Complementary DNA cloning and characterization of rat spergen-3,a spermatogenic cell-specific gene-3,expressed in mammalian spermatogenic cells

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By use of differential display in combination with cDNA cloning approach, we isolated a novel gene termed spergen-3,which had an open reading frame of 1,500-length nucleotides encoding a protein of 500 amino acids. Spergen-3 contains ankyrin repeat motifs and a putative nuclear localization signal. We found that the expression of spergen-3 was developmentally up-regulated and that it was exclusively expressed in testis. In situ hybridization revealed that spergen-3 mRNA was expressed in spermatocytes and round spermatids (step 1〜6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-3 protein was predominantly expressed in nuclei of late spermatocytes (stage IX〜XIV) and spermatids (step 1〜11), indicating the restricted expression of spergen-3 during spermatogenesis. In nucleoplasm of spermatogenic cell's nuclei, spergen-3 tended to localize in tide interchromosome space with relatively low density of DNA. Extraction experiments of spergen-3 from isolated nuclei of seminiferous tubules demonstrated that spergen-3 is released from nuclei by DNase I in the presence of NaCl but not by RNase. Affinity column of DNA could bind spergen-3 in the presence of NaCl and it was released from the column by decreasing the concentration of NaCl. These data suggested that spergen-3 might be associated with chromatin DNA in spermatogenic cells. We interpreted these data as a potential role of spergen-3 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermati differentiation.