SPERM PROGESTERONE RECEPTOR, K+ CHANNEL & PB EXPOSURE

描述：（改编自研究者摘要）在设盲研究中 在接受体外受精（IVF）的夫妇中，超过40%的 男性的血清铅水平高于职业 暴露的工人 血清和精浆铅含量与 IVF中的卵母细胞受精率。 精浆铅也与 与精子经历胆固醇刺激的能力相反， 顶体反应（PSAR）和NNPR的聚集/易位到 精子胞吐作用前的赤道区。 初步 实验中，我们证明了人的PSAR被特定的 电压门控性K+通道抑制剂，PSAR增强， 增加K+，表明在PSAR早期K+通道打开。 到 显示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 从大鼠睾丸mRNA中扩增产物。 北分析使用此序列 当探针在合并的大鼠睾丸mRNA中鉴定2.4和4.2kB转录物时， 但在其它任何测试的组织中没有。 原位RT/PCR发现这些转录本 在IX期和X期大鼠精母细胞的细胞质中， 精子细胞 本提案的目标是描述K+通道的特征 与NPR相关的 研究者将使用特定离子进行剂量反应研究 通道抑制剂以进一步表征人精子的K+通道， 目的是在可能的亚型之间进行区分（例如， 延迟整流器电压门控，Ca++激活）。 终点为PSAR 用罗丹明标记的PES凝集素进行测定。 研究者将通过标准品制备生物素化的卡律巴毒素 协议，并试图可视化K+通道上完整的人类精子使用 我们的产品结合抗生物素抗体。 研究者 将从汇集的人精子中克隆和测序人精子K+通道的cDNA， 人精子RNA，从基于人电压门控K+的引物开始 频道 它们将决定人类特定阶段的表达 用原位RT/PCR方法研究了人睾丸组织K+通道。

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.