RHOX action in Sertoli development and function

项目总结/摘要 生殖同源框X连锁（Rhox）基因编码一个新的转录因子家族， 是调节精子发生和排卵过程中事件的极好候选者。RHOX同源结构域 蛋白质家族有几个成员（13个不同的基因，其中一些存在于多个拷贝中）。但只有 其中两种在支持细胞中表达，支持细胞是促进睾丸发育和养育睾丸的主要控制细胞。 生殖细胞的分化和存活。去除这两个基因Rhox 5和Rhox 8， 以附睾精子数量减少、活动力缺陷和生育力降低为特征的生育力低下。后代 来自Rhox 5缺失小鼠，与RHOX 8体内RNAi敲除小鼠杂交，具有独特的表型， 比任何一个突变体都严重虽然这两个鼠标线是有用的工具，发现的作用， RHOX 5和RHOX 8在青春期后，它们不能阐明RHOX 8在性腺中的潜在作用 发育，因为抑制性Rhox 8转基因直到出生后第7天才开启， 信令通常开启Rhox 5。为了解决这个问题，一个新的完整的Rhox 8删除模型将在 用于评估RHOX 8在胚胎支持细胞特化和功能中的功能。表征 这些小鼠是非常感兴趣的，因为：1）Rhox 8是在小鼠的Sertoli细胞中表达的唯一Rhox基因。 胚胎性腺因此，将不会有功能冗余的问题，这一直是Rhox的问题 过去的研究。该簇的所有其他成员仅限于原始生殖细胞，包括 Rhox 5基因仅在生后支持细胞中表达。2)Rhox 8出生后体内敲低的分析 mic和胚胎睾丸培养物中RHOX 8的瞬时敲低，表明RHOX 8调节Sox 9。第三章 SOX 9是SRY下游的主要调节因子，SRY是指定雄性与雌性分化的因子。 因此，RHOX 8可能在SRY和SOX 9之间的性别决定途径中。4)性别决定之后， SOX 9控制着测试发展中的多个关键事件，包括睾丸索的形成，男性- 特定的脉管系统，以及支持细胞和间质细胞群的分化。如果这些青春期后的 在胚胎中也是保守的，那么RHOX 8可能是几个基因的关键辅因子或调节因子。 性腺发育过程中发生的事件。本提案中概述的实验旨在系统地定位 Rhox 8在男性性发育环境中的作用，并发现受其控制的基因网络。 由于RHOX 5和RHOX 8在生后支持细胞中可能具有互补功能，我们将 检查Rhox 5/Rhox 8双敲除动物的精子发生。初步数据显示， 小鼠具有与任一单一突变体不同且更严重的表型。Rhox 8的解释 支持细胞的功能是重要的，因为它将提供一个重要的“基石”， 目的是了解睾丸中所有Rhox基因的独立和协作功能。

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.