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Testis |
Horizontal Medical Research Organization3
Department of Pathology and Biology of Diseases,4 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gametogenesis, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The purification of spermatogonial stem cells has been most successful in mice [5]. Using spermatogonial transplantation, we found that 
6- and ß1-integrin are expressed on spermatogonial stem cells [6], and using flow cytometry with multiparameter selection, we successfully enriched the stem cell population to 1 in 30 testis cells (166-fold enrichment compared with the unselected testis cell population) [5]. Another research group recently achieved 700-fold enrichment using a transgenic donor mouse expressing a specific marker for undifferentiated spermatogonia [7]. Using this strategy, mouse spermatogonial stem cells can now be routinely isolated for various purposes. Similar studies have begun with rats using xenogeneic transplantation of rat testis cells into immunodeficient nude mice [8]. Rat spermatogonial stem cells can complete spermatogenesis in the mouse testis environment following introduction into the seminiferous tubules [9]. Using this system, selection with laminin-coated plates resulted in 8.5-fold enrichment, similar to mouse spermatogonial stem cells [8, 10]. Although several molecules are expressed on rat gonocytes [11, 12], such as Epithelial Cellular Adhesion Molecule (EpCAM) or Neural Cell Adhesion Molecule (NCAM) antigens, it is not clear whether they are expressed on stem cells, because gonocytes are heterogeneous populations of precursor cells for spermatogonia [1] and their expression was not confirmed by functional transplantation assay.
In this investigation, we searched for new antigens that are expressed on spermatogonial stem cells. We hypothesized that stem cells of many self-renewing tissues share a common molecular machinery and examined the expression of CD9, which is expressed on other types of stem cells [13–15]. Mouse and rat testis cells were selected by magnetic bead technique and examined for the stem cell activity by spermatogonial transplantation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first set of experiments using mice, cells were collected from the testes of a transgenic mouse line B6-TgR(ROSA26)26Sor (designated ROSA; Jackson Laboratory, Bar Harbor, ME) [16]. This mouse line expresses the Escherichia coli LacZ transgene in all seminiferous tubule cells [16]. In a second set of experiments using rats, cells were collected from the testes of wild-type SD rats (Japan SLC, Shizuoka, Japan) or from the transgenic rat line TgN(act-EGFP)Osb4 (designated Green; Dr. M. Okabe, Osaka University, Osaka, Japan). These rats express the Enhanced Green Fluorescent Protein (EGFP) gene ubiquitously under the control of the ß-actin promoter [17]. Cells for transplantation were obtained from the testes of 8- to 12-wk-old animals. In both experiments, cells were collected from intact testes. Single-cell suspensions from testes were prepared by two-step enzymatic digestion [18].
Mouse cells were transplanted into the testes of WBB6F1-W/Wv mice (designated W; Japan SLC), whereas rat cells were transplanted into immunodeficient Imperial Cancer Research nude mice (designated nude; Charles River Japan, Atsugi, Japan). W mice are histocompatible with the donor cells and are congenitally infertile because they lack all stages of differentiating germ cells because of mutations in the c-kit receptor tyrosine kinase [19, 20]. Cells were transplanted into W mice when the recipients were 6–10 wk of age. Nude mice were injected with busulfan (44 mg/kg i.p.) at 6 wk of age and used for the experiments at least 1 mo after treatment. For the testicular injections, approximately 3 µl of the donor cell suspension was introduced into the seminiferous tubules of a W mouse testis and 10 µl was introduced into the tubules of a nude mouse testis, because the nude mouse testes are larger. Transplantation was by efferent duct injection [18], which filled 75%–85% of the tubules in each recipient testis.
The Institutional Animal Care and Use Committee of Kyoto University approved all of the animal experimentation protocols.
Flow Cytometry
Flow cytometric analyses were performed using a standard protocol [6]. Aliquots of 106 testis cells were suspended in 0.1 ml of PBS containing 1% fetal bovine serum (PBS/FBS) and incubated with primary antibodies. To detect CD9-positive cells, mouse or rat testis cells were incubated with 10 µg/ml biotin-conjugated anti-mouse CD9 antibody (clone KMC8; BD Biosciences, Franklin Lakes, NJ) or mouse anti-rat CD9 antibody (clone RPM.7; BD Biosciences), respectively. The primary antibodies were detected using 5 µg/ml of fluorescein isothiocyanate (FITC)-conjugated streptavidin (BD Biosciences) or Cy5-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), respectively, for mouse and rat testis cells. Control cells were not treated with primary antibodies.
To analyze selected testis cell populations, CD9-selected mouse testis cells were incubated with 5 µg/ml FITC-conjugated streptavidin, phycoerythrin (PE)-conjugated anti-6-integrin antibody (clone GoH3; BD Biosciences), or allophycocyanin (APC)-conjugated anti-c-kit antibody (clone 2B8; BD Biosciences). Control cells were not treated with streptavidin or the two antibodies. Likewise, CD9-selected rat testis cells were incubated with 5 µg/ml Cy5-conjugated anti-mouse IgG antibody, and control cells were not incubated with this antibody. Cells were kept in the dark on ice until analysis on a Becton Dickinson FACSCalibur (BD Biosciences). At least 10 000 events were acquired for each sample.
Immunohistochemistry
The technique for immunohistochemistry was previously described [21]. Testes were fixed with 10% neutral formalin and embedded in paraffin. Antigen was retrieved by autoclaving in 10 mM citrate buffer, pH 6.0, at 121°C for 10 min. The sections were stained with 10 µg/ml biotin-conjugated anti-mouse CD9 antibody (clone KMC8). The avidin-biotin complex method combined with the TSA Biotin system (PerkinElmer Life Sciences, Boston, MA) was applied for detection. Liquid diaminobenzidine (DAB; Dako Japan, Kyoto, Japan) was used as a substrate for peroxidase.
Selection of Testis Cell Subpopulation
The magnetic bead selection technique was described previously [22]. Aliquots of 2 x 107 testis cells were suspended in 1 ml of PBS/FBS with 10 µg/ml primary antibody. The cells plus antibody were incubated for 15 min on ice and washed three times with PBS/FBS, and magnetic beads were added. Biotin-conjugated rat anti-mouse CD9 antibody was used with streptavidin microbeads (25 µl; Miltenyi Biotec, Gladbach, Germany) to select cells expressing mouse CD9 molecules. Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 µl; Miltenyi Biotec) to select cells expressing rat CD9 molecules. The cells were further incubated for 15 min at 4°C and passed through an MS+ separation column (Miltenyi Biotec) to select cells, according to the manufacturer's instructions. Selected or unselected cells were suspended in Dulbecco modified Eagle medium supplemented as described previously [18].
Analysis of Testes
To evaluate colony number, recipient mice were examined 2 mo (first experiment) or 3 mo (second experiment) after the transplantation of donor cells. Transplanted donor cells, from ROSA mice or Green rats, were detected by staining for LacZ with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal; Wako Pure Chemical Industries, Osaka, Japan) [16] or by ultraviolet (UV) light excitation [17], respectively. These methods specifically identify donor cells, because host cells do not stain positively for LacZ and lack endogenous fluorescence. A cluster of germ cells was defined as a colony when it occupied >50% of the basal surface of the tubule and was 
0.1 mm long [16]. The efficiency of colonization was evaluated by counting the total number of colonies under a stereomicroscope. Because donor testis cell concentrations varied, colony number was normalized to 107 cells/ml. All sections were fixed in 10% neutral buffered formalin (Wako) and stained with hematoxylin and eosin. Statistical analysis was performed using the Student t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because CD9 is expressed on mouse embryonic stem (ES) cells [13], hematopoietic stem cells [14], and neural stem cells [15], we hypothesized that CD9 is also expressed on spermatogonial stem cells. In a previous study, expression of CD9 was found along the basement membrane of the seminiferous tubules in human testis [23]. However, these researchers did not examine whether CD9 is similarly expressed in the mouse testis. To test this possibility, we initially examined the presence of the CD9 molecule in a mouse testis cell population using flow cytometry. Testes of ROSA mice were dissociated enzymatically and stained with anti-mouse CD9 antibody. The expression of CD9 was confirmed in the mouse testis by flow cytometry, and approximately 4.7% of the testis cells expressed CD9 (Fig. 1A). Furthermore, immunohistochemical analysis of wild-type mouse testis also showed that CD9 is predominantly expressed on cells of the basement membrane, including spermatogonia, in the seminiferous tubule and interstitial cells (Fig. 1B).
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We next used a magnetic bead selection procedure to enrich cells expressing CD9. The selected cells were analyzed for the degree of enrichment and for the expression of 6-integrin (a stem cell marker) and c-kit (a marker for differentiating spermatogonia) [5, 6]. In these experiments, 2 x 107 testis cells from ROSA mice were used for the selection, and approximately 1.5 x 106 cells (7.5% of the total) were recovered with this procedure. Flow cytometric analysis revealed that 28% of the selected cells expressed CD9, indicating a 6-fold enrichment from the control unselected testis cells. Staining for spermatogonial markers revealed that the selected cells were relatively enriched for spermatogonia: the percentage (mean ± SEM) of cells expressing 6-integrin or c-kit increased from 3.1% ± 0.7% to 7.4% ± 0.7% (2.4-fold increase, n = 3) or from 7.7% ± 1.6% to 17.1% ± 8.7% (2.2-fold increase, n = 3), respectively, suggesting that the concentration of stem cells is increased after CD9 selection (Fig. 1A).
To examine whether stem cells express CD9, we next performed spermatogonial transplantation experiments [3]. Because the LacZ transgene is expressed in donor spermatogenic cells at all stages [16], spermatogenesis from donor stem cells can be identified by staining with X-gal following transplantation into recipient testes. In three experiments, 2 x 107 testis cells were selected using the magnetic bead procedure, and 1.1–1.5 x 106 cells were recovered. Equal numbers of selected and unselected cells were transplanted into the seminiferous tubules of infertile W mice at concentrations of 2.3–3.0 x 107 cells/ml. Two months after transplantation, the recipient mice were killed and their testes were stained for LacZ activity. The numbers of blue seminiferous tubules (colonies) were counted in each experimental and control recipient testis. Each colony is believed to arise from a single stem cell [16]. Therefore, the number of colonies reflects the concentration of stem cells in the injected testis cell population.
The number of blue colonies was increased by a factor of 6.9 for anti-CD9 antibody-selected cells compared with unselected control cells (5.5 vs. 0.8 colonies per 3 x 104 cells), and the difference was significant (P < 0.001) (Fig. 2, A and B). Histological analysis of the blue region of the seminiferous tubules revealed normal-appearing spermatogenesis and the presence of mature spermatozoa in the host seminiferous tubules (Fig. 2A). Therefore, the population of mouse testis cells with CD9 surface antigen can be enriched for spermatogonial stem cells.
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Expression of CD9 on Rat Spermatogonial Stem Cells
Based on the results of the first experiments with mouse testis cells, we investigated whether CD9 is expressed on rat spermatogonial stem cells. Testis cells from wild-type SD rats were dissociated enzymatically and stained with anti-rat CD9 antibody. Flow cytometric analysis revealed the presence of CD9 in the rat testis cell population, and approximately 17.5% of the testis cells expressed CD9 (Fig. 3A), a value higher than that in mouse testis cells (4.7%) (Figs. 1 and 3A). Using the same magnetic bead selection protocol as in the first experiment, 2 x 107 rat testis cells were selected and the degree of enrichment was examined. Approximately 1.1 x 106 cells (5.5% of the total) were recovered using this procedure. Flow cytometric analysis indicated that 87% of the selected cells expressed CD9, a percentage significantly greater than that in the first experiments using mouse cells (28%) (Fig. 3A). However, CD9-selected rat cells showed 4.9-fold enrichment compared with the unselected cells, a value comparable to that in the mouse experiment.
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To examine whether rat spermatogonial stem cells express CD9, we performed xenogeneic transplantation using immunodeficient nude mouse recipients [9]. As donors in this experiment, we used Green rats in which the EGFP gene is expressed ubiquitously [17]. Therefore, donor rat cells fluoresce under UV light after transfer into nude mouse recipients. Testis cells were collected from Green rats and isolated using the magnetic bead purification procedure. In these experiments, 2 x 107 cells were used for selection in each experiment and 0.8–1.6 x 106 cells were recovered. Unselected and selected cells were transplanted into busulfan-treated nude mouse testes. Because colonies from rat stem cells are significantly larger than those from mouse stem cells [8], the CD9-selected testis cells were injected at lower concentrations (2.7–5.6 x 106/ml) to avoid the merging of colonies. Unselected cells were injected at higher concentrations (0.8–1.7 x 107/ml). The recipients were analyzed 3 mo after donor cell transplantation, because rat spermatogenesis takes longer than mouse spermatogenesis (53 vs. 35 days) [9].
Xenogeneic rat spermatogenesis was apparent under UV light, and the numbers of colonies in each experimental and control recipient testis were counted (Fig. 3B). The number of colonies derived from CD9-selected cells showed 5.1-fold enrichment of stem cells compared with unselected control testis cells (15.8 vs. 3.1 colonies per 105 cells), and the difference was significant (P < 0.001) (Fig. 3C). These results demonstrate that rat spermatogonial stem cells also express CD9.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6-integrin+, ß1-integrin+, CD24+, thy-1+, v-integrin-, c-kit-, MHC- [5, 6, 24]. These stem cells also express Oct-4 and Stra8 [7, 25]. Some of these molecules are expressed on other types of stem cells. For example, thy-1 and ß1-integrin are expressed on hematopoietic stem cells [26, 27], and Oct-4 and 6-integrin are expressed on ES cells [28]. However, spermatogonial stem cells and other stem cells are not identical. Hematopoietic stem cells express 4-integrin and c-kit [27, 29], which are absent on spermatogonial stem cells [6]. Oct-4, a marker on ES cells, is not expressed on hematopoietic stem cells [30]. Nonetheless, the search for common molecules on different stem cells has facilitated the identification of stem cell surface markers [6, 24]. CD9 can now be added to the list of molecules that are commonly expressed on several types of stem cells. CD9 is a type III membrane protein with four transmembrane domains and is involved in cell adhesion, migration, proliferation, and fusion [31–35]. It is expressed on many types of cells, including bone marrow, brain, and muscle cells [31–35]. It is also expressed on oocytes and plays an important role in fertilization; disruption of the CD9 molecule by gene targeting results in female infertility [36, 37]. In the testis, expression of CD9 is not specific to stem cells. The CD9-selected cells showed significant amount of c-kit, which is expressed on differentiating spermatogonia [5, 6]. Nonspecific expression of CD9 on stem cells was corroborated by the results of immunohistochemical staining. However, fractionation of CD9-positive cells is possible by combining with other cell-specific markers, and identification of a new surface antigen on stem cells will likely improve stem cell enrichment protocols in mice.
An important question that arises from this study concerns the role of CD9 in spermatogonial stem cells. CD9 associates with integrins, including ß1- and 6-integrin [38], and it may play a role in signal transduction and in regulating cellular adhesion [39]. Because these integrin molecules are involved in the binding of cells to the basement membrane, this complex of molecules may be expressed on spermatogonial stem cells and may regulate cell adhesion. In a previous study, the addition of leukemia inhibitory factor (LIF) or STAT3 activation induced CD9 expression in ES cells [13], which indicates that the LIF/STAT3 pathway is critical for maintaining CD9 expression. The LIF/STAT3 pathway is essential for maintaining the undifferentiated state of ES cells [40, 41]. Given that spermatogonial stem cells in Drosophila also depend on the STAT pathway for maintenance of the undifferentiated state [42, 43], one possible scenario is that a similar pathway regulates CD9 expression in mammalian spermatogonial stem cells, thereby tethering stem cells to a niche so that they remain undifferentiated. Although CD9-null male mice lack an apparent reproductive phenotype, other closely related tetraspan transmembrane molecules, such as CD81, may serve this function [31–35]. Further functional study is required to investigate the role of CD9 in mammalian spermatogenesis.
Our identification of a rat spermatogonial stem cell antigen will help in the development of a new enrichment strategy for this species. To our knowledge, this is the first demonstration of a rat stem cell surface antigen using a functional assay. Rats are important in the study of spermatogenesis because regeneration of spermatogenesis after irradiation or chemotherapy in humans is more similar to the process in rats than to the process in mice [44]. However, there are several problems to be resolved before an efficient protocol for enriching rat spermatogonial stem cells can be developed. Stem cell purification in many self-renewing tissues is based on a combination of several enrichment techniques, such as multiparameter selection of cells using a cell sorter or centrifugation, as initially performed with mouse testis cells [5]. In mice, a simple cryptorchid operation enriches spermatogonial stem cells in vivo to 1/200 cells [45]. This procedure greatly facilitates subsequent multiparameter selection of stem cells using antibodies with a cell sorter [5]. The greater sensitivity of differentiated germ cells to body core temperature results in massive apoptosis, leaving only undifferentiated spermatogonia [46]. Unfortunately, however, previous attempts using this procedure with rats met with little success [8], and it was not possible to achieve an enrichment level comparable to that in mice [47, 48]. Another approach is to use vitamin A-deficient animals, because they have only undifferentiated spermatogonia in the testis [49, 50]. However, the degree of enrichment achieved with mouse models was unexpectedly modest [51]. It remains to be determined whether testes from vitamin A-deficient rats have a high stem cell concentration.
At present, the most reliable method for enriching rat spermatogonial stem cells depends on the ability of stem cells to bind laminin [8, 10]. This laminin selection technique is based on the observation that spermatogonial stem cells adhere to laminin more rapidly than do other somatic cells [45]. The degree of selection with this method was 3- to 8-fold for mouse and rat spermatogonial stem cells [6, 8, 45]. Although the technique may be extended to other species, the absence of surface markers limits application of this multiparameter selection strategy to testis cells of other species. Another study showed that it is possible to select neonatal gonocytes with high stem cell activity by micromanipulation [52]. However, the selection procedure is based on the size and morphology of stem cells (i.e., the presence of pseudopods, low intracellular complexity, and prominent round nuclei) and is therefore subject to interoperator differences and is extremely laborious. In addition, the number of stem cells in the neonatal testis is smaller than that in the adult testis [53]. In this context, our identification of a new antigen for rat spermatogonial stem cells is an important step for applying the concept and technique established in mice to enrich stem cells in large numbers.
Further studies should also be directed to identifying new molecules on the stem cells of other animal species. Xenogeneic transplantation of stem cells from hamsters, cattle, pigs, primates, and humans has been reported [47, 48, 54–59]. These reports indicate that stem cells of distantly related species can still attach to the basement membrane of mouse seminiferous tubules and proliferate. Although these studies indicate striking conservation of stem cell properties between species and suggest that they share many molecules, little progress has been made in characterizing stem cells from these species, and no surface molecules on stem cells have been found. Given our results, it is worth examining whether CD9 is also expressed on the stem cells of other animal species.
Our successful identification of a spermatogonial stem cell antigen supports the hypothesis that spermatogonial stem cells share the same molecule with stem cells in other self-renewing systems. The identification of a rat spermatogonial stem cell antigen provides a basis for establishing an efficient enrichment protocol. It is worth examining whether a similar approach is useful for identifying new antigens on spermatogonial stem cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Takashi Shinohara, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. FAX: 81 75 753 9281; takashi{at}mfour.med.kyoto-u.ac.jp
Received: 1 July 2003.
First decision: 21 July 2003.
Accepted: 2 September 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999 96:5504-55093ß1 in normal human tissues. J Histochem Cytochem 2001 49:439-4446, 3 and ß1. Mol Hum Reprod 2000 6:252-257This article has been cited by other articles:
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  T. Muller, K. Eildermann, R. Dhir, S. Schlatt, and R. Behr Glycan stem-cell markers are specifically expressed by spermatogonia in the adult non-human primate testis Hum. Reprod., July 10, 2008; (2008) den253v1. [Abstract] [Full Text] [PDF]
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 J.-B. Stukenborg, J. Wistuba, C. M. Luetjens, M. A. Elhija, M. Huleihel, E. Lunenfeld, J. Gromoll, E. Nieschlag, and S. Schlatt Coculture of Spermatogonia With Somatic Cells in a Novel Three-Dimensional Soft-Agar-Culture-System J Androl, May 1, 2008; 29(3): 312 - 329. [Abstract] [Full Text] [PDF]
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 K. E. Orwig, B.-Y. Ryu, S. R. Master, B. T. Phillips, M. Mack, M. R. Avarbock, L. Chodosh, and R. L. Brinster Genes Involved in Post-Transcriptional Regulation Are Overrepresented in Stem/Progenitor Spermatogonia of Cryptorchid Mouse Testes Stem Cells, April 1, 2008; 26(4): 927 - 938. [Abstract] [Full Text] [PDF]
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 M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Yoshida, S. Toyokuni, J. Lee, A. Ogura, and T. Shinohara Leukemia Inhibitory Factor Enhances Formation of Germ Cell Colonies in Neonatal Mouse Testis Culture Biol Reprod, January 1, 2007; 76(1): 55 - 62. [Abstract] [Full Text] [PDF]
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M. Tokuda, Y. Kadokawa, H. Kurahashi, and T. Marunouchi CDH1 Is a Specific Marker for Undifferentiated Spermatogonia in Mouse Testes Biol Reprod, January 1, 2007; 76(1): 130 - 141. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, K. Inoue, J. Lee, H. Miki, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara Anchorage-Independent Growth of Mouse Male Germline Stem Cells In Vitro Biol Reprod, March 1, 2006; 74(3): 522 - 529. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, H. Miki, K. Inoue, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara Long-Term Culture of Mouse Male Germline Stem Cells Under Serum-or Feeder-Free Conditions Biol Reprod, April 1, 2005; 72(4): 985 - 991. [Abstract] [Full Text] [PDF]
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 L. Cui, K. Johkura, F. Yue, N. Ogiwara, Y. Okouchi, K. Asanuma, and K. Sasaki Spatial Distribution and Initial Changes of SSEA-1 and Other Cell Adhesion-related Molecules on Mouse Embryonic Stem Cells Before and During Differentiation J. Histochem. Cytochem., November 1, 2004; 52(11): 1447 - 1457. [Abstract] [Full Text] [PDF]
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