Spermatogenesis is a tightly regulated process characterized by spermatogonial stem cells undergoing mitotic expansion, meiosis and postmeiotic differentiation. Distinct morphological and biological characteristics of germ cells at different stages of spermatogenesis allow preparation of these cells in relatively pure form. Animal models are available which permit study of arrest and restart of spermatogonial differentiation. This makes spermatogenesis a unique model for studying stem cells and the genetic factors that regulate cellular proliferation and differentiation in general. The transcriptome of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) purified by the STAPUT technique were determined by sequencing of the respective SAGE (Serial Analysis of Gene Expression) libraries. A total of 444,015 SAGE tags derived from one Spga, two Spcy, and one Sptd libraries were analyzed. A total of 34,619 different species of transcripts were identified among these SAGE tags. Among these transcripts, 42.6% were derived from known genes, 42.1% were uncharacterized cDNAs and 15.3% were not described previously. Analysis of the spermatocyte transcriptome indicates the size of germ cell transcriptome could comprise of more than 30,000 transcripts, different from what was observed for other cells (Zhang et al., Science, 276[1997]1268). For each type of germ cells analyzed, 8-14 % of the transcriptomes are novel transcripts, 44-46% are uncharacterized cDNAs and 42-46% are transcripts of known genes. 12-19.5% of the transcriptomes consist of cell-specific transcripts while approximately 6,000 transcripts are shared by the three types of cells. It is worth noting that the most abundant tag matched with three UniGene clusters, the second most abundant tag represented an uncharacterized cDNA, while the third most abundant tag had no match in the SAGEmap database. Differential expression between the SAGE libraries was validated by quantitative realtime polymerase chain reaction (QPCR) of randomly selected transcripts. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy, transcription factors are abundantly expressed while chromosome remodeling genes and testis-specific genes are prominent in Sptd. Comparative analysis of SAGE and QPCR data were used to reveal the presence of alternatively spliced variants. The present study represents the first global study of gene expression in mouse germ cells. Based on this study it is evident that a core set of genes may be required for basic biological activities in all germ cells. However, cell-specific stages of spermatogenesis (mitotic, meiotic, and post-meiotic) require the concerted action of distinct sets of genes. It has been reported that aberrant expression of some X-linked genes would cause failure in spermatogenesis as well as improper sexual development. A recent report also indicated that spermatogonia harbor a substantial number of X-linked genes related to spermatogenesis. We have identified a number of X-linked genes that escape transcription inactivation at meiosis and in the post-meiotic stage in the SAGE study. In order to have a better understanding of the regulation of expression of X-linked genes, we clustered the expression of all X-linked genes. Two of such genes, the Testis expressed gene 13 (Tex13) and Sex comb on midleg-like 2 (Scml2), were found to express predominantly in type A spermatogonia. In silico studies indicated the existence of a potential antisense transcript of Tex13, which is complementary to the 3!| end of the Tex13 sense transcript. Reverse transcription-polymerase chain reaction (RT-PCR) studies revealed that the trend of change in the expression of the sense and antisense transcripts were similar, but the relative expression levels of the transcripts in different stages of germ cells were dissimilar. The regulation of expression, biological activities, as well as the relationship between the sense and antisense transcripts of Tex13 are currently under study. Scml2 is a member of the Polycomb group genes, which encode transcriptional repressors essential to appropriate development in the fly and in mammals. At least 3 different Scml2 transcripts were found in mouse testes, with each of them having a different 5!| untranslated region. Alternative use of exons in the coding region was also observed. By RT-PCR analyses, we found that the various forms of Scml2 transcripts were expressed at different levels during spermatogenesis, implying preferential use of the transcripts during germ cell development. In vivo as well as in vitro gene knockdown experiments are planned to study the functional roles of these X-linked genes during spermatogenesis. A number of novel as well as uncharacterized cDNAs identified to be differentially expressed in germ cells by SAGE as well cDNA microarray experiements performed previously were cloned and characterized. A novel ~1.1kb transcript shown to be preferentially expressed in round spermatids was cloned by cDNA library screening and 5!| RACE. Another spermatid-specific EST identified by cDNA microarray experiment was also cloned. SAGE analyses of germ cell transcriptomes identified a number of novel transcripts. The more abundant novel transcripts are being cloned using RT-PCR with extended primers. Characterization and developmental studies of these cDNAs are expected to yield information of their roles in spermatogenesis.