RHOX action in Sertoli development and function

RHOX 在支持细胞发育和功能中的作用

• 批准号: 10218674
• 负责人: JAMES Arthur MACLEAN
• 金额: $ 32.2万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218674
• 关键词: Ablation; Address; Adult; Affect; Androgens; Antibodies; Apoptosis; Birth; Blood-Testis Barrier; CRISPR/Cas technology; Carrying Capacities; Cell Count; Cell Differentiation process; Cell Survival; Cells; ChIP-seq; Couples; Data; Defect; Development; Down-Regulation; Embryo; Event; Exhibits; Family; Female; Fertility; Genes; Genetic; Genetic Transcription; Germ Cells; Goals; Gonadal Ridge; Gonadal structure; Health; Homeobox; Homeobox Genes; Homeodomain Proteins; Human; Infertility; Knock-out; Knockout Mice; Knowledge; Light; Link; Mammals; Meiosis; Modeling; Mus; Mutation; Nurses; Oligospermia; Output; Ovulation; Pathway interactions; Phenotype; Population; Positioning Attribute; Production; Puberty; Publishing; RNA Interference; Reproduction; Reproductive History; Research; Role; Sexual Development; Signal Transduction; Somatic Cell; Specific qualifier value; Sperm Count Procedure; Sperm Motility; Spermatogenesis; Structure of primordial sex cell; Supporting Cell; Testicular Dysgenesis Syndrome; Testing; Testis; Testosterone; Time; Tissues; Transgenes; United States National Institutes of Health; X Chromosome; blastomere structure; cell motility; cell type; cofactor; experimental study; fetal; fetus cell; gene discovery; gene product; gonad development; idiopathic infertility; in vivo; interest; knock-down; knockout animal; leydig interstitial cell; male; male fertility; member; mutant; novel; postnatal; promoter; protein function; reproductive; selective expression; self-directed learning; sertoli cell; sex determination; sperm cell; stem; stem cell niche; subfertility; tool; transcription factor; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT The Reproductive Homeobox X-linked (Rhox) genes encode a novel family of transcription factors that are excellent candidates to regulate events during spermatogenesis and ovulation. The RHOX homeodomain protein family has several members (13 distinct genes, with some existing in multiple copies). However, only two are expressed in Sertoli cells, the master control cells that promote testis development and nurse the differentiation and survival of germ cells. Ablation of these two genes, Rhox5 and Rhox8, results in male subfertility characterized by lower epididymal sperm numbers, motility defects, and reduced fecundity. Progeny from Rhox5-null mice, crossed with RHOX8 in vivo RNAi knockdown mice possess a distinct phenotype that is more severe than either single mutant. While these two mouse lines are useful tools for discovering the role of RHOX5 and RHOX8 after puberty, they are incapable of shedding light on RHOX8’s potential role in gonad development as the inhibitory Rhox8 transgene does not turn on until after post-natal day 7 when androgen signaling normally turns on Rhox5. To address this issue, a new complete Rhox8 deletion model will be employed to assess RHOX8’s function in embryonic Sertoli cell specification and function. Characterization of these mice is of great interest because: 1) Rhox8 is the only Rhox gene expressed in Sertoli cells of the embryonic gonad. Thus, there will be no concerns of functional redundancy which has been an issue for Rhox studies in the past. All of the other members of the cluster are restricted to primordial germ cells, including Rhox5 which is only found in Sertoli cells postnatally. 2) Analysis of the Rhox8 postnatal in vivo knockdown mic, and transient knockdown of RHOX8 in embryonic testis cultures, show that RHOX8 regulates Sox9. 3) SOX9 is a master regulator downstream of SRY – the factor that specifies male vs. female differentiation. Thus, RHOX8 may be in the sex determination pathway between SRY and SOX9. 4) After sex-determination, SOX9 governs multiple key events in the development of the tests including the formation of testis cords, male- specific vasculature, and differentiation of Sertoli and Leydig cell populations. If these post-puberty relationships are also conserved in the embryo, then RHOX8 may be a key cofactor or regulator of several events during gonad development. The experiments outlined in this proposal seek to systematically position Rhox8 in the milieu of male sexual development and discover the gene networks that are under its control. Because RHOX5 and RHOX8 may possess complementary functions in postnatal Sertoli cells, we will examine spermatogenesis in Rhox5/Rhox8 double knockout animals. Preliminary data suggests that these mice have a distinct and more severe phenotype than either single mutant. The elucidation of Rhox8’s function in Sertoli cells is important because it will provide an important “building block” towards the long-term goal of learning the independent and collaborative functions of all the Rhox genes in the testes.

项目摘要/摘要 生殖同源X连锁（Rhox）基因编码一个新的转录因子家族， 是在精子发生和排卵过程中调节事件的出色候选者。 Rhox同源域 蛋白质家族有几个成员（13个不同的基因，有些具有多个副本）。但是，只有 两个在Sertoli细胞中表达 生殖细胞的分化和存活。这两个基因Rhox5和Rhox8的消融导致男性 副作用为较低的附睾精子数，运动缺陷和繁殖力降低。后代 从Rhox5-null小鼠中，与Rhox8在体内RNAi敲低小鼠中的交叉具有独特的表型 比任何一个突变体都更严重。虽然这两条鼠标线是发现的作用的有用工具 青春期后的Rhox5和Rhox8，它们无法阐明Rhox8在性腺中的潜在作用 发展为抑制性Rhox8变换，直到第7天后的雄激素才能打开 信号通常打开Rhox5。为了解决这个问题，将是一个新的完整的Rhox8删除模型 用于评估Rhox8在胚胎Sertoli细胞规范和功能中的功能。表征 这些小鼠引起了极大的兴趣，因为：1）rhox8是在sertoli细胞中唯一表达的rhox基因 胚胎性腺。那就不关心功能冗余，这是rhox的问题 过去的研究。集群的所有其他成员都仅限于原始生殖细胞，包括 Rhox5仅在产后在Sertoli细胞中发现。 2）分析Rhox8产后体内敲低的 MIC和Rhox8在胚胎睾丸培养物中的瞬时敲低，表明Rhox8调节SOX9。 3） Sox9是SRY下游的主要调节剂 - 指定男性与女性差异化的因素。 这就是Rhox8可能处于SRY和SOX9之间的性别确定途径中。 4）性别确定后， Sox9控制着测试开发中的多个关键事件，包括睾丸绳的形成，男性 特定的脉管系统以及Sertoli和Leydig细胞种群的分化。如果这些后堆 关系在胚胎中也是保守的，然后Rhox8可能是几个的关键辅助因子或调节器 性腺发展期间的事件。该提案中概述的实验试图系统地定位 Rhox8在男性性发展的环境中，发现其控制的基因网络。 由于Rhox5和Rhox8可能在产后Sertoli细胞中具有互补功能，我们将 检查Rhox5/Rhox8双基因敲除动物中的精子发生。初步数据表明这些 小鼠比单个突变体具有明显和更严重的表型。 Rhox8的阐明 Sertoli细胞中的功能很重要，因为它将为长期提供重要的“构件” 学习测试中所有Rhox基因的独立和协作功能的目标。