Effects of glial cell line-derived neurotrophic factor on isolated developing mouse Sertoli cells in vitro

Abstract

Cell proliferation is a key factor in sex determination where a size increase relative to the XX gonad is one of the first signs of testis differentiation. Moreover, proliferation of Sertoli cells during development is important in building up the stock of supporting cells necessary for subsequent successful fertility. Because proliferation is such an essential part of testis development, the hypothesis under long-term investigation is that it is under fail-safe control by multiple alternative growth factors. This study was undertaken to investigate the role of glial cell-derived neurotrophic factor (GDNF) on developing mouse Sertoli cells in vitro. Sertoli cells, isolated from mouse embryos at three stages of testis development, were maintained for 2-7 days in vitro (div) in the presence or absence of GDNF at 1, 10 and 100 ng mL(-1). Overall the presence of extracellular matrix gel had little effect on proliferative activity, but encouraged expression of the epithelial phenotype. A statistically significant difference in proliferation, assessed by immunocytochemical staining for proliferating cell nuclear antigen, was seen with GDNF at embryonic day (E)12.5 after 2 div (at both 10 and 100 ng mL(-1), P < 0.001) and 7 div (at both 10 and 100 ng mL(-1), P < 0.05); at E13.5 after 3 div (at both 10 and 100 ng mL(-1), P < 0.05) and at E14.5 after 7 div (100 ng mL(-1), P < 0.01), compared with controls cultured without growth factor. In conclusion, GDNF stimulates mitosis throughout this critical developmental window. The in vitro approach used here is a useful adjunct to the knockout mouse model and has been applied to show that GDNF exerts a proliferative effect on developing mouse Sertoli cells.

Fig. 1

Phase contrast micrograph of ovary (A) and testis (B) isolated from E12.5 mouse embryos. Abbreviations: o = ovary, t = testis, m = mesonephros. Arrows in B indicate blood vessels between testicular cords and testicular cords. Scale bars, 200 µm.

Fig. 2

(A,B) Phase contrast micrographs of cells from E12.5 supernatant 1 (A) and supernatant 2 (B) after 2 div. Note red blood cell (rbc) in A, cells with peritubular phenotype (arrows) in B. Scale bars represent 20 µm. (C,D) Immunolocalization of AMH (C) and α-SMA (D) in E13.5 Sertoli cells cultured for 7 div in the presence of 10 ng mL⁻¹ GDNF. Arrows indicate areas of positive staining. Scale bars, 100 µm.

Fig. 3

Morphology of cultured Sertoli cells. (A) After 1 div some E13.5 cells without GDNF remain rounded (arrows) whilst others are flattening to the uncoated substrate (arrowheads). (B) E13.5 cells cultured on ECM gel substrate align together (arrows) and begin to form cord-like aggregations after 2 div without GDNF. (C) E12.5 cells cultured on ECM gel substrate without GDNF, 1 div. Note cord-like cluster of cells at c, with apposition of cell membranes; other cells remain mesenchymal (m). (D) Cord-like aggregations of cells (arrows) seen after 2 div on ECM gel and PCNA immunolabelling. Scale bars represent 100 µm (A,B), 50 µm (C,D).

Fig. 4

(A–D) Immunolocalization of PCNA in E13.5 Sertoli cells cultured for 3 div in the absence or presence of ECM gel or GDNF. Arrows indicate positive nuclear staining. (A) Cells cultured without GDNF on ECM gel-coated substrate. (B) Cells cultured without GDNF on uncoated substrate. (C) Cells cultured with 10 ng mL⁻¹ GDNF on ECM gel-coated substrate. (D) Cells cultured with 10 ng mL⁻¹ GDNF on uncoated substrate. (E) Immunolocalization of PCNA in E14.5 Sertoli cells cultured on ECM gel-coated substrate for 7 div; note cell division in progress (asterisk). (F) Immunolocalization of PCNA in E13.5 Sertoli cells cultured with 10 ng mL⁻¹ GDNF on uncoated substrate for 3 div; negative control with omission of primary antibody. Note absence of immunostaining in nuclei (arrows). Scale bars, 50 µm.

Fig. 5

Mean percentage of PCNA-positive Sertoli cells cultured on ECM gel-coated substrate: (A) E12.5 cells cultured for 2 div; (B) E12.5 cells cultured for 7 div; (C) E13.5 cells cultured for 3 div; (D) E13.5 cells cultured for 7 div; (E) E14.5 cells cultured for 7 div. Data were analysed using Kruskall–Wallis analysis and Dunn's post hoc analysis. *Significantly different from 0 ng mL⁻¹ GDNF cultures, P  < 0.05; **significantly different from 0 ng mL⁻¹ GDNF cultures, P  < 0.01.

Fig. 6

Mean percentage of PCNA-positive Sertoli cells cultured on uncoated substrate: (A) E12.5 cells cultured for 2 div; (B) E12.5 cells cultured for 7 div; (C) E13.5 cells cultured for 3 div; (D) E13.5 cells cultured for 7 div. *Significantly different from 0 ng mL⁻¹ GDNF cultures, P  < 0.05; **significantly different from 0 ng mL⁻¹ GDNF cultures, P  < 0.01; ***significantly different from 0 ng mL⁻¹ GDNF cultures, P  < 0.001.