaction between the different cell types that constitute the epididymal epithelium, mainly the clear and principal cells. We plan to dissect the factors involved in the establishment and maintenance of the mature phenotypes of these cells, as well as factors that coordinate the acid/base transport activities of these differentiated cells. SPECIFIC AIM 1 will examine cell-specific patterns of mRNA and protein expression in the epididymis during: A) post-natal development; and B) hormonal treatment or orchidectomy of adult animals. We will use our novel transgenic (Bl-EGFP) mice, in which enhanced green fluorescent protein (EGFP) is expressed specifically in narrow and clear cells. Selected clear and principal cells will be harvested by fluorescence assisted laser capture microdissection (FLCM), and mRNA expression will be examined by real-time PCR. Selected genes related to acid/base transport (H+ATPase, CAII, NHE3, NBC, etc.) and its regulation (PLC, PKA, PKC, gelsolin, etc) will be studied. Selected genes involved in epithelial cell differentiation, cell shape determination and the retention of specific cell types within the epithelium (cytoskeletal proteins, extracellular matrix proteins and cell adhesion molecules, proteins involved in apoptosis) will also be examined. Protein expression will be analyzed by immunofluorescence and Western blotting. SPECIFIC AIM 2 will characterize cell specific signaling pathways in the epididymis. A) As a sensitive real-time marker of activation of key cell-specific responses to stimuli (e.g. receptor activation leading to H+ATPase exocytosis), intracellular calcium will be measured by spectrofluorometry in positively identified clear and principal cells from our Bl-EGFP mice using Fura-2. B) We will also use a novel assay, developed using these mice, to follow in real time, cell shape variations in individual living clear cells, following activation or inhibition of proton secretion. These studies will help to elucidate key biological mechanisms that establish the proper luminal acid/base balance that leads to the optimum environment for sperm maturation and storage.

描述（由申请人提供）：在美国，数百万对夫妇受到不孕症的影响。在这些夫妇中，有很大一部分不孕症是由男性缺陷引起的，这种缺陷不仅与少精子症有关，也与弱精子症有关。精子在穿过附睾时获得其活动力和受精能力，但我们对睾丸后机制对这些关键精子功能的贡献的了解很少。附睾和输精管（VD）的内腔维持在酸性pH值，并具有低碳酸氢盐浓度：两者对于精子成熟和维持精子在储存期间处于静止状态都很重要。因此，附睾酸化缺陷可能会损害男性生殖能力。管腔酸化涉及构成附睾上皮的不同细胞类型（主要是透明细胞和主细胞）之间的协同相互作用。我们计划解剖参与这些细胞的成熟表型的建立和维持的因素，以及协调这些分化细胞的酸/碱运输活动的因素。特异性目的1将检查在以下过程中附睾中mRNA和蛋白质表达的细胞特异性模式：A）出生后发育;和B）成年动物的激素治疗或附睾切除术。我们将使用我们的新的转基因（Bl-EGFP）小鼠，其中增强型绿色荧光蛋白（EGFP）的表达，特别是在狭窄和透明的细胞。通过荧光辅助激光捕获显微切割（FLCM）收获选定的透明和主细胞，并通过实时PCR检查mRNA表达。选择与酸/碱转运相关的基因（H+ ATP酶、CAII、NHE 3、NBC等）并对其调控机制（PLC、PKA、PKC、凝溶胶蛋白等）进行了研究。还将检查参与上皮细胞分化、细胞形状决定和上皮内特定细胞类型保留的选定基因（细胞骨架蛋白、细胞外基质蛋白和细胞粘附分子、参与细胞凋亡的蛋白）。将通过免疫荧光和Western印迹分析蛋白质表达。特异性AIM 2将表征附睾中的细胞特异性信号通路。A）作为对刺激的关键细胞特异性应答的激活（例如受体激活导致H+ ATP酶胞吐）的灵敏实时标记物，将使用Fura-2通过荧光分光光度法在来自我们的Bl-EGFP小鼠的阳性鉴定的透明和主细胞中测量细胞内钙。B）我们还将使用使用这些小鼠开发的新测定法来真实的跟踪在激活或抑制质子分泌后单个活透明细胞中的细胞形状变化。这些研究将有助于阐明建立适当的内腔酸/碱平衡的关键生物学机制，从而为精子成熟和储存提供最佳环境。