ANALYSIS OF THE ROLE OF GCNF IN SPERMATOGENESIS

男性特发性不育是男性不育的一种形式 最不为人所知是因为它是由多种原因造成的。通常，受影响 男性在精子功能上表现出明显或轻微的异常。因此， 了解男性特发性不育需要了解 分子水平上的精子发生。精子发生是一种高度 协调调节的复杂的分化过程 睾丸/脑垂体轴。在睾丸内控制涉及许多 旁分泌信号通路主要在间质或支持层发出 细胞。睾酮和维甲酸激素已被证明是重要的 在这些过程中起作用的调节分子。核子 受体介导这些激素的转录效应。我们有 最近克隆了核受体超家族的一个新成员 Northern分析表明，在睾丸中有非常高的表达 作为两条信息。原位杂交分析显示高水平的 GCNF仅在人的生精细胞中表达。 精母细胞到精子细胞阶段。这种核受体并不紧密地 与先前克隆的成员有关，被指定为孤儿受体 因为它的配体还没有确定。因此，这种核受体具有 有可能成为尚未确定特征的细胞内或细胞内 睾丸内的信号通路，调节某些方面的 精子发生。在本应用程序中，我们建议分析以下几个方面 GCNF的职能，长期目标是理解 调节该因子转录活性的信号通路， 无论它是依赖于配体还是不依赖于配体。 我建议确定该因子是否存在GCNF亚型。每个人 物种将被克隆。GCNF及其相关基因的发育性表达 将在精子发生过程中检查异构体，以确定是否有 不同或重叠的表达结构域暗示单独的功能 在此过程中的每个表单。免疫检测，使用产生的抗体 根据GCNF，将确定GCNF转换是否与表达式匹配 或者是否有延迟，这是一种常见的方法 精子发生过程中的调控。然后，我建议分析一下 GCNF的分子生物学，包括对其DNA的全面表征 结合属性，这涉及到与扩展的半结合部位的结合 TCAAGGTCA。随后对其转录功能进行了分析 瞬时转染法。了解了GCNF函数的这些方面后， 这些知识将被应用于目标基因的分析，其中之一 其中鱼精蛋白2被认为是一个很好的候选者。用这个武装起来 知识，我们想要了解GCNF在皮损中的作用 导致不育的精子发生。为此，我们采取双管齐下的方法 都会被带走。一种方法是分析突变小鼠模型 GCNF的参与。这将涉及到识别染色体 GCNF基因定位以确定候选突变菌株。另外， 将获得突变的小鼠品系，并将它们的睾丸切片 分析GCNF的异常表达。这种类型的分析可能会发现 一种分析GCNF及其在不孕症中作用的模型小鼠系统。第二 方法包括直接对患有特发性疾病的男性进行筛查 以确定GCNF表达是否存在任何异常或 在这些子群中可以与这种综合征相关的功能 男性。这些分析应该有助于我们理解精子发生。 以及所涉及的信令过程。理解GCNF的功能 将有助于对假定的配体进行长期鉴定。 然后，配基的鉴定可用于产生激动剂和 可用于调节ITS的GCNF功能拮抗剂 活动和治疗特发性不孕症，如适用。

Male idiopathic sterility is the form of male infertility about which least is known because it is due to multiple causes. Generally, affected males display gross or subtle abnormalities in sperm function. Thus, understanding male idiopathic infertility requires an understanding of spermatogenesis at the molecular level. Spermatogenesis is a highly complex differentiation process that is coordinately regulated by the testicular/pituitary axis. Within the testis control involves many paracrine signaling pathways primarily emanating in the Leydig or Sertoli cells. Testosterones and retinoid hormones have been shown to be important regulatory molecules that play a role in these processes. Nuclear receptors mediate the transcriptional effects of these hormones. We have recently cloned a novel member of the nuclear receptor superfamily which by Northern analysis was shown to be very highly expressed in the testis as two messages. In situ hybridization analysis showed that high level GCNF expression was detected only in the spermatogenic cells from the spermatocyte to spermatid stages. This nuclear receptor is not closely related to previously cloned members and is designated an orphan receptor as its ligand has not been identified. Thus, this nuclear receptor has the potential to be part of an as yet uncharacterized inter- or intra-cellular signaling pathway within the testis that regulates some aspect of spermatogenesis. In this application we propose to analyze several aspects of the function of GCNF with the long term goal of understanding the signaling pathway regulating the transcriptional activity of this factor, whether it be ligand-dependent or -independent. I propose to identify whether GCNF isoforms exist for this factor. Each species will be cloned. The developmental expression of GCNF and its isoforms will be examined during spermatogenesis to determine if there are distinct or overlapping expression domains suggesting individual functions for each form during the process. Immunodetection, using antibodies raised against GCNF, will determine whether GCNF translation matches expression of the gene or whether there is a delay, which is a common method of regulation during spermatogenesis. Then, I propose to analyze the molecular biology of GCNF which involves fully characterizing its DNA binding properties, which involves binding to the extended half site TCAAGGTCA. Subsequently to analyze its transcriptional function in transient transfections. Having understood these aspects of GCNF function, this knowledge will be applied to the analysis of target genes, one of which protamine 2 has been proposed as a good candidate. Armed with this knowledge, we want to understand the role of GCNF in lesions of spermatogenesis that lead to sterility. For this a two pronged approach will be taken. One approach is to analyze mutant mouse models for involvement of GCNF. This will involve identifying the chromosomal localization of the GCNF gene to identify candidate mutant strains. Also, mutant mice strains will be obtained and sections of their testis will be analyzed for aberrant GCNF expression. This type of analysis may uncover a model mouse system to analyze GCNF and its role in sterility. The second approach involves directly screening males suffering from idiopathic sterility to determine if there are any aberrations in GCNF expression or function that can be correlated with this syndrome in a subgroup of these males. These analyses should aid in our understanding of spermatogenesis and the signaling processes involved. Understanding the function of GCNF will aid in the long-term identification of the putative ligand. Identification of the ligand can then be used to generate agonists and antagonists of GCNF function which can be used to used to modulate its activity and treat idiopathic sterility where applicable.