HUMAN SPERM ZONA ACCEPTOR--ENVIRONMENTAL EFFECTS

人类精子带受体——环境影响

• 批准号: 2693848
• 负责人: SUSAN H BENOFF
• 金额: $ 32.22万
• 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 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2693848
• 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.

我们有兴趣开发金属暴露影响的测试 对人类精子功能的影响 我们建议跟进 目前对接受体外受精的夫妇的男性进行的前瞻性研究 体外受精（IVF），42/95例受试者的血液和精液水平升高 Pb~（2+）> 40 μ g/dcl者，女性伴染率 IVF组卵母细胞受精率与IVF组呈负相关（r =-448，P < 0.01）， 0.001）与这些Pb +2水平。 在这个队列中，精浆Pb +2 相关系数r =-0.523，P <0.01）， 精子进行孕酮刺激的顶体反应（PSAR）。 当用非核孕酮受体（NNPR）探针可视化时 和甘露糖凝集素（ML），Pb +2阻断的主要位点位于 PSAR的早期阶段。 获能，由表达决定 NNPR和ML的水平受到影响，但与对照组相比影响较小， 但NNPR和ML从精子头部前部向 精子赤道段（通常先于Ca +2通过 电压门控钙通道，进而触发顶体融合 和细胞膜）被大量阻止。 血Pb +2与 激素水平，表明Pb +2的影响不是通过介导的 内分泌干扰物 当添加微摩尔Pb +2或特定的 延迟整流电压门控K+通道的抑制剂， 大鼠睾丸cDNA也提示人精子中存在这种通道。 在北方杂交中发现的多态性表明， 对类似的[Pb +2]挑战的生产性反应不同 可能有不同的K+通道亚型。 我们将完成人类精子延迟整流器的测序 电压门控K+通道，并将试图克隆人类形式的 仓鼠钙激活钾通道。 我们将开发DNA探针， 表征选择性剪接的K+通道。 我们会做第二个 对150名男性IVF患者进行的前瞻性研究证实了我们的发现， 寻求确定Pb2+影响的进一步环境风险因素， 人类精子功能，与变异K+表达水平相关 精子与血液和精浆[Pb2 +]和 精子NNPR表达和PSAR。 我们还将探讨Pb~（2+）对细胞增殖的可能机制。 对男性精子的影响，影响精子K+通道功能，或 干扰膜去极化后的早期信号转导。 我们将使用金属离子感应荧光探针， 抑制剂来探测PB 2+对离子通量的影响。 我们会委聘 磷脂酶C测定和竞争测定，以探测 Pb2+对磷酸肌醇次级信息的影响。 本申请的目的是确定损伤的机制 对男性生殖道造成的环境和 重金属和过渡金属离子的职业暴露。 重点 铅暴露对顶体反应功能不全的影响 和男性不育。 实力应用包括专业知识 首席研究员Benoff博士和她的研究 团队，分析问题的新颖方法。