ANALYSIS OF THE ROLE OF GCNF IN SPERMATOGENESIS

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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.