Distribution of intracellular and extracellular expression of transforming growth factor-b1 (TGF-b1) in human testis and their association with spermatogenesis

Masaki Dobashi, Masato Fujisawa, Takafumi Yamazaki, Hiroshi Okada, Sadao Kamidono

Division of Urology, Department of Organs Therapeutics, Graduate School of Medicine, Kobe University, Japan

Asian J Androl  2002 Jun; 4:  105-109             

Keywords: transforming growth factor-b1; fibrosis; spermatogenesis; testis

Abstract

Aim: Spermatogenic dysfunction may result from thickening of seminiferous tubular basement membrane (BM) with tubular sclerosis. Transforming growth factor b1 (TGF-b1) plays an important role in fibrogenesis. The intracellular and extracellular expression of TGF-b1 in the testis were immunohistochemically determined, using LC antibody (LC) for intracellular TGF-b1 and CC antibody (CC) for extracellular TGF-b1. Methods: Twenty-three testicular biopsy specimens were obtained from varicocele and five from Sertoli-cell-only (SCO) patients, and five from normal volunteers. The relative area involved by the expression of TGF-b1 for CC or LC (TGF-b1 index for CC or LC) was examined, and semen parameters and serum hormonal levels and TGF-b1 were analyzed. The Johnson score (JS), the BM thickness, and the tubular diameter were also determined. Results: Immunoreactivity for CC was hardly detected. That for LC was detected in the Sertoli and germ cells. The TGF-b1 index for LC was significantly higher in the varicoceles than in the normal testes. Interestingly, that for LC was significantly higher in the varicoceles than in the SCO. The level of serum TGF-b1 was significantly higher in varicoceles than in the normal testes. Conclusion: The distribution of the intracellular and extracellular expression of TGF-b1 in human testis was demonstrated. It suggests that TGF-b1 is related to fibrosis of seminiferous tubules and may lead to spermatogenic disruption.

Testicular development and function are under the control of hormones, cytokines and growth factors [1]. Among the growth factors related to spermatogenesis, there are various peptide families, including insulin-like growth factors, fibroblast growth factors, epidermal growth factor and transforming growth factors (TGF). TGF-b, a major initiator of fibrotic reaction, induces fibroblast cell growth and stimulates the expression, synthesis and release of collagen, fibronectin and tissue inhibitors of metalloproteinases [2]. The basement membrane (BM) of seminiferous tubules contains a common set of proteins that includes laminin, type IV collagen, various heparan sulfate proteoglycans and ectatin/nidogen. Previous studies in the rat demonstrated that TGF-b influenced peritubular cell function and migration, increasing extracellular matrix (ECM) production and promoting colony formation [3]. The synthesis and secretion of ECM components by peritubular cells are involved in the formation of the BM of seminiferous tubules and are important in maintaining the structural integrity of seminiferous tubules and in promoting structural differentiation of the cells and spermatogenesis [4]. The testes of elderly males and patients with idiopathic male infertility show histopathological changes with thickening of the seminiferous tubular BM and increment in the interstitial connective tissue [5].

In mammals, there have been many studies which demonstrated the expression of TGF-b1 in testis. The aim of the present study was to determine the distribution of the intracellular and extracellular expression of TGF-b1 in human testis and their association with spermatogenesis.

Twenty-three testicular biopsy specimens were obtained from men with left idiopathic varicocele (mean age 34.0 years), five from men with SCO (mean age 31.0 years) and five from normal volunteers (mean age 39.4 years). Informed consent was obtained from all the subjects. Patients with varicocele were diagnosed after careful physical examination and a scrotal thermography.

Semen samples were obtained by masturbation after 3 to 5 days of sexual abstinence and were liquefied at room temperature. After liquefaction, the sperm concentration was estimated using procedures of the World Health Organization (WHO, 1992). The blood follicle-stimulating hormone (FSH, 1.6-10.6 mIU/mL), luteinizing hormone (LH, 1.8-9.1 mIU/mL), testosterone (T,2.4-10.4 ng/mL), prolactin (PRL, 3.4-16.2 ng/mL) and estradiol (E2) (16-71 pg/mL) were analyzed using chemiluminescent immunoassay (CLIA). The level of serum TGF-b1 was measured by the AN'ALYZA Immunoassay System (GENZYME-TECHNE, Minneapolis, USA).

2.3 Immunohistochemistry

The intracellular and extracellular expressions of TGF-b1 were determined immunohistochemically with the avidin-biotin-peroxidase kit (Vector Laboratories, Burlin-game, CA), using LC antibody (LC) for the intracellular TGF-b1 and CC antibody (CC) for the extracellular TGF-b1 [6]. Two rabbit polyclonal antibodies against TGF-b1 (LC and CC) were employed, which were kindly provided by Dr KC Flanders of the Laboratory of Chem-oprevention at the US National Cancer Institute. Paraffin-embedded sections 4 mm in thickness were cut from the biopsy specimens, deparaffinised in xylene, rehydrated in graded ethanol solutions and washed in phosphate-buffered saline (PBS; 140 mM NaCl, 8 mM Na₂HPO₄, 2 mM NaH₂PO₄, pH 7.2). The endogeneous peroxidase activity was blocked by incubating the slides in fresh 0.3 % H₂O₂ in methanol for 20 min. After the nonspecific binding was blocked with 1.5 % normal goat serum/ 0.5 % PBS, sections were incubated overnight at 4 with each first antibody. Bound antibody was detected using biotinylated goat anti-rabbit IgG and avidin-peroxidase complex. Sections were stained with 3,3'-diaminoben-zidine (DAB) (Sigma, St Louis, USA) and counterstained with 1% methyl green. As a negative control, the primary antibodies was omitted.

All sections were viewed under a microscope with an attached video camera. The camera was linked to Photograb-300 (Fuji, Tokyo, Japan) within a Macintosh Power Mac G3 microcomputer (Apple, Cupertino, USA). Images were transferred using the 10 objective, moving along the longitudinal axis of the biopsy specimen. Each image was imported directly to the Photoshop 4.0J program (Adobe Systems, San Jose, USA) and then converted to gray-scale images abandoning the red and green channels. Expression of TGF-b1 stained brown by DAB was extinguished to a greater extent than other areas. Processed images were exported to a freeware image-analysis program, NIH Image 1.62f. To calculate the fraction of area occupied by the expression of TGF-b1, a threshold was applied to each image at a constant level that distinguished between the expression of TGF-b1 (rendered black) and the remaining area (rendered white). Black pixels were regarded as the expression of TGF-b1. More than 20 seminiferous tubular sections per testis were analyzed. The total area of the expression of TGF-b1 was divided by that of seminiferous tubular sections in order to calculate the TGF-b1 index. Another sections were stained with haematoxylin and eosin to measure the BM thickness, the tubule diameter, and the JS with an inner piece (Olympus, Tokyo, Japan). More than 20 seminiferous tubular sections per testis were examined. The JS was determined as previously described [7].

The statistical significance of difference was evaluated by using the two-sample t-test, and that of correlation by Spearman's correlation coefficient. P values of < 0.05 were considered statistically significant.

FSH (P<0.01), LH (P<0.05) and E2 (P<0.01) of patients with SCO were significantly higher than those of the normal subjects. The levels of serum TGF-b1 of the normal, varicoceles and SCO subjects were 308.562.5, 1813.2177.2 and 1078.6254.6 pg/mL, respectively. The level of serum TGF-b1 of the vari-coceles was significantly higher than that of the normal subjects (P<0.01).

In the pathological study, the BM thickness of the SCO (P<0.01) or varicocele patients (P<0.05) was significantly higher than that of the normal subjects. The tubular diameter of SCO patients was significantly lower than that of the varicocele (P<0.01) or normal subjects (P<0.01). The JS of SCO patients was significantly lower than that of the varicocele (P<0.01) or normal subjects (P<0.01), and that of the varicocele was also significantly lower than that of the normal subjects (P<0.05) (Table 1).

Table 1. Clinical data in the patients with normal, varicocele, and SCO.

LC staining was seen in the Sertoli and the germ cells. On the other hand, CC staining could hardly been detected. Cross-reaction between LC and CC was not detected (Figure 1).

Figure 1. Distribution of the intracellular and extracellular expression of TGF-b1 in the testis with varicocele. A: expression of TGF-b1 of CC; B: expression of TGF-b1 of LC; C: control (100 Bar=50 mm)

The TGF-b1 indices for LC in the normal, varicocele, and SCO subjects were 3.71.5 %, 9.34.1% and 4.91.1 %. There were significant differences between the varicocele and the SCO subjects (P<0.05), and between the varicocele and the normal subjects (P<0.01). Interestingly, the TGF-b1 index for LC in the varicocele patients was significantly higher than that in the SCO patients (Figure 2).

Figure 2.  The TGF-b1 index for LC in patients with normal testis, varicocele, and SCO. ^bP<0.05 SCO vs varicocele, ^cP<0.01 varicocele vs normal.

TGF-b superfamily is a large group of structurally related proteins involved in the growth and differentiation of both vertebrates and invertebrates [8]. In the testis, TGF-b1 is known to stimulate fibronectin and collagen and to increase the production of ECM by peritubular cells. The synthesis and secretion of ECM components are involved in the formation of seminiferous tubular BM and are important in maintaining the structural integrity of the seminiferous tubules. During puberty, the expression of TGF-bs is regulated by hormonal influences and is involved in steroidogenesis and spermatogenesis [9,10], suggesting their potential role as mediators of cell-cell interactions within the seminiferous tubules [11]; the factor plays an autocrine/paracrine role in the regulation of testicular function and differentiation [12]. TGF-bs are potent inhibitors of Leydig cell function and have some effects on Sertoli cell function in vitro. TGF-bs also affect peritubular myoid cell migration, shaping and contractibility [13].

In mammals, there are some reports concerning the distribution of TGF-b1 in the testis. In rats, TGF-b1 expresses in the germ, Sertoli, and Leydig cells [14]. In other studies, TGF-b1 is detected in rat Sertoli and peritubular cells [12,15-20] or in Leydig cells, spermatocytes and weakly in Sertoli cells, but not in peritubular cells [12].

In rat primordial Sertoli cells, TGF-b1 expresses on fetal day 14.5; the expression increases until day 16.5 and becomes faint from fetal day 18.5 onwards, whereas the staining in Leydig cells appears on day 16.5, becomes very intense during late fetal life and persists until postnatal day 20. No immunoreactivity for TGF-b1 was found in germ cells and peritubular cells on any day [12,16,21].

In pigs, it was reported that the Leydig and Sertoli cells from immature testis express TGF-b1 mRNA and protein both in vivo and in vitro [17]. In the adult mouse testis, TGF-b1 mRNA is expressed in both the somatic and germ cells [12,17]. In adult boar testis, TGF-b1 is mainly detected in young spermatocytes [15].

In the present study, TGF-b1 of LC was expressed in the Sertoli and germ cells. The TGF-b1 index for LC was higher in the varicocele than in the normal subjects. In addition, the seminiferous tubular BM was thicker in the varicocele than in the normal subjects, while the tubular diameter in the varicoceles was lower. The result suggests that seminiferous tubular sclerosis is caused by TGF-b1. In addition, the TGF-b1 index for LC was lower in SCO than in varicocele subjects. The reason may be that there are no germ cells in SCO and that Sertoli cell-germ cell interactions regulate TGF-b1 expression and secretion [17].

In conclusion, we demonstrated the distribution of the intracellular and extracellular expression of TGF-b1 in human testes. It is suggested that TGF-b1 is related to seminiferous tubular fibrosis and may lead to spermatogenic disruption.

References

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Correspondence to: Dr Masato Fujisawa, Division of Urology, Department of Organs Therapeutics, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-Cho, Chuo-ku, Kobe 650-0017, Japan.
Tel: +81-78-382 6155, Fax: +81-78-382 6169
E-mail: masato@med.kobe-u.ac.jp
Received 2002-05-14      Accepted 2002-05-28