MICROARRAY ANALYSIS OF DIFFERENTIAL CELL CYCLE GENE EXPRESSION DURING THE SPERMATOGONIUMSPERMATOCYTE TRANSITION IN THE MOUSE

Abstract

Mammalian spermatogenesis is a continuum of cellular differentiation in which three principal phases can be discerned: 1) a mitotically active phase of spermatogonial renewal, proliferation and initiation of differentiation, 2) a meiotic phase in spermatocytes to facilitate reduction to haploidy and genetic recombination, and 3) a postmeiotic phase in spermatids where the differentiative process of spermiogenesis leads to formation of spermatozoa. The goal of the present study was to use recently acquired microarray data from the mouse to identify changes in expression of cell cycle and related genes that accompany the transition from mitotically active spermatogonia to meiotically active spermatocytes. We used microarray data previously reported (GEO accession number GSE4193) as well as unpublished microarray data (Griswold lab) to compare gene expression in type A spermatogonia and pachytene spermatocytes. After eliminating pixel intensities below a threshold level designed by Affymetrix, raw data was transformed in GeneSpring 7.2 using GC-RMA preprocessor software and then normalized for inter-array and intra-array comparison. Of the 39,000 transcripts represented on the Affymetrix MOE430 2.0 chip, 1,611 were annotated by Gene Ontology to be involved in cell cycle control. A majority of these were expressed at similar levels in both spermatogonia and spermatocytes, indicating ongoing activity of the cell cycle network in both cell types. A minimum fold change of 1.5 was set to select genes that are differentially expressed between mitotic and meiotic spermatogenic cells, respectively. Further refinement using a one-way ANOVA parametric test identified 430 cell cycle genes that were differentially expressed in a statistically significant manner. Of these, 239 were downregulated and 191 were upregulated in spermatocytes when compared to spermatogonia. Independent validation of differential expression of a subset of these genes is underway. Overall, our results indicate that the transition from mitosis to meiosis during spermatogenesis is characterized by a switch from relatively high activity of parts of the cell cycle network that promote initiation into and progression through the G1 phase in spermatogonia, to relatively high activity of parts of the network that promote exit from the G2 phase and entry into meiosis-specific regulation in spermatocytes. Specifically, in spermatogonia we observed higher expression of genes in the MAP kinase pathway, the protein biosynthesis pathway, and the Wnt receptor signaling pathway, and lower expression of cell cycle inhibitors that act on Cyclin D – a combination that favors continual progression through the cell cycle in mitotic cells. In spermatocytes, we observed relatively higher expression of the prostaglandin biosynthesis and ubiquitin pathways, as well as the anaphase regulating complex and cell cycle checkpoint proteins. We also observed expression of several spermatogenesis-specific members of the cell cycle network, including four in spermatogonia and five in spermatocytes. Of these, two of those expressed in spermatogonia, and three of those expressed in spermatocytes have not previously been reported. These results provide useful insight into key differences in cell cycle regulation that facilitate the transition from mitotic spermatogonia to meiotic spermatocytes. This work was supported by a grant from the NIH to JRM (HD46637). (platform)