Abnormal Accumulation of Collagen Type I Due to the Loss of Discoidin Domain Receptor 2 (Ddr2) Promotes Testicular Interstitial Dysfunction

Abstract

Background: Loss of functional allele for discoidin domain receptor 2 (Ddr2) results in impaired Leydig cell response to luteinizing hormone (LH), low testosterone production and arrested spermatogenesis in older male Ddr2slie/slie mice. However, the underlying mechanism responsible for this phenotype remains unknown. Herein, we reported for the first time that the deregulated expression of Ddr2 cognate ligand, namely collagen type I (COL1), may account for the disruption of the testicular steroidogenesis in Ddr2slie/slie mutant testes.

Methodology/principal findings: Expression of Ddr2 increased gradually along postnatal development, whereas COL1 expression became negligible from adulthood onwards. In Ddr2slie/slie mutant testis, however, in contrast to the undetectable staining of Ddr2, COL1 expression was constantly detected, with the highest values detected during adulthood. In the experimental vasectomy model, Ddr2slie/slie mutant mice exhibited an early androgen deficiency than wild-type mice, along with the accumulation of fibrotic tissue in the interstitium. Functionally, ablation of endogenous Ddr2 resulted in a significant decrease of testosterone (T) level in TM3 cells in the presence of higher concentration of COL1 treatment. Conversely, overexpression of Ddr2 could help TM3 cells to maintain a normal testicular steroidogenesis even in the presence of high concentration of COL1. Additionally, attenuated expression of Ddr2 correlates to the deregulated level of serum T levels in human pathological testes.

Conclusions: Abnormal accumulation of interstitial COL1 may be responsible for the steroidogenic dysfunction in Ddr2slie/slie mutant testes.

Fig 1. Analysis of DDR2 and COL1 expression in wild-type and Ddr2^slie/slie mutant testes along postnatal development.

A Assessment of DDR2 and COL1 protein expression in wild-type and Ddr2^slie/slie mutant developing testes by western blotting analyses. Immunolabeling of Actin is shown as the loading control. B Immunolocalization of DDR2 and COL1 protein in developing wild-type and Ddr2^slie/slie mutant testes. The arrows indicate DDR2 in LCs and the arrow heads indicate COL1 in testicular interstitium. Bar = 25 μm

Fig 2. In vitro effects of collagen accumulation on DDR2 expression in TM3 cells.

A Effects of different concentrations of COL1 on DDR2 expression in TM3 cells was illustrated using immunofluorescence. Nuclear were demonstrated using DAPI staining. Bar = 10 μm. B Relative fluorescence intensity of DDR2 in panel A was analyzed using Image J. *P < 0 .05 vs 0 μg/cm² group.

Fig 3. Establishment of murine vasectomy model and its effects on endogenous DDR2 and COL1 expression.

A Establishment of murine vasectomy model was evaluated using Hematoxylin and eosin staining and Masson staining. Collagen fibers inside testicular interstitium displays blue coloration. B Immunolocalization of DDR2 and COL1 protein in testicular tissues from murine vasectomy model was carried out by immunofluorescence. Nuclear were demonstrated using DAPI staining.

Fig 4. Effects of vasectomy on testicular COL1 expression and steroidogenesis.

A The experimental murine vasectomy model was established as described in the Materials and Methods section. Animals were sacrificed at Postoperative 3 and 5 month, and serum testosterone (T) level was assessed by radioimmunoassay. Each data point represents the mean ± S.E.M. Bars with different letters in this panel indicate statistically significant differences in mean values from each other (P < 0.05), as determined by ANOVA and Tukey’s test. B Effects of vasectomy on testicular COL1 expression were determined using immunoblotting analyses in different experimental groups. Actin was served as internal control. C Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in Group 0, which was arbitrarily set at 1.

Fig 5. Effects of Ddr2 knockdown or Ddr2 overexpression on androgen production of TM3 Leydig cells in response to different concentrations of COL1 treatment.

A Verification of Ddr2 knock down effect by siRNA treatment was performed using western blotting analyses. Actin was used as an internal control. B T concentrations in the culture medium of Ddr2-knockdown TM3 cells were determined using radioimmunoassay. C-E Expression levels of the testicular mRNAs encoding several key factors in the steroidogenic route were evaluated by real-time PCR. The data are expressed as the mean ± S.E.M. from three independent experiments. Parallel amplification of Gapdh was used as the internal control. *P < 0 .05 vs control siNC group. ^# P < 0 .05 vs. control siDDR2, **P < 0.05, 24 μg/cm² siNC group vs. 96 μg/cm² siNC group, ^## P < 0.05, siDDR2 24 μg/cm² vs. siDDR2 96 μg/cm², ^#*P < 0.05, siNC 96μg/cm² vs. siDDR2 96 μg/cm². F Expression level of StAR in the presence of different concentrations of COL1 treatment was evaluated using western blotting analyses. G Verification of Ddr2 overexpression was performed using western blotting analyses. H T concentrations in the culture medium of Ddr2-overexpressing TM3 cells were determined using radioimmunoassay. I-K Expression levels of the testicular mRNAs encoding several key factors in the steroidogenic route were evaluated by real-time PCR. The data are expressed as the mean ± SEM from three independent experiments. Parallel amplification of Gapdh was used as the internal control. *P < 0 .05 vs control Ad-EGFP group. ^# P < 0 .05 vs. control Ad-DDR2, **P < 0.05, 24 μg/cm² Ad-EGFP group vs. 96 μg/cm² Ad-EGFP group, ^## P < 0.05, Ad-DDR2 24 μg/cm² vs. Ad-DDR2 96 μg/cm², ^#*P < 0.05, Ad-EGFP 96μg/cm² vs. Ad-DDR2 96 μg/cm². L Expression level of StAR in the presence of different concentrations of COL1 treatment was evaluated using western blotting analyses. Immunolabeling of Actin is shown as loading control.

Fig 6. Deregulated testicular expression of Ddr2 positively correlates with serum T level in human infertile patients.

A Immunohistochemical analysis of Ddr2 expression in human pathological testes. The arrows and asterisks denote the positive staining of Ddr2 in LCs and of COL1 in testicular interstitium, respectively. Bar = 25 μm. B correlation of relative Ddr2 immunoreactive content in human testis with serum T level was determined based on Pearson’s correlation coefficient with the aid of SPSS 15.0 software.

Fig 7. Summary diagram of the possible mechanisms related to deregulated Ddr2 expression contributing to the impairment of testicular steroidogenesis.