HUMAN SPERM ZONA ACCEPTOR: ENVIRONMENTAL EFFECTS

人类精子带受体：环境影响

• 批准号: 6547671
• 负责人: SUSAN H BENOFF
• 金额: $ 36.72万
• 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 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6547671
• 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（由申请人提供）：我们专注于了解人类睾丸中的有毒金属作用。男性因素存在于约60%的不育夫妇中，但其潜在的分子机制在很大程度上尚未被描述。我们目前工作的令人兴奋的结果暗示了一种机制。铅水平显着升高，睾丸和精浆（在25%的男性在四个独立的人群）。高铅与特定的钾和钙离子通道亚型的表达相关，与精子受精潜力生物标志物差和IVF，人工授精和性交的生育力低相关。研究的受试者中有很大一部分从高铅状态纵向转换为低铅状态，同时生物标志物从不育转换为可育，并转换钾通道亚型表达。这表明，铅epigerietically修改睾丸基因表达（在转录和mRNA剪接水平），钾通道亚型可以开发为铅暴露的生物标志物。一个初步的DNA微阵列研究铅处理的“耐铅”大鼠品系确定了许多铅影响的基因参与钙介导的诱导细胞凋亡，包括钾通道。目前的体细胞凋亡机制的支持下，这促使我们的假设，铅暴露产生男性不育通过改变钙稳态，以及相关的详细机制的铅行动。这些将在铅处理的铅敏感”大鼠品系和人类中进行测试。我们将使用微阵列来探测大鼠受影响的睾丸基因与CAMP反应元件和其他基因参与钙/钙调蛋白依赖性蛋白激酶IV信号。对照包括通过原子吸收进行的金属检测、凋亡的TUNEL估计、通过组织学和通过细胞类型特异性mRNA水平进行的细胞类型水平以及通过蛋白质印迹进行的蛋白质表达。与“耐铅”菌株的比较应该可以确定铅敏感性”基因。我们将在人类临床人群中探测相同的基因，并进行类似的控制。我们还将探测与上述钾通道共调节的基因。结果将测试几个具体步骤，在我们提出的机制：验证，否定或修改it. Because微阵列不能检测差异钙通道剪接事件与铅对人类睾丸的影响，该基因和其他钙转运蛋白将研究免疫细胞化学，RT原位PCR和实时PCR。结果是假设的检验，以及解释与低精子计数或特发性男性不育相关的不育的可能机制，诊断工具和治疗希望。