SPERM PROGESTERONE RECEPTOR, K+ CHANNEL & PB EXPOSURE

描述：(改编自《调查者摘要》)一项盲目研究 在接受体外受精(IVF)的夫妇中，超过40%的 男性的血清铅水平超过了职业行动极限。 暴露的工人。血清和精浆铅均与 体外受精中卵母细胞受精率。精浆铅也与此相关 与精子接受黄体酮刺激的能力成反比 顶体反应(Psar)与NNPR的聚集/转位 在胞吐作用之前的精子的赤道区域。在预赛中 实验中，我们证明了人类Psar被特定的 电压门控K+通道的抑制剂，Psar被增强 K+增加，提示Psar早期有K+通道开放。至 表明通道存在于雄性生殖细胞中，研究人员使用 基于大鼠神经皮质电压依赖性K+通道的聚合酶链式反应 从大鼠睾丸中扩增一种产物。使用该序列进行北向分析 作为一种探针，在汇集的大鼠睾丸mRNA中鉴定出2.4和4.2kB的转录本， 但不是在任何其他被测试的组织中。原位RT/PCR发现了这些转录本 在第IX期和第X期大鼠精母细胞的细胞质中以及伸长 精子细胞。这项提议的目标是描述K+通道的特征 与NNPR相关联。 研究人员将对特定离子进行剂量-反应研究 通道抑制剂以进一步表征人类精子的K+通道， 目的是区分可能的子类型(例如 延迟整流电压门控，钙离子激活)。端点将为Psar 罗丹明标记的PES凝集素AS法。 研究人员将按标准制备生物素化白纹藻毒素 方案和尝试可视化完整的人类精子上的K+通道 我们的产品结合了抗生物素抗体。调查员 将从池中克隆并测序人精子通道K+的cdna 人类精子RNA，从基于人类电压门控K+的引物开始 频道。它们将决定人类的特定阶段的表达 人睾丸活检切片中精子K+通道的原位RT/PCR研究

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.