Molecular Bases Committing Primate Spermatogonia to a Pathway of Differentiation.

描述(申请人提供)：这个项目的长期目标是了解恒河猴精原细胞分化的细胞和分子机制，恒河猴是一种具有代表性的高等灵长类动物。这一建议背后的策略部分是基于这样的发现：虽然精原细胞在没有促性腺激素刺激的情况下发生增殖，但灵长类精原细胞的分化只有在促性腺激素驱动的情况下才会发生。这两个过程的内分泌需求的差异将被用来询问睾丸基因、基因网络和任何决定精原细胞更新或分化的新转录本。这里有三个主要问题。首先，精原细胞决定分化而不是自我更新的基因和途径是什么。其次，是什么是促性腺激素依赖的支持细胞旁分泌信号，指示未分化的精原细胞沿着分化的道路前进。第三，精原细胞的分化在多大程度上依赖维生素A。为了开始回答这些问题，将使用RNA-SEQ来评估生殖细胞和支持细胞基因表达和转录组的全球变化，这些变化与将未分化的A型精原细胞置于促性腺激素依赖的分化途径上的初始步骤相关。经qRT-PCR证实后，将通过免疫组织化学(IHC)和/或原位杂交(ISH)检测与精原细胞分化相关的上调或下调基因及其相关转录本的细胞特异性表达。维生素A缺乏(VAD)对精原细胞增殖的影响将通过喂养维生素A缺乏的饲料来确定。这项工作将针对两个具体目标：1鉴定生殖细胞和支持细胞基因、基因网络和与未分化的AP精原细胞决定致力于分化途径有关的新转录本；2确定未分化的AP精原细胞致力于猴子分化途径的决定是否依赖于维生素A。这项工作将在一个互动的智力环境中进行，该环境由一组对精原生物学和支持细胞功能相关方面感兴趣的研究人员产生。预计拟议的工作将提供对精原细胞分化的基本见解：精子发生的一个关键方面，将与人类男性不育的治疗和新型男性避孕药的开发相关。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the cellular and molecular mechanisms that control spermatogonial differentiation in the rhesus monkey, a representative higher primate. The strategy underlying the proposal is based, in part, on the finding that while spermatogonial proliferation occurs in the absence of gonadotropin stimulation, differentiation of primate spermatogonia occurs only in the presence of a gonadotropin drive. This difference in the endocrine requirements of the two processes will be used to interrogate the testicular genes, gene networks and any novel transcripts that determine the decision of spermatogonia to renew or to differentiate. There are three major questions. First, what genes and pathways within spermatogonia underlie the decision of the cell to differentiate rather than undergo self-renewal. Second, what are the gonadotropin dependent Sertoli cell paracrine signals that instruct undifferentiated spermatogonia to proceed down the path of differentiation. Third, to what extent is spermatogonial differentiation dependent on Vitamin A. To begin to answer these questions, RNA-seq will be used to assess global changes in germ cell and Sertoli cell gene expression and transcriptome profiles associated with the initial step in placing undifferentiated type A spermatogonia on the gonadotropin dependent pathway of differentiation. Following confirmation by qRTPCR, cell specific expression of those genes and relevant transcripts that are shown by qRTPCR to be up- or down-regulated in association with spermatogonial differentiation will then be determined using immunohistochemistry (IHC) and/or in situ hybridization (ISH). The effects of Vitamin A deficiency (VAD) on spermatogonial proliferation will be determined by feeding the animals a Vitamin A deficient diet. Two Specific Aims will be addressed; 1 to identify germ cell and Sertoli cell genes, gene networks and novel transcripts associated with the decision of undifferentiated type Ap spermatogonia to commit to a pathway of differentiation, and 2 To determine whether the decision of undifferentiated Type Ap spermatogonia to commit to the path of differentiation in the monkey is dependent on Vitamin A. This work will be conducted in an interactive intellectual environment that is generated by a group of investigators interested in related aspects of spermatogonial biology and Sertoli cell function. It is anticipated that the proposed work will provide fundamental insight into spermatogonial differentiation: a critical aspect of spermatogenesis that will be relevant to the treatment of infertility in the human male and to the development of novel male contraceptives.