Testicular activin and follistatin levels are elevated during the course of experimental autoimmune epididymo–orchitis in mice

Figures

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  Figure 1. Testicular weight (a) and induction rate (b) of EAEO in animals used in the study. Paired testicular weight of untreated, adjuvant control and EAEO mice 30, 50 and 80 days after the first immunisation (a). Data are expressed as mean ± SEM (n = 4–7 animals per group, numbers of animals per group are shown in the respective columns); ****P < 0.0001, all other comparisons are not statistically significant. Representative image shows macroscopic difference in testicular size from control and EAEO mice 50 days after the first immunisation (c).

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  Figure 2. Azo-carmine and aniline blue staining of collagen fibres in paraffin sections from adjuvant control (a,d,g), low grade (b,e,h), and severe EAEO (c,f,i) mouse testes at 30 (a–c), 50 (d–f) and 80 (g–i) days after first immunisation. An increase in collagen fibres is visible in low grade EAEO in the areas with lymphocytic infiltrates. A strong peritubular fibrotic response is detectable in EAEO mouse testes at 50 (f) and 80 (i) days after the first immunisation. Scale bars represent 100 μm.

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  Figure 3. Double staining for CD206 (AlexaFluor546, orange) (a,e,i,m) and the macrophage marker F4/80 (AlexaFluor488, green) (b,f,j,n) as well as MHC class II single staining (d,h,l,p) in testicular cryosections from untreated (a–d), adjuvant control (e–h), low grade EAEO (i–l) and severe EAEO (m–p) mice. Nuclei were counterstained with DAPI (blue). Under non-inflammatory conditions, co-localized CD206 and F4/80 positive macrophages (c,g) and MHC class II positive cells (d–h) were present in low numbers in the interstitial space. An accumulation of double positive F4/80 and CD206 macrophages and MHC class II positive cells was observed in inflamed low grade (k,l, respectively) and severe (o,p, respectively) 50 day EAEO testis, with a higher number of F4/80 positive only (CD206-negative) cells. In low grade 50 day EAEO testis (k), the accumulation of macrophages was present in areas with reduced tubule diameter, whereas in severe 50 day EAEO testis (m–p) macrophages and MHC class II positive cells were more evenly distributed in the interstitial space. Scale bars represent 100 μm.

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  Figure 4. Representative flow cytometry plots for testicular CD45+ leukocytes (a) evaluated in the testicular single cell suspension. Percentage of CD45+ leukocytes (a,b), CD3+ T cells within CD45+ leukocytes (c) and different subtypes of CD3+ T cells such as CD4 − CD8+, CD4 + CD8+, CD4 + CD8− and CD4 + CD25+ T cells (d) as well as ratio of CD4+/CD8+ T cells (e) was analysed in untreated, adjuvant control and EAEO testicular single cell suspension 50 days after the first immunisation, by flow cytometry. After gating out cell debris, doublets and nonviable cells, the population of CD45+ leukocytes and CD3+ T cells was selected for further analysis. Data are expressed as mean ± SEM (n = 5 animals per group); *P < 0.05, **P < 0.01, ***P < 0.001, all other comparisons are not statistically significant.

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  Figure 5. Tnf (a), Ccl2 (MCP-1) (b), Il10 (c) and Il6 (d) mRNA expression was measured in untreated, adjuvant control and inflamed mouse testis at 30, 50 and 80 days after first immunisation. Relative mRNA levels in mouse testes were analysed using quantitative RT-PCR. Data are represented as mean ± SEM (n = 4–5 animals per group); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, all other comparisons are not statistically significant.

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  Figure 6. Distribution of α-smooth muscle actin (αSMA) in paraffin sections from untreated (a,e,i,m), adjuvant control (b,f,j,n), low grade (c,g,k,o) and severe EAEO (d,h,l,p) testis at 30 (a–d), 50 (e–h) and 80 (i–l) days after the first immunisation. Panels m–p represent a higher magnification of images i–l. In the testis from untreated and adjuvant controls, αSMA is localised in the peritubular cells as a thin layer, but in low grade and severe EAEO testis, the αSMA is diffusely distributed within the cell. αSMA is also seen in the blood vessels (asterisks) within the testis. Scale bars represent 100 μm.

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  Figure 7. Localisation of activin βA subunit in a testis from EAEO, adjuvant and untreated control mice. Immunofluorescence staining of activin βA subunit using the E4 antibody on paraffin sections from untreated (a,e,i) adjuvant control (b,f,j), low grade (c,g,k) and severe EAEO (d,h,l) at 30 (a–d), 50 (e–h) and 80 (i–l) days after the first immunisation. The activin βA subunit is localised in the cytoplasm of Sertoli cells (arrowheads), peritubular cells and some interstitial cells (arrows) in untreated, adjuvant controls and low grade EAEO. In severe EAEO testis, the staining was present in Sertoli cells and individual immune cells (d,h,l). Scale bars represent 100 μm.

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  Figure 8. Protein levels of activin A (a), activin B (b), inhibin (c) and follistatin (d) were measured in testicular homogenates from untreated, adjuvant control and EAEO animals 30, 50 and 80 days after the first immunisation. Data are represented as mean ± SEM of 4–5 animals per group; *P < 0.05, all other comparisons are not statistically significant.

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  Figure 9. Relative mRNA expression of Inhba (inhibin βA) (a), Inhbb (inhibin βB) (b), Inha (inhibin α) (c), Acvr1b (activin receptor, type IB) (d) and Acvr2b (activin receptor, type IIB) (e) in testes from untreated, adjuvant control and EAEO mice 30, 50 and 80 days after the first immunisation analysed by quantitative RT-PCR. Gene expression levels were similar between untreated, adjuvant and EAEO testis at 30 days. Data are represented as mean ± SEM of 4–5 animals per group; *P < 0.05, **P < 0.01, ***P < 0.001, all other comparisons are not statistically significant.