To understand the mechanisms that regulate the transition of primordial germ cells to gonocytes and the initiation of sexual dimorphism, we profiled the genes expressed in 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 using SAGE. We completed the analysis of ~152,000 SAGE tags for each of the male E10.5, E11.5, E12.5, and E17.5 gonads. The 10 most abundant tags are Cybb cytochrome b-245, beta polypeptide, Cyp2e1 cytochrome P450, COX5b, Translationally-controlled tumor protein Tctp1, Hbb-y hemoglobin Y, beta-like embryonic chain, Tuba2 tubulin, alpha 2, 4 ribosomal proteins (X-linked S4, L26, 29, and L10A) and one uncharacterized cDNA. Among the 10 most abundant tags present in embryonic gonads and absent in germ cells, are four genes encoding hemoglobin chains, namely Hba-X, Hbb-b1, and Hbb-Y, and Hba-a1. The role of hemoglobin genes in early embryonic gonad development is not presently known. However, in a recent paper that examined gene expression in embryonic lens, the expression of hemoglobin isoforms (Hba-a1, Hba-X, Hbb-b1, Hbb-b2, and Hbb-Y) was also demonstrated. These preliminary observations have important implications in understanding the regulation of gonad development and germ cell differentiation. Continued analysis of expressed genes in male gonads at later embryonic ages and female gonads and mesonephros are underway. Sexual dimorphism is known to occur in the brain, but the time of divergence is poorly understood. A better understanding of the relationship between brain and gonad development may give us hints of the cause of gender differences in development. Expressed genes in paired gonad and brain samples at different embryonic days (E10.5, E11.5, E12.5, E13.5, E15.5 and E17.5) were profiled using Affymetrix oligonucleotide gene chips. We had also generated human cDNA sex-linked gene microarrays of 724 X-linked and 28 Y-linked human genes on glass slides. These microarrays will be used to profile expressed genes in the brain and gonad 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 brains and gonads at different time points will be compared. The relationship between gonad development and onset of sexual dimorphism of the brain will be examined. Epigenetics of Testicular Germ Cell Tumors The objective of this study is to examine the global epigenetic alteration in human tumors, in particular testicular germ cell tumors (TGCT). Despite sharing similar regional genomic disruptions, recent studies of seminoma and non-seminoma testicular germ cell tumors (TGCT) suggested different epigenotypes. CpG island methylation is virtually absent in seminomas, while the methylation level in non-seminomas are similar to that of other solid tumors. Aberrant methylation of genes including CDKN2A, RUNX3, and MGMT hve been reported. However, the global picture of epigenetic alteration with reference to gene function category, expression pattern and prevalence in TGCT genome remains unclear. To study the global epigenetic alteration in TGCT, we examined the methylation profile of several testicular cancer cell lines using oligonucleotide based methylation microarrays. We have collected a number of testicular tumor tissue samples including archived frozen or paraffin samples of nonseminomas, seminomas, carcinoma in situ and normal testis. To minimize the effect of normal cell contamination in tumor tissue, Laser Capture Microdissection (LCM) will be performed. Epigenetic profile of TGCT will be obtained by hybridizing DNA with oligonucleotide based methylation microarray, while RNA will be used for expression profiling using affymetrix platform. Bisulfite sequencing/Methylation specific PCR (MSP) and real-time RT-PCR will be performed to validate methylation and expression data from the arrays. The data obtained from the cells will be compared with those from the tissues. The epigenetic profile and expression profile of the various tissues will be compared to identify differences among different histological types and stages of TGCT. The epigenetic and global expression profile information may allow a better characterization and molecular classification of TGCT and facilitate the discovery of specific biomarkers for early detection. Role of the TSPY-1 Oncogene in Prostate and Testis Cancers The testis-specific protein, Y-encoded (TSPY) gene has been mapped to the vicinity of the gonadoblastoma (GBY) locus on the Y chromosome and is considered to be a candidate gene for GBY. It is expressed in normal spermatogonial cells and carcinoma in situ (CIS) of the testis. Recently TSPY has been shown to express aberrantly in tumor epithelial cells in prostate cancers of low and high Gleason grades. This project aims to understand the genetic role of TSPY. HeLa cells harboring either a stably integrated TIG-TSPY or TIG vector were examined. Differential gene expression between the transfectant and the control was analyzed by the Significance Analysis of Microarrays. The biological processes represented by the differentially expressed genes were identified by analyzing the ontology of these genes using Gene Ontology Tree Machine (GOTM). The results showed 3 specific processes, i.e. cell cycle regulation, phosphate transport and neuromuscular development, to be altered. Among the genes up-regulated by the expression of TSPY were several oncogenes (epidermal growth factor receptor [ERBB], and members of the WNT and RAS oncogenes), growth factors (PDGFC, EGF-related, ANKRD15, RGC32, NANOS1) cyclin D2 (CCND2), and a co-factor for the hypoxia inducible factor 1A (p300), an apoptosis inhibitor (GSPT1) and an antigen (CD24) highly expressed small cell lung carcinoma. The down-regulated genes included CDK4/CDK6, transforming growth factor b3 (TGFb3), a pro-apoptotic factor (IGFB3), and an inhibitor of MAP kinases. In particular, the CCND2 gene resides on chromosome 12p that is frequently amplified and expressed at high levels in TGCTs. CCND2 complexes with CDK4 or CDK6 to mediate G1/S transition and promote cell proliferation. These preliminary data suggested that TSPY could be a candidate oncogene in male-specific tissues.
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