Stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and Sertoli cells

Abstract

Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes, and spermatids suggests the existence of precise programs of gene expression in these cells and in their neighboring somatic Sertoli cells. The objective of this study was to identify the genes that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, and XIII-XIV of the cycle were isolated by microdissection, whereas Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes, and round spermatids were purified from enzymatically dispersed testes. Microarray analysis by using Rat Genome 230 2.0 arrays identified 16,971 probe sets that recognized testicular transcripts, and 398 of these were identified as testis-specific. Expression of 1,286 probe sets were found to differ at least 4-fold between two cell types and also across the stages of the cycle. Pathway and annotated cluster analyses of those probe sets predicted that entire biological pathways and processes are regulated cyclically in specific cells. Important among these are the cell cycle, DNA repair, and embryonic neuron development. Taken together, these data indicate that stage-regulated gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells.

Fig. 1.

Relative expression levels of 1,286 probe sets 4-fold regulated in both testicular cell types and during the cycle of the seminiferous epithelium. These probe sets were ordered by cell type and by stage with the highest expression. Data were visualized in Spotfire 8.0. Each probe set was z-scored normalized and color-coded (red = high expression, white = median expression, blue = low expression).

Fig. 2.

Transcriptional expression of cell cycle genes by pachytene spermatocytes, round spermatids, Sertoli cells, spermatogonia plus early spermatocytes, and by seminiferous tubules at defined stages of the cycle. In this figure and in Figs. 3 and 4, the testicular cell-type with the highest level of expression was defined as having a mean level of expression of 10 units. Data for the other cell-types and for staged tubules were normalized accordingly. Cdk2 and Ccnb1 encode the subunits of MPF. Data are expressed as the mean + SD. The stages of the cycle are defined by germ cell morphology and different stages persist for different but precise periods of time (12, 46).

Fig. 3.

Transcriptional expression of genes involved in base excision repair, nucleotide excision repair, recombinational repair, and/or single-strand break repair by pachytene spermatocytes, round spermatids, Sertoli cells, spermatogonia and early spermatocytes, and seminiferous tubules at defined stages of the cycle of the seminiferous epithelium. Shown are messenger RNA levels for Polk, Pold3 (A); Mlh3, Parp2 (B); Ercc1, Nbs1, Rad50 (C); Apex1, Xrcc1, Aptx (D); and Tdp1 and Pnpk (E) as determined by microarray analysis. Data are expressed as the mean + SD.

Fig. 4.

Transcriptional expression by Sertoli cells and by spermatogenic cells of genes previously shown to regulate embryonic neuron development. Shown are messenger RNA levels for Cxcr4, Gpc4, Dpysl4 (A) and Dcx and Map1b (B) as determined by microarray analysis. Data are expressed as the mean + SD.