:: Asian Journal of Andrology ::

Stage-specific localization of transforming growth factor 1 and 3 and their receptors during spermatogenesis in men

Yuan-Qiang Zhang¹, Xiao-Zhou He¹, Jin-Shan Zhang¹, Rui-An Wang¹, Jie Zhou¹, Ruo-Jun Xu²

¹Department of Histology & Embryology, Fourth Military Medical University, Xi'an 710032, China
²Department of Zoology, University of Hong Kong, Hong Kong, China

Asian J Androl 2004 Jun; 6: 105-109

Keywords: transforming growth factor; transforming growth factor receptors; human testis

Abstract

Aim: To investigate the stage-specific localization of transforming growth factor (TGF) 1 and 3 during spermatogenesis in adult human testis. Methods: The localization of TGF1 and 3 was investigated by immunohistochemical staining method employing specific polyclonal antibodies. Results: Both TGF1 and 3 and their receptors were preponderant in the Leydig cells. TGF1 could not be detected in the seminiferous tubules. TGF3 and TGF-Receptor (R) I were mainly seen in the elongated spermatids, while TGF-RII in the pachytene spermatocytes and weak in the spermatogonia, spermatids and Sertoli cells. Only TGF-RII was detected in the Sertoli cells. TGF3, TGF-RI and TGF-RII showed a staining pattern dependent upon the stages of the seminiferous epithelium cycle. Conclusion: TGF isoforms and their receptors are present in the somatic and germ cells of the adult human testis, suggesting their involvement in the regulation of spermatogenesis.

1 Introduction

Testicular function is controlled not only by gonadotropins but also by cytokines and growth factors [1-3]. Three isoforms of transforming growth factor (TGF) , TGF1, TGF2 and TGF3 have been identified in mammals. They have been shown to be paracrine/autocrine regulators of testicular function [4-7].

TGFs are produced by the testis and are potent inhibitors of the Leydig cell function [8, 9] with certain effects on Sertoli cell function in vitro [10]. TGFs also affect the migration and the shape and contractibility of peritubular myoid cells. Olaso et al [11] indicated that TGF1 induced apoptosis in the gonocytes of foetal testis in vitro and Hakovirta et al [12] found that TGF1 increased the level of DNA synthesis in preleptotene spermatocytes in cultured seminiferous tubules. However, all these results are from experimental animals, the function of TGFs in human spermatogenesis is poorly understood. The present work was designed to localize the TGF ligands and their receptors in adult human testis and their roles in spermatogenesis.

2 Materials and methods

2.1 Tissue collection

Fourteen testes were collected from orchiectomy patients due to prostate cancer and autopsy cases following accidental deaths (20-55 yr old). The testicular tissue was fixed in Bouin's acid and processed for routine paraffin embedding. Normal tissue and spermatogenesis were identified in accordance with the criteria by Suares-Quian et al [13] and only 8 testes were selected for immunohistochemical analysis. Tissue collection was approved by the Human Research Committee of the University.

2.2 Antibodies

TGF1 and TGF3 rabbit polyclonal antibodies (Santa Cruz Biotechnology, USA) were raised against peptides mapping the carboxy terminus of the precursor forms of human TGF1 and TGF3, respectively. These antibodies have been recommended for the detection of precursor and mature TGF1 and TGF3 in mouse, rat and human and showed no cross-reactivity with each other. TGF- RI (sc-398, Santa Cruz), a polyclonal and affinity purified antibody raised against a peptide corresponding to the human and rat intracellular domain of TGF-RI, was used. TGF-RII (sc-220 Santa Cruz) was an affinity purified rabbit polyclonal antibody raised against a peptide mapping within the carboxy terminal domain of TGF-RII of human origin (differing from corresponding rat sequence by a single amino acid). TGF-RI and TGF-RII showed no cross-reactivity with each other.

2.3 Immunohistochemistry

Serial sections, 3 m in thickness, were processed for immunolocalization. The standard streptavidin-biotin-peroxidase complex was used. Briefly, the sections were de-waxed in xylene and rehydrated through descending concentrations of ethanol and the endogenous peroxidases were blocked with 3 % H₂O₂/methanol for 15 min at 37 . The sections were placed in a moist chamber and pre-incubated with a protein blocker solution containing 0.1 % BSA and 2 % non-immune rabbit serum to minimize non-specific staining. The sections were incubated for 2 h at 37 with the primary antibody diluted in an antibody diluting buffer (TGF 1:100, TGF2 1:100, TGF-RI 1:100, TGF-RII 1:100) and then with the biotinylated antibody (goat anti-rabbit IgG 1: 300, DAKO ChemMate^TM, Japan) and the streptavidin-biotin-peroxidase complex (1: 300, DAKO ChemMateTM) according to the manufacturer's recommendation. Peroxidases were observed with 0.7 mg/mL 3-3'-diaminobenzidine tetrahydrochloride (Sigma) in 1.6 mg/mL urea hydrogen peroxide, 60 mmol/L Tris buffer, pH 7.6, RT as the chromogen and the sections were briefly counter-stained with hematoxylin. Negative controls were prepared by using 0.1 % PBS/BSA and non-immune rabbit serum instead of the primary antibody.

2.4 Analysis of seminiferous epithelium stages

The criteria described by Clermont [14] was used to identify the germ cell types and define the stages of seminiferous epithelium cycle. The histology of the seminiferous epithelium was investigated on H-E and PAS (periodic acid-Schiff-hemotoxylin) stained sections. Typical germ cell associations of fixed composition were described with the nuclear morphology of the germ cell and the topographical arrangement of spermatids as the principal criteria. The human seminiferous epithelium cycle are divided into six stages (Figure 1 and Figure 2). Briefly, stage I is characterized by the presence of two generations of spermatids and pachytene spermatocytes, stage II by the elongated spermatids moving to the luminal aspect of the seminiferous epithelium, stage III by only one generation of round spermatids, stage IV by spermatids with nuclei showing initial signs of elongation, stage V by one generation of elongating spermatids having typically pointed and deeply stained nuclei directed toward the limiting membrane and stage VI by primary and secondary spermatocytes undergoing first and second maturation divisions.

Figure 1 and Figure 2. Testicular cross-sections showing stages I-VI human seminiferous epithelium cycle (400, HE and PAS staining) .

3 Results

3.1 Immunolocalization of TGF1 and 3 (Figure 3 and Figure 4)

The Leydig cells exhibited an intense TGF1 staining. However, even at higher magnification it was not conclusive whether seminiferous tubules were positive for TGF1. There appeared to be more widespread staining of TGF3 in the Leydig cells, Sertoli cells, primary spermatocytes and spermatids. No distinct positive staining was observed in spermatogonia. TGFb3 showed weak staining in the Sertoli cells and primary spermatocytes, while spermatids exihibited intense TGF3 staining. The TGF3 staining of the Sertoli cells and primary spermatocytes did not exhibit a cyclic pattern. On the contrary, the staining of the spermatids was dependent upon the stages of the seminiferous epithelium cycle. TGF3 first appeared in the spermatids with nuclei showing the initial signs of elongation at stage IV, weak staining at stages V-VI and reached the highest intensity at stages I-II. There was no positive staining in the round spermatids at Stage III.

Figure 3. Distribution of TGFb1 in human testis with Leydig cells showing an intense TGFb1 staining (400).
Figure 4. Distribution of TGFb3 in testis. TGFb3 in spermatids with nuclei showing initial sign of elongation at stage IV, weak staining at stages V-VI and reaching the highest intensity at stages I-II (400).

3.2 Immunolocalization of TGFb-RI and RII (Figure 5 and Figure 6)

The staining for TGF-RI protein was stronger in the testicular interstitium, including the Leydig cells, weaker in the Sertoli cells and was not detectable in the spermatogonia and primary spermatocytes. The staining for TGF-RII was clearly visible in all the spermatids at the whole seminiferouse cycle. The TGF-RII protein was localized at the Leydig cells and nearly all the cells in the seminiferous epithelium except the peritubular cells. Their staining pattern was dependent upon the stages of the seminiferous cycle. TGF-RII showed intense staining in the pachytene spermatocytes at stages II-VI and was faint or undetectable in the primary spermatocytes, which had just entered the pachytene step at stage I. In spermatids, from round to elongating, the staining for TGF-RII could also be detected at all stages.

Figure 5 and Figure 6. Distribution of TGFb-RI and -RII in testis. Staining for TGFb-RI protein is strong in testicular interstitium, including Leydig cells, weak in Sertoli cells and not detectable in spermatogonia and primary spermatocytes. TGFb-RII showing intense staining in pachytene spermatocyte at stages II-VI and faint or undetectable in primary spermatocytes just entering the pachytene step at stage I. Staining for TGFb-RII in spermatids can be detected at all stages (400) .

4 Discussion

There were a few papers concerning the distribution and expression of TGFs in human testicular tumor [15,16], yet to our knowledge, this is the first study reporting the distribution of TGFs and their receptors during spermatogenesis in normal human testis.

In the current study TGF1 was only detected in the Leydig cells, while in previous papers, it was mainly seen in the spermatocytes and early round spermatids in rats [12] and in young spermatocytes in boars [17]. To eliminate the difference in TGF1 antibody, we repeated the same experiment in rats and the results obtained were similar to the previous papers. Thus the distribution and expression of TGF1 may be species-specific. The physiological significance of this observation warrants further investigation.

It has been shown that TGF3 may regulate the opening and closing of the inter-Sertoli cell tight junctions so that fully developed spermatids can be released to the tubular lumen at spermiation [5]. In the present study it was shown that spermatids exhibited a strong TGF3 immunostaining that was dependent upon the stages of the seminiferous epithelium cycle. The results may indicate that TGF3 may play certain roles in spermatid maturation.

The present study shows that both the TGF-RI and -RII are present in the Leydig and germ cells, while only TGF-RII is found in the Sertoli cells. Caussanel et al [17] and Olaso et al [18] also failed to detect TGF-RI in pig and rat Sertoli cells. However, it is well known that TGFs are important factors regulating lactate production, a-inhibin mRNA level, proteoglycan synthesis, calcium metabolism and aromatase activity, so the failure to detect TGF-RI in the Sertoli cells is puzzling. One may suggest that the expression of TGF-RI is below the detection limit of the immunohistochemical method.

It is not known whether TGFs affect germ cell differentiation. A suitable spermatogenesis in vitro system is not available. Transgenic animals with an inactivated TGF1, 2 or 3 gene cannot be used, as all such animals die before puberty and TGF1, 2 and -RII knockout could result in embryonic lethality [19]. The sophisticated genetic strategies embracing techniques for tissue-specific regulation and temporal switching of transgene expression in conjunction with tissue transplantation may provide important information.

The present study suggests that the TGF isoforms and their receptors may modulate differentiation of the somatic and germ cells in human testis. The changes in staining intensity and the expression pattern of TGF-Rs during the seminiferous cycle make us to believe that TGF may control two key steps in spermatogenesis: the meiotic process and the onset of spermiogenesis. In conclusion, this study provides new evidence for the potential role of TGF in the regulation of adult human spermatogenesis.

References

[1] Weinbauer GF, Wessels J. 'Paracrine' control of spermato-genesis. Andrologia 1999; 31: 249-62.
[2] Mather JP, Moore A, Li RH. Activins, inhibins, and follistatins: further thoughts on a growing family of regulators. Proc Soc Exp Biol Med 1997; 215: 209-22.
[3] Anderson RA, Wallace EM, Groome NP, Bellis AJ, Wu FC. Physiological relationships between inhibin B, follicle stimulating hormone secretion and spermatogenesis in normal men and response to gonadotrophin suppression by exogenous testosterone. Hum Reprod 1997; 12: 746-51.
[4] Haagmans BL, Hoogerbrugge JW, Themmen AP, Teerds KJ. Rat testicular germ cells and sertoli cells release different types of bioactive transforming growth factor beta in vitro. Reprod Biol Endocrinol 2003; 1: 3.
[5] Lui WY, Lee WM, Cheng CY. TGF-betas: their role in testicular function and Sertoli cell tight junction dynamics. Int J Androl 2003; 26: 147-60.
[6] Bernard DJ. Editorial commentary: SMAD expression in the testis predicts age- and cell-specific responses to activin and TGFbeta. J Androl 2003; 24: 201-3.
[7] Dickson C, Webster DR, Johnson H, Cecilia Millena A, Khan SA. Transforming growth factor-beta effects on morphology of immature rat Leydig cells. Mol Cell Endocrinol 2002; 195:65-77.
[8] Gautier C, Levacher C, Saez JM, Habert R. Transforming growth factor beta1 inhibits steroidogenesis in dispersed fetal testicular cells in culture. Mol Cell Endocrinol 1997; 131: 21-30.
[9] Olaso R, Pairault C, Saez JM, Habert R. Transforming growth factor beta3 in the fetal and neonatal rat testis: immunoloca-lization and effect on fetal Leydig cell function. Histochem Cell Biol 1999; 112: 247-54.
[10] Lui WY, Lee WM, Cheng CY. TGF-betas: their role in testicular function and Sertoli cell tight junction dynamics. Int J Androl 2003; 26: 147-60.
[11] Olaso R, Pairault C, Boulogne B, Durand P, Habert R. Transforming growth factor beta1 and beta2 reduce the number of gonocytes by increasing apoptosis. Endocrinology 1998; 139:733-40.
[12] Hakovirta H, Kaipia A, Soder O, Parvinen M. Effect of avidin-A, inhibin-A, and transforming growth fctor beta1 on stage specific deoxyribonucleic acid synthesis during rat seminiferous epithelial cycle. Endocrinology 1993; 133: 1664-8.
[13] Suarez-Quian CA, Martinez-Garcia F, Nistal M, Regadera J. Androgen receptor distribution in adult human testis. J Clin Endocrinol Metab 1999; 84: 350-8.
[14] Clermont Y. The cycle of the seminiferous epithelium in man. Am J Anat 1963; 112: 35-51.
[15] Dobashi M, Fujisawa M, Yamazaki T, Okada H, Kamidono S. Distribution of intracellular and extracellular expression of transforming growth factor-beta1 (TGF-beta1) in human testis and their association with spermatogenesis. Asian J Androl 2002; 4: 105-9.
[16] Devouassoux-Shisheboran M, Mauduit C, Tabone E, Droz JP, Benahmed M. Growth regulatory factors and signalling proteins in testicular germ cell tumours. APMIS 2003; 111: 212-24.
[17] Caussanel V, Tabone E, Hendrick JC, Dacheux F, Benahmed M. Cellular distribution of transforming growth factor betas 1, 2, and 3 and their types I and II receptors during postnatal development and spermatogenesis in the boar testis. Biol Reprod 1997; 56: 357-67.
[18] Olaso R, Pairault C, Habert R. Expression of type I and II receptors for transforming growth factor beta in the adult rat testis. Histochem Cell Biol 1998; 110: 613-8.
[19] Ingman WV, Robertson SA. Defining the actions of transforming growth factor beta in reproduction. Bioessays 2002; 24:904-14.

Correspondence to: Prof. Y. Q. Zhang, Department of Histology and Embryology, Fourth Military Medical University, Xi'an 710032, China.
Tel/Fax: +86-29-8337 4508
E-mail: zhangyq@fmmu.edu.cn
Received 2004-01-15 Accepted 2004-04-14