Biology of Reproduction 61, 999-1004 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.
Transforming Growth Factor ß Signal Transducer Smad2 Is Expressed in Mouse Meiotic Germ Cells, Sertoli Cells, and Leydig Cells During Spermatogenesis1
Rui-An Wanga, and
Guang-Quan Zhao2,a
a Department of Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211
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ABSTRACT
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Although previous studies have shown that members of the transforming growth factor ß (TGFß) family are expressed in the seminiferous tubules, the functions of these growth factors in spermatogenesis remain elusive. In order to shed light on the mechanisms of TGFß action in spermatogenesis, it is crucial to determine whether and where their downstream signaling molecules are expressed in the testis. We examined the expression of Smad2, an intracellular signal transducer of the TGFßs, in mouse testes by in situ hybridization and immunohistochemistry. Both Smad2 mRNA and protein were detected in meiotic germ cells, from preleptotene to pachytene spermatocytes, but not in postmeiotic germ cells. Smad2 expression was also observed in interstitial cells and Sertoli cells. Therefore, our data provide molecular evidence for TGFß signal transduction during spermatogenesis.
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INTRODUCTION
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Mammalian spermatogenesis is a unique and complex developmental process. Spermatogonia, the stem cell population, undergo mitosis and differentiate into primary spermatocytes. These cells in turn undergo meiosis, and differentiate into secondary spermatocytes and spermatids. However, the molecular mechanisms underlying these processes are poorly understood.
The transforming growth factor ß (TGFß) superfamily of growth factors contains a growing number of structurally related but functionally diverse polypeptides, including TGFßs, activins/inhibins, decapentaplegic proteins, and bone morphogenetic proteins (BMPs) [1]. They exert a wide range of biological effects on cell proliferation, differentiation, and survival [1, 2]. These proteins function as homodimers or heterodimers and bind to serine/threonine kinase receptor complexes. Upon ligand binding, type II receptors activate the type I receptor subunits by phosphorylation [1, 2]. The type I receptors then phosphorylate SMAD proteins, a group of recently identified proteins responsible for intracellular signal transduction of the TGFß superfamily [3–6]. At least 10 Smad genes have been identified in vertebrates. All SMAD proteins share two highly conserved amino- and carboxy-terminal dominants separated by a more diverse proline-rich region [3]. SMAD1, SMAD5, and SMAD8 mediate the action of BMPs [7–10], while SMAD2 and SMAD3 are specific for TGFß and activin signaling [1, 2, 11–16]. SMAD6 and SMAD7 negatively regulate other SMAD proteins by preventing their phosphorylation [1, 2, 17–19].
Despite the fact that TGFßs are expressed in the seminiferous tubules [20, 21], their roles in spermatogenesis are far from clear. To shed some light on this issue, we have investigated the expression of Smad2 in developing and adult mouse testes.
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MATERIALS AND METHODS
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Animals and Tissue Preparation
Male mice of outbred genetic backgrounds (129SvEv x Swissblack) of 1, 2, 3, 4, and 8 wk of age were used for testis collection. Testes were dissected out and fixed in 4% paraformaldehyde/PBS, or Bouin's solution for 2–12 h according to the size. After dehydration, testes were embedded in Paraplast (Fisher Scientific Co., Pittsburgh, PA). Testes fixed in 4% paraformaldehyde/PBS were cut into 7-µm-thick sections and mounted onto slides for in situ hybridization, and those fixed with Bouin's solution were cut into 4-µm-thick sections for immunohistochemistry.
In Situ Hybridization
In situ hybridization was done essentially as previously described [22]. A 1-kilobase DNA fragment corresponding to the 3' untranslated region of murine Smad2 was subcloned into pBluescript (Stratagene, La Jolla, CA) and used for riboprobe synthesis. RNA probes were labeled with [
-35S]UTP to a specific activity of 1.0 x 109 cpm/µg. Hybridization was carried out at 60–65°C for 16–20 h in buffer containing 2 x 104 cpm/µl riboprobes, 50% formamide, 300 mM NaCl, 10 mM Tris (pH 7.4), 10 mM NaH2PO4 (pH 6.8), 5 mM EDTA (pH 8.0), 0.2% Ficoll 400, 0.2% polyvinyl pyrolidone, 10% dextran sulfate, 200 µg/ml yeast total RNA, and 50 mM dithiothreitol. Two rounds of 30-min high-stringency washes were carried out in double-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 50% formamide at 60–65°C. Slides were dipped in Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY) for 3 wk at 4°C, developed, fixed, stained in Mayer's hematoxylin for 3–5 min, and then mounted with Permount (Fisher Scientific Co.) for photography. Sense probe hybridization was used as control for background levels.
Immunohistochemistry
The standard avidin-biotin-peroxidase complex (ABC) staining method was adopted. Briefly, the sections were dewaxed in xylene and rehydrated through descending concentrations of ethanol, immersed in 0.3% H2O2/methanol for 30 min, and washed with PBS. The sections were then sequentially incubated with 1% normal rabbit serum for 1 h, goat anti-SMAD2 IgG (2 µg/ml; cat. no. SC-6200, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 h, biotin-labeled rabbit anti-goat IgG (1:100; Vector Labs., Burlingame, CA) for 1 h, and ABC (1:100; Vector Labs.) for 30 min, with intervening PBS washes after each incubation. Finally, the antigen sites were visualized with diaminobenzidine (DAB)/hydrogen peroxide solution, and the sections were counterstained with Mayer's hematoxylin for 30 sec. For specificity control, the primary antibody was incubated at a working dilution with 20 µg/ml synthesized SMAD2 peptides for 2 h and then used to stain the adjacent sections. All the reactions were carried out at room temperature.
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RESULTS
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Smad2 Expression in Mouse Testes During Postnatal Development
As shown in Figure 1, Smad2 mRNA messages were detected in the seminiferous tubules of postnatal mouse testes at all ages. In the testes of 1-wk-old mice, Smad2 mRNA signals were observed in germ cells from preleptotene to zygotene spermatocytes (Fig. 2A) and also in the center of the seminiferous tubules, an area corresponding to the cytoplasm of Sertoli cells (Figs. 1A and 2A). In the testes of 2- to 3-wk-old mice, strong Smad2 mRNA signals were detected in germ cells from preleptotene to pachytene spermatocytes (Fig. 2, B and C). From 4-wk-old on, high levels of Smad2 hybridization signals were observed at the periphery of the seminiferous tubules (Fig. 1, C and D), where preleptotene to pachytene meiotic germ cells reside (Fig. 2D). No obvious signals were detected in cells with characteristics of spermatogonia, small nuclei, and localization near the basement membrane (Fig. 2, A, B, and D).
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FIG. 1. Developmental expression of Smad2 in mouse testes. Left panels, darkfield photomicrographs; right panels, brightfield photomicrographs of the corresponding darkfield. A) A section through a testis of a 1-wk-old mouse. Strong signals are observed in all the seminiferous tubules, especially in the center. B) A section through a testis of a 2-wk-old mouse. Every layer of the germinal epithelium shows hybridization signals. C, D) Sections from 4- and 8-wk-old mice, respectively. Strong signals are detected at the periphery of the seminiferous tubules, and the signals are much weaker in the areas toward the lumen. Scale bar = 120 µm in A, 240 µm in B and C, 480 µm in D.
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FIG. 2. High-power magnification of seminiferous tubules to show Smad2 expression at cellular levels. A) A section through a testis of a 1-wk-old mouse. Signals were observed in early meiotic germ cells, as well as in the center of the seminiferous tubules (Sertoli cell cytoplasm). B) A section through a testis of a 2-wk-old mouse. Signals were detected from preleptotene to zygotene spermatocytes (arrows) and early stages of pachytene spermatocytes (large arrowheads). C). A section through a testis of a 3-wk-old mouse. Strong signals were detected in the pachytene spermatocytes (large arrowheads). D) A section through a testis of a 4-wk-old mouse. Signals were detected in leptotene, zygotene, and pachytene spermatocytes. No obvious signals were associated with spermatids. Small arrowheads point to cells at the periphery of the seminiferous tubules that did not show detectable Smad2 expression and were probably spermatogonia. Scale bar = 24 µm
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Smad2 Expression in Relationship to the Cycling of Seminiferous Tubules
Russell's staging system was adopted for our studies [23]. As shown in Figure 3, Smad2 mRNAs were detected in every stage of the cycle, but in different cell groups. From stages I to VII, high levels of silver grains were observed in pachytene spermatocytes (Fig. 3, A–F). A reduced level of Smad2 signals was detected in pachytene spermatocytes of stage XIII–X (Fig. 3, G and H). Smad2 messages were also detected in meiotic germ cells close to the basement membrane (from preleptotene to zygotene stages; Fig. 3, A and G–J). Hybridization signals were barely detectable in diplotene (Fig. 3I) and metaphase (Fig. 3J) spermatocytes, spermatids, and the small cells close to the basement membrane with characteristics of spermatogonia (Fig. 3, A–C and I). In summary, Smad2 messages were mainly detected in preleptotene, leptotene, zygotene, and pachytene spermatocytes.
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FIG. 3. Smad2 expression in relationship with the cycling seminiferous epithelium. A, B) Stage I to II–III seminiferous tubules. Large arrowheads indicate early stage pachytene spermatocytes with strong signals. C–G) Stage IV to VIII seminiferous tubules. Strong signals were found in pachytene spermatocytes (large arrowheads). In stage VIII, note the reduction of signals in pachytene spermatocytes when compared to previous stages and the higher levels of expression in preleptotene spermatocytes (small arrow in G). H–J) Stage X to XII seminiferous tubules. Signals were detected in leptotene and zygotene (arrows) spermatocytes. The last-stage pachytene spermatocytes (arrowheads in H) showed a low signal level, while signal levels detected in the diplotene (arrowheads in I) and metaphase (arrowheads in J) stages were not significant. Small arrowheads in A, B, C, and I point to cells at the periphery of the seminiferous tubules that did not show detectable Smad2 expression and were probably spermatogonia. Scale bar = 24 µm
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Immunohistochemical staining with anti-SMAD2 antibodies showed a similar result of Smad2 expression at the protein level in the adult mouse testes. Strong staining was found in the meiotic germ cells, and weak or no staining was observed in spermatids. Negative control staining with peptide-preabsorbed antibodies produced a negligible background staining (Fig. 4).
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FIG. 4. Immunohistochemical detection of SMAD2 in mouse testes. A) A section through a testis of a 4-wk-old mouse, stained with anti-SMAD2 antibodies, shows a positive staining in all seminiferous tubules. Arrows point to the Sertoli cell staining pattern. B) A section adjacent to A stained with antibodies preabsorbed with SMAD2 peptides produced a negligible background staining. C, E) Higher magnifications showing Smad2 proteins in pachytene spermatocytes (small arrowheads) and Leydig cells (large arrowheads). D, F) Sections adjacent to C and E stained with antibodies preabsorbed with SMAD2 peptides. No clear staining was detected in round and elongated spermatids (in C and E). However, a strong nonspecific staining was detected in an unidentified structure in the cytoplasm of pachytene spermatocytes and round spermatids (small arrow in C), which was also observed in controls (small arrow in D). Such nonspecific staining could be observed with all antibodies we purchased from Santa Cruz Biotechnology Inc., including antibodies against SMAD1, SMAD3, SMAD4, SMAD5, BMP2, BMP4, BMP7, BMP8, and TGFß receptors. Scale bar = 60 µm in A and B, and 24 µm in C–F.
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Smad2 Expression in Sertoli and Interstitial Cells
Smad2 messages were found in the center of seminiferous tubules at 1 wk of age, which corresponds to the cytoplasm of Sertoli cells. To further verify Smad2 expression in Sertoli cells, we used Bmp8b null mutant testes for in situ hybridization. Strong signals were detected in both the germ cell-free seminiferous tubules and the tubules with normal morphology (Fig. 5). Immunohistochemistry with SMAD2 antibodies also showed positive staining in Sertoli cells (Fig. 4). Furthermore, Smad2 expression was detected in interstitial cells by both in situ hybridization (Figs. 2 and 3) and immunohistochemistry (Fig. 4).
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FIG. 5. Smad2 expression in the Bmp8b null mutant mouse testes. Brightfield (A) and darkfield (B) photomicrographs of a Bmp8b mutant testis. Strong signals were detected in both the germ cell-free seminiferous tubules (asterisk) and the tubules with normal morphology. Scale bar = 480 µm
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DISCUSSION
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Although members of the TGFß superfamily are expressed in the testes [20–22, 24–28], their functions in male reproduction are not yet clear. Thus far, Smad1, the intracellular signal transducer of BMPs, has been the only documented case of Smad expression in male germ cells, from pachytene spermatocytes to stage 1 spermatids [29]. In this study, we present the expression pattern of Smad2, the intracellular signaling molecule of both TGFßs and activin, in postnatal mouse testes and during the cycling of the seminiferous epithelium. Smad2 expression during postnatal development and the cycling seminiferous epithelium was detected much earlier than Smad1 expression. Smad2 messages could be detected at 1 wk of age, while Smad1 expression is barely detectable until 2 wk of age. Smad2 expression was detected from preleptotene to pachytene spermatocytes, while Smad1 expression is observed from mid-stages of pachytene spermatocytes to the early stage of round spermatids [29].
Smad2 has been found to exert a wide spectrum of functions in regulating cell growth and differentiation [3–6, 30–33]. For example, during early development, Smad2 plays a pivotal role in patterning the left-right, and anterior-posterior axis formation of the embryo disc, as well as in inducing the formation of mesoderm [30–32]. Later, Smad2 functions during the morphogenesis of the lung and during craniofacial development [31, 33]. Furthermore, mutations of Smad2 have been found in human cancers such as colorectal carcinoma [34]. These obviously suggest that Smad2 plays an important role in inhibiting cell proliferation and promoting cell differentiation.
Our findings of Smad2 expression in preleptotene, leptotene, zygotene, and pachytene spermatocytes provide direct evidence for the molecular mechanism of TGFß action during spermatogenesis. The different expression patterns of Smad2 and Smad1 suggest that BMPs, TGFßs, and activin may be involved in different aspects of spermatogenesis.
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ACKNOWLEDGMENTS
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This work was initiated when G.-Q. Zhao was a research associate in Dr. Brigid Hogan's laboratory at Vanderbilt University. The authors are grateful to Dr. Hogan for her support and encouragement.
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FOOTNOTES
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1 This research was supported by NIH grant No. HD 36218. 
2 Correspondence. FAX: 573 884 5414; zhaog{at}missouri.edu
Accepted: May 19, 1999.
Received: March 29, 1999.
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ABSTRACT
INTRODUCTION
