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

通过差异显示和克隆相结合的方法，我们克隆了一个新的基因，命名为SERGEN-3，其开放阅读框架为1,500个核苷酸，编码一个500个氨基酸的蛋白质。Spergen-3含有Ankyrin重复基序和推测的核定位信号。我们发现精子-3的表达在发育过程中上调，并且只在睾丸中表达。原位杂交结果显示，精原细胞和圆形精子细胞中均有SERGEN-3mRNA的表达(步骤1~6)。激光共聚焦显微镜免疫组织化学分析显示，精子-3蛋白主要表达在晚期精母细胞(第IX~XIV期)和精子细胞(第1~11步)的胞核中，提示精子发生过程中精子-3的表达受到限制。在生精细胞核的核质中，精子-3倾向于定位于染色体间隙，DNA密度相对较低。从生精小管细胞核中提取精子-3的实验表明，在氯化钠存在的情况下，DNase I可以将精子-3从细胞核中释放出来，而RNase不能。DNA亲和层析柱在氯化钠存在下能与精子-3结合，并通过降低氯化钠浓度将其释放。这些数据表明，精子-3可能与生精细胞中的染色质DNA有关。我们将这些数据解释为精子-3在精子发生中的潜在作用，特别是在从粗线期晚期精母细胞到精子细胞的细胞分化或早期精子分化中。

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.