Challenging the role of retinoic acid in meiotic initiation

项目摘要/摘要 减数分裂对于有性繁殖是必不可少的，其特殊的分子和细胞程序 在低等真核生物和哺乳动物中得到了广泛的研究。然而，在哺乳动物中，从 有丝分裂的精原细胞进入减数分裂程序，称为减数分裂开始，几乎没有受到关注。 减数分裂的开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，为减数分裂重组和分离做好准备。维甲酸(RA)已被 建议作为减数分裂诱导物质。在出生后的睾丸中，尽管这是明确要求的 对于精原细胞的分化，我们令人兴奋的初步数据显示，8.6天后RA是必不可少的 进行减数分裂的启动。因此，RA在减数分裂中的作用还没有得到适当的研究，真正的减数分裂-- 诱发因素(S)尚不明确。这项提议代表了盖耶尔和 辛德勒实验室，他们将共同努力，揭示RA在减数分裂中的真实作用(S饰)。在目标1中，我们将确定 RA通过减数分裂形成单倍体精子细胞的起始和进展的特殊要求。 在目标2中，我们将使用一种新的同步精子发生的转基因小鼠模型来 在减数分裂开始前和减数分裂开始期间，以荧光为基础分离数百万生殖细胞。使用这个 独一无二的资源，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) 减数分裂开始时的水平。这项工作的结果是识别了这一关键的新调节器 在细线期前期精母细胞中上调的睾丸特异蛋白将代表 假定的男性避孕目标。事实上，前细线期精母细胞代表了一种理想的细胞类型 男性避孕药的开发，有两个原因：1)它们位于血-睾丸屏障之外 (Btb)，这是药物输送的重要障碍；和2)它们与精原干细胞不同。 细胞(SSC)池，因此可以成为靶标，而不会不可逆转地损害雄性生殖系。结果是 该提案将为定义RA和 对男性生育能力至关重要的减数分裂。

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.