Cytogenetic analysis has identified 12p gain as the most frequent abnormality in human testicular germ cell tumors (TGCTs). It has been suggested that amplification and overexpression of stem cell-associated genes, including cyclin-D2, on the human chromosome 12p region are involved in germ cell tumorigenesis. By subtractive cDNA analysis, we identified Ddx1, a member of the DEAD-box protein family, as a gene predominantly expressed in the primordial germ cells of mouse embryos. Knockdown of Ddx1 in a mouse spermatogonia-derived cell line, GC-1spg, by short interference RNA repressed the expression of cyclin-D2, CD9 and GDF3 genes. In the mouse cyclin-D2 gene, a genomic DNA region between À348 and À329 was responsible for transcriptional activation by DDX1 based on reporter and gel shift assays. Similarly, DDX1 knockdown in the human TGCT cell line NEC8 repressed the expression of stem cell-associated genes localized on chromosome 12p13.3, including cyclin-D2, CD9 and NANOG. DDX1-knockeddown TGCT cells could not form solid tumors in nude mice. Furthermore, in situ hybridization revealed that DDX1 mRNA was produced in both seminoma and nonseminoma types of human TGCT samples. We conclude that DDX1 is a critical factor for testicular tumorigenesis.

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We recently established a technique to expand male germ line stem (GS) cells in long-term culture without losing their spermatogenic capacity. To gain insight into the genetic program of these cells, we compared the mRNA expression profile of GS cells with that of embryonic stem (ES) cells using DNA microarrays. We found 79 genes that were upregulated in GS cells compared to ES cells, including synaptonemal complex protein-1, deleted in azoospermia-like, ubiquitin-conjugating enzyme E2B, and ubiquitin carboxy-terminal hydrolase L1, all of which are functionally important for spermatogenesis. In addition, we identified a cDNA encoding the mouse ortholog of capillary morphogenesis gene (CMG)-1. CMG-1 transcripts were predominantly produced in spermatogonia and spermatocytes in mouse testis. When CMG-1 expression was attenuated in a mouse spermatocyte-derived cell line, GC-2spd(ts), by a target-specific short interfering RNA, the morphology of the cells was changed and the expression of cyclin D2 was abrogated. A reporter assay using a genomic region upstream of the mouse cyclin D2 gene revealed that this downmodulation occurs at the transcriptional level. We detected FLAG-tagged CMG-1 protein in the nuclei of transfected COS7 cells, suggesting that CMG-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene. The upregulated GS genes identified in this study will provide useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation.

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