Expression and localization of Smad1, Smad2 and Smad4 proteins in rat testis during postnatal development

Jing HU¹, Yuan-Qiang ZHANG¹, Xin-Ping LIU², Rui-An WANG¹, Yan JIN³, Ruo-Jun XU⁴

Asian J Androl 2003 Mar; 5: 51-55             

Keywords: TGF-b; Smad1; Smad2; Smad4; testis

Abstract

Aim: To study the expression and regulation of Smad1, Smad2 and Smad4 proteins (intracellular signaling molecules of transforming growth factor-b family) in rat testis during postnatal development. Methods: The whole testes were collected from SD rats aged 3, 7, 14, 28 and 90 (adult) days. The cellular localization and developmental changes were examined by immunohistochemistry ABC method with the glucose oxidase-DAB-nickel enhancement technique. Quantitative analysis of the immunostaining was made by the image analysis system. The Smads proteins coexistence in the adult rat testis was tested by the double immune staining for CD14-Smad4 and Smad2-Smad4. The protein expression of Smad during rat testicular development was examined by means of Western blots. Results: Smad1, Smad2 and Smad4 were present throughout testicular development. The immunostaining of Smad1 and Smad2 were present in spermatogenic cells. A positive immunoreactivity was located at the cytoplasm, but the nucleus was negative. Smad1 was immunolocalized at the d14, d28 and adult testes, while Smad2, at the d7, d14, d28 and adult testis. There was positive immunoreaction in the Sertoli cells and Leydig cells as well. The immunolocalization of Smad4 was exclusively at the cytoplasm of Leydig cells and the nuclei were negative throughout the testicular development. No expression was detected in the germ cells. The results of image and statistical analysis showed that generally the expression of Smad1, Smad2 and Smad4 in the testis tended to increase gradually with the growth of the rat. Conclusion: The present data provide direct evidences for the molecular mechanism of TGF-b action in rat testes during postnatal development and spermatogenesis.

1 Introduction

Normal testicular development and maintenance of spermatogenesis during fetal and postnatal life is controlled not only by hormone but also by cytokines and growth factors [1]. The transforming growth factor - b (TGF-b) superfamily is one such important local regulator of the testicular function.

The TGF-b superfamily members, including TGF-bs, activins/inhibins and the bone morphogenetic proteins (BMPs), are structurally related cytokines found in species ranging from worms and insects to mammals. A wide spectrum of cellular functions such as proliferation, apoptosis, differentiation and migration are regulated by them [2, 3]. These factors signal through heteromeric complexes of transmembrane type I and type II serine/threonine kinase receptors. Within this complex the type II receptor kinase activates the type receptor kinase, which subsequently propagates signals to the Smad pathway [3]. Smad proteins are recently identified proteins that mediate intracellular signaling of the TGF-b superfamily from the cell-surface to the nucleus [3, 4]. The name Smad originates from a fusion between Drosophila mothers against dpp (Mad) and C. elegans Sma [5]. The Smad family can be divided into three distinct subfamilies depending on their structures and functions. The receptor-regulated Smads (R-Smads) are phosphorylated by specific type I receptors on a carboxyl-terminal SSXS motif. Thus, the TGF-b and activin type I receptors activate Smad2 and -3, whereas the BMPs type I receptors target Smad1, -5, and -8. Then the R-Smads combine with the common-mediator Smads (Co-Smads) forming heteromeric complexes in the nucleus. Nuclear Smads complexes bind to DNA directly or indirectly through other DNA-binding proteins and regulate the transcription of target genes. Smad4 is the only vertebrate Co-Smad identified up to now. The inhibitory Smads (I-Smads) inhibit the activation of R-Smads [4, 6-8].

Several studies have investigated the expression and effects of members of the TGF-b superfamily and their receptors in the mammal testes during postnatal development. Both the Leydig cells and germ cells in fetal and neonatal pigs and rats bear types I and II TGF-b receptors, which are involved in signaling [9-12]. Members of the TGF-b superfamily may be involved during spermatogenesis and testicular development as the autocrine or paracrine factors, but their roles are far from clear. In order to shed light on the mechanisms of TGF-b action in spermatogenesis, it is crucial to determine whether and where their downstream signaling molecules are expressed in the testis. In this paper the expression and localization of Smad1, Smad2 and Smad4 proteins in the developing and adult rat testes were studied by Western blot and immunohistochemistry.

2 Materials and methods

Healthy male Sprague-Dawley rats of 3, 7, 14, 28 and 90 days of age (corresponding to neonatal, pre-pubertal, pubertal and adult, respectively) with 5 animals in each age group, were obtained from the Animal House of the Fourth Military Medical University. Rats were housed in a 12 h light/12 h dark and temperature-controlled room with food and water provided ad libitum. Rats were anesthetized by intraperitoneal injection of 4 mg/kg sodium pentobarbital and both testes were dissected out. One testis was fixed in Bouin's solution for 12 h, embedded in paraffin, cut into 4 mm sections and mounted onto 2 % 3-aminopropyltriethoxysilane-coated slides for immunohistochemistry; the other was stored at -70 until proteins isolation.

Goat anti-Smad1, Smad2, Smad4 and CD14 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Smad1 (T-20) was raised against a peptide mapping within the amino terminal domain of human origin Smad1, Smad2 (S-20) against a peptide mapping near the amino terminus of human origin Smad2 and Smad4 (C-20) against a peptide mapping at the carboxyl terminus of human origin Smad4. The other reagents were purchase from the following vendors: biotinylated rabbit anti-goat IgG antibody from Santa Cruz, USA; avidin biotin-peroxidase complex (ABC) from Dakopatts, Diaminobenzedin (DAB) from Sigma, D.P.X. mountant from BDH, UK; Enhanced Chemiluminescence (ECL) Western blotting system and Nitrocellulose filter (NC) from Sino-American Biotechnology Co., China.

After extracting the total protein from the testis, the samples were subjected to electrophoresis on a 12 % SDS polyacrylamide gel and transferred onto nitrocellulose filter. Then the filters were made Western blot [13]. The proteins were detected by two methods: ECL Western blotting system (protocols supplied by manufacturer) and DAB [11].

The standard ABC staining method was used [14]. The antigen sites were visualized with the glucose oxidase-DAB-Nickel enhancement technique [15]. The sections were dehydrated in graded ethanol, cleared in xylene and coverslipped using DPX. Sections were observed and photographed using a BH-2 microscope (Olympus, Japan). The specificity of the antibody was controlled by using PBS instead of the primary antibody.

Immunostaining of Smads was performed by the densitometric test with the image analysis system Q-500 (Leica). Taking 5 pieces of tissue sections from each rat and 4 units (each 0.015 mm²) out of a piece. The optical density of 4 randomly selected positive cells was detected at a unit, thus there were 16 data in a piece of section. After homogeneity of variance by the Bartlett test, the data were analyzed by two-tailed ANOVA using SPSS 10.0 for Windows software program. The results were expressed in meanSEM and P<0.05 was considered statistically significant.

The sections were incubated at 4 with goat anti-CD14 and Smad2 IgG (1:100) overnight, with biotinylated rabbit anti-goat IgG antibody (1:200) for 2 h and with ABC complex (1:100) for 1 h. Then, the antigenic sites were visualized with DAB (0.05 % diaminobenzidine in PBS containing 0.035 % hydrogen peroxide) for 3~5 min. Four PBS washes each for 15 min were done in order to clear away the first antibody, the anti-CD14 and the Smad2 IgG. The sections were then incubated with goat anti-Smad4 IgG and visualized by the glucose oxidase-DAB-nickel enhancement technique. Images were observed and photographed with an Olympus BH-2 optical microscope.

3 Results

The three Smads were expressed throughout the testicular development, i.e., there was one band in the testes of rats of each age group. Smad1 and Smad2 antibodies revealed a 52 kDa protein and Smad4 antibody a 62 kDa protein (Figure 1).

Figure 1. Expression of Smad proteins in rat testis during postnatal development by means of Western Blot (A: Smad1, B: Smad2, C: Smad4; 1: d3, 2: d7, 3: d14, 4: d28, 5: Adult).

Immunoreactivity of Smad1 and Smad2 was present in the spermatogenic cells and a positive immunoreaction was located at the cytoplasm, but not the nucleus. Immunolocalization of Smad1 was found in the d14, d28 and adult testis (Figure 2: 1, 2, 3, 4), while Smad2 in the d7, d14, d28 and adult testes (Figure 2: 5, 6, 7, 8). There was a positive immunoreaction in the Sertoli and Leydig cells (Figure 2: 9, 10) as well. Smad4 immunoreactivity was localized at the cytoplasm of Leydig cells in all the age groups. No expression was detected in the germ cells (Figure 3, 4). Nothing was observed in the negative controls.

Figure 2. Expression of Smad1 and Smad2 proteins in rat testis during postnatal development.
1. d28 (hspermatogenic cells) 200
2. Adult (hspermatogenic cells) 200
3. Adult 200
4. Spermatogenic cells of Smad1 protein positive immunoreaction (*meiotic germ cells and spermatids nearer to the lumen of seminiferous tubule; hspermatogonia and primary spermatocytes at periphery of seminiferous tubule) 200
5. d14 (hspermatogenic cells) 200
6. d28 (hspermatogenic cells) 200
7. Adult (hspermatogenic cells) 200
8. Adult (hspermatogenic cells) 100
9. Sertoli cells showing Smad2 protein immunoreactivity (h) 400
10. Leydig cells showing Smad2 protein immunoreactivity (h) 400

As shown in Table 1, Smad1, Smad2 and Smad4 were all present in rats starting at d3. Both Smad1 (P< 0.05) and Smad2 (P<0.01) expressions increased progressively thereafter, while Smad4 (P<0.01) expression increased progressively except at d14.

Table 1. Smad protein immunoreactive products in rat testis (n=16).

The immuoreactivity of CD14 and Smad4 was present in the loose connective tissue between the seminiferous tubules. Macrophages showed a brown staining of CD14, while Leydig cells, an indigo staining of Smad4. Double stained cells could also be found (Figure 5: a, b).

Smad2 was present in the spermatogenic cells and Leydig cells and the brown substance in the cytoplasm. Smad4 was exclusively localized at the cytoplasm of the Leydig cells and its indigo staining was stronger than the brown staining of Smad2. Double staining could be found in the Leydig cells (Figure 5: c, d).

Figure 5. Result of goat anti-CD14-Smad4 and goat anti-Smad2-mad4 double staining immunohistochemistry in adult rat testis.
a. Goat anti-CD14-mad4 double staining 200
b. Goat anti-CD14-mad4 double staining (rMacrophages showing immunostaining of CD14, pLeydig cells showing immuno-staining of Smad4, hdouble staining cells) 400
c. Goat anti-Smad2-mad4 double staining 200
d. Goat anti-Smad2-mad4 double staining (rGerm cells showing immunostaining of CD14, pLeydig cells showing immunostaining of Smad4, hdouble staining cells) 400

4 Discussion

TGF-b superfamily members are a group of polypeptides that regulate spermatogenesis and steroidogenesis through autocrine or paracrine mechanism [9-12]. The mechanism of TGF-b action on male reproduction is not clear. Smad proteins are a group of important intracellular signaling molecules of the TGF-b superfamily. Smad1, the intracellular signal transducer of BMPs, has been documented as the only Smad expression in male germ cells, from pachytene spermatocytes to stage 1 spermatids in mice [16]. As the intracellular signaling molecule of both TGF-bs and activins, Smad2 was detected from preleptotene to pachytene spermatocyte during postnatal development and cycling of seminiferous epithelium at the age of 1 week [17], but other Smads are not clearly known.

In the present study, the result of Western blot showed that Smad1, Smad2 and Smad4 proteins expressed in the testis of rats at d3, which was earlier than the results of Zhao & Hogen [16] and Wang & Zhao [17] obtained by in situ hybridization and immuno-histochemistry. The Smad4 protein was present in the Leydig cells of rats throughout the whole observation period. In our experiment, the results obtained by Western blot and immunohistochemistry were different; perhaps this may be due to that the antibody appropriate for Western blot is not sensitive to immunohistochemistry.

Both Smad1 and Smad2 are R-Smads and Smad1 mediates the intracellular signaling of BMP, whereas Smad2, the intracellular signaling of TGF-b and activin. Though they were mainly located in the spermatogenic cells, Smad2 is also expressed in the Sertoli cells and Leydig cells and the expression of Smad2 was more intense and occurred earlier than Smad1. Thus we believe that Smad2 may play a more important role in testicular regulation.

Smad4 is a Co-Smad, which works together with Smad1 and Smad2 to control gene transcription. We found that Smad4 protein expressed in the clustered or scattered Leydig cells throughout postnatal development with increasing intensity except at d14. It is believed that after birth the fetal Leydig cells do not transform into their adult counterparts, but are replaced by new adult-type Leydig cells differentiated from the stem cells [18]. In rats, by about d14 after birth, very few Leydig cells are found in the testis and when the new adult Leydig cells first appear, they are scattered between the tubules and not in clusters as the fetal Leydig cells. The immunoreactivity of Smad4 in adult Leydig cells was weaker than that in the fetal cells. Smad4 may regulate spermatogenesis indirectly through its action on Leydig cells. Macrophages are relatively plentiful in the interstitium of testis, but immunoreactivity of Smad4 was mainly found in the Leydig cells and not in the macrophages. There were a few double stained cells, thus we assume that macrophages might also express Smad4, which however, is awaiting further documentation.

In conclusion, TGFbs, activin and BMPs may influence different aspects of spermatogenesis and testicular development, which provides direct evidence for the action mechanism of TGF-b superfamily members on male reproduction.

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Correspondence to: Prof. Y. Q. Zhang, Department of Histology and Embryology, Fourth Military Medical University, Xi'an 710032, China.
Tel: +86-29-337 4508,     Fax: +86-29-337 4508
E-mail: zhangyq@fmmu.edu.cn
Received 2002-04-28      Accepted 2002-12-05
The work was supported by the National Science Foundation of China (39870109) and the Military Medical Foundation ( 98M106, 01MA183).