Microarray-based analysis of differential expression during spermatogenesis in the mouse

Abstract

Mammalian spermatogenesis is a continuum of cellular differentiation in a lineage that features three principal stages: (1) a mitotically active stage in spermatogonia (2) a meiotic stage in spermatocytes, and (3) a post-replicative stage in spermatids. We used a microarray-based approach to identify changes in expression of (1) cell cycle, (2) DNA repair and apoptosis and (3) chromatin remodeling and epigenetic regulation that distinguish type A spermatogonia from pachytene spermatocytes and pachytene spermatocytes from post-replicative round spermatids. Despite the rigorous protection of the genome, genetic integrity is observed to decline with increased maternal and paternal age. We analyzed newly acquired microarray data from pachytene spermatocytes and round spermatids from different ages of mice to characterize expression of different pathways in analysis of expression of genes as a function of age.

We detected expression of 550 genes related to cell cycle function, 259 DNA repair genes and 470 apoptotic genes, and 661 chromatin remodeling and epigenetic regulation genes that were expressed during adult spermatogenesis in one or more of these cell types.

Our cell cycle analysis suggests that distinct cell cycle gene regulatory networks or sub-networks are associated with each phase of the cell cycle in each spermatogenic cell type. In addition, we observed expression of different members of certain cell cycle gene families in each of the three spermatogenic cell types investigated. We report expression of 221 cell cycle genes that have not previously been annotated as part of the cell cycle network expressed during spermatogenesis, including eight novel genes that appear to be testis-specific.

Our DNA repair and apoptosis pathway analysis suggests that genes up-regulated in spermatogonia include a majority of genes annotated as members of SSB repair, whereas genes up-regulated in spermatocytes included members of DSB repair. On the other hand, genes involved in TLS and direct repair were up-regulated in spermatids. Among the proapoptotic and antiapoptotic networks, some are up-regulated uniquely in spermatogonia showing there is a stringent regulation of apoptotic genes during mitosis. Most of the proapoptotic or antiapoptotic genes up or down-regulated in spermatocytes retain similar expression patterns in spermatids, indicating these pathways are similarly emphasized in meiotic and postmeiotic spermatogenic cells.

During adult spermatogenesis, we observed that even though a majority of the chromatin remodeling and epigenetic modification genes are expressed in all three spermatogenic cell types, there is a subset of genes that are unique to each cell type. On the other hand, many of the genes affected by these modifications were up-regulated in spermatogonia and then dramatically down-regulated in spermatocytes.

With age, there are subtle changes in expression of genes encoding pathways responsible for maintaining genetic integrity in male germ cells as a function of age, and that these changes appear to progress in opposite directions (up-regulation of DNA repair genes and down-regulation of apoptosis/cell death genes) as aging progresses. We also found that with increasing age, there is an age-associated loss of repression of genes affected by chromatin remodeling and epigenetic modifications in pachytene spermatocytes. This is the first report of an age-related reduction in epigenetic silencing of gene expression in germ cells.

Description
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Keywords
cell cycle, DNA repair, Epigeneitcs, mammal, Microarray, Spermatogenesis
Citation
Department
Integrative Biology