Identification of Molecular Biomarkers in Rat Sperm and Testis Exposed to BPA

描述(申请人提供)：双酚A(BPA)是一种内分泌干扰性化学物质，在发达国家造成普遍的暴露。动物研究和最近的流行病学证据都表明，BPA针对男性生殖道，导致睾丸损伤和精子参数改变。在啮齿动物模型中，基于标准良好实验室操作规范(GLP)的方法在检测双酚A诱导的雄性生殖道异常方面相对不敏感。研究人员建议使用成熟的睾丸形态计量学评估，包括对保留的精子细胞头部和dUTP缺口末端标记阳性生殖细胞的发生率进行量化，以提高检测BPA暴露对睾丸影响的敏感性。此外，研究人员将通过检查全球mRNA和miRNA水平以及全基因组DNA甲基化来识别影响睾丸和精子的分子生物标记物。测量精子中的这些分子生物标志物被认为是特别敏感和特异的，因为精子是一种同质的细胞群体，在其整个生命历史中在生精上皮内发育。GLP设计提供了多剂量和阳性雌二醇对照，允许对双酚A诱导效应的存在进行强有力的测试。此外，研究人员将评估父本F0大鼠及其后代，以解决世代之间的差异。该项目的目标是检验以下工作假设：在GLP设计中增加增强的形态和分子终点将提供更敏感和更特异的BPA诱导的男性生殖毒性生物标志物。GLP研究将通过以下具体目标通过形态计量评估和分子生物标记物得到加强：具体目标1将利用先进的组织学和形态计量终点加强对双酚A引起的睾丸损伤的评估。特异性目标2将表征尾部精子和睾丸信使以及microRNA(分别为mRNA和miRNA)转录本的内容。特异靶3将发育尾部精子和睾丸甲基组。

DESCRIPTION (provided by applicant): Bisphenol A (BPA) is an endocrine disrupting chemical causing ubiquitous exposures in the developed world. Both animal studies and recent epidemiological evidence indicate that BPA targets the male reproductive tract, producing testicular injury and alterations in sperm parameters. Standard good laboratory practices (GLP)-based approaches in rodent models have been relatively insensitive in detecting BPA-induced abnormalities in the male reproductive tract. The investigators propose the use of well-established morphometric assessments of the testis, including quantitation of retained spermatid heads and the incidence of terminal dUTP nick end-labeling (TUNEL)- positive germ cells, to enhance the sensitivity of detecting testicular effects of BPA exposure. In addition, the investigators will identify molecular biomarkers of effect in the testis and sperm, by examining global mRNA and miRNA levels and genome-wide DNA methylation. Measuring these molecular biomarkers in sperm is expected to be particularly sensitive and specific, since sperm are a homogeneous population of cells that developed throughout their life history within the seminiferous epithelium. The GLP design provides multiple doses and a positive estradiol control, allowing robust testing for the presence of a BPA-induced effect. In addition, the investigators will assess both the paternal F0 rats and their progeny, addressing differences across generations. The goal of this project is to test the following working hypothesis: addition of the enhanced morphological and molecular endpoints with the GLP design will provide more sensitive and specific biomarkers of BPA-induced male reproductive toxicity. The GLP study will be enhanced by morphometric assessments and molecular biomarkers of effects through the following Specific Aims: Specific Aim 1 will enhance the assessment of BPA-induced testicular injury using advanced histological and morphometric endpoints. Specific Aim 2 will characterize the caudal sperm and testis messenger and microRNA (mRNA and miRNA, respectively) transcript content. Specific Aim 3 will develop the caudal sperm and testis methylome.