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

描述（由申请人提供）：双酚A （BPA）是一种内分泌干扰化学物质，在发达国家普遍存在。动物研究和最近的流行病学证据都表明，BPA以男性生殖道为目标，造成睾丸损伤和精子参数的改变。在啮齿类动物模型中，基于标准良好实验室规范（GLP）的方法在检测bpa诱导的男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，包括保留精子头的定量和末端dUTP nick end-labeling （TUNEL）阳性生殖细胞的发生率，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甲基化来确定睾丸和精子中影响的分子生物标志物。测量精子中的这些分子生物标志物预计将特别敏感和特异性，因为精子是一个均匀的细胞群，在其整个生活史中都在精原上皮内发育。GLP设计提供多剂量和阳性雌二醇控制，允许对bpa诱导效应的存在进行稳健的测试。此外，研究人员将评估父系F0大鼠及其后代，以解决代际差异。该项目的目标是验证以下工作假设：添加增强的形态和分子端点与GLP设计将提供bpa诱导的男性生殖毒性更敏感和特异性的生物标志物。通过以下特定目的，GLP研究将通过形态计量学评估和分子生物标志物的作用来加强：特异性目的1将使用先进的组织学和形态计量学终点来加强对bpa诱导睾丸损伤的评估。特异性Aim 2将表征尾端精子和睾丸的信使和microRNA（分别为mRNA和miRNA）转录物的含量。特异性Aim 3将发展尾端精子和睾丸甲基化。

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.