The transcriptome of male germ cells at different stages of development, i.e. mitosis, meiosis and post-meiosis as represented by type A spermatogonia, pachytene spermatocytes and round spermatids, respectively, was profiled by Serial Analysis of Gene Expression (SAGE) and cDNA microarray hybridization previously. The SAGE data were deposited in our publicly accessible website (URL:www.nichddirsage.nichd.nih.gov/publicsage/). A number of genes with similar profile of change among the germ cells using both techniques were selected for further studies. We hypothesize the distinct expression pattern of a gene reflects the specific role of the respective gene in different stages of spermatogenesis. Currently we are investigating three such genes, namely, Testis expressed gene 13 (Tex13), the mouse Lin-28 homolog, and a novel isoform of the mouse Ard-1 gene which encodes a putative co-subunit of the murine N-terminal acetyltransferase 1 complex. Anti-sense Transcription in Differentiating Germ Cells Anti-sense transcripts have been shown to be involved in transcriptional and post-transcriptional gene regulation including genomic imprinting, X-inactivation, RNAi, RNA editing, and mRNA processing, splicing, stability, transport, and translation. Computational analysis had identified substantial number of antisense transcripts in the mouse and the human genome recently. However, with the exception of the anti-sense transcript of the SPEER2 gene, no other example of sense-antisense transcript pairs in mammalian germ cell has been reported. Examination of 64 differentially expressed genes identified in mouse type A spermatogonia, pachytene spermatocytes, and round spermatids showed the presence of anti-sense transcripts of 41 genes (66%). Among the 41 genes, 29 genes with appreciable number of SAGE tags in the germ cells were picked for confirmation using orientation specific RT-PCR. The presence of anti-sense transcripts was confirmed experimentally for 17 genes. Examination of tissue distribution of 9 selected genes including Uba52, Calm2, Ppp1cc, Ppic, Tsg1, Tcte3, Pdcl2, Prm 1 and Prm2 showed a wide spectrum of tissue-specificity of the expression of the sense and the anti-sense transcripts. The antisense transcripts of four genes, namely Uba52, Tcte3, Prm1 and Prm2 were cloned and characterized. Alignment of the nucleotide sequence of the anti-sense transcripts with the genomic sequence of the genes encoding the sense transcripts allowed localization of the anti-sense transcripts to exons and introns of the sense gene, psuedogene, intronic as well as intergenic sequences. This study shows that anti-sense transcription occurs more frequently in differentiating germ cells than in somatic cells. We are in the process of identifying in vitro and in vivo systems suitable for testing functional activities of the anti-sense transcripts cloned. Novel Activity of Cytochrome C Oxidase in Germ Cell Development Extensive apoptosis occurs during spermatogenesis, particularly in spermatogonia and spermatocytes. The mechanism of apoptosis in germ cells has not been totally worked out. Recent studies suggested that cytochrome c plays a critical role in apoptosis occurring in germ cells. A search of the germ cell SAGE libraries revealed that all 13 subunits of cytochrome c oxidase, complex IV of the respiratory chain are expressed at appreciable levels and follow a comparable differential expression pattern in these cells. On the other hand, some of the subunits of complexes II, III, and V of the respiratory chain are absent and only 12 of the 43 subunits of complex I have the same differential expression pattern. These results imply that cytochrome c oxidase may not function as a component of the respiratory chain in the germ cells. Thus, we hypothesize that cytochrome c oxidase oxidizes cytochrome c causing its release from the inner mitochrondrial membbrane . Transfer of the solubilized cytochrome c into the cytosol results in the amplification of calcium-dependent apoptosis. We are in the process of proving this hypothesis using in vitro cell models. Functional Genomic Studies of Gonad Development and Sexual Dimorphism of the Brain In order to understand the mechanism that regulates the transition of primordial germ cells to gonocytes and the initiation of sexual dimorphism, we have undertaken the task of profiling the expressed genes in embryonic gonads of the mouse. We apply SAGE to male and female embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5, and E17.5 and mesonephros at E13.5, E15.5 and E17.5. We have so far completed the analysis of ~152,000 SAGE tags for each of the male E10.5, E11.5, and E12.5 gonads. These tags identify 24,460, 214,762, and 26,378 genes in the E10.5, E11.5, and E12.5 gonads, respectively. The 10 most abundant tags represent Cybb, Cyp2e1, Cox5b, Tctp1, Hbb-y, Tuba2, alpha 2, 4 ribosomal proteins (X-linked S4, L26, 29, and L10A) and one uncharacterized cDNA. Among the 10 most abundant tags that are present in the embryonic gonads and absent in germ cells, 4 represent genes encoding hemoglobin chains, namely Hba-X, Hbb-b1, and Hbb-Y, and Hba-a1. The roles of the hemoglobin genes in early embryonic gonad development are not clear at this point. In spite of the fact that less tags were sequenced for the germ cell SAGE libraries (~ 111,000 tags for each germ cell type versus ~152,000 tags for each embryonic gonad stage), more specific genes are found in germ cells than in embryonic gonads (4,946 germ cell specific-gene tags versus 4,755 embryonic gonad specific-gene tags). These preliminary observations have important implication on the regulation of gonad development and germ cell differentiation. We will continue to analyze expressed genes in male gonads at later embryonic ages as well as female gonads and mesonephros. We are also interested in the role of sex chromosome-linked genes in sexual dimorphism of the brain. To that end, we have generated human sex-linked gene cDNA microarrays on which the cDNA of 724 X-linked and 28 Y-linked human genes were spotted on a glass slide. These microarrays will be used to profile expressed genes in brains and gonads of male and female mice at E10.5, E13.5, E15.5, E17.5, newborn mice, and adult mice. The expression profile of male and female brain and gonad at different time point will be compared. The relationship between gonad development and onset of sexual dimorphism of the brain will be examined. The Role of Dby in Mouse Gonad and Germ Cell Development Dby (also known as Ddx3y) has been considered as a strong candidate mediating the function of the Y chromosome in spermatogonial proliferation. In the mouse the Dby gene gave rise to two alternative transcripts that differ only in the length of 3' untranslated region as a consequence of alternative polyadenylation signals. Both transcripts were ubiquitously expressed and were present in male germ cells and Sertoli cells. They were more abundant in type A spermatogonia compared to pachytene spermatocytes and round spermatids. Expression of Dby in the embryonic gonad increased from E10.5 and reached a peak at E17.5. The expression level of Dby decreased after birth and remained low in adult male gonads. Testicular expression of Dby was comparable to its X chromosome homolog, Ddx3. In contrast, D1Pas1, the autosomal homolog of Dby, was predominantly expressed in pachytene spermatocytes and round spermatids. The differential expression patterns of Dby and its X- and autosome-homologs suggests that though the three genes are highly homologous they participate in different regulatory pathways. Dby in mouse, unlike that in humans, may only affect earlier stages of spermatogenesis.
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