Spermatogenesis is a tightly regulated process characterized by spermatogonial stem cells undergoing mitotic expansion and differentiation. Distinct morphological and biological characteristics of germ cells at different stage 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. One of the goals of this research is to delineate the network of genes that regulate spermatogenesis. To achieve this goal, we study the global changes in gene expression in type A spermatogonia (Sg), pachytene spermatocytes (Sc) and round spermatids (Sd) of the mouse using cDNA microarrays. Two types of microarrays were employed, namely the Nylon-membrane based Mouse GeneFilters that contain 5,184 mouse genes (ResGen) and the glass-slide microarrays that were printed with the NIA 15K mouse cDNA clone set. In the experiment with GeneFilters, 79 differentially expressed genes and ESTs were identified among the three types of germ cells. Quantitative Real-Time PCR was used to confirm the differential expression of a number of these genes. In the experiment using glass-slide microarrays, we focused on studying the changes in gene expression in the transition of Sc to Sd. A total of 161 differentially expressed genes were identified. More than one-fourth (43/161) of these were uncharacterized genes. A larger number of genes (110/161) were found to be preferentially expressed in Sd. Functional categorization indicated that genes responsible for signal transduction, energy metabolism, biosynthesis and cellular transport were preferentially expressed in Sd, while genes for chromatin remodeling were expressed only in Sc. Several testis-specific genes (feminization 1b homolog; phosphatidylcholine transfer protein-like; sperm specific antigen 1; cDNA moderately similar to casein kinase) were found to be expressed preferentially in Sd. Confirmation of differential expression of these genes was achieved by both Quantitative Real-Time PCR and Serial Analysis of Gene Expression (SAGE). SAGE was performed on Sc and Sd using the I-SAGE kit (Invitrogen Corp). 101,068 and 106,212 tags of the Sc and Sd library respectively were sequenced. Excluding singletons, these represented 10,717 and 10,135 genes respectively. In the Sc library, 4 tags were present at more than 0.5%. These tags matched a mitochondrial sequence (0.65%), t-complex-associated testis expressed 3 (0.57%), and Y box protein 2 (0.50%). The third abundant tag (0.56%) had multiple hits in the SAGEmap database. In the Sd library, 4 tags were present at more than 0.5%. The most abundant tag matched protamine 2 (1.31%), followed by that matching FK506 binding protein (1.18%), and a tag that matched a mitochondrial sequence (0.64%). The fourth abundant tag (0.56%) had multiple hits. Virtual subtraction of the two libraries yielded 4,344 Sc-specific tags and 4,155 Sd-specific tags. The majority of these cell stage specific tags were present at less than 5 copies. 353 Sc-specific tags were present at more than 5 copies and only 38 of these were present at more than 10 copies. The corresponding figures for Sd-specific tags were 266 and 27. The most abundant Sc-specific tag matched Janus kinase 3 (43 tags, 0.040% of library), followed by that matching WW domain binding protein 4 (37 tags, 0.034%) and dynein (28 tags, 0.026%). Two of the 3 most abundant Sd-specific tags, CAGAAGGCGG and TATTAAAGCT, both at 18 copies (0.017%) were novel with no hit in the SAGEmap database. The other tag also present at 18 copies matched a RIKEN cDNA. Comparison of the results obtained by cDNA microarray hybridization and SAGE indicated a high degree of concordance (80%) between the two methods. Discordance was limited only to genes of low expression level. Our work succeeded in identifying a large number of genes previously unknown to be expressed in germ cells (characterized genes + ESTs more than 12,270) as well as novel genes. It is also the first in its kind to compare in detail the gene expression pattern in mouse germ cells. Results obtained provide the foundation for investigation of genetic regulation of spermatogenesis as well as abnormalities of such process in pathological conditions.
Showing the most recent 10 out of 14 publications
