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， 最少的是已知的，因为它是由于多种原因。一般来说，受 雄性在精子功能上表现出明显或细微的异常。因此，在本发明中， 了解男性特发性不孕症需要了解 精子发生的分子水平。 精子发生是一个高度 复杂的分化过程，由 睾丸/垂体轴睾丸内的控制涉及许多 旁分泌信号通路主要起源于Leydig或Sertoli 细胞睾丸酮和维甲酸类激素已被证明是重要的 在这些过程中发挥作用的调节分子。 核 受体介导这些激素的转录作用。我们有 最近克隆了核受体超家族的一个新成员， 通过北方分析，显示在睾丸中非常高表达 两个信息。 原位杂交分析表明，高水平的 GCNF的表达仅在睾丸生精细胞中检测到， 精母细胞到精子细胞阶段。这种核受体与 与先前克隆的成员相关，并被指定为孤儿受体 因为它的配体还没有被鉴定出来。因此，这种核受体具有 可能是尚未表征的细胞间或细胞内 睾丸内的信号通路，调节某些方面， 精子发生在本申请中，我们提出分析几个方面 GCNF的功能，长期目标是了解 调节该因子转录活性的信号通路， 无论它是配体依赖性的还是非依赖性的。 我建议，以确定是否存在GCNF亚型的这个因素。 每个 物种将被克隆。GCNF的发育性表达及其与发育的关系 将在精子发生过程中检查同种型，以确定是否存在 不同的或重叠的表达域，表明个体功能 在过程中的每一种形式。免疫检测，使用产生的抗体 针对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， 在精子发生过程中的调节。然后，我建议分析 GCNF的分子生物学，包括完全表征其DNA 结合特性，包括与延伸半位点的结合 TCAAGGTCA。 随后分析其转录功能， 瞬时转染。在理解了GCNF功能的这些方面之后， 这些知识将被应用于靶基因的分析， 其中鱼精蛋白2已被提议为良好的候选物。 有了这些 知识，我们想了解GCNF在病变中的作用， 导致不育精子发生。为此， 会被带走一种方法是分析突变小鼠模型， GCNF的参与。 这将涉及识别染色体 定位GCNF基因以鉴定候选突变株。还有， 将获得突变小鼠品系，并将其睾丸切片 分析异常GCNF表达。这种分析可能会揭示 一种分析GCNF及其在不育中作用的模型小鼠系统。第二 方法涉及直接筛查患有特发性 无菌性，以确定GCNF表达中是否存在任何畸变， 功能，可以与这种综合征在一个小组，这些 男性。这些分析有助于我们理解精子发生 以及相关的信号传递过程。 了解GCNF的功能 将有助于长期鉴定推定的配体。 然后，配体的鉴定可用于产生激动剂， GCNF功能的拮抗剂，其可用于调节其 活性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.