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BOR - Papers in Press, published online ahead of print October 11, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.053181
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BIOLOGY OF REPRODUCTION 76, 130–141 (2007)
DOI: 10.1095/biolreprod.106.053181
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

CDH1 Is a Specific Marker for Undifferentiated Spermatogonia in Mouse Testes1

Masutaka Tokuda 3 4, Yuzo Kadokawa 2 3 4, Hiroki Kurahashi 4 5, and Tohru Marunouchi 3 4

Division of Cell Biology, Institute for Comprehensive Medical Science,3 21st Century COE Program, Development Center for Targeted and Minimally Invasive Diagnosis and Treatment,4 Division of Molecular Genetics, Institute for Comprehensive Medical Science,5 Fujita Health University, Toyoake 470-1192, Japan

ABSTRACT

In the mammalian testis, spermatogenesis is initiated from a subset of stem cells belonging to undifferentiated type A spermatogonia. In spite of the biologic significance of undifferentiated type A spermatogonia, little is known about their behavior and properties because of a lack of specific cell surface markers. Here we show that CDH1 (previously known as E-cadherin) is expressed specifically in undifferentiated type A spermatogonia in the mouse testis. Histologic analysis showed that CDH1-positive cells had all the characteristics of undifferentiated type A spermatogonia. Whole-mount immunohistochemistry showed that CDH1-positive cells made clusters mainly comprising one, two, four, or eight cells. They survived after administration of the cytotoxic agent busulfan to mice, and then regenerated seminiferous epithelia. Transplantation experiments showed that only CDH1-positive cells had colonizing activity in the recipient testis. Our data clearly demonstrated that spermatogenic stem cells reside among undifferentiated type A spermatogonia, which express CDH1.

CDH1 (E-cadherin), developmental biology, spermatogenesis, spermatogenic stem cells, testis, undifferentiated spermatogonia

INTRODUCTION

In mammalian testes, large numbers of mature sperm are continuously produced in an organized manner. Spermatogenesis is largely divided into the mitosis of spermatogonia, meiosis of spermatocytes, and subsequent spermiogenesis. Spermatogonia are classified as type A, intermediate (IN), or type B according to their amount of nuclear heterochromatin. Type A spermatogonia are the most primitive spermatogonia and have little heterochromatin. They are subdivided into seven categories according to their development. The most primitive spermatogonium is the Asingle (As). As gives rise to Apaired (Apr) and then divides to make chains of 4, 8, and 16 cells known as Aaligned (Aal). As, Apr, and Aal spermatogonia together have been called undifferentiated type A spermatogonia. Most Aal and some Apr spermatogonia differentiate into A1 spermatogonia at a certain stage, go through a series of six divisions, and become primary spermatocytes via A2, A3, A4, IN, and B spermatogonia [1–3]. The mitosis of differentiating spermatogonia, the meiosis of spermatocytes, and spermiogenesis proceed synchronously along the seminiferous tubules, and they constitute the cycle of the seminiferous epithelium, which is divided into 12 stages (stages I–XII) in mice [4].

Many lines of investigation suggest that the spermatogenic stem cell activity resides in undifferentiated type A spermatogonia. Undifferentiated type A spermatogonia showed random division independent of the cycle of the seminiferous epithelium, and the length of their cell cycle was much longer than that of the other spermatogonia [5], which is characteristic of stem cells. Undifferentiated type A spermatogonia were resistant to cytotoxic agents such as irradiation, whereas other spermatogonia were radiosensitive. Undifferentiated type A spermatogonia could repopulate the seminiferous epithelia and entire spermatogenic cell population after irradiation [6–10]. Indeed, the stem cell population was enriched in experimental cryptorchid testes [11], in which only undifferentiated type A spermatogonia accumulated because of an arrest of differentiation [12]. Furthermore, undifferentiated type A spermatogonia were also abundant in stem cell factor (KITL)-deficient mice (KitlSl/KitlSl-d), because the KITL signal was required for survival and/or differentiation of the differentiating spermatogonia, so spermatogenesis was arrested at the point of undifferentiated type A spermatogonia in the mutant mice [13, 14]. However, these cells retained stem cell activity when transplanted into the testis of wild-type mice [11, 15].

In spite of their biologic significance, the behavioral and molecular mechanisms underlying the behavior of undifferentiated type A spermatogonia have not been elucidated largely because of the lack of a specific biologic marker. POU5F1 (previously known as Oct4), a homeobox transcription factor that is required for the maintenance of totipotency of embryonic stem cells [16, 17], is expressed in undifferentiated spermatogonia [18]. Recently, a helix-loop-helix transcription factor, Neuog3 (previously known as neurogenin3), and an Sry-related HMG box transcription factor, Sox3, were found to be expressed specifically in undifferentiated type A spermatogonia [19, 20]. It was thought that Neuog3 was regulated by the Sox3 signal, because targeted disruption of Sox3 in mice causes repression of Neuog3. Another nuclear protein, promyelocytic leukemia zinc-finger (ZBTB16; previously known as Plzf), which functions as a transcriptional repressor, also was reported to be expressed only in undifferentiated type A spermatogonia [21, 22]. These molecules are candidate markers for undifferentiated type A spermatogonia; however, the cells expressing these markers cannot be separated alive by immunologic methods because of the markers' nuclear localization.

Many investigations have identified stem cell populations using surface markers. Flow cytometry-based transplantation experiments showed that stem cell activity was concentrated in fractions of spermatogonia positive for ITGB1 (previously known as ß1-integrin), ITGA6 (previously known as {alpha}6-integrin) [23], CD9 [24], and TACSTD1 (previously known as Ep-CAM) [25], and negative for KIT [26]. All of these cell surface markers are expressed in every type of spermatogonium, except KIT, a tyrosine kinase receptor for KITL, which is expressed in some Aal spermatogonia, differentiating spermatogonia, and the earliest preleptotene spermatocytes [14]. Therefore, multiple combinations of these markers have been needed to concentrate the spermatogenic stem cells.

In this study, we showed that CDH1, a homophilic cell-cell adhesion molecule spanning the cell membrane, is expressed specifically in undifferentiated type A spermatogonia in mouse testes. This new cell surface marker enabled us to identify the morphology and topographic arrangement of undifferentiated type A spermatogonia in seminiferous tubules. Moreover, we demonstrated that spermatogenic stem cells belong to the population of CDH1-positive cells by conducting a transplantation assay.

MATERIALS AND METHODS

Immunohistochemistry

ICR, C57BL/6J, WBB6F1-KitW/KitW-v, and WBB6F1-KitlSl/KitlSl-d mice were purchased from SLC (Hamamatsu, Japan). Testes were removed from the tunica albuginea and fixed with 10% neutral buffered formalin containing 10% methanol for 1 h at 4°C with tilting and rotation. After treatment with a sucrose gradient (10%, 15%, and 18%: 1 h at each concentration), the testes were embedded in Cryo Mount (Muto Pure Chemicals, Tokyo, Japan), frozen in liquid nitrogen, and stored at –80°C. Specimens were sectioned at a thickness of 8 µm using a cryostat (CM1800; Leica, Heerbrugg, Switzerland). The cryosections were then washed with Dulbecco phosphate-buffered saline (D-PBS). After treatment with 0.3% H2O2 in methanol at –20°C for 15 min, the sections were blocked with Protein Block Serum Free (DAKO Cytomation, Glostrup, Denmark) for 30 min at room temperature (RT). The samples were incubated with 2 µg/ml goat anti-mouse CDH1 antibody (AF748; R&D Systems, Minneapolis, MN) in "Can Get Signal immunostain solution A" (Toyobo, Osaka, Japan) overnight at 4°C. They were then washed with PBS containing 0.1% Triton X-100 (PBST) and were incubated with 0.2 µg/ml horse radish peroxidase (HRP)-conjugated donkey anti-goat immunoglobulin G (IgG; sc-3851; Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at RT. After being washed with PBST, samples were stained with diaminobenzidine (DAB; metal-enhanced DAB substrate kit; Pierce Biotechnology, Rockford, IL).

For immunofluorescence microscopy, mice were fixed by perfusion with 10% neutral buffered formalin containing 10% methanol. After the fixation, both testes were recovered. The testes were cut in two at the center, then postfixed with 4% paraformaldehyde in D-PBS at 4°C overnight. The cryosections were blocked with Protein Block Serum Free for 10 min at RT and incubated with 10 µg/ml rat anti-mouse CDH1 antibody (ECCD-2, M108; Takara Bio, Kyoto, Japan) in D-PBS for 30 minutes at 37 °C. They were then washed with D-PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rat IgG antibody (all Alexa-conjugated antibodies were purchased from Molecular Probes, Carlsbad, CA) in D-PBS for 30 min at 37°C. After further washing with D-PBS, the sections were mounted in VECTASHIELD with 4',6'-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA).

For double staining with anti-CDH1 and other antibodies we did not use perfusion fixation. Testes were removed from the tunica albuginea and fixed with 10% neutral buffered formalin containing 10% methanol for 1 h at 4°C with tilting and rotation. After sucrose treatment, samples were embedded in Cryo Mount. The cryosections were incubated with 2 µg/ml goat anti-mouse CDH1 antibody or 10 µg/ml rat anti-mouse CDH1 and either rat anti-human ITGA6 (MCA 699; Serotec, Kidlington, UK), rat anti-mouse ITGB1 (MAB 1977; Chemicon, Temecula, CA), goat anti-mouse GATA4 (sc-1237; Santa Cruz Biotechnology), rabbit anti-rat CYP11A1 (AB1244; Chemicon), goat anti-human VIMENTIN (sc-7557; Santa Cruz Biotechnology), or rat anti-TACSTD1 (Developmental Studies Hybridoma Bank, Iowa City, IA) antibody. After being washed with D-PBS, the sections were incubated with Alexa Fluor 488-conjugated donkey anti-goat IgG or Alexa Fluor 488-conjugated donkey anti-rat IgG and either Alexa Fluor 594-conjugated donkey anti-rat IgG, Alexa Fluor 594-conjugated donkey anti-goat IgG, or Alexa Fluor 594-conjugated donkey anti-rabbit IgG, antibodies.

Whole-Mount Immunohistochemistry

Testes were removed from the tunica albuginea and digested with 1 mg/ml collagenase type 2/Hanks Balanced Salt Solution modified (HBSS; both from Sigma-Aldrich) for 5 min at RT with manual agitation until the seminiferous tubules were dispersed. Almost all interstitial cells were removed during this digestion. After being washed with D-PBS, they were fixed with 10% neutral buffered formalin containing 10% methanol for 1 h on ice. After the blocking of nonspecific reactions with Protein Block Serum Free for 1 h at RT, the samples were incubated with 2 µg/ml goat anti-mouse CDH1 antibody or 10 µg/ml rat anti-CDH1 antibody, and either rat anti-mouse KIT (clone ACK2, CBL1360; Chemicon), rat anti-TACSTD1, mouse anti-human ZBTB16 (OP128; Calbiochem), goat anti-rat GFRA1 (previously known as GFR{alpha}-1; AF560; R&D Systems), mouse anti-ACTA2 (previously known as {alpha}-smooth muscle actin; A5228; Sigma-Aldrich), goat anti-human VIMENTIN (sc-7557; Santa Cruz Biotechnology), or rabbit anti-human DESMIN (sc-14026; Santa Cruz Biotechnology) antibody in 1% BSA, 0.25% casein, and D-PBS for 1 h at 37°C. After further washing, the samples were incubated with Alexa Fluor 350-conjugated donkey anti-goat IgG, Alexa Fluor 488-conjugated donkey anti-goat IgG, Alexa Fluor 488-conjugated donkey anti-rat IgG, Alexa Fluor 594-conjugated donkey anti-rat IgG, Alexa Fluor 594-conjugated donkey anti-goat IgG, Alexa Fluor 594-conjugated donkey anti-rabbit IgG, and Alexa Fluor 594-conjugated donkey anti-mouse IgG antibodies for 1 h at 37°C. After more washing, several pieces of seminiferous tubule were mounted on slide glasses and enclosed in VECTASHIELD with DAPI or 1 µg/ml Hoechst 33258 (23491-44-3; Polyscience, Warrington, England). For double staining with CDH1 and POU5F1, the samples were incubated with 2 µg/ml goat anti-mouse CDH1 antibody and 4 µg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in "Can Get Signal Immunostain Solution A" overnight at 4°C. After being washed with PBST, the samples were incubated with Alexa Fluor 488-conjugated donkey anti-goat IgG antibody and Alexa Fluor 594-conjugated donkey anti-rabbit IgG antibody for 1 h at RT. For double staining with CDH1 and RET, the samples were treated with LAB solution (Polyscience) for 5 min at RT and were washed by D-PBS. The samples were incubated with 10 µg/ml rat anti-CDH1 antibody and 10 µg/ml mouse anti-RET antibody (AF482; R&D Systems). After washing, the samples were incubated with Alexa Fluor 488-conjugated donkey anti-rat IgG antibody and Alexa Fluor 594-conjugated donkey anti-goat IgG for 1 h at 37°C. The stage in the cycle of the seminiferous epithelium was determined from the nuclear morphology of spermatids by DAPI staining and was supported by the cell shape and size of differentiating spermatogonia stained with KIT antibody.

Alkaline Phosphatase Activity

The cryosections were incubated for 15 sec in 0.1 M Tris-HCl (pH 8.5) solution containing 1 mg/ml naphthol AS-MX phosphate (N4875; Sigma-Aldrich), 1 mg/ml Fast Red TR salt hemi (zinc chloride) salt (F8764; Sigma-Aldrich), and 8% N,N-dimethylformamide. After washing with D-PBS, the stained samples were incubated with goat anti-mouse CDH1 antibody and then Alexa Fluor 488-conjugated donkey anti-goat IgG antibody, as previously mentioned. As the reaction product of alkaline phosphatase has fluorescence activity near around 600 nm, the double-stained samples could be observed by fluorescence microscopy.

Western Blot Analysis

Western blot analysis was performed as previously described [27]. Briefly, 20 testes were recovered from 1-wk-old male ICR mice, removed of the tunica albuginea, and homogenized with 100 µl lysis buffer (25 mM Tris-HCl, pH 7.5; 100 mM NaCl; 2 mM EDTA; 1 mM PMSF; 10 µg/ml leupeptin; and 10 µg/ml aprotinin) in a 1.5-ml Eppendorf tube. COS7 cells were transfected with mouse CDH1 cDNA in a Miw vector driven by a combination of the chicken ß-actin promoter and RSV LTR sequence [28] using FuGENE6 (Invitrogen, Carlsbad, CA). Forty-eight hours after the transfection, the cells were harvested and lysed in lysis buffer. The lysates were sonicated and clarified by centrifugation, analyzed on 7.5% SDS-polyacrylamide gels, and electroblotted onto polyvinylidene fluoride membranes (FluorTrans W; Pall, Glen Cove, NY). The membranes were probed with anti-CDH1 monoclonal antibody or polyclonal antibody and were incubated with HRP-conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) or HRP-conjugated anti-goat IgG (Rockland, Gilbertsville, PA). The signals were visualized using a chemiluminescence system (ECL; Amersham Bioscience, Little Chalfont, UK).

Busulfan Treatment and Bromodeoxyuridine Labeling

To deplete the proliferating spermatogenic cells in seminiferous tubules, 40 mg/kg busulfan/dimethyl sulfoxide (busulfan from Sigma-Aldrich) was injected i.p. once into the 4-wk-old male ICR mice. The busulfan-treated mice were killed 2, 3, 4, or 6 wk after the injection, and both testes were recovered. Bromodeoxyuridine (BrdU; 1 mg/ml; Sigma-Aldrich) was given to ICR mice in their drinking water for 1, 2, or 3 wk. The testes were fixed with 4% paraformaldehyde in D-PBS overnight at 4°C. The cryosections were stained with rat anti-mouse CDH1 antibody and Alexa Fluor 488-conjugated donkey anti-rat IgG antibody. Then, the stained samples were postfixed with 4% paraformaldehyde in D-PBS for 20 minutes at 4°C. After being treated with 2 N hydrochloric acid for 20 min at RT and washed with D-PBS, the samples were stained with 10 µg/ml sheep anti-BrdU antibody (P00013; Capralogics, Hardwick, MA) and Alexa Fluor 594-conjugated donkey anti-sheep IgG. The sections were mounted in VECTASHIELD with 4',6'-diamidino-2-phenylindole (DAPI). We counted 100, 53, and 58 CDH1-positive cells and 903, 802, and 702 CDH1-negative spermatogonia in the BrdU-labeled samples after 1-, 2-, and 3-wk administrations, respectively.

Transplantation Assay

The donor cells for transplantation were prepared from C57BL/6-Tg (CAG-EGFP) C15–001-FJ001Osb x ICR hybrid F1 male mice at 6–10 wk of age. The testes were sterilized by treatment with 70% ethanol for 20 sec. After the tunica albuginea was removed, the seminiferous tubules were minced with a surgical knife in HBSS. To remove the cell debris, the samples were centrifuged three times at 120 x g for 5 min at 4°C. Minced samples were treated with 1 mg/ml collagenase type 2 in HBSS for 10 min with occasional agitation at 37°C. After 50 times the volume of HBSS was added, the cell suspension was filtered through nylon mesh 70 µm in pore size (BD Falcon, Bedford, MA) to remove the tissue debris. The filtrate was centrifuged at 120 x g for 5 min at 4°C three times, and the cell suspension was filtered again through nylon mesh 45 µm in pore size (BD Falcon). A total of 0.75 µg rat anti-mouse antibody against CDH1 (ECCD-2), as well as 12.5 µl (5 x 106 beads) of Dynabeads M-450 conjugated with sheep anti-rat IgG (Dynal Biotech ASA, Oslo, Norway) were preincubated in 500 µl HBSS at 4°C overnight before being mixed with cells. The cell suspension then was incubated with a complex of ECCD-2 and Dynabeads for 45 min at 4°C with gentle mixing. The CDH1-positive cells were separated with Dynal MPC (Dynal AS, Oslo, Norway) and washed four times for 5 min at 4°C. Control cells for each experiment were taken from the remaining of immunoselection. Cells for injection into testes were suspended in HBSS, and 3 µl cell suspension containing about 0.9 x 104 to 4.1 x 104 cells was injected into seminiferous tubules through the efferent ducts. WBB6F1-KitW/KitW-v male mice at 8–10 wk of age were used as recipients. One testis was injected with the CDH1-positive cells with magnetic beads, and the other testis of the same mouse was injected with the control cells. The recipient mice were killed 8 wk after the transplantation, and recovered testes were observed under a fluorescence microscope to count the number of colonies. The testis is one of several immune-privileged organs and has the ability to support allogeneic tissue transplants. It was reported that allogeneic germ cells were able to make colonies for at least 8 wk without rejection when transplanted into testes [29]. All animals were maintained, operated, killed, and dissected according to guidelines set down by Fujita Health University.

RESULTS

CDH1 Is Expressed in Undifferentiated Type A Spermatogonia

Immunohistochemical analysis of the adult mouse testis showed that CDH1 was expressed in a small number of cells sparsely distributed in the seminiferous tubules (Fig. 1, a and b). To exclude the possibility of nonspecific reactions of the antibodies, we performed Western blotting using cell lysate from P7 mouse testes. Both monoclonal (ECCD-2) and polyclonal antibodies against mouse CDH1 showed a single band of approximately 124 kDa in mouse testes, the size of which was coincident with that of COS7 cells transfected with mouse CDH1 cDNA (Fig. 1c). The monoclonal and polyclonal antibodies both yielded similar results in immunohistochemstry (Fig. 1d). To confirm whether Leydig cells express CDH1 or not, we performed DAB-based immunohistochemistry, because Leydig cells showed strong autofluorescence (Fig. 1b). DAB staining clearly showed that Leydig cells were negative for CDH1 expression (Fig. 1a). Not only Leydig cells but also Sertoli cells that were stained with anti-GATA4 antibody [30, 31] were negative for CDH1 expression (Fig. 1b). The CDH1-positive cells attached to the basement membrane of the seminiferous epithelia and often appeared flattened. Their nuclei showed no heterochromatin staining with DAPI (Fig. 1e, arrowhead). Double-staining immunohistochemistry showed that CDH1-positive cells also expressed ITGA6, ITGB1, and TACSTD1 (Fig. 1, f–h), all known markers of spermatogonia [23, 32]. We also checked somatic cell markers other than GATA4 in seminiferous tubules. CDH1-positive cells did not express CYP11A1 (previously known as cytochrome P450 side chain cleavage enzyme), a marker for Leydig cells [33], VIMENTIN, a marker for somatic cells [34], and alkaline phosphatase, a marker for peritubular myoid cells [35] (supplemental Fig. 1 available online at www.biolreprod.org). These morphologic and immunohistochemical characteristics showed that CDH1-positive cells are type A spermatogonia.


Figure 76011501
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  		FIG. 1. CDH1 was expressed in a small subset of cells in the seminiferous tubules of mouse testes. a) Immunohistochemical analyses of seminiferous tubules in mature mouse testes showed that CDH1 was expressed in a small fraction of germ cells (arrows). The section was counterstained with hematoxylin. Bar = 20 µm. b) Double staining with CDH1 (green) and GATA4 (red). GATA4 was expressed in the nuclei of Sertoli cells and Leydig cells. CDH1-positive cells (arrows) were located on the basement membrane in seminiferous tubules and were negative for GATA4. The strong green fluorescence shown in the cytoplasm of Leydig cells was autofluorescence. Bar = 20 µm. c) Western blot analysis of CDH1 (arrow, about 124 kDa) from mouse testes. Lysate from testes (lane 1) and the COS7 cells transfected with the CDH1 cDNA expression vector (lane 2) and control COS7 (lane 3) were electrophoresed. Left panel shows the Western blot with the monoclonal antibody (ECCD-2), and right panel shows the Western blot with the polyclonal antibody. The extra band of about 90 kDa shown in lane 3 of the right panel was probably the degradation product of ectopically expressed CDH1. d) Double staining of CDH1 with polyclonal antibody (left), monoclonal antibody (middle), and merged images (right). e) CDH1-positive cells had oval-shaped nuclei displaying no heterochromatin with DAPI staining (middle, arrowhead). f–h) Double staining of CDH1 (left) and ITGA6 (f, middle), ITGB1 (g, middle), or TACSTD1 (h, middle) and merged images (right). CDH1-positive cells also were positive for ITGA6, ITGB1, and TACSTD1. Bar = 10 µm.

To investigate the profile of CDH1 expression during testicular development, we performed an immunohistochemical analysis of seminiferous tubules from neonatal to aged mice. In newborn mice, the seminiferous tubules contained only gonocytes and Sertoli cells. Gonocytes showed a uniformly rounded shape and were separated from the basement membrane. CDH1 was expressed in gonocytes but not in Sertoli cells (Fig. 2a). Within several days postpartum, gonocytes started proliferating and migrated to the basement membrane expressing CDH1 (Fig. 2b). After 1 wk postpartum, germ cells attaching to the basement membrane were defined as prospermatogonia, and in morphologic phenotype they resembled undifferentiated type A spermatogonia. Almost all prospermatogonia expressed CDH1 on their surface (Fig. 2c). Two weeks postpartum, meiotic division had already started, and the seminiferous tubules were occupied by many spermatocytes. A subset of spermatogonia was positive for CDH1, but other spermatogonia were negative for CDH1 (Fig. 2d). The number of CDH1-positive spermatogonia per section of a seminiferous tubule gradually decreased in older mice (Fig. 2, e and f). However, CDH1-positive spermatogonia were detected even in a 480-day-old mouse testis (Fig. 2g). We also examined CDH1 expression in the testes of KitlSl/KitlSl-d mice deficient in KITL signaling and KitW/KitW-v mice deficient in the KIT receptor. In both mice, spermatogenesis was arrested at the point of undifferentiated type A spermatogonia because of a deficiency in KITL signaling. The cells in the seminiferous tubules of these mice were only undifferentiated spermatogonia and Sertoli cells. Almost all spermatogonia were stained with CDH1 in both mice at 4 wk of age (Fig. 2, h, i, and supplemental Fig. 2 available online at www.biolreprod.org).


Figure 76011502
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  		FIG. 2. CDH1 was expressed continuously in seminiferous tubules of the mouse testis from the postnatal stage to old age: P1 (a), P4 (b), 1 wk (c), 2 wk (d), 4 wk (e), 8 wk (f), and 480 days (g) of age, respectively. h–i) CDH1 expression was observed in all spermatogonia of both KitlSl/KitlSl-d (h) and KitW/KitW-v (i) mice. Bar = 50 µm. A higher magnification of the cells indicated by the arrows is shown in each inset. Bar = 10 µm.

To investigate the topographic arrangement of CDH1-positive spermatogonia in seminiferous tubules, we performed whole-mount immunohistochemistry. Although a small number of CDH1-positive cells were observed as single cells, the majority had attached to other such cells to form small clusters. Whole-mount immunostaining of CDH1 clearly revealed the shape of the cells, because CDH1 demarcated the entire cell boundary through its localization at the cell surface. CDH1-positive cells usually extended several fine processes (Fig. 3, b–d). In the clusters consisting of CDH1-positive cells, some pairs of cells were connected to each other via fine cellular processes. These processes occasionally extended quite a distance (Fig. 3d). Other pairs of cells adhered to each other via broad cell boundaries where the expression of CDH1 was concentrated (Fig. 3b, arrowheads). The number of cells comprising the clusters was mainly two, four, or eight (Fig. 3, a, b, e). This staining pattern strongly suggested that CDH1 was expressed in undifferentiated type A spermatogonia. To exclude the possibility that these CDH1-positive cells were residual Leydig cells, we performed the double staining with anti-CDH1 and anti-GATA4 antibodies in whole-mount samples. Residual Leydig cells were easily identified with their cytoplasmic autofluorescence and the nuclear staining with anti-GATA4 antibody [31] (Fig. 1b). CDH1-positive cells were not stained with anti-GATA4 antibodies (supplemental Fig. 3 available online at www.biolreprod.org).


Figure 76011503
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  		FIG. 3. CDH1 was expressed in undifferentiated type A spermatogonia. a) Whole-mount immunohistochemistry revealed the cell shape and the topographical arrangement of CDH1-positive cells. b) A higher magnification of some clusters comprising one, two, or eight cells outlined in a CDH1-positive spermatogonia were connected to each other via intercellular bridges in the cluster, whereas some cells adhered to neighboring cells via extensive cell boundaries where CDH1 staining was concentrated (arrowheads). c) Paired cells extending long filopodia resembling leading processes. d) Paired cells connected with a long, fine cellular process. e) Frequency of the clusters with different numbers of CDH1-positive cells. The vertical axis shows the number of colonies of different sizes. The horizontal axis shows the number of cells comprising each cluster. Bars = 20 µm.

Expression of CDH1 in Undifferentiated Type A Spermatogonia Disappears after the Transformation into A1 Spermatogonia

It has been reported that the numbers of As and Apr are relatively constant during all stages, whereas the number of Aal spermatogonia increases from stage I and maximizes at stage VIII. During stages VII and VIII, almost all Aal spermatogonia and some Apr spermatogonia transform into A1 spermatogonia, then synchronously divide and differentiate into A2 spermatogonia at stage IX [1, 36–38]. We examined the distribution of As, Apr, and Aal cells throughout the stages by using whole-mount immunohistochemistry for CDH1 in long seminiferous tubules and obtained the result previously reported; the numbers of As and Apr were constant from stages IV to IX, whereas the number of Aal peaked at stage VIII and declined from stage IX.

To investigate the expression pattern of CDH1 during the transformation from undifferentiated type A spermatogonia into A1 spermatogonia, we performed the double staining of CDH1 and KIT, a marker of differentiating spermatogonia [14]. At stages V to VI, type B spermatogonia and preleptotene spermatocytes were stained with the anti-KIT antibody; however, CDH1-positive spermatogonia were not (Fig. 4b). From stages VII to VIII, besides small preleptotene spermatocytes, connected large cells were stained with the anti-KIT antibody, and some of them also expressed CDH1 (Fig. 4c). It is likely that these KIT and CDH1 double-positive cells are transforming from Aal into A1 spermatogonia. At stage IX, KIT and CDH1 double-positive cells were rarely observed (Fig. 4d). These findings support the model put forward by Schrans-Stassen et al., who proposed that Aal spermatogonia gradually change from being KIT negative to KIT positive before their differentiation into A1 spermatogonia [39]. Our observations indicate that the expression of CDH1 in undifferentiated type A spermatogonia disappears after the differentiation into A1 spermatogonia.


Figure 76011504
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  		FIG. 4. Whole-mount double immunostaining of CDH1 and KIT in long seminiferous tubules. a) Panoramic view of a CDH1-stained seminiferous tubule. Roman numerals show the stages of spermatogenic epithelia. Bar = 5 mm. b–d) Higher magnification of panel a indicated by arrows with each character. Left panels show KIT staining, middle panels show CDH1 staining, and right panels show merged images of the left and middle panels. b) KIT staining showed IN spermatogonia (larger cells in the left half of panel) and type B spermatogonia (smaller cells in the right half of panel). No Aal cells were stained with anti-KIT antibody. c) KIT staining showed preleptotene spermatocytes (smaller cells) and differentiating Aal spermatogonia (larger cells). Many Aal cells were stained with the anti-KIT antibody at this stage. Note that some cells expressed CDH1 only (arrowheads). d) KIT staining showed A1 spermatogonia. Most A1 cells lost CDH1 expression, whereas some A1 cells retained a weak CDH1 expression (arrowheads). e–g) Double staining of CDH1 and KIT (e–f) or CDH1 and TACSTD1 (g) showed heterogeneity of KIT or TACSTD1 expression in a cluster. e) Two of four cells in an Aal cluster were KIT negative (arrowheads), whereas the other two cells were KIT positive (arrows). f) One cell of an Apr cluster was KIT negative (arrowhead), and the other was KIT positive (arrow). g) Only two cells (arrow and arrowhead) showed strong CDH1 expression in a TACSTD1-positive cluster. One showed strong TACSTD1 expression (arrow), whereas the other showed weak TACSTD1 expression (arrowhead). Bars = 20 µm.

At stages VII to VIII, the majority of As and some of the Apr cells did not express KIT (arrowheads in Fig. 4c). Interestingly, part of the cells comprising a single cluster of Apr or Aal spermatogonia expressed KIT, whereas the remainder did not (Fig. 4, e and f). Figure 4e shows that two of four CDH1-positive cells in a cluster were stained with the anti-KIT antibody, and Figure 4f shows that one of two CDH1-positive cells was stained with the antibody. A similar pattern was observed when the expression of CDH1 and TACSTD1 was examined at these stages (Fig. 4g). Double staining with CDH1 and TACSTD1 showed that the spermatogonia expressing CDH1 weakly or not at all expressed high levels of TACSTD1, whereas those expressing CDH1 strongly expressed TACSTD1 weakly. These two types of cells were observed occasionally in a single cluster, as was the case for KIT and CDH1 (Fig. 4, e–g).

Stem Cell Markers Were Expressed in a Subset of CDH1-Positive Cells

We performed whole-mount immunohistochemistry with stem cell markers to investigate the relationship between CDH1-positive spermatogonia and spermatogenic stem cells. POU5F1 is expressed in undifferentiated spermatogonia [18, 21]. Double staining of POU5F1 and CDH1 showed that these two proteins were co-expressed; however, the expression level of POU5F1 was low in As spermatogonia (Fig. 5a, arrowheads). Double staining of POU5F1 and CDH1 clearly showed that CDH1-positive cells were not peritubular somatic cells but germ cells (Fig. 5a and supplemental Fig. 4 available online at www.biolreprod.org). The glial cell line-derived neutrophic factor (GDNF) is known to be essential for the maintenance of spermatogenic stem cells in mice. Thus, the receptors of GDNF, RET, and GFRA1 are reasonably thought to be stem cell markers [40–42]. Double staining for RET and CDH1 showed that all of the RET-positive cells were CDH1 positive (Fig. 5b). RET was expressed in almost all single and paired CDH1-positive cells; however, its expression was gradually reduced in clusters of more than four cells and rarely observed in CDH1-positive cell clusters comprising more than eight cells. The expression pattern of GFRA1 was similar to that of RET (Fig. 5c).


Figure 76011505
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  		FIG. 5. Stem cell markers were expressed in a subset of CDH1-positive cells. a) Double staining of CDH1 and POU5F1. The nuclei of all CDH1-positive cells were stained with POU5F1, but the expression level of POU5F1 was low in some As spermatogonia (arrowheads). b) Double staining of CDH1 and RET. RET was expressed in almost all As and Apr cells but rarely expressed in Aal cells. c) Double staining of CDH1 and GFRA1. The staining pattern of GFRA1 was similar to that of RET. d) Double staining of CDH1 and ZBTB16. The nuclear staining of ZBTB16 was mosaic in CDH1-positive cell clusters. Nonspecific binding of the secondary antibody stained the peritubular fibroblast cells (asterisks). Bar = 20 µm. e) Schematic view of the expression level of several surface antigens and POU5F1 through the early development of spermatogonia. The expression of CDH1 is reduced during the transition from undifferentiated type A spermatogonia to A1 spermatogonia. By contrast, the expression of KIT and TACSTD1 is upregulated. RET and GFRA1 are expressed mainly in As and Apr cells and expressed rarely in Aal cells.

The expression pattern of ZBTB16, which was found to play an important role in the regulation of the self-renewal of spermatogonial stem cells, was curious. Double staining with ZBTB16 and CDH1 showed that all of the ZBTB16-positive cells were CDH1 positive, but some CDH1-positive cells were ZBTB16 negative. As shown in Figure 5d, a mosaic pattern of staining of ZBTB16 was observed in As, Apr, and Aal cells at all stages. Heterogeneity in ZBTB16 staining was observed in the clusters of CDH1-positive cells, and in total 30% of the cells in these clusters were negative for ZBTB16. The ZBTB16 staining does not seem to correlate with the cell cycle of CDH1-positive cells, because most of the cells comprising a cluster are synchronized in their cell cycles. At the transition stage, triple staining of ZBTB16, KIT, and CDH1 showed that ZBTB16 staining was mosaic even in KIT and CDH1 double-positive cell clusters (supplemental Fig. 5 available online at www.biolreprod.org). Although the reason ZBTB16 staining showed a mosaic pattern in CDH1-positive cells is unknown, it was confirmed that RET, GFRA1, and ZBTB16 were expressed in a subset of CDH1-positive cells.

In summary, a schematic figure of the expression pattern of several cell markers during the early developmental phase of spermatogonia is shown in Figure 5e. The expression level of CDH1 was high in As, Apr, and Aal cells and then declined with development into A1 spermatogonia. The expression pattern of KIT was complementary to that of CDH1, and both markers were expressed simultaneously during the transition from Aal to A1 cells. TACSTD1 was expressed in both undifferentiated and differentiating spermatogonia; however, its expression was upregulated in differentiating spermatogonia. POU5F1 was expressed in all undifferentiated spermatogonia; however, the expression level was low in As cells and some Apr cells. By contrast, the expression levels of RET and GFRA1 were high in As and Apr cells, and gradually declined in Aal cells. The expression of GFRA1 occasionally overlapped with that of KIT (supplemental Fig. 6 available online at www.biolreprod.org).


Figure 76011506
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  		FIG. 6. Continuous BrdU labeling showed that the cell cycle of CDH1-positive spermatogonia was much longer than that of other spermatogonia. a–b) After 1 wk of labeling, a CDH1-positive cell (green) labeled with no BrdU (a), and a CDH1-positive cell labeled with BrdU (red; b). c) After 3 wk of labeling, all the CDH1-positive and CDH1-negative cells were labeled with BrdU. The insets show a higher magnification of the cells indicated by arrows in each panel. Bars = 10 µm. d) Ratios of BrdU-positive cells among CDH1-negative spermatogonia (red line) and CDH1-positive spermatogonia (blue line) are shown after 1, 2, and 3 wk of continuous BrdU labeling.

To exclude the possibility that some population of CDH1-positive cells may be peritubular cells, we performed double staining of seminiferous tubules for CDH1 and the markers of peritubular cells. No CDH1-positive cells expressed ACTA2, DESMIN, or VIMENTIN, which are markers for peritubular myoid cells [34, 43–46] (supplemental Fig. 7 available online at www.biolreprod.org). The myoid cells arranged in a continuous monolayer of polygonal cells and showed abundant filaments crossing the cytoplasm containing ACTA2, DESMIN, and VIMENTIN. Besides myoid cells, anchor-shaped fibroblastlike cells located between myoid cells were stained with anti-VIMENTIN antibody (supplemental Fig. 7k available online at www.biolreprod.org). These cells also were stained with anti-mouse IgG antibodies (Fig. 5d and supplemental Fig. 7, b and e available online at www.biolreprod.org). They are probably the peritubular interstitial fibroblast cells, judging from their location and the expression of VIMENTIN [34]. These results demonstrated that neither peritubular myoid cells nor peritubular fibroblast cells express CDH1.


Figure 76011507
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  		FIG. 7. Kinetics of CDH1-positive cells in seminiferous tubules after busulfan treatment. a) Two weeks postinjection, most spermatogonia had disappeared, but CDH1-positive cells were not so affected. b) Three weeks postinjection, most spermatocytes had disappeared, but Sertoli cells, spermatids, and CDH1-positive cells survived. c) Some CDH1-positive cells started to proliferate after 4 wk. d) Six weeks postinjection, many spermatocytes appeared in some seminiferous tubules, and many spermatogonia were regenerated, but most of them lost CDH1 expression. Left panels show CDH1 staining, and right panels show DAPI staining. Arrowheads indicate CDH1-positive cells. Bar = 10 µm.

The Cell Cycle of CDH1-Positive Spermatogonia Was Longer Than That of the Other Spermatogonia

Undifferentiated type A spermatogonia are known to divide randomly, and their cell cycle is much longer than that of other spermatogonia [5, 47]. To compare the cell cycle lengths of CDH1-positive and CDH1-negative spermatogonia, we performed a DNA labeling experiment by administering BrdU to mice. After 1-, 2-, or 3-wk administration, a labeling index of BrdU in CDH1-positive and CDH1-negative spermatogonia was determined. Within 1 wk, almost all CDH1-negative spermatogonia had taken up BrdU, but only 36% of CDH1-positive spermatogonia were labeled with BrdU. After 3 wk, the labeling index reached 100% in both CDH1-negative and CDH1-positive spermatogonia (Fig. 6). This result showed that the cell cycle of CDH1-positive spermatogonia was much longer than that of CDH1-negative spermatogonia.

CDH1-Positive Spermatogonia Survived and Proliferated in Busulfan-Treated Testes

To study the behavior and kinetics of CDH1-positive cells during regeneration of the seminiferous epithelium, we administered busulfan to mice by i.p. injection and performed a sequential immunohistochemical analysis of their testes. It is well established that mammalian testes treated with a cytotoxic agent like busulfan or with irradiation lose almost all spermatogenic cells; however, spermatogonial stem cells and Sertoli cells survive. If stem cells survived after these treatments, they could repopulate the seminiferous epithelium and regenerate the entire spermatogenic cell population [48, 49]. We examined the expression pattern of CDH1 in the regenerating seminiferous epithelium for up to 6 wk following the injection of busulfan. Two weeks after injection, the number of spermatogonia was reduced in the experimental testes compared with the normal testes, but spermatids were not affected as much (Fig. 7a). After 3 wk, most spermatocytes were lost from the treated testes, but Sertoli cells, spermatids, and a few spermatogonia were alive. Almost all surviving spermatogonia showed CDH1 expression; however, the number of CDH1-positive cells was much lower in the treated testes than normal testes (Fig. 7b). After 4 wk, most spermatids were lost from the treated testes, whereas the number of CDH1-positive cells gradually increased (Fig. 7c). Six weeks later, many spermatogonia were regenerated, but most of them had lost CDH1 expression (Fig. 7d). These results showed that some CDH1-positive spermatogonia were resistant and survived transient treatment with busulfan and were then able to proliferate and regenerate the seminiferous epithelia.

Spermatogonial Stem Cells Were Included in the CDH1-Positive Cell Population

To investigate their stem cell activity, CDH1-positive cells were transplanted into the seminiferous tubules of an infertile mouse (KitW/KitW-v). We isolated CDH1-positive cells from green fluorescent protein (GFP)-transgenic mice as a donor by using immunomagnetic beads, and we injected 0.9 x 104 to 4.1 x 104 cells into a recipient testis through the efferent duct. For the control experiments, an equivalent number of CDH1-negative cells, which were remaining after depletion of CDH1-positive cells, were injected into the other testis of the same recipient mouse. Eight weeks after the transplantation, recipient testes were analyzed for donor cell-derived colonies. The number of GFP-positive colonies was counted in each experimental and control recipient testis. The average colony number in the experimental recipient was 3.47 per 104 injected cells. By contrast, no GFP-positive colony was found in control recipients (Table 1). This result indicates that the population of CDH1-positive cells includes the spermatogenic stem cells.


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  		TABLE 1. Spermatogenic stem cell activity of CDH1-positive and CDH1-negative cells.

DISCUSSION

CDH1 Is a Specific Marker for Undifferentiated Type A Spermatogonia

In this study we demonstrated that CDH1 was expressed only in undifferentiated type A spermatogonia in mouse testes based on the following properties of the CDH1-positive cells. First, CDH1-positive cells were small in number and showed a characteristic flattened cell shape and heterochromatin-deficient nuclei. Second, whole-mount immunohistochemistry clearly showed that the CDH1-positive cells made small clusters comprising one, two, four, or eight cells, which is a characteristic feature of undifferentiated type A spermatogonia. Third, CDH1-positive cells also expressed several cell surface proteins known to be markers of spermatogonia, such as ITGA6, ITGB1, and TACSTD1. Fourth, all germ cells in KitlSl/KitlSl-d and KitW/KitW-v mice were stained with CDH1. The germ cells of these mutant mice were arrested as undifferentiated type A spermatogonia because of a deficiency in KITL signaling. Finally, the cell cycle of CDH1-positive cells was much longer than that of other spermatogonia. The length of the cell cycle of type A2 through type B spermatogonia has been estimated to be 28.5 h in mice and 42 h in rats [47, 50], and that of As, Apr, and Aal spermatogonia has been estimated to be 56 h in rats [5]. Huckins suggested that there is a long-cycling subpopulation in As spermatogonia that has a cell cycle length longer than 13 days [51]. Our experiment in mouse testes showed that 3 wk was necessary to label all of the CDH1-positive cells with BrdU, which is consistent with the result observed by Huckins.

By using a new cell surface marker, CDH1, we were able to identify the topographical arrangement of undifferentiated type A spermatogonia easily. For example, we could determine the paired cells connected with long, fine cellular processes as Apr cells, such as the case in Figure 3d. Thus, this method allowed us to identify clonal cells easier than the conventional method based on hematoxylin staining [37]. Furthermore, we were able to observe the complicated cell shape of undifferentiated type A spermatogonia. Most CDH1-positive cells extend several fine processes looking like filopodia or leading processes. We were able to observe occasionally very fine cell processes longer than 20 µm. These cell processes seemed not to be intercellular bridges, because they were not always connecting two cells (Fig. 3, c and d, and supplemental Fig. 4 available online at www.biolreprod.org). It is unlikely that these cellular processes are the results of a fixation artifact, because we were able to find them not only in whole-mount samples but also in cryosectioned samples when seminiferous tubules were sliced tangentially. It also is unlikely that these CDH1-positive cells were somatic cells, because all of these cells expressed POU5F1, a germ cell marker (supplemental Fig. 4 available online at www.biolreprod.org), and did not express any peritubular cell markers, such as alkaline phosphatase, ACTA2, DESMIN, and VIMENTIN (supplemental Figs. 1 and 7 available online at www.biolreprod.org).

There has been much inconsistency regarding the expression of CDH1 and the localization of CDH1-positive cells in rodent testes. Some investigators reported that CDH1 was not found in the testis [52, 53], whereas several recent papers reported otherwise [54–58]. In this report, we clearly showed that CDH1 was expressed in a subset of germ cells in the mouse testes throughout life. The number of CDH1-positive cells was very small in the adult mouse testis, which might be why CDH1 staining was missed in some previous papers. It is reasonable that Wu et al. were able to show the localization of CDH1 in the P8 mouse testis by immunohistochemistry [58], and Munro and Blaschuk could detect CDH1 mRNA until P8 but not in the P21 mouse testis [57], because the population of CDH1-positive cells reaches a peak at around 1 wk of age, as shown in Figure 2. The expression of CDH1 both in Sertoli and germ cells has been reported in rat testes [54, 55], but we have not detected CDH1 staining of Sertoli cells in mouse testes. The possibility cannot be ruled out that the CDH1 expression in Sertoli cells was too weak to identify using our method.

The CDH1-Positive Cell Population Contains the Spermatogenic Stem Cells

The regeneration of spermatogenic cells or the formation of colonies from the transplanted testicular cells in the busulfan-treated testes is believed to start from the division and differentiation of spermatogenic stem cells. We were able to find only Sertoli cells and CDH1-positive spermatogonia in the seminiferous tubules of the busulfan-treated testes 3 wk after injection. CDH1-positive cells proliferated after 4 wk, and then differentiating spermatogonia and spermatocytes were regenerated 6 wk after injection. This result suggests that spermatogenic stem cells express CDH1. van Keulen and de Rooij reported the proliferation of repopulating stem cells occurred from 10 to 15 days after the injection of busulfan [48]. In our experiment, the repopulation of CDH1-positive cells started slightly later and was similar to the result of rat testes [49]. This may be due to the difference between mouse strains in susceptibility to busulfan treatment.

The transplantation experiment clearly demonstrated that the CDH1-positive cell population contains the spermatogenic stem cells, because we found colonies in the testes transplanted with only CDH1-positive cells. As far as we know, CDH1-positive cells are the smallest population having stem cell activity that can be isolated easily from normal mouse testes and transplanted to recipient testes. CDH1-mediated isolation of spermatogenic stem cells would improve the recently described culture of these cells [59]. The receptors of GDNF, such as RET and GFRA1, are other candidates for sorting the smallest population containing the stem cells, because they were expressed mainly in As and Apr spermatogonia. However, transplantation analyses showed that the cells sorted by antibodies against RET or GFRA1 had low stem cell activity [60, 61]. These results may be due to low antibody-epitope recognition or low-level expression of the epitopes in stem cells. Another possibility is the functional inhibition of GDNF signaling by antibodies against GDNF receptors, which suppress the stem cell activity of the sorted cells.

Regulation of the Differentiation and Self-Renewal of Undifferentiated Type A Spermatogonia

The most widely accepted model concerning the spermatogenic stem cell is the As model, which was proposed originally by Huckins [36] and Oakberg [4]. According to this model, As is the only cell type that can act as a stem cell, whereas Apr and Aal are irreversibly committed to differentiation [1–3, 62]. However, several results from our experiments are inconsistent with the As model. First, we observed that both RET and GFRA1, which are thought to be stem cell markers, were not only expressed in As cells but were substantially expressed in a subset of Apr and Aal cells. Second, we observed heterogeneity in KIT or TACSTD1 expression within a single cluster of Apr or Aal cells when undifferentiated type A spermatogonia transformed into A1 spermatogonia. Not all CDH1-positive cells in a cluster expressed KIT or showed strong TACSTD1 expression. Based on our observations, we propose another hypothesis, that along with As cells, some Apr or Aal cells maintain stem cell activity. The CDH1-positive and KIT-negative cells in Aal clusters at the transition stage would not transform into A1, but they might restart proliferating as As cells. Indeed, we found a considerable number of CDH1-positive clones comprising a moderate odd number of cells, such as three or seven (Fig. 3e). The clones consisting of odd numbers of cells might result from Aal clusters from which single cells were pinched off, and these cells might start clonal cell division elsewhere at a later stage. The possibility of single cells pinching off from Apr or Aal spermatogonia or the fragmentation of an Aal clone was discussed in a previous study as a way to increase the number of stem cells when irradiation occurred or in some mutant mice [2, 63]. However, since we observed this phenomenon under standard conditions, we believe it might not only be for emergencies.

Another theory about the origin of spermatogenic stem cells was proposed by Clermont et al. [64, 65], in which they consider the As and the Apr cells to be resting reserve cells and the A1 cells to be derived from the A4 cells as renewing stem cells. Their idea is that a small number of A4 cells divide to form A1 cells, whereas most of the A4 cells go forward to form IN cells. However, much of the data from our experiments are not consistent with their model. Our transplantation experiment showed that only CDH1-positive cells have stem cell activity. This means that the stem cell activity resides in As, Apr, or Aal cells and not in A4 cells that are CDH1 negative. We cannot rule out the possibility that CDH1 and KIT double-positive cells have stem cell activity, because whole-mount double staining showed that the CDH1 and KIT double-positive cells were differentiating spermatogonia at stages VII and VIII. It is difficult to classify these cells as Aal cells or A1 cells, but we did not find double-positive cells at stages I to II when the transition from A4 cells to A1 cells should take place, according to the Clermont et al. model. Moreover, it is proposed that As and Apr cells are normally quiescent cells in their model, but BrdU labeled all CDH1-positive cells within 3 wk in our experiment.

What, then, is the function of CDH1 in undifferentiated type A spermatogonia? Both the number of stem cells and colony size of Aal clusters in the mouse testis are important factors regulating the germ cell density in seminiferous tubules. CDH1 might control them through CDH1-mediated cell adhesion. Our observations suggest that it is likely that the cells detached from their sibling cells of the clusters have stem cell activity, whereas the cells staying in the cluster are destined to differentiate into A1 cells. When As cells perform self-renewal cell division, the daughter cells should separate from each other and still express CDH1. On the other hand, the daughter cells of As cells do not separate when they differentiate into Apr cells. Thus, the signals that maintain the stem cell activity might also regulate the adherens junctions through CDH1 and determine the colony size of undifferentiated type A spermatogonia. Wang et al. reported recently an interesting mechanism for the maintenance of stem cells in the Drosophila testis: spermatogenic stem cells remain anchored to their niche to maintain their stem cell activity by regulating DCDH1-mediated cell adhesion [66]. A similar regulation through CDH1 might control the maintenance of stem cells in the mouse testis. Disturbing the function of CDH1 using an anti-CDH1 antibody in vivo or in vitro would help us to understand the role of CDH1 expression.

Furthermore, the advantages of the CDH1-mediated isolation of spermatogenic stem cells also would apply to the therapeutic treatment of male infertility, especially the cryopreservation of spermatogenic stem cells from young cancer patients undergoing intensive chemotherapy.

ACKNOWLEDGMENTS

The authors thank Dr. M. Okabe (Osaka University, Genome Information Research Center) for generously providing the GFP transgenic mice. We also thank Dr. T. Kameyama (Fujita Health University, ICMS) for critical reading of the manuscript and constant encouragement.

FOOTNOTES

1Supported in part by a grant-in-aid for the 21st Century Center of Excellence Program of Fujita Health University, the Collaboration with Local Communities Project, and a grant-in-aid for Scientific Research on Priority Areas (18051015) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

Correspondence: 2 Yuzo Kadokawa, Division of Cell Biology, Institute for Comprehensive Medical Science, Fujita Health University, 1–98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan. FAX: 81 562 93 8834; e-mail: ykado{at}fujita-hu.ac.jp

Received: 13 April 2006.

First decision: 4 May 2006.

Accepted: 5 October 2006.

REFERENCES

  1. de Rooij DG. Stem cells in the testis. Int J Exp Pathol 1998; 79:67–80[CrossRef][Medline]
  2. de Rooij DG. Proliferation and differentiation of spermatogonial stem cells. Reproduction 2001; 121:347–354[Abstract]
  3. de Rooij DG and Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Androl 2000; 21:776–798[Medline]
  4. Oakberg EF. A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am J Anat 1956; 99:391–413[CrossRef][Medline]
  5. Huckins C. The spermatogonial stem cell population in adult rats. II. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet 1971; 4:313–334[Medline]
  6. Dym M and Clermont Y. Role of spermatogonia in the repair of the seminiferous epithelium following x-irradiation of the rat testis. Am J Anat 1970; 128:265–282[CrossRef][Medline]
  7. van Beek ME, Meistrich ML, de Rooij DG. Probability of self-renewing divisions of spermatogonial stem cells in colonies, formed after fission neutron irradiation. Cell Tissue Kinet 1990; 23:1–16[Medline]
  8. van den Aardweg GJ, de Ruiter-Bootsma AL, Kramer MF, Davids JA. Growth of spermatogenetic colonies in the mouse testis after irradiation with fission neutrons. Radiat Res 1982; 89:150–165[CrossRef][Medline]
  9. van der Meer Y, Huiskamp R, Davids JA, van der Tweel I, de Rooij DG. The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation. Radiat Res 1992; 130:289–295[CrossRef][Medline]
  10. van der Meer Y, Huiskamp R, Davids JA, van der Tweel I, de Rooij DG. The sensitivity to X rays of mouse spermatogonia that are committed to differentiate and of differentiating spermatogonia. Radiat Res 1992; 130:296–302[CrossRef][Medline]
  11. Shinohara T, Avarbock MR, Brinster RL. Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models. Dev Biol 2000; 220:401–411[CrossRef][Medline]
  12. de Rooij DG, Okabe M, Nishimune Y. Arrest of spermatogonial differentiation in jsd/jsd, Sl17H/Sl17H, and cryptorchid mice. Biol Reprod 1999; 61:842–847[Abstract/Free Full Text]
  13. Ohta H, Yomogida K, Dohmae K, Nishimune Y. Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF. Development 2000; 127:2125–2131[Abstract]
  14. Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S, Kunisada T, Fujimoto T. Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 1991; 113:689–699[Abstract]
  15. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat Med 2000; 6:29–34[CrossRef][Medline]
  16. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998; 95:379–391[CrossRef][Medline]
  17. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24:372–376[CrossRef][Medline]
  18. Pesce M, Wang X, Wolgemuth DJ, Scholer H. Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 1998; 71:89–98[CrossRef][Medline]
  19. Yoshida S, Takakura A, Ohbo K, Abe K, Wakabayashi J, Yamamoto M, Suda T, Nabeshima Y. Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev Biol 2004; 269:447–458[CrossRef][Medline]
  20. Raverot G, Weiss J, Park SY, Hurley L, Jameson JL. Sox3 expression in undifferentiated spermatogonia is required for the progression of spermatogenesis. Dev Biol 2005; 283:215–225[CrossRef][Medline]
  21. Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 2004; 36:647–652[CrossRef][Medline]
  22. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig KE, Wolgemuth DJ, Pandolfi PP. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004; 36:653–659[CrossRef][Medline]
  23. Shinohara T, Avarbock MR, Brinster RL. ß1- and {alpha}6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999; 96:5504–5509[Abstract/Free Full Text]
  24. Kanatsu-Shinohara M, Toyokuni S, Shinohara T. CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 2004; 70:70–75[Abstract/Free Full Text]
  25. Ryu BY, Orwig KE, Kubota H, Avarbock MR, Brinster RL. Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev Biol 2004; 274:158–170[CrossRef][Medline]
  26. Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, Tsuchiya H, Kuwana T, Kehler J, Abe K, Scholer HR, Suda T. Identification and characterization of stem cells in prepubertal spermatogenesis in mice small star, filled. Dev Biol 2003; 258:209–225[CrossRef][Medline]
  27. Mori S, Kadokawa Y, Hoshinaga K, Marunouchi T. Sequential activation of Notch family receptors during mouse spermatogenesis. Dev Growth Differ 2003; 45:7–13[CrossRef][Medline]
  28. Suemori H, Kadodawa Y, Goto K, Araki I, Kondoh H, Nakatsuji N. A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous ß-galactosidase expression. Cell Differ Dev 1990; 29:181–186[CrossRef][Medline]
  29. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Honjo T, Shinohara T. Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis. Biol Reprod 2003; 68:167–173[Abstract/Free Full Text]
  30. McCoard SA, Lunstra DD, Wise TH, Ford JJ. Specific staining of Sertoli cell nuclei and evaluation of Sertoli cell number and proliferative activity in Meishan and White Composite boars during the neonatal period. Biol Reprod 2001; 64:689–695[Abstract/Free Full Text]
  31. Tadokoro Y, Yomogida K, Ohta H, Tohda A, Nishimune Y. Homeostatic regulation of germinal stem cell proliferation by the GDNF/FSH pathway. Mech Dev 2002; 113:29–39[CrossRef][Medline]
  32. Anderson R, Schaible K, Heasman J, Wylie C. Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line. J Reprod Fertil 1999; 116:379–384[Abstract]
  33. Wang Y, Newton DC, Miller TL, Teichert AM, Phillips MJ, Davidoff MS, Marsden PA. An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells. Am J Pathol 2002; 160:369–380[Abstract/Free Full Text]
  34. Kierszenbaum AL, Crowell JA, Shabanowitz RB, Smith EP, Spruill WA, Tres LL. A monoclonal antibody recognizes a form of intermediate filament protein in rat Sertoli cells that is not present in seminiferous peritubular cells. Biol Reprod 1986; 35:227–238[Abstract]
  35. Palombi F and Di Carlo C. Alkaline phosphatase is a marker for myoid cells in cultures of rat peritubular and tubular tissue. Biol Reprod 1988; 39:1101–1109[Abstract]
  36. Huckins C. The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat Rec 1971; 169:533–557[CrossRef][Medline]
  37. Lok D, Weenk D, De Rooij DG. Morphology, proliferation, and differentiation of undifferentiated spermatogonia in the Chinese hamster and the ram. Anat Rec 1982; 203:83–99[CrossRef][Medline]
  38. Tegelenbosch RA and de Rooij DG. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res 1993; 290:193–200[CrossRef][Medline]
  39. Schrans-Stassen BH, van de Kant HJ, de Rooij DG, van Pelt AM. Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology 1999; 140:5894–5900[Abstract/Free Full Text]
  40. Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287:1489–1493[Abstract/Free Full Text]
  41. Naughton CK, Jain S, Strickland AM, Gupta A, Milbrandt J. Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol Reprod 2006; 74:314–321[Abstract/Free Full Text]
  42. Yomogida K, Yagura Y, Tadokoro Y, Nishimune Y. Dramatic expansion of germinal stem cells by ectopically expressed human glial cell line-derived neurotrophic factor in mouse Sertoli cells. Biol Reprod 2003; 69:1303–1307[Abstract/Free Full Text]
  43. Virtanen I, Kallajoki M, Narvanen O, Paranko J, Thornell LE, Miettinen M, Lehto VP. Peritubular myoid cells of human and rat testis are smooth muscle cells that contain desmin-type intermediate filaments. Anat Rec 1986; 215:10–20[CrossRef][Medline]
  44. Tung PS and Fritz IB. Characterization of rat testicular peritubular myoid cells in culture: {alpha}-smooth muscle isoactin is a specific differentiation marker. Biol Reprod 1990; 42:351–365[Abstract]
  45. Tripiciano A, Filippini A, Giustiniani Q, Palombi F. Direct visualization of rat peritubular myoid cell contraction in response to endothelin. Biol Reprod 1996; 55:25–31[Abstract]
  46. Maekawa M, Kamimura K, Nagano T. Peritubular myoid cells in the testis: their structure and function. Arch Histol Cytol 1996; 59:1–13[Medline]
  47. Monesi V. Autoradiographic study of DNA synthesis and the cell cycle in spermatogonia and spermatocytes of mouse testis using tritiated thymidine. J Cell Biol 1962; 14:1–18[Medline]
  48. van Keulen CJ and de Rooij DG. Spermatogenetic clones developing from repopulating stem cells surviving a high dose of an alkylating agent. Cell Tissue Kinet 1975; 8:543–551[Medline]
  49. Jiang FX. Behaviour of spermatogonia following recovery from busulfan treatment in the rat. Anat Embryol (Berl) 1998; 198:53–61[CrossRef][Medline]
  50. Hilscher B, Hilscher W, Maurer W. Autoradiographic studies on the modus of proliferation and regeneration of the seminiferous epithelium of Wistar rats. Z Zellforsch Mikrosk Anat 1969; 94:593–604[CrossRef][Medline]
  51. Huckins C. The spermatogonial stem cell population in adult rats. 3. Evidence for a long-cycling population. Cell Tissue Kinet 1971; 4:335–349[Medline]
  52. Cyr DG, Blaschuk OW, Robaire B. Identification and developmental regulation of cadherin messenger ribonucleic acids in the rat testis. Endocrinology 1992; 131:139–145[Abstract]
  53. Byers SW, Sujarit S, Jegou B, Butz S, Hoschutzky H, Herrenknecht K, MacCalman C, Blaschuk OW. Cadherins and cadherin-associated molecules in the developing and maturing rat testis. Endocrinology 1994; 134:630–639[Abstract]
  54. Johnson KJ, Patel SR, Boekelheide K. Multiple cadherin superfamily members with unique expression profiles are produced in rat testis. Endocrinology 2000; 141:675–683[Abstract/Free Full Text]
  55. Lee NP, Mruk D, Lee WM, Cheng CY. Is the cadherin/catenin complex a functional unit of cell-cell actin-based adherens junctions in the rat testis? Biol Reprod 2003; 68:489–508[Abstract/Free Full Text]
  56. Mackay S, Nicholson CL, Lewis SP, Brittan M. E-cadherin in the developing mouse gonad. Anat Embryol (Berl) 1999; 200:91–102[CrossRef][Medline]
  57. Munro SB and Blaschuk OW. A comprehensive survey of the cadherins expressed in the testes of fetal, immature, and adult mice utilizing the polymerase chain reaction. Biol Reprod 1996; 55:822–827[Abstract]
  58. Wu JC, Gregory CW, DePhilip RM. Expression of E-cadherin in immature rat and mouse testis and in rat Sertoli cell cultures. Biol Reprod 1993; 49:1353–1361[Abstract]