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

View Article: PubMed Central - PubMed

ABSTRACT

Experimental autoimmune epididymo-orchitis (EAEO) is a model of chronic inflammation, induced by immunisation with testicular antigens, which reproduces the pathology of some types of human infertility. Activins A and B regulate spermatogenesis and steroidogenesis, but are also pro-inflammatory, pro-fibrotic cytokines. Expression of the activins and their endogenous antagonists, inhibin and follistatin, was examined in murine EAEO. Adult untreated and adjuvant-treated control mice showed no pathology. All mice immunised with testis antigens developed EAEO by 50 days, characterised by loss of germ cells, immune cell infiltration and fibrosis in the testis, similar to biopsies from human inflamed testis. An increase of total CD45+ leukocytes, comprising CD3+ T cells, CD4 + CD8− and CD4 + CD25+ T cells, and a novel population of CD4 + CD8+ double positive T cells was also detected in EAEO testes. This was accompanied by increased expression of TNF, MCP-1 and IL-10. Activin A and B and follistatin protein levels were elevated in EAEO testes, with peak activin expression during the active phase of the disease, whereas mRNA expression of the inhibin B subunits (Inha and Inhbb) and activin receptor subunits (Acvr1b and Acvr2b) were downregulated. These data suggest that activin–follistatin regulation may play a role during the development of EAEO.

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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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f2: 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.

Mentions: Successful induction of EAEO was estimated based on testicular weight and histopathological changes of testicular architecture. In the EAEO group, a more than 2-fold decrease of testis weight collected at 50 (n = 7) and 80 (n = 5) days after the first immunisation compared to untreated (n = 5) and adjuvant control (n = 5 for 50 days or n = 4 for 80 days) groups was observed (Fig. 1a). The mean testis weight 30 days after the first immunisation was similar in all investigated animals (Fig. 1a). Histopathological changes and the fibrotic response were assessed by azo-carmine and aniline blue staining (azan), as shown in Fig. 2. The changes in the inflamed testis included reduced diameter of the seminiferous tubules, germ cell sloughing leading to Sertoli cell only tubules, leukocytic infiltrates in the interstitium and thickening of the lamina propria of seminiferous tubules (Fig. 2). Notably, the grade of EAEO development was variable between individual animals, therefore the EAEO groups were subdivided into animals with severe disease symptoms (severe EAEO) and mice showing only mild symptoms of EAEO (low grade EAEO). Testes from the severe EAEO group at 50 (Fig. 2f) and 80 (Fig. 2i) days after the first immunisation represented a complete destruction of testicular morphology with a reduction of the size of the seminiferous epithelium, tubular atrophy and thickening of the seminiferous lamina propria. An increase of the immune cell infiltrates in EAEO testes was observed. In contrast, in the mild form of the disease, some of the seminiferous tubules were still normal. Moreover, the morphological changes were accompanied by a strong fibrotic response in the testes represented by an increase of the collagen fibres around the remaining empty tubules, and in EAEO testes the tunica albuginea was thicker compared to controls (data not shown). Similar changes were also observed in human testicular biopsies with impaired spermatogenesis and inflammatory infiltrates (Supplementary Fig. S1). Moreover, testes from untreated and adjuvant control mice (Fig. 2a,d,g) showed a normal morphology with seminiferous tubules containing Sertoli cells as well as germ cells at all stages of spermatogenesis. At 50 and 80 days after the first immunisation, 100% of animals (7/7 and 5/5, respectively) developed EAEO, whereas in the 30 days EAEO group only 33% (2 of 6) animals showed histological signs of the disease (Fig. 1b). Representative macroscopic difference in testis size between EAEO and control group is shown in Fig. 1c.


Testicular activin and follistatin levels are elevated during the course of experimental autoimmune epididymo – orchitis in mice
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5304336&req=5

f2: 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.
Mentions: Successful induction of EAEO was estimated based on testicular weight and histopathological changes of testicular architecture. In the EAEO group, a more than 2-fold decrease of testis weight collected at 50 (n = 7) and 80 (n = 5) days after the first immunisation compared to untreated (n = 5) and adjuvant control (n = 5 for 50 days or n = 4 for 80 days) groups was observed (Fig. 1a). The mean testis weight 30 days after the first immunisation was similar in all investigated animals (Fig. 1a). Histopathological changes and the fibrotic response were assessed by azo-carmine and aniline blue staining (azan), as shown in Fig. 2. The changes in the inflamed testis included reduced diameter of the seminiferous tubules, germ cell sloughing leading to Sertoli cell only tubules, leukocytic infiltrates in the interstitium and thickening of the lamina propria of seminiferous tubules (Fig. 2). Notably, the grade of EAEO development was variable between individual animals, therefore the EAEO groups were subdivided into animals with severe disease symptoms (severe EAEO) and mice showing only mild symptoms of EAEO (low grade EAEO). Testes from the severe EAEO group at 50 (Fig. 2f) and 80 (Fig. 2i) days after the first immunisation represented a complete destruction of testicular morphology with a reduction of the size of the seminiferous epithelium, tubular atrophy and thickening of the seminiferous lamina propria. An increase of the immune cell infiltrates in EAEO testes was observed. In contrast, in the mild form of the disease, some of the seminiferous tubules were still normal. Moreover, the morphological changes were accompanied by a strong fibrotic response in the testes represented by an increase of the collagen fibres around the remaining empty tubules, and in EAEO testes the tunica albuginea was thicker compared to controls (data not shown). Similar changes were also observed in human testicular biopsies with impaired spermatogenesis and inflammatory infiltrates (Supplementary Fig. S1). Moreover, testes from untreated and adjuvant control mice (Fig. 2a,d,g) showed a normal morphology with seminiferous tubules containing Sertoli cells as well as germ cells at all stages of spermatogenesis. At 50 and 80 days after the first immunisation, 100% of animals (7/7 and 5/5, respectively) developed EAEO, whereas in the 30 days EAEO group only 33% (2 of 6) animals showed histological signs of the disease (Fig. 1b). Representative macroscopic difference in testis size between EAEO and control group is shown in Fig. 1c.

View Article: PubMed Central - PubMed

ABSTRACT

Experimental autoimmune epididymo-orchitis (EAEO) is a model of chronic inflammation, induced by immunisation with testicular antigens, which reproduces the pathology of some types of human infertility. Activins A and B regulate spermatogenesis and steroidogenesis, but are also pro-inflammatory, pro-fibrotic cytokines. Expression of the activins and their endogenous antagonists, inhibin and follistatin, was examined in murine EAEO. Adult untreated and adjuvant-treated control mice showed no pathology. All mice immunised with testis antigens developed EAEO by 50 days, characterised by loss of germ cells, immune cell infiltration and fibrosis in the testis, similar to biopsies from human inflamed testis. An increase of total CD45+ leukocytes, comprising CD3+ T cells, CD4 + CD8− and CD4 + CD25+ T cells, and a novel population of CD4 + CD8+ double positive T cells was also detected in EAEO testes. This was accompanied by increased expression of TNF, MCP-1 and IL-10. Activin A and B and follistatin protein levels were elevated in EAEO testes, with peak activin expression during the active phase of the disease, whereas mRNA expression of the inhibin B subunits (Inha and Inhbb) and activin receptor subunits (Acvr1b and Acvr2b) were downregulated. These data suggest that activin–follistatin regulation may play a role during the development of EAEO.

No MeSH data available.


Related in: MedlinePlus