ANALYSIS OF THE ROLE OF GCNF IN SPERMATOGENESIS

GCNF在精子发生中的作用分析

• 批准号: 2392466
• 负责人: Austin J Cooney
• 金额: $ 10.15万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392466
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; disease /disorder model; gel mobility shift assay; genetic transcription; laboratory mouse; laboratory rabbit; mutant; spermatogenesis; transcription factor

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.

雄性特发性无菌性是男性不育症的形式 至少是因为这是由于多种原因所致。通常，受影响 雄性在精子功能中表现出严重或微妙的异常。因此， 了解男性特发性不育症需要了解 分子水平的精子发生。 精子发生是高度 复杂的分化过程由 睾丸/垂体轴。在睾丸控制中涉及许多 旁分泌信号通路主要是在leydig或sertoli中发出的 细胞。已经证明睾丸激素和类视黄素激素很重要 在这些过程中起作用的调节分子。 核 受体介导这些激素的转录作用。我们有 最近克隆了一个新型核受体超家族成员 通过北方分析显示在睾丸中非常高度表达 作为两个消息。 原位杂交分析表明高水平 仅在来自的生理细胞中检测到GCNF的表达 精子至精子阶段。这个核受体不密切 与先前克隆的成员有关，并被指定为孤儿受体 由于尚未确定其配体。因此，该核受体具有 可能成为尚未表征的细胞间或细胞内的潜力 睾丸中的信号传导途径调节 精子发生。在此应用中，我们建议分析几个方面 GCNF功能的长期目标是理解 信号通路调节该因素的转录活性， 无论是配体依赖性还是非依赖性。 我建议确定该因素是否存在GCNF同工型。 每个 物种将被克隆。 GCNF及其的发展表达 将在精子发生过程中检查同工型，以确定是否存在 暗示单个功能的独特或重叠的表达域 对于过程中的每种形式。免疫检测，使用抗体提出 针对GCNF，将确定GCNF翻译是否匹配表达式 基因或是否有延迟，这是一种常见的方法 精子发生过程中的调节。然后，我建议分析 GCNF的分子生物学涉及完全表征其DNA 绑定特性，涉及与延长一半位点结合 TCAAGGTCA。 随后分析其转录函数 瞬态转染。了解GCNF功能的这些方面后， 这些知识将应用于目标基因的分析，这是 已提出了哪种精神蛋白2作为好候选者。 用这个 知识，我们想了解GCNF在病变中的作用 可导致无菌性的精子发生。为此，一种两条脚步的方法 将被带走。一种方法是分析突变小鼠模型的模型 GCNF的参与。 这将涉及识别染色体 GCNF基因的定位以鉴定候选突变菌株。还， 将获得突变小鼠菌株，其睾丸的部分将是 分析了异常的GCNF表达。这种类型的分析可能会发现 一种模型的小鼠系统来分析GCNF及其在不育中的作用。第二个 方法涉及直接筛查患有特发性的雄性 不育以确定GCNF表达中是否存在任何畸变或 可以在这些子组中与该综合征相关的功能 男性。这些分析应有助于我们理解精子发生 以及涉及的信号过程。 了解GCNF的功能 将有助于长期识别推定的配体。 然后可以使用配体的识别来产生激动剂和 GCNF功能的拮抗剂，可用于调节其 在适用的情况下，活动和治疗特发性不育。