ABSTRACT
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.
Subject(s)
Cytoskeletal Proteins/genetics , Gene Expression Regulation , Germ Cells/physiology , Stem Cells/physiology , Animals , Cell Line , Cyclin D2 , Cyclins/genetics , Cyclins/metabolism , Cytoskeletal Proteins/metabolism , Gene Expression Profiling , Genes, Reporter , Germ Cells/cytology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Spermatocytes/cytology , Spermatocytes/physiology , Spermatogonia/cytology , Spermatogonia/physiology , Stem Cells/cytologyABSTRACT
In spermatogenesis, Sertoli cells serve as supporting cells for the proliferation and differentiation of germ cells. However, it appears that Sertoli cell function is regulated by adjacent spermatogonial cells in the testis because expression of lipocalin-2 mRNA, which encodes an iron-siderophore-binding protein, is barely detectable in Sertoli cells of germ cell-deficient W/Wv mice, and more abundantly expressed in jsd/jsd mice. By employing a coculture system comprising immortalized Sertoli cells (designated as Sertoli-B) and c-Kit(+) spermatogonial cells from 7-d-old mouse testis, we found that lipocalin-2 gene transcription in Sertoli cells is induced by a factor secreted from spermatogonial cells. Transfection of Sertoli-B cells with a series of reporter constructs encompassing an upstream region of the mouse lipocalin-2 gene revealed that a nuclear factor (NF)-kappaB binding consensus sequence in the proximal region of lipocalin-2 gene is responsible for transcriptional activation. A major NF-kappaB component, p65, bound to this region and translocated from the cytoplasm to the nucleus upon stimulation with spermatogonial cell-conditioned medium. Moreover, short interference RNA directed to p65 or a dominant-negative form of IkappaBalpha suppressed the spermatogonial cell factor-mediated transcription of lipocalin-2. However, NF-kappaB-activating inflammatory molecules, such as IL-1beta and lipopolysaccharide, did not induce lipocalin-2 mRNA in Sertoli-B cells and the expression of lipocalin-2 was unaffected in the testis of IkappaBzeta-deficient mice. These results demonstrate that spermatogonial cells regulate lipocalin-2 gene expression in Sertoli cells in a manner distinct from that employed by immune cells.
