Department of Urology, Kobe University School of Medicine, Kobe, Japan
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Abstract |
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Key words: cathepsin G/heterogeneity/male infertility/mast cell/tryptase
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Introduction |
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It has been shown that MC can be identified in the normal human testes, and that there is an increase in the number of MC in the testes of infertile men (Maseki et al., 1981; Nagai et al., 1992
). However, the role of MC in the human testis remains unknown. Using immunohistochemical techniques, we examined the heterogeneity of MC in the human testes in an attempt to relate it to spermatogenetic disorders.
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Materials and methods |
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The specimens were divided into two pieces. One piece was fixed in 10% formalin and another in Bouin's solution for 12h at room temperature. The tissues were prepared in an automatic tissue processor using ascending ethanol concentrations, xylene and paraffin wax. Serial paraffin sections (4 µm thick) were mounted on glass slides for staining.
To identify the mast cell subtypes, immunostaining was performed for tryptase, and cathepsin G in place of chymase because MCTC contained cathepsin G in addition to chymase of which immunoreactivity was lost in formalin-fixed tissue. The formalin-fixed sections were deparaffinized in xylene through ethanol to phosphate-buffered saline (PBS; pH 7.2). To block endogenous peroxidase activity, 0.3% hydrogen peroxide in methanol was applied for 20 min. All slides were then incubated in normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 40 min to block non-specific binding. Primary antibodies were added to the slides and incubated for 60 min in a moist chamber at room temperature. Biotinylated anti-rabbit immunoglobulin G (IgG) was applied and incubated for 1h. Then avidinbiotinperoxidase complex (ABC; Vector Laboratories) was applied and incubated for 30 min. Both incubations were carried out in a moist chamber at room temperature. The final colouring agent was diaminobenzidine tetrahydrochloride. The tissue was counterstained with Methyl Green. The primary antibodies were anti-human tryptase (IgG rabbit monoclonal; BioPur AG, CH, Bubendorf, Switzerland; diluted 1:400 in PBS) and anti-human neutrophil cathepsin G (rabbit polyclonal; Dako Corporation, Carpinteria, CA, USA; diluted 1:500 in PBS). All slides were covered with a cover-slip after mounting in buffered glycerin.
The slides in which there were at least 20 seminiferous tubular sections were examined with an Olympus light microscope equipped with a x40 objective. The cells positive for tryptase were counted as the total number of MC. The cells positive for both tryptase and cathepsin G were counted as the number of MCTC. The number of MCT was calculated by subtracting the number of MCTC from the total number of MC. The number of seminiferous tubular sections was also counted. The average numbers of MC, MCT and MCTC per seminiferous tubular section, and the ratio of MCTC to total MC were calculated for each case.
Haematoxylineosin staining was performed using the specimens fixed in Bouin's solution. The stained slides were examined with a light microscope equipped with a x20 objective. More than 20 seminiferous tubular sections per testis were each given a Johnsen's score from 1 to 10 as described previously (Johnsen, 1970). To calculate the Johnsen's score, the sum of all scores was divided by the total number of seminiferous tubular sections. The ratio of tubules with Sertoli cell-only per total tubules was also calculated. Azan staining was also performed to assess fibrosis of seminiferous tubules (Miyata et al., 1997
). The number of sclerotic seminiferous tubules with thickened lamina propria was counted under microscopic examination, and the ratio of tubules with sclerosis to total tubules was calculated. In addition, the area of seminiferous tubular section and that of fibrosis in the same section were evaluated respectively using computer-assisted colour image analysis. More than 20 seminiferous tubular sections per testis were analysed. The total area of fibrosis was divided by that of seminiferous tubular sections in order to calculate the fibrosis index.
In the patient groups, hormonal analysis (FSH, LH, and testosterone) was performed. Statistical analysis was performed using the non-parametric MannWhitney U-test to reveal differences among the control and patient groups. Correlations were tested for by Pearson's correlation coefficient; P < 0.05 was considered to be statistically significant.
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Results |
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Discussion |
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Recent studies have shown that a large number of human MC can be generated from human cord blood mononuclear cells cultured in the presence of stem cell factor and IL-6 (Saito et al., 1996; Igarashi et al., 1996
). At first, cultured MC contained only tryptase (MCT). Subsequently, MC positive for chymase (MCTC) appeared. Finally, 25% of the total were mature MCTC (Igarashi et al., 1996
). Using a co-culture system with murine 3T3 fibroblasts and cord blood cells, almost all of the cultured MC were changed into MCTC (Furitsu et al., 1989
; Mitsui et al., 1993
). It has been suggested that fibroblast-derived factors, stem cell factor, and other mediators may be required for the development of MC.
The development of MC subtypes depends on the specific tissue environment. In addition, the ratio of the subtypes changes in disease conditions. MCT are the predominant subtype in the normal gastrointestinal mucosa, nasal mucosa, and lung alveoli, whereas MCTC are the predominant subtype in the normal gastrointestinal submucosa, nasal submucosa, and skin (Weidner and Austin, 1993; Irani and Schwartz, 1994
). A selective increase in MCT has been found in the skin of patients with atopic dermatitis (Irani et al., 1989
), in the nasal submucosa of patients with allergic rhinitis (Bentley et al., 1992
), and in breast cancer tissue (Kankkunen et al., 1997
). On the other hand, an accumulation of MCTC has been found in the skin of patients with mastocytosis, which is characterized by a lack of inflammatory infiltration (Irani et al., 1990
). These observations suggest that the MCT subtype is involved in allergic and inflammatory responses, whereas the MCTC subtype is involved predominantly in fibrosis and tissue remodelling.
In the testes, MC are detected in the interstitium and the lamina propria. Using histochemical procedures to identify the proteoglycans of MC, Nagai et al. (1992) have shown that the number of MC is increased and the ratio of MC subtypes is changed in idiopathic azoospermia and oligozoospermia. In the present study, we detected the MC subtypes using immunohistochemical techniques to examine the heterogeneity in diseased human testes. Immunostaining was performed using formalin-fixed tissue. Although Bouin's solution is commonly used for fixation of the testicular specimens, it is not suitable for immunostaining due to the destruction of the specific antigen. To identify MCTC, we used the antibody against cathepsin G which is present in MCTC because the immunoreactivity of chymase is lost in formalin-fixed tissue. Since cathepsin G is also contained in monocytes and neutrophils, it is not specific for MC (Schechter et al., 1990). Therefore, we identified the MCTC as those cells which were positive for both tryptase and cathepsin G.
We have shown that MCT are the predominant subtype in the normal testes. In contrast, MCTC were the predominant subtype in the patients with obstructive azoospermia, idiopathic azoospermia, and varicocele. The numbers of MCTC in the patient groups were significantly increased, while the numbers of MCT did not change. In the patients with idiopathic azoospermia, the number of MC was significantly increased. These findings are largely the result of a selective increase in MCTC. Thus, the change in MC subtypes seems to be the result of increased infiltration of progenitor cells and of increased development of MCTC, rather than an alteration in the subtypes of the MC already present.
As previously mentioned, MCTC appear to be related to the pathogenesis of fibrosis and tissue remodelling without inflammation. In the testes of infertile men, one of the main histological changes is fibrosis in the interstitium and lamina propria of the seminiferous tubules. In the present study, increases in the ratio of tubules with sclerosis and the fibrosis index were found in the patient groups, in which a selective expansion of the MCTC population was also found. In the varicocele group, significant positive correlations were found between the number of MCTC and the ratio of tubules with sclerosis, and between the number of MCTC and the fibrosis index. These results suggest that the proliferation of MCTC increases in proportion to testicular fibrosis.
In conclusion, increases in the number of MCTC and in the MCTC to MC ratio were found in the testes of patients with obstructive azoospermia, idiopathic azoospermia, and varicocele. The total number of MC was increased in the testes of patients with idiopathic azoospermia. The ratio of tubules with sclerosis and the fibrosis index increased in the patient groups. Significant correlations between the number of MCTC and the ratio of tubules with sclerosis, and between the number of MCTC and the fibrosis index were found in the varicocele group. A selective expansion of the MCTC population and an increase in the number of MC are related to spermatogenetic disorders and testicular fibrosis.
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Notes |
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References |
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Blair, R.J., Meng, H. and Marchese, M.J. et al. (1997) Human mast cells stimulate vascular tube formation; tryptase is a novel, potent angiogenic factor. J. Clin. Invest., 99, 26912700.
Bradding, P., Okayama, Y. and Howarth, P.H. et al. (1995) Heterogeneity of human mast cells based on cytokine content. J. Immunol., 155, 297307.[Abstract]
Cairns, J.A. and Walls, A.F. (1997) Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts. J. Clin. Invest., 99, 13131321.
Craig, S.S., Schechter, N.M. and Schwartz, L.B. (1988) Ultrastructural analysis of human T and TC mast cells identified by immunoelectron microscopy. Lab. Invest., 58, 682691.[ISI][Medline]
Feldmann, M.J., Morris, G.P. and Dinda, P.K. et al. (1996) Mast cells mediate acid-induced augmentation of opossum esophageal blood flow via histamine and nitric oxide. Gastroenterology, 110, 121128.[ISI][Medline]
Furitsu, T., Saito, H. and Dvorak, A.M. et al. (1989) Development of human mast cells in vitro. Proc. Natl Acad. Sci. USA, 86, 1003910043.[Abstract]
Gruber, B.L., Kew, R.R. and Jelaska, A. et al. (1997) Human mast cell activate fibroblasts; tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis. J. Immunol., 158, 23102317.[Abstract]
Hatamochi, A., Fujiwara, K. and Ueki, H. (1985) Effects of histamine on collagen gel synthesis by cultured fibroblasts derived from guinea pig skin. Arch. Dermatol. Res., 277, 6064.[ISI][Medline]
Igarashi, Y., Kurosawa, M. and Ishikawa, O. et al. (1996) Characteristics of histamine release from cultured human mast cells. Clin. Exp. Allergy, 26, 597602.[ISI][Medline]
Irani, A.A. and Schwartz, L.B. (1994) Human mast cell heterogeneity. Allergy Proc., 15, 303308.[ISI][Medline]
Irani, A.A., Schechter, N.M. and Craig, S. et al. (1986) Two types of human mast cells that have distinct neutral protease compositions. Proc. Natl Acad. Sci. USA, 83, 44644468.[Abstract]
Irani, A.A., Sampson, H.A. and Schwartz, L.B. (1989) Mast cell in atopic dermatitis. Allergy, 44, 3134.[ISI][Medline]
Irani, A.A., Garriga, M.M. and Metcalfe, D.D. et al. (1990) Mast cells in cutaneous mastocytosis: accumulation of the MCTC type. Clin. Exp. Allergy, 20, 5358.[ISI][Medline]
Irani, A.A., Goldstein, S.M. and Wintroub, B.U. et al. (1991) Human mast cell carboxypeptidase; selective localization to MCTC cells. J. Immunol., 147, 247253.
Jordana, M. (1993) Mast cells and fibrosis: who's on first? Am. J. Respir. Cell. Mol. Biol., 8, 78.[ISI][Medline]
Johnsen, S.G. (1970) Testicular biopsy score count a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones, 1, 225.[Medline]
Kankkunen, J., Harvima, I.T. and Naukkarinen. (1997) Quantitative analysis of tryptase and chymase containing mast cells in benign and malignant breast lesions. Int. J. Cancer, 72, 385388.[ISI][Medline]
Kofford, M.W., Schwartz, L.B. and Schechter, N.M. et al. (1997) Cleavage of type I procollagen by human mast cell chymase initiates collagen fibre formation and generates a unique carboxyl-terminal propeptide. J. Biol. Chem., 272, 71277131.
Malaviya, R., Ikeda, T. and Ross, E. et al. (1996) Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-. Nature, 381, 7780.[ISI][Medline]
Maseki, Y., Miyake, K. and Mitsuya, H. et al. (1981) Mastcytosis occurring in the testes from patients with idiopathic male infertility. Fertil. Steril., 36, 814817.[ISI][Medline]
Mitsui, H., Furitsu, T. and Dvorak, A.M. et al. (1993) Development of human mast cells from umbilical cord blood cells by recombinant human and murine C-Kit ligand. Proc. Natl Acad. Sci. USA, 90, 735739.[Abstract]
Miyata, A., Goto, H. and Niwa, Y. et al. (1997) Histological evaluation of connective tissue components in the healing process of human gastric ulcer. Clin. Exp. Pharmacol. Physiol., 24, 714719.[ISI][Medline]
Nagai, T., Hirabayashi, Y. and Tkaba, H. et al. (1992) Testicular mast cell heterogeneity in idiopathic male infertility. Fertil. Steril., 57, 13311336.[ISI][Medline]
Qu, Z., Liebler, J.M. and Powers, M.R. et al. (1995) Mast cells are a major source of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma. Am. J. Pathol., 47, 564573.
Saito, H., Ebisawa, M. and Nakahata, T. et al. (1996) Selective growth of human mast cells induced by steel factor, interleukin-6 and prostaglandin E2 from cord blood mononuclear cells. J. Immunol., 157, 343350.[Abstract]
Schechter, N.M., Irani, A.A. and Sprows, J.L. et al. (1990) Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell. J. Immunol., 145, 26522661.
Weidner, N. and Austin, K.F. (1993) Heterogeneity of mast cells at multiple body sites; fluorescent determination of avidin binding and immunofluorescent determination of chymase, tryptase and carboxypeptidase content. Pathol. Res. Pract., 189, 156162.[ISI][Medline]
Submitted on December 10, 1999; accepted on March 31, 2000.