Importance of Androgen regulated miRNAs in male fertility

雄激素调节的 miRNA 在男性生育能力中的重要性

基本信息

批准号：
8889281
负责人：
Manjeet Kumar Rao
金额：
$ 7.29万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-15 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8889281
关键词：
Address Adult Androgen Receptor Androgen Response Element Androgen Suppression Androgens Apoptosis Applications Grants Binding Biochemical Biological Assay Blood Tests Cell Adhesion Cell Differentiation process Cell physiology Critical Pathways Development Disease Event Fertility Foundations Gene Targeting Genes Genetic Models Genetic Transcription Germ Cells Giant Cells Goals Health In Vitro Infertility Knock-out Knockout Mice Male Contraceptions Malignant neoplasm of prostate Mediating Meiosis Messenger RNA MicroRNAs Molecular Mus Oligoribonucleotides Pathway interactions Phagocytosis Phenocopy Phenotype Physiology Pilot Projects Play Reproductive Physiology Research Role Site Spermatids Spermatocytes Spermatogenesis Syndrome Testing Testis Testosterone Time Trans-Activators Transcript Transgenic Mice Translating Translations Untranslated RNA Work X Chromosome base in vivo insight male mouse model mutant mouse model novel novel strategies overexpression promoter reproductive sertoli cell

项目摘要

DESCRIPTION (provided by applicant): Even after decades of research, the molecular basis for why spermatogenesis requires testosterone is not completely understood. This is in part because very few androgen-regulated genes have been definitively identified so far. It is likely that most androgen-regulated genes in the testis respond indirectly to androgen via activation or suppression of transcription by other trans-acting factors directly regulated by androgen. Recently, we have shown that one class of such androgen-regulated trans-acting factors in the testis are microRNAs (miRNAs), small non-coding RNA molecules that inhibit the translation of the target transcript by binding to complementary cis-regulatory sites in the mRNA. Specifically, we have shown that a number of these miRNAs are upregulated (suggesting their gene targets would be downregulated) in the absence of androgen. Our long term goal is to understand the in vivo function and mechanism by which these androgen-responsive miRNAs regulate critical pathways essential for male fertility. Toward this goal, we generated transgenic mice expressing miR-471 driven by the Sertoli cell-specific promoter Rhox5Pp that we have previously extensively characterized. Our preliminary analyses of miR-471 transgenic mice reveal that Sertoli cell-specific overexpression of miR-471 resulted in dramatically increased germ cell apoptosis, multinucleated giant cells suggesting abortive meiosis in spermatocytes, impaired postmeiotic germ cell differentiation and extensive germ cell sloughing, a phenotype that correlates with impaired Sertoli cell-Sertoli cell/germ cell adhesion at the blood-test barrier, which is an androgen-dependent event during spermatogenesis. These findings led us to hypothesize that by targeting specific transcripts, miR-471 expression in the Sertoli cells regulates specific androgen-dependent steps critical for germ cell development and progression. Two specific aims are proposed to test our hypothesis: In aim 1, we will extend our preliminary analysis and further characterize miR-471 transgenic mice. We will specifically assess the integrity of Sertoli cell-Sertoli cell/germ cell adhesion as well as germ cell meiotic arrest using molecular and biochemical approaches. This study will be highly significant as miR-471 transgenic mice represent the first genetic model to study the function of specific miRNAs in the Sertoli cells and miR-471 will be the first proven androgen-regulated miRNA important for male fertility. In aim 2, we will validate miR-471 gene targets and address their role in mediating Sertoli cell-specific function of miR-471 using in vitro and ex-vivo approaches. The androgen-regulated miRNA on which this proposal is focused provide an opportunity to begin to address this. The proposed work will provide important leads to clearly understand the molecular mechanism by which androgen regulates critical pathways essential for male fertility. Furthermore, this research initiative may provide insights into the putative role of miRNA in androgen related diseases such as prostate cancer and androgen insufficiency syndrome.

描述（由申请人提供）：即使经过数十年的研究，精子发生需要睾酮的分子基础仍未完全了解。部分原因是迄今为止，很少有雄激素调节基因被明确识别。睾丸中大多数雄激素调节基因很可能通过由雄激素直接调节的其他反式作用因子激活或抑制转录来间接响应雄激素。最近，我们发现睾丸中一类雄激素调节的反式作用因子是 microRNA (miRNA)，它们是一种小非编码 RNA 分子，通过与睾丸中互补的顺式调节位点结合来抑制目标转录本的翻译。 mRNA。具体来说，我们已经证明，在缺乏雄激素的情况下，许多 miRNA 会上调（表明它们的基因靶标会下调）。我们的长期目标是了解这些雄激素反应性 miRNA 调节男性生育能力所必需的关键途径的体内功能和机制。为了实现这一目标，我们产生了表达 miR-471 的转基因小鼠，该小鼠由支持细胞特异性启动子 Rhox5Pp 驱动，我们之前已经对其进行了广泛的表征。我们对 miR-471 转基因小鼠的初步分析表明，支持细胞特异性过度表达 miR-471 导致生殖细胞凋亡显着增加，多核巨细胞表明精母细胞减数分裂失败，减数分裂后生殖细胞分化受损和广泛的生殖细胞脱落（一种表型）与血液测试屏障处的支持细胞-支持细胞/生殖细胞粘附受损相关，这是在检测过程中的雄激素依赖性事件精子发生。这些发现使我们推测，通过靶向特定转录本，支持细胞中的 miR-471 表达调节对生殖细胞发育和进展至关重要的特定雄激素依赖性步骤。提出了两个具体目标来检验我们的假设：在目标 1 中，我们将扩展我们的初步分析并进一步表征 miR-471 转基因小鼠。我们将使用分子和生化方法专门评估支持细胞-支持细胞/生殖细胞粘附的完整性以及生殖细胞减数分裂停滞。这项研究将具有非常重要的意义，因为 miR-471 转基因小鼠代表了第一个研究支持细胞中特定 miRNA 功能的遗传模型，并且 miR-471 将是第一个被证明对男性生育能力重要的雄激素调节 miRNA。在目标 2 中，我们将验证 miR-471 基因靶标并解决其在介导中的作用使用体外和离体方法观察 miR-471 的支持细胞特异性功能。本提案所关注的雄激素调节 miRNA 提供了一个开始解决这个问题的机会。拟议的工作将为清楚地了解雄激素调节男性生育能力所必需的关键途径的分子机制提供重要线索。此外，这项研究计划可能会深入了解 miRNA 在雄激素相关疾病（如前列腺癌和雄激素不足综合征）中的假定作用。