Interdependence of platelet-derived growth factor and estrogen-signaling pathways in inducing neonatal rat testicular gonocytes proliferation

Abstract

We previously found that platelet-derived growth factor (PDGF) and 17beta-estradiol stimulate gonocyte proliferation in a dose-dependent, nonadditive manner. In the present study, we report that gonocytes express RAF1, MAP2K1, and MAPK1/3. Inhibition of RAF1 and MAP2K1/2, but not phosphoinositide-3-kinase, blocked PDGF-induced proliferation. AG-370, an inhibitor of PDGF receptor kinase activity, suppressed not only PDGF-induced proliferation but also that induced by 17beta-estradiol. In addition, RAF1 and MAP2K1/2 inhibitors blocked 17beta-estradiol-activated proliferation. The estrogen receptor antagonist ICI 182780 inhibited both the effects of 17beta-estradiol and PDGF. PDGF lost its stimulatory effect when steroid-depleted serum or no serum was used. Similarly, 17beta-estradiol did not induce gonocyte proliferation in the absence of PDGF. The xenoestrogens genistein, bisphenol A, and DES, but not coumestrol, stimulated gonocyte proliferation in a dose-dependent and PDGF-dependent manner similarly to 17beta-estradiol. Their effects were blocked by ICI 182780, suggesting that they act via the estrogen receptor. AG-370 blocked genistein and bisphenol A effects, demonstrating their requirement of PDGF receptor activation in a manner similar to 17beta-estradiol. These results demonstrate the interdependence of PDGF and estrogen pathways in stimulating in vitro gonocyte proliferation, suggesting that this critical step in gonocyte development might be regulated in vivo by the coordinated action of PDGF and estrogen. Thus, the inappropriate exposure of gonocytes to xenoestrogens might disrupt the crosstalk between the two pathways and potentially interfere with gonocyte development.

FIG. 1.

Identification of PDGFR-associated molecules in PND3 testes and isolated gonocytes. A) The mRNA expression of potential downstream elements of the PDGF signaling cascade were examined in PND3 testes sections by in situ hybridization, using antisense ³⁵S-labeled probes for specific labeling (top panels) and sense probes for background signals (bottom panels). ISH signals and corresponding H&E staining are shown. B) Protein expression was examined in the testes by immunohistochemistry. Nuclear labeling (Hoechst), specific immunoreactions (Ab), merged images, and nonspecific signals obtained by using either a mouse or rabbit IgG or by omitting primary antibodies (NS) are presented. Arrows indicate representative gonocytes. C) Expression of the signaling molecules was examined by immunocytochemistry in gonocytes fixed right after cell isolation (Day 0) or after 1 day in culture following cell isolation (Day 1) and collection by cytospin centrifugation. Specific immunoreaction, DAPI staining of nuclei, and merged images are presented, as well as nonspecific stainings obtained with either a mouse or rabbit IgG or by omitting primary antibodies (2d Ab). Bars = 20 μm (A, B) and 10 μm (C). Representative pictures are shown. D) Protein expression was determined by immunoblot analysis of isolated gonocytes pelleted and extracted right after enrichment on BSA gradient and Sertoli/myoid cell extracts (S/M). Two representative blots from different cell preparations are shown.

FIG. 2.

Role of PDGF signaling cascade in gonocyte proliferation. Isolated gonocytes were incubated overnight in the presence of BrdU used as a proliferation marker in 2.5% FBS ± 10 ng/ml PDGF-BB and ± the following inhibitors: 100 μM AG-370 (A); 1 μM wortmannin (B); 10 μM RAF1 kinase Inhibitor 1 (iRAF1; C); 10 μM UO126 or 25 μM PD98059 (D). The cells were collected by cytospin centrifugation, and BrdU incorporation was determined by immunocytochemistry. The percents of BrdU-positive versus total cell numbers (proliferation rate) were determined. The histograms represent the means ± SEM calculated from three independent experiments in which duplicate or triplicate samples were used. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 3.

Interdependence of PDGF and estrogen pathways in the stimulation of gonocyte proliferation. The possibility of interdependence between the PDGF and estrogen pathways in gonocyte proliferation was examined by incubating isolated gonocytes overnight in the presence of BrdU in 2.5% FBS with or without the following compounds: 1 μM 17β-estradiol (A, C–E); 100 μM ICI 182780 (A, B); 10 ng/ml PDGF-BB (B, C); 100 μM AG-370 (C); 10 μM iRAF1 (D); 10 μM UO126 (E); or 25 μM PD98059 (E). The histograms represent the means ± SEM of proliferation rates calculated from three independent experiments in which duplicate or triplicate samples were used. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 4.

Role of FBS in the effects of PDGF and 17β-estradiol on gonocyte proliferation. PDGF- or steroid-depleted sera were used in order to determine whether serum was a source of PDGF and 17β-estradiol in amounts sufficient to permit each compound to stimulate proliferation when added alone in the culture medium. Isolated gonocytes were incubated overnight in the presence of BrdU with 2.5% of normal FBS (A, B), charcoal-stripped FBS (A), or PDGF-depleted FBS (B) ± 10 ng/ml PDGF-BB ± 1 μM 17β-estradiol (A, B). The histograms represent the means ± SEM of proliferation rates calculated from three independent experiments in which duplicate or triplicate samples were used. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 5.

Comparative effects of xenoestrogens and 17β-estradiol on gonocyte proliferation. The effects of genistein (A, F), BPA (B, F), DES (D), and coumestrol (E) on gonocyte proliferation were compared to that of 17β-estradiol (C) in dose-response experiments performed on isolated gonocytes. The cells were incubated overnight in the presence of BrdU in 2.5% FBS using concentrations of estrogens ranging from 10⁻¹⁵ to 10⁻⁶ M, with or without 100 μM ICI 182780 (A–E) or 100 μM AG-370 (A, B). The histograms represent the means ± SEM of proliferation rates calculated from three independent experiments in which duplicate or triplicate samples were used. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 6.

Effects of steroid hormones, xenoestrogens, and 17β-estradiol on gonocyte proliferation in the absence of serum or the presence of FBS or PDGF-depleted FBS. A) The effects of 10⁻⁶ M testosterone and progesterone on proliferation were examined in the absence or presence of 2.5% FBS. B) Comparison of the dose-response curves of genistein, BPA, and 17β-estradiol added with PDGF to gonocytes in the absence of serum. C) Effects of genistein, BPA, and 17β-estradiol added with PDGF-depleted FBS with or without PDGF to gonocyte suspensions. The histograms represent the means ± SEM of proliferation rates calculated from two to three independent experiments in which duplicate or triplicate samples were used. *P < 0.05; ***P < 0.001.