Study On The Transcriptional Regulation Of Spermatogenesis-related Gene ZNF230 And Mutation Screening Of UBE2B Gene In Patients With Azoospermia

Spermatogenesis is a complex cell division and differentiation process that results in a series of molecular and morphological changes in male germ cells.This process requires the highly regulated expression of a number of genes. Some of the genes on Y chromosome have been studied systematically and two candidate gene families onY chromosome are reported to be associated with azoospermia.They are RNA-binding motif(RMB) genes and Gene deleted in azoospermia(DAZ).In addition,many other autosomal genes are also reported to be involoved in spermatogenesis in both mouse and human including DAZLA,the autosomal DAZ gene homologue),CREM,HSP70-2 and its human homologue HSPA2.My study include 3 parts.Recently the research group at Department of Medical Genetics has cloned spermatogenesis-related cDNA of Human gene ZNF230 and itsmouse homolog znf230 by rapid amplification of cDNA ends (RACE).The protein of znf230 exhibits DNA binding activity and its ring finger domain may function as an activator module in transcription. Therefore, it is postulated that znf230 may function as a testis-specific transcription factor during mouse spermatogenesis.Part I .For better understanding the expression,i.e.the localization and regulation of the ZNF230 protein,we use the green fluorescent protein GFP as a marker to study the localization of the fusion protein. We constructed the plasmids by fusing the gene of ZNF230 with green fluorescent protein(GFP). and then transfected the Cos cell line.As the results,we are able to show that the latter can express both human ZNF230 and mouse znf230 with high efficiency.and the ZNF230 protein localizes mainly in the nucleus.Part II .To better understand the mechanism of the transcriptional regulation of ZNF230,We have cloned a 1.1-kb fragment upstream of the ZNF230 gene and by deletion mapping,we are able to identify the key regions necessary for the promoter activity. Thus the ZNF230 gene expression is controlled by a TATA-less promoter. The region of bp -134 to +149 is the minimal promoter for the transcription of the gene. Several putative cis-acting elements, such as GC box, SRY/Sox, PUbox, API, AP2, and USF elements, are found in the 5' flanking region of the gene. Transcriptional activation and positional mutation demonstrated that the ZNF230 promoter contains a functional Spl response element,These results suggest that ZNF230 gene expression is regulated at the transcriptional level and that the transcription factor Spl plays an important role in regulation of ZNF230 expression.Part III. To identify the other possible germ cell specific transcription factor which may also control the expression of ZNF230, we constructed a recombinant pcDNA3-Sox5 expression vector.Since overexpression of Sox5 protein significantly potentiates ZNF230 promoter activity, the Sox5 protein may also take part in the control of testis specific expression of ZNF230.UBE2B gene encodes a ubiquitin-conjugating DNA-repair enzyme, Previous study show that this gene is necessary for the meiotic division of germ cells in mouse testis. Using DHPLC, screening of the exon-containing genomic segments of the UBE2B gene was performed in 157 infertile patients with severe oligozoospermia or azoospermia. An A→G transition mutation in exon 3 (Second codon) was identified in one case,which was not found in controls. This finding provide a preliminary evidence for the possible role of the autosomal gene UBE2B in human spermatogenesis.