To develop an understanding of the functional significance of protein and regulatory networks requires a complete description of the cellular transcriptome. Following the sequencing of the genes from several mammalian species the idenitification of functional genes within a network is based on in silico predictions substantiated by evidence of transcription in vivo. Conservative estimates suggest that there are about 20 000 protein-encoding genes in the mammalian genome. However, in-depth analyses of the transcriptional outputs from a range of experimental approaches suggests that the information content of the genome is much more complex than previously perceived. We used the developing male germ cells as a model system to study key developmental processes that lead to the transition from one cell type to another. To explore and identify the transcriptome complexity in male germ cells, we previously applied Serial Analysis of Gene Expression (SAGE) to profile the expression signature of the major stages of spermatogenesis, including type A spermatogonia (Spga), pachytene spermatocyte (Spcy) and round spermatid (Sptd). This study successfully identified a large number of novel transcripts, alternative splicing transcript variants and anti-sense transcripts. ? ? To provide an unbiased and higher resolution profile of the transcriptome, we expanded our SAGE findings by incorporating whole-genome 25 bpresolution tiling expression arrays(Affymetrix). With 45 million oligonucleotide probes and 35 bp probe spacing, we generated a high-definition transcriptome map of developing male germ cells with an unbiased and germ cellspecific whole-genome expression map. Preliminary data proved that, combining the SAGE data set with the tiling platform is powerful, and provides new insights into an analysis of the germ cell. In summary we found that more than 45% of transcripts were not annotated; current annotation only accounts for about 30% of our data set, the remainder of the data set mostly contains expressed sequence tags (ESTs). We identified thousands of transcript cluster units located within introns and internal exons of protein-coding genes indicative that promoter sites are common, and that transcriptional organization is complex. This transcriptional architecture implies that most genomic regions serve multiple functions. Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of such transcription remains unknown.? ? To further facilitate data analysis of the high throughput genomic assay generated in our laboratory custom bioinformatics tools were developed. GermSAGE is a comprehensive web-based database generated by the Serial Analysis of Gene Expression (SAGE) of the major stages during mouse male germ cell development with a sequence tag coverage of 150,000 in each SAGE library. A total of 452,095 tags derived from Spga, Spcy and round Sptd were included. GermSAGE provides web-based tools for browsing, comparing and searching male germ cell transcriptome data at different stages with customizable searching parameters. The data can be visualized in tabulated format or further analyzed by alignment with various annotations available in the UCSC genome browser. This flexible platform will be useful for gaining a better understanding of the genetic networks that regulate spermatogonial cell renewal and differentiation, and will allow novel gene discovery. GermSAGE is freely available at http://germsage.nichd.nih.gov/.? ? We are also developing an online tool called TransfragMap to map tiling array data to various gene annotations and mark particular gene features. This tool will accelerate qualitative analysis of tiling microarray data, and is platform independent. It is compatible with tiling microarrays from major tiling array platforms, including Affymetrix and Nimblegen. The tool will support the human and mouse genome in its initial version, and will be expanded to other species. ? ? Previous work has shown that GDNF-receptor-alpha-1 (GFRA1) is specifically expressed in spermatogonial stem cells (SSCs) and is required for their stem-cell properties. To characterize the molecular phenotype of SSCs, GFRA1(+) and GFRA1(-) spermatogonia were isolated from 6-day-old mice using magnetic activated cell sorting with an antibody to GFRA1 and their microarray-based expression profiles compared. The expression of a number of genes were found to be up-regulated in GFRA1(+) spermatogonia, with the most over-expressed one being Csf1r; it encodes the receptor for granulocyte-macrophage colony stimulating factor (GM-CSF), which has a well-established role in hematopoietic stem cell function. A number of chemokine ligands were also highly overexpressed in SSCs. Analysis revealed the potential role of chemokine signaling in SSCs and suggested a common pathway for GFRalpha-1 and Csf1r, which may lead to their self-renewal. ? ? Analyses of antisense transcripts suggested the existence of RNA-dependent RNA polymerase (RdRP) activity in mouse germ cells. Antisense transcripts complementary to multiple coding exons were identified for Tcte3, Ldh3, and Calm2. We focused our studies on Calm2 antisense transcript. The Calm2 antisense transcript was present in mouse testis and 3 mouse cell lines, namely,CRL-2576 (mouse spermatogonia cell line), CRL-1715 (mouse Sertoli cell line) and CRL-6436 (mouse kidney cell line). Confirmation of the antisense transcript as a product of the sense transcript was provided by a knockdown experiment. Knocking down the sense transcript of Calm2 using siRNA demonstrated reduced levels of both sense and antisense transcripts indicating that the synthesis of Calm2 antisense transcript was dependent on the sense transcript. Calm2 antisense was not synthesized starting from the 3 end of the sense mRNA. The sequence representing the potential start site of the action of RdRP was defined. A hybrid RNA containing this sequence ligated to EGFP on its 5 end was generated and introduced into CRL-6436 cells. Orientation specific RT-PCR showed production of an antisense RNA derived from hybrid RNA. The level of Calm2 anitsense transcript appeared to be independent of the stage of growth of cultured cells and was ubiquitous in all mouse tissues examined (testis, overy, liver, lung, kidney, spleen, thymus, heart, brain and embryo). These results provide further proof of the existence of RdRP activity in mammalian cells. Experiments to isolate and purify RdRP activity are underway.? ? Vitamin A deficiency (VAD) is known to cause spermatogenesis arrest. The molecular mechanism of this effect of vitamin A is not known. We plan to employ expression profiling to delineate the genetic basis of VAD-induced arrest of spermatogonial stem cell differentiation and the potential regeneration of spermatogenesis following restoration of vitamin A in the diet. We had initiated a timed study of the VAD diet in which we measure the vitamin A status of the animal, the RBP (Retinol Binding Protein) level in the serum, and the mRNA levels of retinoic acid receptors, RARalpha,beta,and gamma, and RXRalpha, beta, and gamma, in the liver and the testis. There is close relationship between thyroid hormones and vitamin A metabolism. Therefore we will also study the paternally imprinted enzyme deionidase iodothyronine type III (Dio3) during VAD. Dio3 is highly expressed in developmental tissues. However its expression and function in the testis has not been studied. Expression profiling and epigenomic studies of the germ cells will be started once the chronology of VAD is established.
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