Three-week-old male ICR mice were purchased from DBL (Eumseong, Korea) and used as SSC donors. All of the animal housing, handling, and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval No. KW-130307-1) and conducted in accordance with the Animal Care and Use Guidelines of Kangwon National University.

The tunica albuginea and epididymis were removed from the mouse testes, and the seminiferous tubules were digested by incubation for 20 min in Dulbecco’s Modified Eagle’s Medium (DMEM; Welgene Inc., Daegu, Korea) supplemented with 0.5 mg/ml type IV collagenase (Worthington Biochemical, Lakewood, CA) at 37℃. Subsequently, the fragmented seminiferous tubules were washed with DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Welgene), and dissociated with 0.25% trypsin-EDTA (Welgene) for 5 min at 37℃. The dispersed testicular cells were filtered through a 70 μm nylon strainer (SPL, Pocheon, Korea) to eliminate peritubular myoid, sertoli, and leydig cells, and the filtered testicular cells were purified by magnetic-activated cell sorting (MACS; Invitrogen, Carlsbad, CA) using anti-Thy1 antibody (Novus Biologicals, Littleton, CO) according to the manufacturer’s instructions. Subse-quently, the finally sorted cells (herein referred to as SSC population) were assigned to the following experiments.

Total mRNA was extracted from the cells using a Dynabeads mRNA Direct^TM Kit (Ambion, Austin, TX), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA remover kit (Toyobo, Osaka, Japan) in accordance with the respective manufacturer’s instructions. Subsequently, specific gene expression was quantified with THUNDERBIRD^TM SYBR^Ⓡ qPCR Mix (Toyobo) using a 7500 Real-time PCR system (Applied Biosystems, Foster City, CA). PCR specificity was determined by analyzing the melting curve data. The mRNA levels are presented as 2^−ΔCt where Ct=threshold cycle for target amplification and ΔCt=Ct_{target gene} (specific genes for each sample) – Ct_{internal reference} (β-actin for each sample). Primer sequences were designed with Primer3 software (Whitehead Institute/MIT Center for Genome Re-search) with mouse cDNA sequence data obtained from GenBank. General information and sequences of primers are described in Supplementary Table S1.

Cells fixed with 4% (v/v) paraformaldehyde (Junsei, Tokyo, Japan) for 10 min were washed twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene). The fixed cells were stained with fluorescence-conjugated integrin α₁, α₅, α₆, α₉, α_V, α_E, β₁, and β₅ primary antibodies diluted in DPBS for 16 h at 4℃. After rinsing with DPBS, the stained cells were double stained by fluorescence-unconjugated glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 1 (GFRα1; a SSC-specific marker) primary antibody diluted in DPBS, or florescence-unconjugated alpha smooth muscle actin (αSMA; a peritubular myoid cell-specific marker), GATA binding protein 4 (GATA4; a sertoli cell-specific marker), or luteinizing hormone receptor (LHR; a leydig cell-specific marker) primary antibody diluted in Hank’s balanced salt solution (HBSS, Invitrogen) containing 0.1% (w/v) saponin (Sigma-Aldrich, St. Louis, MO) for 16 h at 4℃. Subsequently, the detection of GFRα1 primary antibody was conducted by incubating Alexa Fluor 488- or 546-conjugated secondary antibody diluted in DPBS for 2 h at 4℃, and the detection of αSMA, GATA4, and LHR primary antibodies was conducted by incubating Alexa Fluor 488-, 546- or 568-conjugated secondary antibody diluted in HBSS containing 0.1% (w/v) saponin for 1 h at 4℃. Supplementary Table S2 describes the detailed information and dilution rate of the used antibodies. Then, rinsing twice with DPBS was performed and the double stained cells were counterstained with mounting medium for fluorescence with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, CA). Moreover, the triple-stained cells were monitored under a confocal laser scanning microscope (LSM880; ZEISS, Jena, Germany).

Three thousand three hundred of cells fixed in 4% (v/v) paraformaldehyde (Junsei) for 10 min were rinsed twice with DPBS. Subsequently, the fixed cells were stained for 2 h at room temperature with FITC-conjugated anti-mouse integrin α₅, α₉, α_V, α_E, and β₁ and Alexa Fluor 488-conjugated anti-mouse integrin α₆ antibodies diluted in DPBS. The detailed information and antibody dilutions used are listed in Supplementary Table S2. The stained cells were washed with DPBS, and fluorescence intensity was measured using SoftMax^Ⓡ Pro 6.2.2. (Molecular Devices Corp., Sunnyvale, CA) after adding 100 μl DPBS to the stained cells.

First, 96-well tissue culture plates were coated with the following concentrations of purified ECM proteins overnight at 4℃: 0, 40, and 80 μg/ml fibronectin (Millipore, Burlington, MA); 0, 200, and 400 μg/ml laminin (Sigma-Aldrich); 0, 20, and 40 μg/ml tenascin C (RayBiotech, Peachtree Corners, GA) and 0, 5, and 10 μg/ml vitronectin (R&D Systems, Minneapolis, MN). Then, each well blocked with 1% (w/v) bovine serum albumin (BSA; Sigma) at 4℃ for 1 h was washed three times with DPBS. Aliquots of 1×10⁴ cells were resuspended in SSC culture medium consisting of Stempro-34 medium (Invitrogen) supplemented with insulin–transferrin–selenium (ITS; Invitrogen), 60 μM putrescine dihydrochloride (Sigma), 6 mg/ml D-(+)-glucose (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 0.11 mg/ml sodium pyruvate (Sigma), 1 μl/ml DL-lactic acid (Sigma), 5 mg/ml BSA, 2 mM L-glutamine (Invitrogen), 0.05 mM β-mercaptoethanol (Invitrogen), 1% (v/v) FBS, 1% (v/v) NEAA (Invitrogen), MEM vitamin solution (Sigma), 1% (v/v) antibiotic–antimycotic solution (Welgene), 0.1 mM ascorbic acid (Sigma), 10³ units/ml mouse LIF (Chemicon International Inc., Temecula, CA), 10 ng/ml GDNF (R&D systems), 30 ng/ml β-estradiol (Sigma), 60 ng/ml progesterone (Sigma), 20 ng/ml human epidermal growth factor (EGF; Peprotech, Inc., Rocky Hill, NJ), and 10 ng/ml human basic fibroblast growth factor (bFGF; Peprotech, Inc.), and seeded in each well. After incubation at 37℃ for 2 h, non-adherent cells were removed by washing each well sufficiently, and adherent cells were fixed in 4% (v/v) paraformaldehyde at room temperature for 10 min. Subsequently, the fixed adherent cells were stained with 0.1% (w/v) crystal violet (Sigma) in 20% (v/v) methanol (Sigma-Aldrich) for 5 min and washed twice with distilled water. Levels of adherent cells were quantified at 570 nm using a microplate reader (Epoch Microplate Spectrophotometer; BioTek Instruments Inc., Winooski, VT) after adding 50 μl of 0.2% (v/v) Triton X-100 (Biopure, Cambridge, MA) diluted with distilled water.

The wells of 96-well tissue culture plates were coated with 40 μg/ml fibronectin, 200 μg/ml laminin, 20 μg/ml tenascin C or 5 μg/ml vitronectin overnight at 4℃, and the coated wells were blocked with 1% (w/v) BSA for 1 h at 4℃. Subsequently, to inhibit integrin function, 1×10⁴ cells were resuspended in SSC culture medium containing anti-integrin α₅ (5H10–27 [MFR5]), anti-integrin α₆ (NKI-GoH3), anti-integrin α₉ (Y9A2) or anti-integrin α_V (RMV-7) blocking antibody, and incubated at 37℃ for 2 h. The detailed information regarding the antibodies used is presented in Supplementary Table S2. Then, the functionally blocked cells were plated in each well and incubated at 37℃ for 8 h. To remove non-adherent cells, the wells were washed extensively with DPBS. The adherent cells were fixed in 4% (v/v) paraformaldehyde for 10 min at room temperature, and staining of adherent cells was performed with 0.1% (w/v) crystal violet in 20% (v/v) methanol for 5 min. Finally, the wells were washed twice with distilled water and supplemented with 50 μl of 0.2% (v/v) Triton X-100 diluted with distilled water. The amount of dye was measured at 570 nm using a microplate reader.

The SAS program was used for statistical analysis of the numerical data shown in each experiment. Comparisons among treatment groups were performed by the least-squares or DUNCAN method, and the significance of the main effects was determined by analysis of variance (ANOVA) in the SAS package. In all of the analyses, p< 0.05 was taken to indicate statistical significance.

A MACS technique based on antibody detecting Thy1 expressed on the surface of undifferentiated mouse SSCs did not show high yield in the isolation of undifferen-tiated SSCs from outbred ICR mouse testes. As shown in Supplementary Fig. S1, SSCs stained positively with GFRα1, peritubular myoid cells stained positively with αSMA, sertoli cells stained positively with GATA4, and leydig cells stained positively with LHR were detected in the retrieved SSC population post-MACS. Moreover, among four cell types included in the SSC population, SSCs (24.57±3.12%) and peritubular myoid cells (28.68±3.20%) showed significantly higher rate of abundance than sertoli (16.39±1.06%) and leydig (11.32±4.80%) cells. Therefore, in order to gain insight into the types of integrin subunit expressed on the membrane of outbred ICR mouse SSCs in the undifferentiated state, we investigated the transcriptional levels of integrin subunit genes in the SSC population and the translation of integrin subunit genes showing significantly strong transcription were identified per each type of cells through immunocytochemistry.

In transcriptional analyses of genes encoding a total of 25 integrin subunits, significantly higher levels of expression were observed for integrin α₁, α₅, α₆, α₉, α_V, and α_E (Fig. 1A), and integrin β₁ and β₅ (Fig. 1B) subunit genes. The minimum levels of expression were detected for integrin α₂, α₃, α₄, α₇, α₈, α₁₀, α_L, α_M, α_D, and α_X (Fig. 1A), and integrin β₂, β₃, β₄, and β₇ (Fig. 1B) subunit genes. No expression of integrin α₁₁ (Fig. 1A) or integrin β₆ and β₈ (Fig. 1B) subunit genes was detected in these cells at the transcriptional level.

Translational regulation of integrin α₁, α₅, α₆, α₉, α_V, and α_E, and integrin β₁ and β₅ subunit genes showing increased transcription was examined in each type of cells included in SSC population. As shown in Fig. 2, SSCs stained positively with GFRα1 showed co-localization of integrin α₅, α₆, α₉, α_V, α_E and β₁ subunit proteins on the cell surface, whereas co-localization of integrin α₁ and β₅ subunit proteins were not detected in GFRα1-positive SSCs. Furthermore, the surface of peritubular myoid (Supplementary Fig. S2), sertoli (Supplementary Fig. S3), or leydig (Supplementary Fig. S4) cells stained positively with αSMA, GATA4, or LHR did not show any co-localization of integrin α₁, α₅, α₆, α₉, α_V, α_E, β₁, and β₅ subunit proteins. Justly, co-localization of integrin α₁ subunit protein was detected on the surface of LHR-positive leydig cells. These results demonstrate that dimerization of integrin α and β subunits as active forms of integrins can’t be absolutely formed on the surface of peritubular myoid, sertoli, and leydig cells, indicating the absence of their influence on the following functional assay using the retrieved SSC population post-MACS.

Subsequently, levels of integrin α₅, α₆, α₉, α_V, α_E, and β₁ subunit genes expressed translationally on the surface of undifferentiated SSCs s were quantified (Fig. 3). Among the six integrin subunit genes, integrin α_V subunit gene had significantly the strongest translation expression, and integrin α₅, α₆, and β₁ subunit genes had significantly intermediate translational expression, whereas integrin α₉ and α_E subunit genes showed significantly very weak expression at the translational level. These results indicate that integrin α₅, α₆, α₉, α_V, α_E and β₁ subunits are presented on the surface of outbred ICR mouse SSCs in undifferentiated state.

Based on the results regarding integrin α and β subunits expressed on the cell membrane of undifferentiated outbred ICR mouse SSCs, we speculated that these cells may possess integrin α₅β₁, α₆β₁, α₉β₁, and α_Vβ₁ as active forms of integrin heterodimers as previously described (24). The presence of these integrin heterodimers was examined by estimating levels of adherent SSCs cultured on purified ECM proteins that interact specifically with each integrin heterodimer and adherent levels post-culture of SSCs treated with antibodies specifically blocking each integrin function on each purified ECM protein. Fig. 4 shows significantly improved adhesion for SSCs incubated on fibronectin-, laminin-, tenascin C- and vitro-nectin-coated culture plates, compared to those cultured on purified ECM protein-free culture plates. These observations suggested the presence of the fibronectin-interacting integrin α₅β₁, laminin-interacting integrin α₆β₁, tenascin C-interacting integrin α₉β₁, and vitronectin-interacting integrin α_Vβ₁ on the cell membrane of SSCs in the undifferentiated state. These specific integrin function-blocked SSCs were incubated on 40 μg/ml fibronectin, 200 μg/ml laminin, 20 μg/ml tenascin C, or 5 μg/ml vitronectin as the minimum concentration among those seen in the ECM showing significantly improved adhesion of SSCs (Fig. 5). Significantly weakened adhesion was detected in SSCs with blockade of integrin α₅β₁ (Fig. 5A), α₆β₁ (Fig. 5B), α₉β₁ (Fig. 5C), or α_Vβ₁ (Fig. 5D), compared to those without blocking of these integrin heterodimers. These results confirmed that the undifferentiated outbred ICR mouse SSCs simultaneously show functional expression of integrin α₅β₁, α₆β₁, α₉β₁, and α_Vβ₁ on the cell membrane.