Significance of human testicular mast cells and their subtypes in male infertility

K. Yamanaka, M. Fujisawa1, H. Tanaka, H. Okada, S. Arakawa and S. Kamidono

Department of Urology, Kobe University School of Medicine, Kobe, Japan


    Abstract
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 Materials and methods
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 Discussion
 References
 
The mast cell populations in the human testis were examined using immunohistochemical techniques in five fertile volunteers and 12 patients with obstructive azoospermia, seven patients with idiopathic azoospermia, and 30 patients with varicocele. The number of mast cells per seminiferous tubular section was significantly increased (P < 0.05) in the men with idiopathic azoospermia. In the normal testes, mast cells containing only tryptase were the predominant subtype. In the patient groups, the predominant subtype of mast cell was shifted to that containing both tryptase and chymase. The average number of mast cells containing both tryptase and chymase per seminiferous tubular section was significantly increased (P < 0.05) compared with the controls in patients with obstructive azoospermia, idiopathic azoospermia, and varicocele. The number of mast cells containing only tryptase was not increased in infertile men. The selective expansion of the mast cell population containing both tryptase and chymase may be related to spermatogenetic disorders and testicular fibrosis.

Key words: cathepsin G/heterogeneity/male infertility/mast cell/tryptase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human mast cells (MC) are found in most major organs and tissues of the body and are the major effectors of the immediate type of hypersensitivity reaction. Recent studies have shown that human MC activate fibroblasts and promote collagen synthesis by producing and releasing fibrogenic substances. Hence, MC play a role in the pathogenesis of chronic inflammation and fibrosis (Hatamochi et al., 1985Go; Jordana, 1993Go; Qu et al., 1995Go; Feldmann et al., 1996Go; Gruber et al., 1997Go). MC can be divided into two subtypes based on differences in their neutral serine protease content (Irani et al., 1986Go). MCT contain only tryptase, whereas MCTC contain both tryptase and chymase in addition to other proteases, including cathepsin G (Schechter et al., 1990Go) and carboxypeptidase (Irani et al., 1991Go). This heterogeneity can express itself as differences in histochemical, biochemical, and functional characteristics. The distribution of MCT and MCTC depends on the tissue examined (Weidner and Austin, 1993Go; Irani and Schwartz, 1994Go) and the pathological state.

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., 1981Go; Nagai et al., 1992Go). 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.


    Materials and methods
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Tissue specimens were obtained from the testes of 12 patients with obstructive azoospermia aged 23–39 years (mean ± SD 29.2 ± 5.2), seven patients with idiopathic azoospermia aged 30–40 years (33.7 ± 5.5), and 30 patients with varicocele aged 26–46 years (33.1 ± 4.3). Tissue specimens were collected from five healthy fertile men aged 32–46 years (38.7 ± 5.6) as controls. Among the patients with obstructive azoospermia, eight patients suffered from congenital absence of the vas deferens and four patients suffered from obstructive azoospermia after herniorrhaphy. All patients with idiopathic azoospermia showed azoospermia on first examination and did not suffer from any disease that obviously caused an obstruction of the spermatic tract. All patients in the varicocele group had clinical varicocele and showed oligozoospermia. The patients in the control group were healthy volunteers who had at least one child and showed normozoospermia. Testicular biopsies were performed after obtaining informed consent.

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 avidin–biotin–peroxidase 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.

Haematoxylin–eosin 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, 1970Go). 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., 1997Go). 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 Mann–Whitney 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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The mean age in the control group was significantly higher (P < 0.05) than that in the obstructive azoospermia or varicocele group. The mean age in the varicocele group was significantly higher (P < 0.05) than that in the obstructive azoospermia group. The average FSH and LH concentrations in the idiopathic azoospermia group were higher than normal (normal FSH, 1.6–9.2 mIU/ml; normal LH, 1.8–8.4 mIU/ml; Table IGo). The mean value of the Johnsen's score in the control group was 8.9, which was within the 95% normal limits as described by Johnsen (Johnsen, 1970Go; Table IIGo). The pathological findings in the idiopathic azoospermia group showed Sertoli cell-only or maturation arrest. The Johnsen's score in the control group was significantly higher than that in the obstructive azoospermia (P < 0.05), idiopathic azoospermia, or varicocele groups (P < 0.01). The ratio of tubules with Sertoli cell-only was significantly increased in the idiopathic azoospermia group when compared with the obstructive azoospermia (P < 0.01), varicocele (P < 0.01), or control group (P < 0.05). The ratio of tubules with sclerosis and the fibrosis index were significantly increased in the patient groups when compared with the control group (P < 0.05; Table IIGo). The Johnsen's score was significantly negatively correlated with concentrations of FSH (r = [nbh]0.49; P = 0.0006), LH (r = [nbh]0.49; P = 0.0006), and positively correlated with testosterone (r = 0.34; P = 0.024; normal testosterone concentration, 2.7–10.7 ng/ml) in all patients (Figure 1Go).


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Table I. Hormonal analysis in patient groups. Values are given as means ± SD
 

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Table II. Numbers of mast cells (MC), MC containing only tryptatse (MCT) and MC containing both tryptase and chymase (MCTC) and pathological characteristics in control and patient groups. Values are given as means ± SD

 


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Figure 1. Johnsen's score plotted against the serum concentrations of FSH, LH, and testosterone in all cases.

 
Testicular tissue from a patient with varicocele was immunostained using anti-tryptase antibody (Figure 2AGo) and anti- cathepsin G antibody (Figure 2BGo). The cells positive for tryptase were MC. They were found in the interstitium and lamina propria of seminiferous tubules (Figure 2AGo). The cytoplasm of the MC exhibited a granular staining pattern. The cells positive for both tryptase and cathepsin G were MCTC, whereas those positive for only cathepsin G were considered monocytes (Figure 2BGo). The average number of MC per seminiferous tubular section in the controls was 1.54 ± 0.40 (mean ± SD) (Table IIGo). The MCT subtype was the predominant form, which accounted for 62.2 ± 6.3% of all MC in the testes of normal fertile men. In the group with idiopathic azoospermia, the average number of MC per seminiferous tubular section was significantly increased compared with the control group (P < 0.05). The average number of MCTC per seminiferous tubular section was significantly increased (P < 0.05) in the obstructive azoospermia, idiopathic azoospermia, and varicocele groups when compared with the control group. There were no significant differences in the average number of MCT per seminiferous tubular section between the control and patient groups. The ratio of MCTC to total MC was significantly increased in the obstructive azoospermia (P < 0.01) and varicocele (P < 0.05) groups when compared with the control group. There were no significant correlations between the hormone concentrations and the numbers of MC, MCT, or MCTC per seminiferous tubular section (data not shown).



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Figure 2. Testicular tissue from a patient with varicocele. (A) Mast cells (MC) were stained for tryptase in the interstitium. (B) Immunostaining of cathepsin G. The cells stained for both tryptase and cathepsin G were MCTC (arrows). The cells stained for only cathepsin G were considered monocytes (arrowheads). (C) Immunostaining of cathepsin G. MCTC (arrow) and monocyte (arrowhead) show staining at a greater magnification than in (B).

 
In the varicocele group, there was significant positive correlation between the ratio of tubules with sclerosis and the fibrosis index (r = 0.50; P = 0.011). Significant correlations were observed between the number of MCTC per seminiferous tubular section and the ratio of tubules with sclerosis (r = 0.41; P = 0.022), and between the number of MCTC per seminiferous tubular section and the fibrosis index (r = 0.48; P = 0.013) (Figure 3Go).



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Figure 3. The ratio of tubules with sclerosis and the fibrosis index plotted against the number of mast cells containing both tryptase and chymase (MCTC) per seminiferous tubular section in the patients with varicocele.

 

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 Materials and methods
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It has previously been shown that MC not only play a major role in allergic immune reactions, but also in tissue remodelling and fibrosis (Cairns and Walls, 1997Go; Gruber et al., 1997Go; Kofford et al., 1997Go), host defence against infectious diseases (Malaviya et al., 1996Go), angiogenesis (Blair et al., 1997Go), and maybe even cancer invasion (Kankkunen et al., 1997Go). MC contribute to these processes by producing and secreting bioactive mediators. MC heterogeneity is characterized by differences in protease content, cytokine content (Bradding et al., 1995Go), and by electron microscopic findings (Craig et al., 1988Go). MCT contain tryptase, interleukin (IL)-4, IL-5, and IL-6. MCTC contain tryptase, chymase, cathepsin G, carboxypeptidase, and IL-4. MCT have secretory granules containing discrete scrolls, whereas MCTC have granules with grating and lattice substructures.

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., 1996Go; Igarashi et al., 1996Go). 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., 1996Go). 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., 1989Go; Mitsui et al., 1993Go). 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, 1993Go; Irani and Schwartz, 1994Go). A selective increase in MCT has been found in the skin of patients with atopic dermatitis (Irani et al., 1989Go), in the nasal submucosa of patients with allergic rhinitis (Bentley et al., 1992Go), and in breast cancer tissue (Kankkunen et al., 1997Go). 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., 1990Go). 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., 1990Go). 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.


    Notes
 
1 To whom correspondence should be addressed at: Department of Urology, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: masato{at}med.kobe-u.ac.jp Back


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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 10, 1999; accepted on March 31, 2000.