To elucidate the molecular mechanisms involved with spermiogenesis in testis, we performed differential display screening to isolate genes that are developmentally up-regulated during rat testis development. One of the cDNAs isolated by differential display was highly expressed in testis. Both reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression level of the gene developmentally increased. By screening the rat testis cDNA library, we successfully isolated rat cDNA clones encoding the entire open-reading frame of 462 base pairs coding a small protein of 154 amino acids. Because in situ hybridization revealed that the gene was specifically expressed in haploid spermatids in the rat seminiferous tubules, it was designated as spergen-1 (spermatogenic cell-specific gene-1). The recently opened database of the full-length mouse cDNA collection contains a mouse gene that is homologous to rat spergen-1. Subcellular fractionation followed by immunoblot analysis revealed that spergen-1 protein was associated with mitochondria. The transfection experiments performed in COS-7 cells suggested that spergen-1 has a N-terminal mitochondria-targeting signal. We suggest that spergen-1 might be involved in spermiogenesis by transiently associating with spermatid mitochondria.
How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.
Postnatal development of mammalian seminiferous tubules can be divided into three phases: spermatogonial mitosis, spermatocyte meiosis, and a postmeiotic phase in which drastic morphological changes occur in spermatids (spermiogenesis). In an attempt to elucidate the molecular mechanisms involved in spermiogenesis, we have applied a differential display method to identify genes that are developmentally up-regulated during rat testis development. One of the cDNA fragments isolated by differential display turned out to be iba1, an ionized calcium binding adapter molecule-1, that contains two EF hand-like motifs. Expression of iba1 mRNA in the rat testis was detected first at 4 wk in postnatal development and then increased up to adulthood. Using the antibody against a synthetic peptide corresponding to the N-terminal Iba1 protein, we discovered that Iba1 protein was not detectable by immunohistochemistry in spermatogonia, spermatocytes, and round spermatids in adult rat testis but was specifically expressed in the cytoplasm of elongate spermatids (steps 10-19) as well as in residual bodies that are ultimately engulfed by Sertoli cells. In situ hybridization, on the other hand, revealed that iba1 mRNA is present in round spermatids as well as early elongate spermatids (steps 1-12) but not in late spermatids, suggesting that iba1 mRNA undergoes post-transcriptional regulation. Because Iba1 protein is specifically expressed in the cytoplasm of elongate spermatids, which is finally engulfed as residual bodies into Sertoli cells, we suggest that Iba1 may be involved in the final stage of spermiogenesis (i.e., in elimination of the residual cytoplasm from spermatids).
Spergen-1, a recently identified molecule specifically expressed in haploid spermatids in testis, is a small protein of 154 amino acids with a mitochondria-targeting signal at the N terminus. To examine the localization of spergen-1 protein in germ cells, we performed immunocytochemistry with the anti-spergen-1 antibody on frozen sections of rat testis and purified spermatozoa. Immunolabeling for spergen-1 was detected in mitochondria of elongating spermatids and of the middle pieces of matured spermatozoa. Immunoelectron microscopy revealed that spergen-1 was localized to the surface of mitochondria in the middle piece of spermatozoa. To investigate the properties of spergen-1, COS-7 cells were transfected with vectors encoding various spergen-1 mutants. The transfection experiments showed that spergen-1 expressed in the cells tended to agglutinate mitochondria and assemble them into aggregations and that the C-terminal region of spergen-1 as well as the N-terminal mitochondrial targeting signal was requisite for induction of mitochondrial aggregation. These results suggest that spergen-1, a mitochondria-associated molecule in spermatozoa, has a property to induce mitochondrial aggregation at least in cultured cells. We hypothesize that spergen-1 might function as an adhesive molecule to assemble mitochondria into the mitochondrial sheath around the outer dense fibers during spermiogenesis.
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