HUMAN SPERM ZONA ACCEPTOR: ENVIRONMENTAL EFFECTS

人类精子带受体：环境影响

• 批准号: 6651127
• 负责人: SUSAN H BENOFF
• 金额: $ 36.72万
• 依托单位: FEINSTEIN INSTITUTE FOR MEDICAL RESEARCH
• 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 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6651127
• 关键词: acrosome; biomarker; calcium flux; calmodulin dependent protein kinase; clinical research; environmental exposure; environmental toxicology; fertility; gene expression; human subject; human tissue; laboratory rat; lead; lead poisoning; male; microarray technology; potassium channel; protein isoforms; sperm; terminal nick end labeling

DESCRIPTION (provided by applicant): We focus on developing an understanding of toxic metal action in the human testis. A male factor is present about 60% of infertile couples, but underlying molecular mechanisms are largely uncharacterized. Exciting results from our current work hints at one mechanism. Lead levels were elevated markedly in testes and seminal plasma (in 25% of males in four independent populations). High lead correlated with expression of particular potassium and calcium ion channel isofomis, with poor sperm-fertilization-potential biomarkers and low fertility by IVF, artificial insemination and coitus. A significant fraction of subjects studied longitudinally switched from high lead states to low lead states, with simultaneous conversion of biomarkers from infertile to fertile and switch in potassium channel isoform expression. This suggested lead epigerietically modified testicular gene expression (at the levels of transcription and mRNA splicing) and that potassium channel isoforms could be developed as biomarkers for lead exposure. A preliminary DNA microarray study of a lead-treated "lead-resistant" rat strain identified many lead-affected genes as being involved in calcium-mediated induction of apoptosis, including a potassium channel. Supported by current somatic cell apoptosis mechanisms, this prompted our hypothesis that lead exposures produce male infertility by altering calcium homeostatsis, and a related detailed mechanism of lead action. These will be tested in a lead-treated lead-sensitive" rat strain and in humans. We will use microarrays to probe in rats for affected testicular genes with CAMP response elements and other genes involved in calcium/calmodulin-dependent protein kinase IV signaling. Controls include metal testing by atomic absorption, TUNEL estimates of apoptosis, cell type levels by histology and by cell-type-specific mRNA levels, and protein expression by Westerns. Comparison with the "lead-resistant" strain should identify lead-sensitivity" genes. We will probe for the same genes in a human clinical population, with similar controls. We will also probe for genes co-regulated with the potassium channel above. Results will test several specific steps in our proposed mechanism: verifying, negating or modifying it. Because microarrays cannot detect differential calcium channel splicing events correlated with lead effects upon human testes, this gene and other calcium transporters will be studied by immunocytochemistry, RT in situ PCR and real-time PCR. Outcome is test of hypothesis, and possible mechanism explaining infertility associated with low sperm counts or idiopathic male infertility, tools for diagnosis, and hope for treatment.

描述(由申请者提供)：我们专注于发展对人体睾丸中有毒金属行为的理解。大约60%的不孕不育夫妇存在男性因素，但潜在的分子机制在很大程度上尚不清楚。我们目前工作的令人兴奋的结果暗示了一种机制。睾丸和精浆中的铅水平显著升高(在四个独立人群中，25%的男性)。高铅与特定的钾和钙离子通道异构体的表达有关，与精子受精潜力生物标志物和体外受精、人工授精和性交的低生育率相关。很大一部分受试者从高铅状态纵向切换到低铅状态，同时生物标记物从不育转换为可育，并切换到钾通道亚型表达。这表明，铅在转录和mRNA剪接水平上改变了睾丸基因的表达，钾通道亚型可以作为铅暴露的生物标志物。对经铅处理的“耐铅”大鼠品系的初步DNA微阵列研究发现，许多受铅影响的基因参与了钙介导的细胞凋亡诱导，包括钾通道。在目前体细胞凋亡机制的支持下，这促使我们提出了铅暴露通过改变钙稳态导致男性不育的假说，并提出了相关的详细机制。这些测试将在铅处理过的敏感大鼠品系和人类身上进行。我们将使用微阵列在大鼠中探测带有cAMP反应元件的受影响的睾丸基因，以及其他参与钙/钙调蛋白依赖的蛋白激酶IV信号转导的基因。控制措施包括原子吸收金属测试、TUNEL对细胞凋亡的估计、组织学细胞类型水平和特定细胞类型的mRNA水平，以及Western ns的蛋白质表达。与“耐铅”品系进行比较应确定铅敏感“基因”。我们将在人类临床人群中探索相同的基因，并进行类似的对照。我们还将探索与上述钾通道共同调节的基因。结果将测试我们提出的机制中的几个具体步骤：验证、否定或修改它。由于基因芯片不能检测到与铅对人类睾丸的影响相关的钙通道剪接差异事件，该基因和其他钙转运蛋白将通过免疫细胞化学、RT-原位聚合酶链式反应和实时定量聚合酶链式反应进行研究。结果是对假设的检验，以及解释与低精子数或特发性男性不育相关的不育的可能机制，诊断工具，以及治疗的希望。