HUMAN SPERM ZONA ACCEPTOR: ENVIRONMENTAL EFFECTS

描述（由申请人提供）：我们专注于了解有毒金属在人类睾丸中的作用。男性因素存在于大约60%的不育夫妇中，但潜在的分子机制在很大程度上是未知的。我们目前工作中令人兴奋的结果暗示了一种机制。睾丸和精浆中的铅含量显著升高（在四个独立人群中有25%的男性）。高铅与特定钾和钙离子通道异构体的表达相关，精子受精潜力生物标志物差，体外受精、人工授精和交媾的生育能力低。相当一部分受试者从高铅状态纵向切换到低铅状态，同时生物标志物从不育状态转换到可育状态，钾通道异构体表达也发生了变化。这表明铅修饰睾丸基因表达（在转录和mRNA剪接水平上），钾通道异构体可以作为铅暴露的生物标志物。一项对铅处理的“抗铅”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，我们提出了铅暴露通过改变钙稳态导致男性不育的假设，以及铅作用的相关详细机制。这些将在铅处理的铅敏感大鼠品系和人类身上进行测试。我们将使用微阵列在大鼠中探测受影响的睾丸基因与CAMP反应元件和其他参与钙/钙调素依赖性蛋白激酶IV信号传导的基因。对照包括通过原子吸收检测金属，TUNEL估计细胞凋亡，通过组织学和细胞类型特异性mRNA水平检测细胞类型水平，以及western检测蛋白质表达。与“抗铅”菌株比较，应确定“铅敏感”基因。我们将在人类临床人群中以类似的控制方法探测相同的基因。我们还将探索与上述钾通道共调控的基因。结果将测试我们提议的机制中的几个具体步骤：验证、否定或修改它。由于微阵列无法检测与铅对人类睾丸的影响相关的钙通道剪接差异事件，因此该基因和其他钙转运蛋白将通过免疫细胞化学、RT原位PCR和实时PCR进行研究。结果是对假设的检验，以及解释与低精子计数或特发性男性不育症相关的不育症的可能机制，诊断工具和治疗希望。

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.