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

描述（由申请方提供）：双酚A（BPA）是一种内分泌干扰化学品，在发达国家引起普遍暴露。动物研究和最近的流行病学证据都表明，BPA的目标是男性生殖道，造成睾丸损伤和精子参数的改变。 在啮齿动物模型中，基于标准药物非临床研究质量管理规范（GLP）的方法在检测BPA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学评估，包括保留精子细胞头部的定量和末端dUTP缺口末端标记（TUNEL）阳性生殖细胞的发生率，以提高检测BPA暴露对睾丸影响的灵敏度。此外，研究人员还将通过检查全球mRNA和miRNA水平以及全基因组DNA甲基化来确定睾丸和精子中的分子生物标志物。预计测量精子中的这些分子生物标志物是特别敏感和特异的，因为精子是在生精上皮内的整个生命史中发育的同质细胞群体。GLP设计提供了多次给药和阳性雌二醇对照，允许对BPA诱导效应的存在进行稳健检测。此外，研究者将评估父代F0大鼠及其后代，以解决各代间的差异。本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学和分子学终点将为BPA诱导的雄性生殖毒性提供更灵敏和特异的生物标志物。 GLP研究将通过以下特定目的的影响的形态学评估和分子生物标志物来增强：特定目的1将使用先进的组织学和形态学终点增强BPA诱导的睾丸损伤的评估。 特异性目标2将表征尾部精子和睾丸信使和microRNA（分别为mRNA和miRNA）转录物含量。 具体目标3将开发尾部精子和睾丸甲基化组。

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.