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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00981

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Expression and role of PDGF-BB and PDGFR-ß during testis morphogenesis in the mouse embryo

Department of Public Health and Cell Biology, Section Histology and Embryology, University of Rome `Tor Vergata', Via Montpellier 1, 00133 Rome, Italy

View larger version (66K): [in a new window]   Fig. 1. PDGFR-ß expression in the male urogenital ridge between 11.5 and 13.5 dcp. (A) Immunohistochemical detection of PDGFR-ß localization in cryostat sections of 11.5 and 13.5 dpc male urogenital ridges. At 11.5 dpc no immunoreactivity can be detected in the gonad (a), while at 13.5 dpc cells with a strong immunoreactivity fill the interstitial compartment of the gonad (c). (b,d) control sections. m, mesonephros; t, testis. Bar, 100 µm. (B) Immunoblot analysis of PDGFR-ß expression, showing the presence of a 190-kDa immunoreactive band that reacts to the same antibody as used for immunolocalization. The presence of equal amounts of proteins in each lane is confirmed by quantitatively similar ß-actin immunoreactive bands.

View larger version (97K): [in a new window]   Fig. 2. Expression of p75NTR and PDGFR-ß by mesonephric sorted cells. Immunoreactivity for p75NTR (a) and PDGFR-ß () in immunomagnetically sorted mesonephric cells. Comparison of antibody (a,c) and Hoechst staining (b,d) shows that the cells are immunoreactive for both antibodies. Bar, 50 µm.

View larger version (38K): [in a new window]   Fig. 3. PDGF-BB expression in the fetal gonad. (A) Northern-blot analysis, performed on 20 µg of total RNA extracted from 12.5 and 13.5 dpc male and female gonads. An erythroleukaemic cell line (K562) was used as a positive control, which, after treatment with TPA expresses high levels of PDGF-BB mRNA; a human lymphoma cell line (MOLT-4) was used as a negative control. Lower panel: filter reprobed with ß-actin cDNA to check for the presence of equal amounts of mRNA in each lane. (B) Immunoblot analysis showing a PDGF-BB immunoreactive band of 30 kDa, detected in a nonreducing gel, which represents the PDGF-BB dimeric molecule present in fetal testis.

View larger version (89K): [in a new window]   Fig. 4. Expression of Pdgf-B in 12.5 dpc male urogenital ridge. The in situ hybridization (a,a') shows that the growth factor is mainly expressed by cells in the interstitial compartment of the developing testis, as indicated by arrowheads; b, phase contrast. c, hybridization with sense probe and, d, its phase contrast. Bar, 100 µm.

View larger version (12K): [in a new window]   Fig. 5. Effect of PDGF-BB on mesenchymal mesonephric cell proliferation. Immunoselected quiescent mesenchymal cells were treated for 16-18 hours with PDGF-BB (various concentrations) and [3H]TdR (1 µCi/well). Results are mean±s.d. of four independent experiments, each done in quadruplicate. *P<0.01 vs untreated control.

View larger version (14K): [in a new window]   Fig. 6. Effect of PDGF-BB on mesenchymal mesonephric cell migration. Assays were performed by incubating cells for 16-18 hours in cell culture inserts with 8 µm porosity membrane. Cells that had migrated to the lower surface of the filters were counted. (A) Dose-response curve. Various concentrations of PDGF-BB were added to the lower wells only. (B) Chemotaxis vs chemokinesis analysis. PDGF-BB (10 ng/ml) was added to the upper well, to the lower well, or to both set of wells. Results are mean±s.d. of three independent experiments, each performed in duplicate. *P<0.01 vs untreated control.

View larger version (76K): [in a new window]   Fig. 7. Effect of PDGF-BB on microfilament reorganization, as revealed by phalloidin staining. Quiescent mesonephric mesenchymal cells (A) were stimulated for 15 minutes (B) with PDGF-BB (10 ng/ml). Note that treated cells exhibit a rapid change in microfilament reorganization and in cellular morphology (phalloidin staining at the leading edge of the cell and extensions of lamellipodia). Bar, 20 µm.

View larger version (28K): [in a new window]   Fig. 8. Erk1/2 and Akt phosphorylation in PDGF-BB-stimulated mesonephric cells: inhibition by U0126 and Ly294002. Quiescent cells were incubated with U0126 or Ly294002 and then stimulated with different concentrations of PDGF-BB for 15 minutes or overnight, as described in Materials and Methods. Lysed cells were separated by SDS-PAGE and blotted filters were incubated with (A) antibody for phospho-Erk1/2 (upper panel) and for nonphosphorylated forms as control (lower panel); or (B) antibody for phospho-Akt (upper panel) and for nonphosphorylated form as control (lower panel).

View larger version (18K): [in a new window]   Fig. 9. Effect of U0126 and Ly294002 on proliferation and migration of mesonephric mesenchymal cells. (A) Quiescent cells were pretreated with U0126 (U0) or Ly294002 (Ly) and then stimulated with PDGF-BB. [3H]TdR incorporation was measured as an index of cell proliferation. Results are means±s.d of four independent experiments, each done in quadruplicate. *P<0.01 vs its respective control (no inhibitor). (B) Quiescent cells were pretreated with U0126 or Ly294002 and then tested for directed migration towards 10 ng/ml PDGF-BB. Results are means±s.d. of four independent experiments, each done in duplicate. *P<0.01 vs PDGF-BB 10 ng/ml.

View larger version (57K): [in a new window]   Fig. 10. Male urogenital ridge organ culture. Photomicrographs illustrating morphological changes in male urogenital ridges cultured for 4 days in the presence of serum-free medium alone (a) or also with 100 ng/ml PDGF-BB (b). Data are representative of three separate experiment, which showed similar results. Bar, 0.5 mm. m, mesonephros; t, testis.

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