Oncostatin-M inhibits luteinizing hormone stimulated Leydig cell progenitor formation in vitro

Abstract

Background: The initial steps of stem Leydig cell differentiation into steroid producing progenitor cells are thought to take place independent of luteinizing hormone (LH), under the influence of locally produced factors such as leukaemia inhibitory factor (LIF), platelet derived growth factor A and stem cell factor. For the formation of a normal sized Leydig cell population in the adult testis, the presence of LH appears to be essential. Oncostatin M (OSM) is a multifunctional cytokine and member of the interleukin (IL)-6 family that also includes other cytokines such as LIF. In the rat OSM is highly expressed in the late fetal and neonatal testis, and may thus be a candidate factor involved in Leydig cell progenitor formation.

Methods: Interstitial cells were isolated from 13-day-old rat testes and cultured for 1, 3 or 8 days in the presence of different doses of OSM (range: 0.01 to 10 ng/ml) alone or in combination with LH (1 ng/ml). The effects of OSM and LH on cell proliferation were determined by incubating the cultures with [3H]thymidine or bromodeoxyuridine (BrdU). Developing progenitor cells were identified histochemically by the presence of the marker enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD).

Results: OSM, when added at a dose of 10 ng/ml, caused a nearly 2-fold increase in the percentage of Leydig cell progenitors after 8 days of culture. Immunohistochemical double labelling experiments with 3beta-HSD and BrdU antibodies showed that this increase was the result of differentiation of stem Leydig cells/precursor cells and not caused by proliferation of progenitor cells themselves. The addition of LH to the cultures consistently resulted in an increase in progenitor formation throughout the culture period. Surprisingly, when OSM and LH were added together, the LH induced rise in progenitor cells was significantly inhibited after 3 and 8 days of culture.

Conclusion: Taken together, the results of the present study suggest that locally produced OSM may not only play a role in the regulation of Sertoli cell proliferation and the initiation of spermatogenesis but may also play a role in the regulation of Leydig cell progenitor formation by keeping the augmenting effects of LH on this process in abeyance.

Figure 1

Effects of OSM (10 ng/ml) either alone or in combination with LH (1 ng/ml) on the proliferation of interstitial cells isolated from 13-day-old rat testes and cultured for 1, 3 or 8 days. Proliferation was determined by [³H]-thymidine incorporation and scintilation counting (see Materials and Methods). Statistical analysis revealed that there was no significant difference among the different experiments. Therefore, the results are represented as mean ± SD of three different experiments that were combined. Each single experiment was carried out in quadruplicate. Differences among groups were considered to be significantly different when p < 0.05. a – significantly different from control; b – significantly different from OSM 10 ng/ml; c – significantly different from LH 1 ng/ml.

Figure 2

Effect of OSM (10 ng/ml) either alone or in combination with LH (1 ng/ml) on the presence of 3β-HSD positive Leydig cell progenitors. Interstitial cells were isolated from 13-day-old rat testes and cultured for 1, 3 or 8 days. The presence of 3β-HSD was determined by enzyme histochemistry or immunohistochemistry using a polyclonal antibody against 3β-HSD (for details see Materials and Methods). Statistical analysis revealed that there was no significant difference among the different experiments and thus the results are presented as mean ± SD of three different experiments that were combined. Each single experiment was carried out either in duplicate, triplicate or quadruplicate. Differences among groups were considered to be significantly different when p < 0.05. a – significantly different from control; b – significantly different from OSM 10 ng/ml; c – significantly different from LH 1 ng/ml.

Figure 3

Immunohistochemical labelling for the presence of OSM in vivo and in vitro. A) Section of the testis of a rat 8 days after EDS administration. All Leydig cells have disappeared from the interstitium. Under these conditions no OSM positive cells could be detected by immunohistochemistry; B) Interstitial cell were isolated from testes of 13-day-old rats and cultured for 3 or 8 days without any additions or in the presence of 10 ng/ml OSM and/or 1 ng/ml LH (for more details see Materials and Methods). Percentage of OSM positive cells in culture determined under the indicated culture conditions. The experiment was carried out in quadruplicate. Values are represented as mean ± SD; statistical analysis was carried by comparing the treated cultures with their respective controls. Means were considered to be significantly different at p < 0.05 (indicated by (a)). C) Cells cultured in the presence of LH for 8 days and immunohistochemically stained with an antibody against OSM. Some positively stained cells are indicated by arrows. Scale bar in (A) represents 21 μm, in (C) 20 μm.

Figure 4

Immunohistochemical labelling for the presence of LIF-R in the testis of 13-day-old rats (A-C). Faint brown staining for the presence of OSM type I receptor/LIF receptor is present in a cluster of fetal-type Leydig cells (A; asterisk). The staining is much stronger in stem Leydig cells/precursor cells identified by their spindle-shaped nucleus, located in close vicinity of the basal membrane of the seminiferous tubules (A, B; filled arrows) and in Leydig cell progenitors identified by their oval nucleus which are either found close to the seminiferous tubules (A, B) or blood vessels (C; arrowhead). Staining in spermatogonia was faint (B, C; open arrow). (D) Control in which the first antibody was replaced by rabbit serum. Scale bar represents 11 μm.