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

Abstract

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.

FIG. 1.

Confocal microscopic analysis of the distribution of GDNF within the rat seminiferous epithelium. Fixed seminiferous tubule segments were incubated with anti-GDNF IgG, the bound antibody was detected by use of a Alexaflour 488 (green)-goat anti-rabbit IgG, and the tubules were counterstained with DAPI (blue). All images are optical sections taken through the middle of a segment of a seminiferous tubule. The bar in the lower right corner of each image in this figure is equal to 10 μm in the tissue. Optical sections are either 320 nm thick (A) or 600 nm thick (B–F). The basement membrane of a tubule is located toward the upper left-hand corner of the image (A) or toward the top of the image (B–F). A) A high-resolution optical section showing the distribution of GDNF in a Sertoli cell within the basal half of a stage VII seminiferous tubule. The arrow points to a Sertoli cell nucleus and its distinctive nucleolus. B) An optical section through a stage XIII–I seminiferous tubule. C) An optical section through a stage II–V seminiferous tubule. D) An optical section through a stage VII seminiferous tubule. E) An optical section through a stage XI–XII seminiferous tubule. F) An optical section through a stage XI–XII tubule that had been incubated with anti-GDNF IgG that was preadsorbed with its antigen.

FIG. 2.

Quantitative analysis of GDNF concentration in Sertoli cells within XIII–I and VII tubules. The relative concentration of GDNF in Sertoli cells at both stages was assayed by immunocytochemistry, confocal microscopy, and image analysis. Results were obtained from five independent sets of tubules. The relative concentrations (mean ± SEM) of GDNF in Sertoli cells within tubules at the two stages are expressed as average pixel intensities per Sertoli cell.

FIG. 3.

RT-PCR analysis of the expression of Gdnf mRNA by pachytene spermatocytes, round spermatids, and Sertoli cells; 150 ng of RNA from each cell type was used as a template in an RT reaction or in a negative control reaction, which lacked RT. One microliter, or the equivalent of 20 nanoliters of the RT or negative control reaction, was used as template for PCR reactions for Gdnf cDNA or 18S rRNA, respectively. The products of the PCR reactions were fractionated on 1% agarose gels along with a 100-bp DNA ladder (right-hand lane), and the DNA was stained with ethidium bromide. These results are representative of results obtained from three independent isolates of pachytene spermatocytes, round spermatids, and Sertoli cells.

FIG. 4.

Stage-specific changes in expression of Gdnf mRNA and cathepsin L (Ctsl) mRNA. Seminiferous tubules at defined stages of the cycle were isolated by transillumination-assisted microdissection, and RNA was purified and used as template for cDNA synthesis. Expression of both Gdnf and Ctsl mRNAs were normalized for 18S rRNA. Data (mean ± SEM) are obtained from three independent samples of tubules and are expressed as the ratio of expression of Gdnf and Ctsl mRNA at each stage compared to the expression of those two transcripts in the whole testis. Means marked with different superscripts are statistically different.