SPERM PROGESTERONE RECEPTOR, K+ CHANNEL & PB EXPOSURE

精子黄体酮受体，K 通道

• 批准号: 6080595
• 负责人: ASHA JACOB
• 金额: $ 4.1万
• 依托单位: NORTH SHORE UNIVERSITY HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6080595
• 关键词: RNA; biopsy; calcium channel; gene expression; human genetic material tag; human tissue; lead; molecular cloning; nucleic acid sequence; polymerase chain reaction; potassium channel; progesterone receptors; sperm; voltage gated channel

DESCRIPTION: (Adapted from the Investigator's Abstract) In a blinded study of couples undergoing in vitro fertilization, (IVF) more than 40% of the males had serum levels of lead above the action limit for occupationally exposed workers. Both serum and seminal plasma Pb correlated inversely with rates of oocyte fertilization in IVF. Seminal plasma Pb also correlated inversely with the ability of the sperm to undergo a progesterone-stimulated acrosome reaction (PSAR) and the aggregation/translocation of NNPR to the equatorial region of sperm which precedes exocytosis. In preliminary experiments, we demonstrated that the human PSAR was blocked by specific inhibitors of voltage-gated K+ channels, and that PSAR was augmented by increasing K+, suggesting that a K+ channel opened early in the PSAR. To show that the channels are present in male germ cells, the investigator used PCR primers based on a rat neural cortex voltage-dependent K+ channel to amplify a product from rat testes mRNA. North analysis using this sequence as a probe identified 2.4 and 4.2kB transcripts in pooled rat testes mRNA, but not in any other tissue tested. In situ RT/PCR found these transcripts in the cytoplasm of stage IX and X rat spermatocytes as well as elongating spermatids. The goal of this proposal is to characterize the K+ channel associated with the NNPR. The investigator will perform dose-response studies with specific ion channel inhibitors to further characterize the K+ channel of human sperm, with the aim of distinguishing among possible subtypes (e.g. delayed-rectifier voltage-gated, Ca++ activated). The endpoint will be PSAR as assay with rhodamine-labeled pes agglutinin. The investigator will prepare biotinylated charybdotoxin by standard protocols and attempt to visualize K+ channels on intact human sperm using our product in conjunction with anti-biotin antibodies. The investigator will clone and sequence the cDNA of the human sperm channel K+ from pooled human sperm RNA, starting from primers based on the human voltage-gated K+ channel. They will determine the stage-specific expression of the human sperm K+ channel by in situ RT/PCR on human testes biopsy sections.

描述：（改编自研究者摘要）在一项盲法研究中 接受体外受精 (IVF) 的夫妇超过 40% 男性血清铅水平高于职业行为限值 暴露的工人。 血清和精浆 Pb 均与 IVF 中卵母细胞的受精率。 精浆 Pb 也相关 与精子接受黄体酮刺激的能力成反比 顶体反应（PSAR）和 NNPR 的聚集/易位 胞吐作用之前的精子赤道区域。 在初步 实验中，我们证明人类 PSAR 被特定的 电压门控 K+ 通道抑制剂，并且 PSAR 被增强 K+ 增加，表明 K+ 通道在 PSAR 早期打开。 到 表明这些通道存在于男性生殖细胞中，研究人员使用 基于大鼠神经皮层电压依赖性 K+ 通道的 PCR 引物 扩增大鼠睾丸 mRNA 的产物。 使用此序列进行北分析 作为探针，在混合的大鼠睾丸 mRNA 中识别出 2.4 和 4.2kB 转录本， 但在任何其他测试的组织中都没有。 原位 RT/PCR 发现了这些转录本 在 IX 期和 X 期大鼠精母细胞的细胞质中以及延长 精子细胞。 该提案的目标是描述 K+ 通道的特征 与 NNPR 相关。 研究人员将使用特定离子进行剂量反应研究 通道抑制剂进一步表征人类精子的 K+ 通道， 目的是区分可能的亚型（例如 延迟整流器电压门控，Ca++ 激活）。 终点将是 PSAR 用罗丹明标记的 pes 凝集素进行测定。 研究者将按标准制备生物素化的charybdotoxin 协议并尝试使用可视化完整人类精子上的 K+ 通道 我们的产品与抗生物素抗体结合使用。 调查员 将克隆并测序来自汇集的人类精子通道 K+ 的 cDNA 人类精子 RNA，从基于人类电压门控 K+ 的引物开始 渠道。 他们将确定人类特定阶段的表达 通过对人类睾丸活检切片进行原位 RT/PCR 检测精子 K+ 通道。