Luminal Acidification in the Male Reproductive Tract

男性生殖道管腔酸化

• 批准号: 7236225
• 负责人: SYLVIE BRETON
• 金额: $ 31.94万
• 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/7236225
• 关键词: Acid-Base Equilibrium; Acids; Acute; Address; Adenosine; Adrenergic Agonists; Adult; Affect; Agonist; Androgens; Animal Model; Apical; Apoptosis; Area; Bicarbonates; Biological; Biological Assay; Bradykinin; Calcium; Calcium Signaling; Cations; Cell Adhesion Molecules; Cell Differentiation process; Cell Shape; Cell Signaling Process; Cells; Chronic; Clear Cell; Condition; Couples; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Defect; Development; Diethylstilbestrol; Doctor of Philosophy; Environment; Epididymis; Epithelial; Epithelial Cells; Epithelium; Esters; Ethinyl Estradiol; Exocytosis; Extracellular Matrix Proteins; Fertility; Fluorescence; Fluorescence Spectrometry; Flutamide; Funding; Fura-2; Gelsolin; Gene Activation; Genes; Genitourinary system; Gland; Goals; Harvest; Heavy Metals; Hormonal; Hormones; ICI 182780; Immunofluorescence Immunologic; Individual; Infertility; Intervention; Ion Transport; Ions; Knowledge; Laboratories; Life; Maintenance; Measures; Messenger RNA; Microscope; Modeling; Mus; Neurotransmitters; Orchiectomy; Pattern; Phenotype; Play; Polymerase Chain Reaction; Population; Process; Production; Progress Reports; Proteins; Proton-Translocating ATPases; Protons; Rate; Receptor Activation; Regulation; Reproductive Physiology; Request for Applications; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; SNAP receptor; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Sperm Maturation; Steroids; Stimulus; Study models; Surface; Testosterone; Time; Tissues; Transgenic Mice; Transgenic Organisms; United States; United States National Institutes of Health; Variant; Vas deferens structure; Vasopressins; Western Blotting; Work; base; cell motility; cell type; cellular microvillus; enhanced green fluorescent protein; extracellular; gastrointestinal microvillus; laser capture microdissection; mRNA Expression; male; mature animal; miniaturize; myosin VI; novel; postnatal; programs; protein expression; reconstruction; reproductive; response; solute; sperm cell; sperm function; steroid hormone; tool

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）：美国有数百万夫妇受到不孕不育的影响。在这些夫妇中，很大一部分不育源于男性缺陷，这种缺陷不仅与少精症有关，还与弱精子症有关。精子在穿过附睾时获得活力和受精能力，但我们对睾丸后机制对这些关键精子功能的贡献知之甚少。附睾和输精管 (VD) 的管腔保持酸性 pH 值并具有较低的碳酸氢盐浓度：两者对于精子成熟以及精子在储存期间维持静止状态都很重要。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。我们计划剖析参与这些细胞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/碱转运活动的因素。具体目标 1 将检查附睾中 mRNA 和蛋白质表达的细胞特异性模式：A) 产后发育； B) 成年动物的激素治疗或睾丸切除术。我们将使用我们的新型转基因 (Bl-EGFP) 小鼠，其中增强型绿色荧光蛋白 (EGFP) 在狭窄且透明的细胞中特异性表达。将通过荧光辅助激光捕获显微切割 (FLCM) 收获选定的透明细胞和主细胞，并通过实时 PCR 检查 mRNA 表达。将研究与酸/碱转运相关的选定基因（H+ATPase、CAII、NHE3、NBC 等）及其调节（PLC、PKA、PKC、凝溶胶蛋白等）。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）。将通过免疫荧光和蛋白质印迹分析蛋白质表达。 SPECIFIC AIM 2 将表征附睾中的细胞特异性信号传导途径。 A) 作为对刺激的关键细胞特异性反应激活的敏感实时标记（例如，受体激活导致 H+ATP 酶胞吐作用），将使用 Fura-2 在我们的 Bl-EGFP 小鼠中通过分光荧光法对阳性识别的透明细胞和主要细胞进行测量。 B) 我们还将使用一种利用这些小鼠开发的新型测定法，在激活或抑制质子分泌后，实时跟踪单个活透明细胞的细胞形状变化。这些研究将有助于阐明建立适当的管腔酸/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。