HUMAN SPERM ZONA ACCEPTOR--ENVIRONMENTAL EFFECTS

人类精子带受体——环境影响

• 批准号: 6178327
• 负责人: SUSAN H BENOFF
• 金额: $ 32.44万
• 依托单位: NORTH SHORE UNIVERSITY HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 2002-08-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178327
• 关键词: acrosome; calcium flux; clinical research; environmental toxicology; fertility; human subject; in vitro fertilization; lead; male; molecular cloning; nucleic acid probes; nucleic acid sequence; occupational hazard; phospholipase C; potassium channel; progesterone receptors; protein isoforms; receptor expression; sperm; voltage gated channel

We are interested in developing tests for effects of metal exposures upon human sperm function. We propose to follow up findings from our current prospective study of males of couples undergoing in vitro fertilization (IVF) that 42/95 subjects had elevated blood and semen Pb2+ (greater than 40 microgram/dcl), and that rates of female partner oocyte fertilization in IVF correlated inversely (r=-448, P less than 0.001) with these Pb+2 levels. In this cohort, seminal plasma Pb+2 correlated inversely (r=-.523, P less than 0.01) with the ability of sperm to undergo a progesterone-stimulated acrosome reaction (PSAR). When visualized with probes for non-nuclear progesterone receptor (NNPR) and mannose lectin (ML), the principal site of Pb+2 blockade lay at an early step of the PSAR. Capacitation, as determined by expression levels of NNPR and ML was affected, but slightly compared to controls, but the relocation of NNPR and ML from the anterior sperm head to the sperm equatorial segment (which normally precedes Ca+2 influx through a voltage-gated calcium channel and in turn triggers fusion of acrosomal and cell membranes) was largely arrested. Blood Pb+2 was unrelated to hormone levels, suggesting Pb+2 effects were not mediate through endocrine disruptors. As adding either micromolar Pb+2 or specific inhibitors of delayed-rectifier voltage-gated K+ channels from human and rat testes cDNA also suggested such channels exist in human sperm. Polymorphisms found in Northern hybridizations suggest that individuals who differed in their productive response to similar [Pb+2} challenge may have variant K+ channel isoforms. We will complete sequencing of the human sperm delayed-rectifier voltage-gated K+ channel, and will attempt to clone the human form of the hamster Ca2+-activated K+ channel. We will develop DNA probes to characterize alternatively spliced K+ channels. We will make a second prospective study of 150 male IVF patients to confirm our findings and seek to identify further environmental risk factors for Pb2+ effects on human sperm function, correlating levels of expression of variant K+ channel mRNAs in sperm with blood and seminal plasma [Pb2+] and with sperm NNPR expression and the PSAR. We will also investigate a possible mechanism for Pb2+'s reproductive effects on male sperm, that it affects sperm K+ channel function or interferes with early signal transduction after membrane depolarization. We will use metal-ion-sensing fluorescent probes, and specific inhibitors to probe the effects of PB2+ upon ion fluxes. We will employ phospholipase C assays and competition assays to probe the effects of Pb2+ upon inositol phosphate secondary messages. The goal of this application is to determine the mechanisms of injury to the male reproductive tract resulting from environmental and occupational exposure to heavy and transition metal ions. The focus being on the effects of lead exposure on acrosome reaction insufficiency and male infertility. The strength application include the expertise and experience of the principal investigator Dr. Benoff and her research team, the novel approach to analysis of the problem.

我们对开发金属暴露影响的测试很感兴趣 对人类精子功能的影响。我们建议跟进我们的调查结果 男性体外受精夫妇的前瞻性研究现状 受精(IVF)：42/95人血液和精液升高 Pb2+(大于40微克/DCL)和女性伴侣比率 卵母细胞受精与体外受精呈负相关(r=-448，P< 0.001)与这些铅+2水平。在这个队列中，精浆中的铅+2 相关系数r=-.523，P<0.01 精子经历黄体酮刺激的顶体反应(Psar)。 当用非核孕酮受体(NNPR)探针进行可视化时 和甘露糖凝集素(ML)，这是铅+2阻断的主要部位。 Psar的早期步骤。获能，由表达式决定 NNPR和ML水平受到影响，但与对照组相比略有下降， 但NNPR和ML从前精子头到精子头的移位 精子赤道段(通常在Ca+2流入之前通过 电压门控钙通道，进而触发顶体融合 和细胞膜)在很大程度上被阻止。血铅+2与血铅无关 激素水平，表明铅+2的影响不是通过 内分泌干扰物。As添加微摩尔Pb2+或特定 人和大鼠延迟整流电压门控钾通道的抑制剂 大鼠睾丸的cDNA也表明人类精子中存在这样的通道。 Northern杂交中发现的多态性表明，个体 谁在对类似的[铅+2]挑战的有效反应上有所不同 可能具有不同的K+通道异构体。 我们将完成人类精子延迟整流的测序 电压门控K+通道，并将尝试克隆人类形式的 金黄地鼠钙激活钾通道。我们将开发DNA探针来 表征选择性拼接的K+通道。我们会制造第二个 150名男性试管受精患者的前瞻性研究，以证实我们的发现和 寻求确定Pb2+影响的进一步环境风险因素 人类精子功能与K+变异体表达水平的相关性 精子与血液和精浆[Pb2+]和[Pb2+]的通道mRNAs 精子NNPR表达与Psar的关系。 我们还将研究Pb2+‘S生殖的可能机制 对男性精子的影响，即它影响精子K+通道功能或 干扰膜去极化后早期的信号转导。 我们将使用金属离子传感荧光探针，以及特定的 探讨Pb2+对离子通量的影响。我们将聘用 磷脂酶C检测和竞争分析 Pb2+对肌醇磷酸二次信息的影响。 这个应用程序的目标是确定损伤的机制 对男性生殖道造成的环境和 职业性接触重金属和过渡金属离子。焦点 铅暴露对顶体反应不全的影响 和男性不育症。力量应用包括专业知识 以及首席研究员贝诺夫博士和她的研究经验 团队，分析问题的新方法。