Stage-specific changes in GDNF expression by rat Sertoli cells: a possible regulator of the replication and differentiation of stem spermatogonia.

In the adult testis, the precise control of the self-renewing replication and differentiation of stem spermatogonia is fundamental to male fertility. Previous studies have shown that the replication of A single (A(s)) spermatogonia, a population that includes the stem cells, is maximal at stage I of the cycle of the rat seminiferous epithelium and minimal at stage VII, while the ratio of A-paired spermatogonia to A(s) spermatogonia increases from stages I to VII. It has been hypothesized that these changes in A(s) spermatogonia replication and differentiation result from changes in the expression of glial cell-line derived neurotrophic factor (GDNF) by Sertoli cells. To directly test this hypothesis, we used immunocytochemistry and confocal microscopy to demonstrate that within intact seminiferous tubules, GDNF is detectable only in Sertoli cells and that its amount and its location within these cells changes with progression of the stages of the cycle. The identification of Sertoli cells as the primary source of GDNF was confirmed by RT-PCR analysis of RNA isolated from purified populations of Sertoli cells, pachytene spermatocytes, and round spermatids. Stage-specific changes in GDNF expression were confirmed by quantifying GDNF mRNA in seminiferous tubules at defined stages of the cycle. Expression of this transcript was maximal at stage I, fell 14-fold by stage VIIc,d, and then increased 12-fold by stages XIII-XIV. This pattern of expression was the opposite of the control, cathepsin L mRNA. Taken together, these data support the hypothesis that cyclical changes in GDNF expression by Sertoli cells are responsible for the stage-specific replication and differentiation of stem spermatogonia, the foundational cells of spermatogenesis.

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

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.

The mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis.

Maturation of spermatozoa, including the acquisition of motility and the ability to undergo capacitation, occurs during transit through the dynamic environment of the epididymis. The microenvironments created along the length of the epididymal tubule are essential to the molecular modifications of spermatozoa that result in fertile gametes. The secretory and resorptive processes of the epithelial cells that line this tubule generate these microenvironments. In the current study, 10 morphologically distinct segments of the mouse epididymis were identified by microdissection. We hypothesized that the changing environments of the epididymal lumen are established by differential gene expression among these segments. RNA isolated from each of the 10 segments was analyzed by microarray analysis. More than 17,000 genes are expressed in the mouse epididymis, compared with about 12,000 genes identified from whole epididymal samples. Screening a panel of normal mouse tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, this study identified 2168 genes that are up-regulated or down-regulated by greater than 4-fold between at least two different segments. The expression patterns of these genes identify distinct patterns of segmental regulation. Using principal component analysis, we determined that the 10 segments form 6 different transcriptional units. These analyses elucidate the changes in gene expression along the length of the epididymis for 17,000 expressed transcripts and provide a powerful resource for the research community in future studies of the biological factors that mediate epididymal sperm maturation.