Challenging the role of retinoic acid in meiotic initiation

项目概要/摘要 减数分裂对于有性生殖至关重要，其专门的分子和细胞程序已 在低等真核生物和哺乳动物中进行了深入研究。然而，在哺乳动物中，从 有丝分裂精原细胞进入减数分裂程序，称为减数分裂起始，很少受到关注。 减数分裂起始发生在减数分裂前期 I 的前细线期，因为这些精母细胞 复制它们的 DNA 并为减数分裂重组和分离做好准备。视黄酸（RA）已被 提议作为“减数分裂诱导物质”。在出生后睾丸中，尽管明确需要它 对于精原细胞分化，我们令人兴奋的初步数据表明 8.6 天后 RA 是可有可无的 用于减数分裂起始。因此，RA 在减数分裂中的作用尚未得到适当的研究，而真正的减数分裂- 诱发因素仍未确定。该提案代表了 Geyer 和 Schindler 实验室将共同努力揭示 RA 在减数分裂中的真正作用。在目标 1 中，我们将确定 RA 在通过减数分裂形成单倍体精子细胞的起始和进展中的具体要求。 在目标 2 中，我们将采用一种具有同步精子发生功能的新型转基因小鼠模型 在减数分裂开始之前和期间基于荧光分离数百万个生殖细胞。使用这个 独特的资源，我们将定义和比较 RA 介导的基因表达变化 转录组（RNA 丰度）、翻译组（翻译 RNA）和蛋白质组（蛋白质丰度） 减数分裂起始期间的水平。这项工作的成果是确定了这一关键的新监管机构 转变，并且在前细线精母细胞中上调的睾丸特异性蛋白质将代表 假定的男性避孕目标。事实上，前细线精母细胞代表了一种理想的细胞类型 男性避孕药物的开发，有两个原因：1）它们位于血睾屏障之外 (BTB)，这是药物输送的一个重要障碍； 2）它们与精原干不同 细胞（SSC）池，因此可以在不不可逆转地损害雄性生殖系的情况下进行靶向。结果 该提案中的内容将为定义 RA 和 RA 之间更广泛的机制关系奠定基础。 减数分裂对于男性生育能力至关重要。

PROJECT SUMMARY/ABSTRACT Meiosis is essential for sexual reproduction, and its specialized molecular and cellular programs have been intensely studied in lower eukaryotes and mammals. In mammals, however, the transition from mitotic spermatogonia into the meiotic program, termed meiotic initiation, has received little attention. Meiotic initiation occurs during the preleptotene phase of meiotic prophase I, as these spermatocytes replicate their DNA and prepare for meiotic recombination and segregation. Retinoic acid (RA) has been proposed to serve as the ‘meiosis inducing substance.’ In the postnatal testis, although it is clearly required for spermatogonial differentiation, our exciting preliminary data reveals RA is dispensable 8.6 days later for meiotic initiation. Thus, the role of RA in meiosis has not been properly examined, and the true meiosis- inducing factor(s) remain undefined. This proposal represents a new collaboration between the Geyer and Schindler labs, who will work together to uncover the true role(s) for RA in meiosis. In Aim 1, we will identify the specific requirement for RA in initiation and progression through meiosis to form haploid spermatids. In Aim 2, we will employ a novel transgenic mouse model with synchronized spermatogenesis for fluorescence-based isolation of millions of germ cells prior to and during meiotic initiation. Using this unique resource, we will define and compare RA-mediated changes in gene expression at the transcriptome (RNA abundance), translatome (translating RNAs), and proteome (protein abundance) levels during meiotic initiation. The outcome of this work is identification of novel regulators of this critical transition, and testis-specific proteins that are upregulated in preleptotene spermatocytes will represent putative male contraceptive targets. Indeed, preleptotene spermatocytes represent an ideal cell type for male contraceptive drug development, for two reasons: 1) they reside outside the blood-testis-barrier (BTB), which is a significant barrier to drug delivery; and 2) they are distinct from the spermatogonial stem cell (SSC) pool, and thus can be targeted without irreversibly damaging the male germline. The results from this proposal will be foundational to defining the broader mechanistic relationship between RA and meiosis that are essential for male fertility.