Integrin Heterodimers Expressed on the Surface of Porcine Spermatogonial Stem Cells

Abstract

To date, in vitro culture systems able to sufficiently expand the small population of spermatogonial stem cells (SSCs), a tool for the development of sperm-mediated gene transfer techniques in transgenic pigs, in the porcine seminiferous tubule have not been reported. Therefore, as a step toward engineering a noncellular niche to support the in vitro maintenance of porcine SSC self-renewal, we investigated the types of integrin heterodimers that are expressed and functional on their membrane. The α and β integrin subunit protein expressions were analyzed using immunocytochemistry and fluorescence immunoassay, and the function of integrin heterodimers was confirmed by attachment and antibody inhibition assays. The integrin subunits, α₃, α₄, α₅, α₆, α₈, α₉, α_V, and β₁, were identified on the surface of them. Moreover, they showed significantly increased adhesion to fibronectin, laminin, and vitronectin, and functional blocking of integrin α₄β₁, α₆β₁, or α_Vβ₁ significantly inhibited adhesion to these molecules. They showed significantly decreased adhesion to tenascin C and functional blocking of integrin α₅β₁ did not significantly inhibit adhesion to fibronectin. Accordingly, we confirmed that the integrin heterodimers α₄β₁, α₆β₁, and α_Vβ₁ actively function on the surface of undifferentiated porcine SSCs, whereas α₃, α₅, α₈, and α₉ are present in inactive forms.

Introduction

Rodents, such as mice and rats, are easy to handle and use in studies, but are remarkably different from humans in many aspects. Among a variety of domestic animals, the pig has been utilized as a laboratory animal because of a number of advantages, including anatomical, physiological, and genomic similarities to humans (Lunney, 2007; Aigner et al., 2010; Nowak-Imialek et al., 2011; Meurens et al., 2012; Swindle et al., 2012; Prather et al., 2013). Moreover, pigs can produce numerous piglets with a relatively short generation period (Lunney, 2007; Nowak-Imialek et al., 2011; Meurens et al., 2012). For these reasons, pigs have been used for the development of technologies to treat human disease, such as organ donors for transplantation, and as bioreactors for producing protein-based drugs through transgenesis. Hence, the pig has been considered a suitable animal model for transgenesis.

There are several methods for generating transgenic animals: DNA microinjection (Niemann and Kues, 2000; Wheeler and Walters, 2001; Houdebine, 2002, 2009; Wheeler et al., 2003; Niemann et al., 2005), somatic cell nuclear transfer (Niemann and Kues, 2000; Wheeler and Walters, 2001; Houdebine, 2002; Wheeler et al., 2003; Niemann et al., 2005; Robl et al., 2007; Vajta and Callesen, 2012), and retrovirus- (Niemann and Kues, 2000; Houdebine, 2002, 2009; Wheeler et al., 2003; Niemann et al., 2005; Robl et al., 2007), biopolymer- (Wheeler et al., 2003; Nitta and Numata, 2013), and embryonic stem cell-mediated gene transfer (Niemann and Kues, 2000; Wheeler and Walters, 2001; Houdebine, 2002, 2009). However, these methods have shown low efficiency in the generation of transgenic progeny (Niemann and Kues, 2000; Houdebine, 2002, 2009; Niemann et al., 2005; Robl et al., 2007) and in transgene integration into the genome (Houdebine, 2002; Wheeler et al., 2003; Niemann et al., 2005). Therefore, sperm-mediated gene transfer has been considered (Maione et al., 1998; Smith and Spadafora, 2005; Lavitrano et al., 2006, 2013; Kurome et al., 2007; García-Vázquez et al., 2011). However, the extremely condensed chromosomes of the sperm head make it difficult to integrate foreign DNA into the genome (Lavitrano et al., 2006; Kurome et al., 2007; García-Vázquez et al., 2011). As a result, scientific interest in the generation of transgenic sperm using spermatogonial stem cells (SSCs) with easy transgene integration and sperm differentiation has gradually increased.

Despite a number of trials, development of an in vitro system capable of sufficiently expanding the small population of SSCs located in the seminiferous tubule of the porcine testis has not yet been successful. Previously, porcine SSCs have been cultured on a feeder cell-based cellular niche, which is difficult to manipulate, but cellular niche-dependent culture systems have not shown effective supports in maintaining self-renewal of porcine SSCs. Therefore, noncellular niches, which are easier to define and manipulate than cellular niches, have taken the spotlight and the development of noncellular niche-dependent culture systems mimicking the seminiferous tubule basement membrane (STBM) in direct contact with porcine SSCs in vivo has been requested.

The STBM, which plays an important role in maintaining the self-renewal of SSCs (Olie et al., 1995; de Rooij et al., 2008; Campos-Junior et al., 2012; Chen et al., 2013), is composed of fibronectin (Skinner et al., 1985; Oatley and Brinster, 2012), laminin (Siu and Cheng, 2004a, 2008; Mazaud Guittot et al., 2008; Cheng et al., 2010; Kanatsu-Shinohara and Shinohara, 2013), collagen IV (Enders et al., 1995; Siu and Cheng, 2004a, 2008; Mazaud Guittot et al., 2008; Cheng et al., 2010; Kim et al., 2011), entactin (Siu and Cheng, 2004b, 2008; Mazaud Guittot et al., 2008), and perlecan (Siu and Cheng, 2004b, 2008; Mazaud Guittot et al., 2008). Structurally, the presence of laminin in the STBM enhances the strength of the entire network by interacting with nidogen and perlecan (Kim et al., 2011), and collagen IV improves the stability by interacting with laminin (Enders et al., 1995; Siu and Cheng, 2004b). Moreover, laminin induces cellular responses by interacting with specific binding sites on membrane proteins embedded in laminin (Olie et al., 1995; Siu and Cheng, 2004b; Oatley and Brinster, 2012), and collagen IV influences adhesion, migration, and cell differentiation (Olie et al., 1995; Siu and Cheng, 2004b; Oatley and Brinster, 2012). The combined stimulation of signals derived from these extracellular matrix (ECM) proteins can regulate the destiny of SSCs through integrins that directly recognize the ECM proteins (Siu and Cheng, 2004b). Integrins are a group of transmembrane cell adhesion molecules constructed as a heterodimer of α and β subunits (Kim et al., 2011; Brizzi et al., 2012; Chen et al., 2013; Silván et al., 2013). A total of 18 α and 8 β subunits can be assembled together, generating at least 24 different heterodimers (Siu and Cheng, 2008; Kim et al., 2011; Brizzi et al., 2012; Silván et al., 2013). These assemblies maintain tissue integrity through cell-to-cell contact (Virtanen et al., 1997; Xi, 2009; Chen et al., 2013; Silván et al., 2013) and communicate extracellular signals to the cytoplasm (Ellis and Tanentzapf, 2010; Brizzi et al., 2012; Kanatsu-Shinohara and Shinohara, 2013; Silván et al., 2013). These heterodimeric receptors interact directly with binding sites in ECM proteins and induce numerous biological processes, including cell attachment (Virtanen et al., 1997; Siu and Cheng, 2004b; Ellis and Tanentzapf, 2010; Chen et al., 2013), spreading (Kim et al., 2011; Brizzi et al., 2012), proliferation (Olie et al., 1995; Ellis and Tanentzapf, 2010; Kim et al., 2011; Brizzi et al., 2012; Silván et al., 2013), survival (Xi, 2009; Ellis and Tanentzapf, 2010; Brizzi et al., 2012; Silván et al., 2013), morphogenesis (Xi, 2009; Geiger and Yamada, 2011), and differentiation (Virtanen et al., 1997; Xi, 2009; Ellis and Tanentzapf, 2010; Kim et al., 2011; Brizzi et al., 2012; Silván et al., 2013).

Despite knowledge of the ECM proteins that make up the STBM in the testis, the integrin family members expressed on the surface of SSCs have remained unknown in domestic animals, with the exception of rodents, making it difficult to define a noncellular niche for in vitro culture of SSCs derived from domestic animals. In this study, we identified the integrin heterodimers expressed on the plasma membrane of SSCs in pigs. The integrin subunits expressed in porcine SSCs were analyzed at the translational level and the functions of integrin heterodimer candidates based on the expressed subunits were identified by attachment and antibody inhibition assays.

Materials and Methods

Animals

One- to 4-day-old crossbred (Landrace × Yorkshire) or purebred (Yorkshire × Yorkshire) male neonatal piglets were generously supplied from Gumbo, Inc. (Wonju, Korea) and collection of testes from them were conducted through routine castration surgery. The Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval No. KW-131106-1) approved all animal experimental procedures, which were performed according to the Animal Care and Use Guideline of Kangwon National University.

Harvest of SSCs from porcine testes

Male neonatal testes were transported from a local farm (Gumbo, Inc.) to our laboratory in ice-cold Dulbecco's phosphate-buffered saline (DPBS; Welgene, Inc., Daegu, Korea) supplemented with 1% (v/v) antibiotic–antimycotic solution (Welgene) within 1 h. Subsequently, to isolate testicular cells from testes, the tunica albuginea and epididymis were removed from testes and the seminiferous tubules were digested by 0.1% (w/v) type IV collagenase (Worthington Biochemical, Lakewood, NJ) in high glucose Dulbecco's modified Eagle's medium (DMEM; Welgene) at 37°C for 15 min. The fragmented seminiferous tubules were dissociated sequentially and separately by 0.1% (w/v) hyaluronidase (Sigma-Aldrich, St. Louis, MO) in high glucose DMEM and 0.25% trypsin-EDTA (Welgene) at 37°C for 10 min. The dissociated testicular cells were washed twice with DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA) and filtered using a 70-μm nylon strainer (SPL, Pocheon, Korea) for eliminating myoid and Sertoli cells. Moreover, red blood cell lysis buffer (Sigma-Aldrich) was used for removing erythrocytes included in the dispersed cells. Then, SSCs were sorted from the isolated testicular cells by Petri dish plating postdifferential plating method described previously (Park et al., 2014). In brief, 5 × 10⁶ testicular cells were resuspended in high glucose DMEM supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) antibiotic–antimycotic solution and these cells were plated on 100-mm Petri dish (SPL) coated with 0.1% (w/v) gelatin (Sigma-Aldrich). After incubating for 16 h at 37°C, 1 × 10⁶ cells suspended in medium were plated on 35-mm Petri dish in high glucose DMEM supplemented with 15% (v/v) heat-inactivated FBS, 0.1 mM β-mercaptoethanol (Gibco), 1% (v/v) nonessential amino acid (NEAA; Gibco), 2 mM l-glutamine (Invitrogen, Carlsbad, CA), 1% (v/v) antibiotic–antimycotic solution, 10³ U/mL mouse leukemia inhibitory factor (LIF; Chemicon International, Inc., Temecula, CA), and 10 ng/mL glial cell-derived neurotrophic factor (GDNF; R&D Systems, Inc., Minneapolis, MN) and incubated at 37°C overnight. Subsequently, the suspended SSC populations were collected and only SSC populations showing above 95% positivity against SSC-specific proteins (OCT4, NANOG, promyelocytic leukemia zinc finger, and GDNF family receptor alpha-1 [GFRα1]) and below 5% positivity against a Leydig cell-specific protein (luteinizing hormone receptor) and a Sertoli cell-specific protein (GATA4) were assigned to the following experiments.

Immunocytochemistry

The SSCs derived from porcine testes were fixed with 4% (v/v) paraformaldehyde (Junsei Chemincal Co., Ltd., Chuo-ku, Japan) for 10 min and washed twice with DPBS. Subsequently, the fixed cells in DPBS were stained with fluorescent-unconjugated GFRα1 primary antibody at 4°C overnight and the detection of GFRα1 primary antibody was conducted by incubating Alexa Fluor^® 546- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies diluted in DPBS at 4°C for 2 h. After rinsing twice with DPBS, the stained cells were double stained by FITC-conjugated anti-mouse integrin α₄, PE-conjugated anti-mouse integrin α₉, and fluorescent-unconjugated anti-human integrin α₃, α₅, α₆, α₈, α_v, and β₁ primary antibodies diluted in DPBS at 4°C overnight, and fluorescent-unconjugated anti-integrin primary antibodies were detected by incubating the double-stained cells with Alexa Flour^® 488- or 546- or FITC-conjugated secondary antibodies at 4°C for 2 h. Table 1 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 DAPI (Vector Laboratories, Inc., Burlingame, CA). Finally, the triple-stained cells were monitored under a confocal laser scanning microscope (LSM880; ZEISS, Jena, Germany).

Table 1.

Primary and Secondary Antibodies

Antibody name	Catalog no.	Company	Application	Dilution rate
Rabbit anti-human GFRα1 (H-70)	sc-10716	Santa Cruz Biotechnology, Inc.	ICC	1:50
Mouse anti-human GFRα1 (E-11)	sc-271546	Santa Cruz Biotechnology, Inc.	ICC	1:50
Goat anti-human integrin α₃ (C-18)	sc-6587	Santa Cruz Biotechnology, Inc.	ICC, FI	1:50
FITC-conjugated rat anti-mouse integrin α₄	103605	BioLegend^®	ICC, FI	1:50
Mouse anti-human integrin α₅ (C-9)	sc-376199	Santa Cruz Biotechnology, Inc.	ICC	1:50
FITC-conjugated rabbit anti-mouse/rat integrin α₅	orb222105	Biorbyt	FI	1:50
Mouse anti-human integrin α₆ (F-6)	sc-374057	Santa Cruz Biotechnology, Inc.	ICC	1:50
Alexa Fluor^® 488-conjugated rabbit anti-human/mouse integrin α₆	313607	BioLegend	FI	1:50
Rabbit anti-human integrin α₈ (H-180)	sc-25713	Santa Cruz Biotechnology, Inc.	ICC, FI	1:50
PE-conjugated goat anti-mouse integrin α₉	FAB3827P	R&D Systems, Inc.	ICC	1:50
FITC-conjugated rabbit anti-mouse integrin α₉	orb188618	Biorbyt	FI	1:50
Mouse anti-human integrin α_v (P2W7)	sc-9969	Santa Cruz Biotechnology, Inc.	ICC	1:50
FITC-conjugated rabbit anti-mouse integrin α_v	orb7231	Biorbyt	FI	1:50
Mouse anti-human integrin β₁ (eBioSAM-1 [SAM-1, SAM1])	14-0496-82	Invitrogen	ICC	1:50
FITC-conjugated rabbit anti-mouse integrin β₁	ab21845	Abcam^®	FI	1:50
FITC-conjugated goat anti-mouse IgG	sc-2010	Santa Cruz Biotechnology, Inc.	ICC	1:50
Alexa Fluor^® 546-conjugated donkey anti-rabbit IgG	A10040	Molecular Probes	ICC	1:50
Alexa Fluor 488-conjugated donkey anti-goat IgG	A11055	Molecular Probes	ICC, FI	1:50
Alexa Fluor 488-conjugated chicken anti-rabbit IgG	A21441	Molecular Probes	FI	1:50
LEAF™ purified rat anti-mouse integrin α₄	103707	BioLegend	AIA	1:10
LEAF purified rat anti-mouse integrin α₅	103807	BioLegend	AIA	1:10
Rat anti-mouse integrin α₆	MAB1378	Chemicon International, Inc.	AIA	1:10
LEAF purified rat anti-mouse integrin α_V	104107	BioLegend	AIA	1:10

AIA, antibody inhibition assay; FI, fluorescence immunoassay; GFRα1, glial cell-derived neurotrophic factor family receptor alpha-1; ICC, immunocytochemistry; FITC, fluorescein isothiocyanate.

Fluorescence immunoassay

The fixation process was conducted by incubating 1 × 10⁵ cells for 10 min in 4% (v/v) paraformaldehyde. After rinsing twice with DPBS, the fixed cells were stained for 2 h at 4°C with FITC-conjugated anti-mouse integrin α₄, α₅, α₆, α₉, α_v, and β₁ antibodies and fluorescent-unconjugated anti-human integrin α₃ and α₈ primary antibodies diluted in DPBS supplemented with 2% (v/v) FBS. Subsequently, primary antibodies were detected using Alexa Flour 488-conjugated secondary antibodies diluted in DPBS supplemented with 2% (v/v) FBS. The detailed information and dilution rate of the used antibodies are listed in Table 1. The stained cells were washed twice 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.

Attachment assay

The attachment of SSCs to ECM ligands was conducted by somewhat modifying previous procedures (Lee et al., 2010). In brief, 96-well tissue culture plates were respectively coated with following concentrations of the purified ECM ligands: 0, 10, 20, 40, 60, and 80 μg/mL fibronectin (Millipore, Billerica, MA); 0, 200, 400, and 600 μg/mL laminin (Sigma-Aldrich); 0, 5, 10, and 20 μg/mL vitronectin (R&D Systems, Inc.); and 0, 1, 5, and 10 μg/mL tenascin C (R&D Systems, Inc.) overnight (minimum 18 h at 4°C). Each well was blocked with 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) at 4°C for 1 h and then the wells were washed three times with DPBS. Around 1 × 10⁵ porcine SSCs resuspended in SSC culture medium (pSSCCM) consisting of StemPro-34 medium (Invitrogen) supplemented with insulin–transferrin–selenium (Invitrogen), 60 μM putrescine dihydrochloride (Sigma-Aldrich), 6 mg/mL D-(+)-glucose (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 0.11 mg/mL sodium pyruvate (Sigma-Aldrich), 1 μL/mL DL-lactic acid (Sigma-Aldrich), 5 mg/mL BSA (Sigma-Aldrich), 2 mM l-glutamine, 0.05 mM β-mercaptoethanol, 1% (v/v) FBS, 1% (v/v) NEAA, minimum essential medium (MEM) vitamin solution (Sigma-Aldrich), 1% (v/v) antibiotic–antimycotic solution, 0.1 mM ascorbic acid (Sigma-Aldrich), 10³ U/mL mouse LIF, 10 ng/mL GDNF, 30 ng/mL β-estradiol (Sigma-Aldrich), 60 ng/mL progesterone (Sigma-Aldrich), 20 ng/mL human epidermal growth factor (PeproTech, Inc., Rocky Hill, NJ), and 10 ng/mL human basic fibroblast growth factor (PeproTech, Inc.) were seeded to each blocked well and incubated at 37°C for 2 h. After the wells were washed sufficiently to remove nonadherent porcine SSCs, adherent cells were fixed in 4% (v/v) paraformaldehyde at room temperature for 10 min and stained with 0.1% (w/v) Crystal Violet (Sigma-Aldrich) in 20% (v/v) methanol (Sigma-Aldrich) for 5 min. Subsequently, washing of the wells was performed 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 (Sigma-Aldrich) diluted with distilled water.

Antibody inhibition assay

Each well of 96-well tissue culture plate was coated with 40 μg/mL fibronectin, 200 μg/mL laminin, and 5 μg/mL vitronectin overnight at 4°C. Blocking of the wells was conducted with 1% (w/v) BSA for 1 h at 4°C. Subsequently, for inhibiting function of integrin heterodimers, 1 × 10⁵ SSCs of porcine neonatal testes were resuspended in pSSCCM, including anti-integrin α₄ [9C10 (MFR4.B)], anti-integrin α₅ [5H10-27 (MFR5)], anti-integrin α₆ (NKI-GoH3), or anti-integrin α_V (RMV-7) blocking antibody and incubated at 37°C for 2 h. The detailed information regarding the used antibodies is shown in Table 1. The functionally blocked porcine SSCs were then plated on each well and incubated at 37°C for 8 h. To remove nonadherent 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 0.2% (v/v) Triton X-100 diluted with distilled water. The amount of dye was measured at 570 nm using a microplate reader (Epoch Microplate Spectrophotometer). Poly-l-lysine- and BSA-coated wells played a role as positive and negative controls, respectively.

Statistical analyses

Statistical analyses of all numerical data derived from each experiment were conducted using the Statistical Analysis System (SAS) program. Furthermore, in detecting a significance of the main effects in the SAS package, control and treatment groups were compared by the PROC-GLM method. The p-values less than 0.05 was indicative of significant differences.

Results

Identification of integrin subunits expressed on the surface of undifferentiated porcine SSCs

To determine the integrin heterodimers expressed on the surface of undifferentiated porcine SSCs, expression of the individual α and β subunits that make up the integrin heterodimers interacting with each ECM protein observed in the testis (Skinner et al., 1985; Paranko et al., 1995; Ozturk et al., 2003; Siu and Cheng, 2004a, 2004b, 2008; Cheng et al., 2010; Oatley and Brinster, 2012) was monitored at the translational level. As shown in Figure 1, expression of the integrin subunits α₅, α₆, α₉, α_V, and β₁ on the surface of undifferentiated porcine SSCs was observed, and the quantitative analysis (Fig. 2) showed that the integrin α₅ subunit had the highest significant expression. Among the integrin subunits with significantly low expression, α₉ and β₁ showed intermediate expression compared with α₆ and α_V. The lowest significant expression was detected with α₆ and the highest was found with α_V. These results demonstrate that α₅, α₆, α₉, α_V, and β₁ are localized on the membrane of porcine SSCs in an undifferentiated state.

FIG. 1.

The expression of the α and β integrin subunits expressed in undifferentiated porcine SSCs. The SSCs were sorted from the testicular cells enzymatically isolated from porcine testis by Petri dish plating post-DP method. Subsequently, the expression of integrin α and β subunit proteins in the sorted porcine SSCs was identified by immunocytochemistry. As the results, integrin α₅ (A), α₆ (B), α₉ (C), α_V (D), and β₁ (E) subunit proteins were localized on the surface of porcine SSCs stained positively against GFRα1, a SSC-specific marker. All figures are representative immunocytochemistry images of integrin subunit proteins expressed on the surface of porcine SSCs, respectively. Nuclear counterstaining was conducted using DAPI. n = 3. Scale bars = 10 μm. DIC, digital image correlation; DP, differential plating; SSC, spermatogonial stem cell; GFRα1, glial cell-derived neurotrophic factor family receptor alpha-1. Color images available online at www.liebertpub.com/dna

FIG. 2.

Quantitative analysis of the α and β integrin subunits expressed in undifferentiated porcine SSCs. The SSCs were sorted from the testicular cells enzymatically isolated from porcine testis by Petri dish plating post-DP method. Subsequently, the expression of integrin α and β subunit proteins in the sorted porcine SSCs was measured using fluorescence immunoassay, and then the expression level of each integrin subunit protein was calculated as the ratio of fluorescence intensity of stained cells to fluorescence intensity of unstained cells. The α₅ integrin subunit showed the highest significant expression. Among the subunits with relatively weak expression, the lowest significant expression was detected with α₆ and the highest significant expression was found with α_V. Moreover, α₉ and β₁ showed intermediate expression. All data shown are mean ± SEM from three independent experiments. *^,**p < 0.05. SEM, standard errors of the mean.

Identification of functional integrin heterodimers on the surface of undifferentiated porcine SSCs

Building off of these results, the previously described integrin heterodimers α₅β₁, α₆β₁, α₉β₁, and α_Vβ₁ (Giebel et al., 1997; Shinohara et al., 1999; Siu and Cheng, 2004b, 2008; Ebata et al., 2005; Xi, 2009; Ellis and Tanentzapf, 2010) were hypothesized to be active integrin heterodimers expressed in undifferentiated porcine SSCs. Their presence was investigated by estimating the levels of adherent porcine SSCs cultured on natural ECM proteins interacting specifically with each integrin heterodimer and the level of adherence of porcine SSCs treated with antibodies blocking the function of individual integrins. Compared with those cultured without ECM proteins, porcine SSCs cultured on fibronectin, laminin, and vitronectin, but not those cultured on tenascin C, showed significantly improved adherence (Fig. 3). These results suggest that undifferentiated porcine SSCs express integrins α₅β₁, α₆β₁, and α_Vβ₁, which specifically interact with fibronectin, laminin, and vitronectin, respectively, on the cell surface.

FIG. 3.

Identification of integrin heterodimers interacting with fibronectin, laminin, vitronectin, and tenascin C on the surface of undifferentiated porcine SSCs. Ninety-six-well tissue culture plates were coated with 0, 10, 20, 40, 60, and 80 μg/mL fibronectin; 0, 200, 400, and 600 μg/mL laminin; 0, 5, 10, and 20 μg/mL vitronectin; and 0, 1, 5, and 10 μg/mL tenascin C, respectively, and 1 × 10⁵ porcine SSCs resuspended in pSSCCM were plated to each well. After incubation for 2 h at 37°C, adherent cells were stained with Crystal Violet and adherent level was quantified using a microplate reader. Porcine SSCs cultured on ECM plates with proteins showed significantly more attachment, regardless of protein concentration, than cultures without proteins. The highest significant rates of attachment were detected in cultures with 40 μg/mL fibronectin (A), 200 μg/mL laminin (B), and 5 μg/mL vitronectin (C), indicating the presence of integrin heterodimers α₅β₁, α₆β₁, and α_Vβ₁. Cultures with tenascin C showed significantly less attachment compared with controls (D), indicating the absence of integrin α₉β₁ on the membranes of porcine SSCs. The percentage of maximum adhesion represents the optical density of cells plated on protein-free ECM plates. All data shown are mean ± SEM from three independent experiments. *^,**p < 0.05. ECM, extracellular matrix; pSSCCM, porcine SSC culture medium.

Next, we blocked the functions of integrin heterodimers of undifferentiated porcine SSCs, and incubated the cells on 40 μg/mL fibronectin, 200 μg/mL laminin, and 5 μg/mL vitronectin, which showed the highest adherence of porcine SSCs (Fig. 3). SSCs in which integrins α₆β₁ or α_Vβ₁ were blocked showed significantly less adherence, whereas those in which integrin α₅β₁ was blocked showed no significant decrease in adherence (Fig. 4). These results confirmed that undifferentiated porcine SSCs exhibit functional expression of the integrins α₆β₁ and α_Vβ₁ on the cell surface, and that both α₅ and α₉ are present in inactive subunit forms and not as functional heterodimers.

FIG. 4.

Functional analysis of the integrin heterodimers thought to function on the surface of undifferentiated porcine SSCs. Porcine SSCs incubated in the absence or presence of anti-integrin α₅ [5H10-27 (MFR5)] (A), anti-integrin α₆ [NKI-GoH3] (B), or anti-integrin α_V [RMV-7] (C) antibody were seeded on 40 μg/mL fibronectin-, 200 μg/mL laminin-, and 5 μg/mL vitronectin-coated wells and incubated for 8 h at 37°C. After staining adherent cells with Crystal Violet, quantification of adherent level was conducted using a microplate reader. As the parameter of functional blocking by antibodies, the percentage of maximum adhesion, which represents as the optical density of cells plated on 1 mg/mL poly-L-lysine-coated wells in the absence of any antibodies, was demonstrated. No significant decrease in attachment was induced by blocking integrin α₅β₁, whereas SSCs treated with integrin α₆β₁ or α_Vβ₁ blocking antibodies showed significantly lower rates of attachment. All data are mean ± SEM from three independent experiments. *p < 0.05.

The adhesion of porcine SSCs to fibronectin in the absence of the functional integrin α₅β₁ (Fig. 3A) led us to hypothesize that porcine SSCs may express additional integrin heterodimers that bind fibronectin. Given that the integrins α₃β₁, α₄β₁, and α₈β₁ can bind fibronectin (Johansson et al., 1997; Aziz-Seible and Casey, 2011; Xu and Mosher, 2011), we assessed the expression of subunits α₃, α₄, and α₈ on the membrane of undifferentiated porcine SSCs (Figs. 5 and 6). Each was expressed on the membrane, but the expressions of α₃ and α₄ were significantly higher than that of α₈. Moreover, SSCs incubated on 40 μg/mL fibronectin with blockade with integrin α₄β₁ showed significantly lower adherence than those in which this integrin was not blocked (Fig. 7). These results confirmed the presence of integrin α₄β₁ on the membrane of undifferentiated porcine SSCs. In this way, we indirectly identified the presence of α₃ and α₈ as individual integrin subunits on the membrane of undifferentiated porcine SSCs.

FIG. 5.

The expression of the integrin subunits α₃, α₄, and α₈ in undifferentiated porcine SSCs. The isolation of SSCs from the testicular cells enzymatically isolated from porcine testis was conducted by Petri dish plating post-DP method. Then, the expression of integrin α₃, α₄, and α₈ subunit proteins associated with binding domains within fibronectin in the sorted porcine SSCs was identified by immunocytochemistry. Integrin subunit α₃ (A), α₄ ( green arrowhead, B), and α₈ (C) proteins were detected on the surface of undifferentiated porcine SSCs stained positively against GFRα1, a SSC-specific marker. All figures are representative immunocytochemistry images of integrin subunit proteins expressed on the surface of porcine SSCs, respectively. DAPI staining was conducted for nuclear counterstaining. n = 3. Scale bars = 10 μm. Color images available online at www.liebertpub.com/dna

FIG. 6.

Quantitative analysis of the integrin subunits α₃, α₄, and α₈ in undifferentiated porcine SSCs. The isolation of SSCs from the testicular cells enzymatically isolated from porcine testis was conducted by Petri dish plating post-DP method. Then, the expression of integrin α₃, α₄, and α₈ subunit proteins associated with binding domains within fibronectin in the sorted porcine SSCs was measured using fluorescence immunoassay. Subsequently, the expression level of each integrin subunit protein was quantified as the ratio of fluorescence intensity of stained cells to fluorescence intensity of unstained cells. Integrin subunits α₃, α_4, and α₈ were expressed on the surfaces of undifferentiated porcine SSCs. The expression of α₈ was significantly lower than those of α₃ and α₄. All data shown are mean ± SEM from three independent experiments. *p < 0.05.

FIG. 7.

Functional analysis of integrin α₄β₁ on the surface of undifferentiated porcine SSCs. Porcine SSCs incubated in the absence or presence of anti-integrin α₄ [9C10 (MFR4.B)] antibody was seeded on 40 μg/mL fibronectin-coated wells and incubated for 8 h in 37°C. After staining adherent cells with Crystal Violet, quantification of adherent level was conducted using a microplate reader. As the parameter of functional blocking by antibody, the percentage of maximum adhesion, which represents as the optical density of cells plated on 1 mg/mL poly-L-lysine-coated wells in the absence of any antibody, was demonstrated. Porcine SSCs treated with integrin α₄β₁ blocking antibody showed a significant decrease in attachment compared with SSCs not treated with blocking antibodies. All data are mean ± SEM from three independent experiments. *p < 0.05.

Discussion

To generate niches that will allow for self-renewal or differentiation of porcine SSCs, it is essential to understand the integrin family members that mediate intracytoplasmic transduction of signals derived from ECM proteins of the STBM in the seminiferous tubule. Therefore, we investigated the types of integrin heterodimers expressed on the membranes of undifferentiated SSCs in pigs. Through translational analysis of eight integrin subunits, followed by confirmation of their attachment to ECM proteins and inhibition with blocking antibodies, we found that the integrin heterodimers α₄β₁, α₆β₁, and α_Vβ₁, and the integrin subunits α₅ and α₉ were expressed on the membranes. We also indirectly identified the localization of α₃ and α₈ on the membranes. These results suggest that the fibronectin-interacting integrin α₄β₁, the laminin-interacting integrin α₆β₁, and the vitronectin-interacting integrin α_Vβ₁ may play an important role in the maintenance of porcine SSC self-renewal, and that fibronectin, laminin, and vitronectin analogs will likely be important for the development of niches customized to the maintenance of SSC self-renewal. In addition, we can speculate that the integrin subunits α₃, α₅, α₈, or α₉ may play a pivotal role in inducing early spermatogenesis of undifferentiated porcine SSCs.

The localizations of α₃, α₄, and α₈ were determined on the surfaces of undifferentiated porcine SSCs (Figs. 5 and 6). The lack of any antibodies that can block α₃ and α₈ made it impossible to define the conformation of these subunits. However, weakened attachment of integrin α₄β₁ function-blocked porcine SSCs was observed on fibronectin (Fig. 7), indicating that integrin α₄β₁ functions as a heterodimer on the cell surface. This result also demonstrates that α₃ and α₈ may exist as inactive subunits on the surfaces of undifferentiated porcine SSCs.

In the seminiferous tubule microenvironment that generates spermatozoa from spermatogonium through spermatogenesis (Siu and Cheng, 2004a, 2004b, 2008; Cheng et al., 2010; Oatley and Brinster, 2012), inactive integrin subunits can induce cytological, chromosomal, and morphological changes in the spermatogonium following detachment of SSCs from the STBM. In this study, the subunits, α₃, α₅, and α₈, were observed in inactive forms on cell membranes, indicating that signals derived from the interaction between fibronectin and integrin heterodimers with subunits α₃, α₅, and α₈ may be important in the differentiation of SSCs into sperm; this was supported by reports that fibronectin is expressed in spermatogenic cells in the seminiferous tubule (Fusi and Bronson, 1992; Schaller et al., 1993). Furthermore, the presence of the inactive integrin subunit α₉ on cell membrane demonstrates that signals derived from the interaction between tenascin C and α₉-containing heterodimers may facilitate the movement of SSCs into the adluminal compartment of the seminiferous tubule during spermatogenesis, which is supported by the fact that tenascin C regulates cell migration (Deryugina and Bourdon, 1996; Zagzag et al., 2002). Therefore, knowledge of the inactive integrin subunits expressed on the membranes of specific cells is important in the construction of microenvironments customized for a particular cell fate.

The integrins α₄β₁ and α₆β₁ were observed on the surfaces of undifferentiated SSCs (Figs. 3 –7), suggesting that the presence of fibronectin and laminin in the STBM may be important for maintaining self-renewal in porcine SSCs. Despite the absence of vitronectin in the STBM, the integrin α_Vβ₁ was expressed on the surfaces of undifferentiated SSCs (Figs. 3 and 4). Therefore, we can speculate that α_Vβ₁ may help maintain self-renewal through an interaction with vitronectin in the ECM of neighboring undifferentiated SSCs or spermatogenesis through an interaction with vitronectin in the ECM of neighboring supporting cells in the seminiferous tubules. There have been no reports of the expression of vitronectin in undifferentiated SSCs, although its expression has been reported in spermatogenic cells of adult testes during normal spermatogenesis (Sawada et al., 1996). The integrin α_Vβ₁ may contribute to spermatogenesis of porcine SSCs, although further studies are required to validate this.

Translation and functional screening demonstrated that the integrins α₄β₁, α₆β₁, and α_Vβ₁ are expressed on the surfaces of undifferentiated porcine SSCs. Expression of the inactive integrin subunits α₃, α₅, α₈, or α₉ was also demonstrated. Identification of these integrins will be useful for determining the identity of undifferentiated SSCs in pigs, and for generating synthetic niches appropriate for the maintenance of SSCs in the undifferentiated state or to induce their differentiation into sperm. Moreover, these achievements will greatly improve the applicability of synthetic niches with respect to the regulation of SSC destiny in pigs.

Footnotes

Acknowledgments

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (IPET) through Agri-Bioindustry Technology Development Program, funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) under Grant IPET117042-3 and IPET112015-4.

Disclosure Statement

No competing financial interests exist.

References

Aigner

, Renner

, Kessler

, Klymiuk

, Kurome

, Wünsch

, et al. (2010). Transgenic pigs as models for translational biomedical research. J Mol Med (Berl), 88, 653–664.

Aziz-Seible

R.S.

, and Casey

C.A.

(2011). Fibronectin: functional character and role in alcoholic liver disease. World J Gastroenterol, 17, 2482–2499.

Brizzi

M.F.

, Tarone

, and Defilippi

(2012). Extracellular matrix, integrins, and growth factors as tailors of the stem cell niche. Curr Opin Cell Biol, 24, 645–651.

Campos-Junior

P.H.

, Costa

G.M.

, Lacerda

S.M.

, Rezende-Neto

J.V.

, de Paula

A.M.

, Hofmann

M.C.

, et al. (2012). The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu). Biol Reprod, 86, 1–10.

Chen

, Lewallen

, and Xie

(2013). Adhesion in the stem cell niche: biological roles and regulation. Development, 140, 255–265.

Cheng

C.Y.

, Wong

E.W.

, Yan

H.H.

, and Mruk

D.D.

(2010). Regulation of spermatogenesis in the microenvironment of the seminiferous epithelium: new insights and advances. Mol Cell Endocrinol, 315, 49–56.

de Rooij

D.G.

, Repping

, and van Pelt

A.M.

(2008). Role for adhesion molecules in the spermatogonial stem cell niche. Cell Stem Cell, 3, 467–468.

Deryugina

E.I.

, and Bourdon

M.A.

(1996). Tenascin mediates human glioma cell migration and modulates cell migration on fibronectin. J Cell Sci, 109, 643–652.

Ebata

K.T.

, Zhang

, and Nagano

M.C.

(2005). Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development. Mol Reprod Dev, 72, 171–181.

10.

Ellis

S.J.

, and Tanentzapf

(2010). Integrin-mediated adhesion and stem-cell-niche interactions. Cell Tissues Res, 339, 121–130.

11.

Enders

G.C.

, Kahsai

T.Z.

, Lian

, Funabiki

, Killen

P.D.

, and Hudson

B.G.

(1995). Developmental changes in seminiferous tubule extracellular matrix components of the mouse testis: alpha 3(IV) collagen chain expressed at the initiation of spermatogenesis. Biol Reprod, 53, 1489–1499.

12.

Fusi

F.M.

, and Bronson

R.A.

(1992). Sperm surface fibronectin. Expression following capacitation. J Androl, 13, 28–35.

13.

García-Vázquez

F.A.

, Ruiz

, Grullón

L.A.

, de Ondiz

, Gutiérrez-Adán

, and Gadea

(2011). Factors affecting porcine sperm mediated gene transfer. Res Vet Sci, 91, 446–453.

14.

Geiger

, and Yamada

K.M.

(2011). Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol, 3, a005033.

15.

Giebel

, Loster

, and Rune

G.M.

(1997). Localization of integrin β1, α1, α5 and α9 subunits in the rat testis. Int J Androl, 20, 3–9.

16.

Houdebine

L.M.

(2002). The methods to generate transgenic animals and to control transgene expression. J Biotechnol, 98, 145–160.

17.

Houdebine

L.M.

(2009). Methods to generate transgenic animals. In Genetic Engineering in Livestock. Engelhard

, Hagen

, and Boysen

, eds. (Springer-Verlag Berlin Heidelberg, New York), pp. 31–48.

18.

Johansson

, Svineng

, Wennerberg

, Armulik

, and Lohikangas

(1997). Fibronectin-integrin interactions. Front Biosci, 2, d126–d146.

19.

Kanatsu-Shinohara

, and Shinohara

(2013). Spermatogonial stem cell self-renewal and development. Annu Rev Cell Dev Biol, 29, 163–187.

20.

Kim

S.H.

, Turnbull

, and Guimond

(2011). Extracellular matrix and cell signaling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor. J Endocrinol, 209, 139–151.

21.

Kurome

, Saito

, Tomii

, Ueno

, Hiruma

, and Nagashima

(2007). Effects of sperm pretreatment on efficiency of ICSI-mediated gene transfer in pigs. J Reprod Dev, 53, 1217–1226.

22.

Lavitrano

, Busnelli

, Cerrito

M.G.

, Giovannoni

, Manzini

, and Vargiolu

(2006). Sperm-mediated gene transfer. Reprod Fertil Dev, 18, 19–23.

23.

Lavitrano

, Giovannoni

, and Cerrito

M.G.

(2013). Methods for sperm-mediated gene transfer. Methods Mol Biol, 927, 519–529.

24.

Lee

S.T.

, Yun

J.I.

, Jo

Y.S.

, Mochizuki

, van der Vlies

A.J.

, Kontos

, et al. (2010). Engineering integrin signaling for promoting embryonic stem cell self-renewal in a precisely defined niche. Biomaterials, 31, 1219–1226.

25.

Lunney

J.K.

(2007). Advances in swine biomedical model genomics. Int J Biol Sci, 3, 179–184.

26.

Maione

, Lavitrano

, Spadafora

, and Kiessling

A.A.

(1998). Sperm-mediated gene transfer in mice. Mol Reprod Dev, 50, 406–409.

27.

Mazaud Guittot

, Vérot

, Odet

, Chauvin

M.A.

, and le Magueresse-Battistoni

(2008). A comprehensive survey of the laminins and collagens type IV expressed in mouse Leydig cells and their regulation by LH/hCG. Reproduction, 135, 479–488.

28.

Meurens

, Summerfield

, Nauwynck

, Saif

, and Gerdts

(2012). The pig: a model for human infectious diseases. Trends Microbiol, 20, 50–57.

29.

Niemann

, Kues

, and Carnwath

J.W.

(2005). Transgenic farm animals: present and future. Rev Sci Tech, 24, 285–298.

30.

Niemann

, and Kues

W.A.

(2000). Transgenic livestock: premises and promises. Anim Reprod Sci, 60–61, 227–293.

31.

Nitta

S.K.

, and Numata

(2013). Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering. Int J Mol Sci, 14, 1629–1654.

32.

Nowak-Imialek

, Kues

, Carnwath

J.W.

, and Niemann

(2011). Pluripotent stem cells and reprogrammed cells in farm animals. Microsc Microanal, 17, 474–497.

33.

Oatley

J.M.

, and Brinster

R.L.

(2012). The germline stem cell niche unit in mammalian testes. Physiol Rev, 92, 577–595.

34.

Olie

R.A.

, Looijenga

L.H.

, Dekker

M.C.

, de Jong

F.H.

, van Dissel-Emiliani

F.M.

, de Rooij

D.G.

, et al. (1995). Heterogeneity in the in vitro survival and proliferation of human seminoma cells. Br J Cancer, 71, 13–17.

35.

Ozturk

, Buyukbayram

, Ozdemir

, Ketani

, Gurel

, Onen

, et al. (2003). The effects of nitric oxide on the expression of cell adhesion molecules (ICAM-1, UEA-1, and tenascin) in rats with unilateral testicular torsion. J Pediatr Surg, 38, 1621–1627.

36.

Paranko

, Haavisto

, Chiquet-Ehrismann

, Aukhil

, and Kaipia

(1995). Sex-dependent expression of tenascin-C in the differentiating fetal rat testis and ovary. Differentiation, 58, 329–339.

37.

Park

M.H.

, Park

J.E.

, Kim

M.S.

, Lee

K.Y.

, Park

H.J.

, Yun

J.I.

, et al. (2014). Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. J Assist Reprod Genet, 31, 983–991.

38.

Prather

R.S.

, Lorson

, Ross

J.W.

, Whyte

J.J.

, and Walters

(2013). Genetically engineered pig models for human diseases. Annu Rev Anim Biosci, 1, 203–219.

39.

Robl

J.M.

, Wang

, Kasinathan

, and Kuroiwa

(2007). Transgenic animal production and animal biotechnology. Theriogenology, 67, 127–133.

40.

Sawada

, Sugawara

, Kitami

, and Hayashi

(1996). Vitronectin in the cytoplasm of Leydig cells in the rat testis. Biol Reprod, 54, 29–35.

41.

Schaller

, Glander

H.J.

, and Dethloff

(1993). Evidence of beta 1 integrins and fibronectin on spermatogenic cells in human testis. Hum Reprod, 8, 1873–1878.

42.

Shinohara

, Avarbock

M.R.

, and Brinster

R.L.

(1999). β1- and α6-Integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A, 96, 5504–5509.

43.

Silván

, Díez-Torre

, Moreno

, Arluzea

, Andrade

, Silió

, et al. (2013). The spermatogonial stem cell niche in testicular germ cell tumors. Int J Dev Biol, 57, 185–195.

44.

Siu

M.K.

, and Cheng

C.Y.

(2004a). Dynamic cross-talk between cells and the extracellular matrix in the testis. Bioessays, 26, 978–992.

45.

Siu

M.K.

, and Cheng

C.Y.

(2004b). Extracellular matrix: recent advances on its role in junction dynamics in the seminiferous epithelium during spermatogenesis. Biol Reprod, 71, 375–391.

46.

Siu

M.K.

, and Cheng

C.Y.

(2008). Extracellular matrix and its role in spermatogenesis. Adv Exp Med Biol, 636, 74–91.

47.

Skinner

M.K.

, Tung

P.S.

, and Fritz

I.B.

(1985). Cooperativity between sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components. J Cell Biol, 100, 1941–1947.

48.

Smith

, and Spadafora

(2005). Sperm-mediated gene transfer: applications and implications. Bioessays, 27, 551–562.

49.

Swindle

M.M.

, Makin

, Herron

A.J.

, Clubb

F.J.

Jr. , and Frazier

K.S.

(2012). Swine as models in biomedical research and toxicology testing. Vet Pathol, 49, 344–356.

50.

Vajta

, and Callesen

(2012). Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs. Theriogenology, 77, 1263–1274.

51.

Virtanen

, Lohi

, Tani

, Korhonen

, Burgeson

R.E.

, Lehto

V.P.

, et al. (1997). Distinct changes in the laminin composition of basement membranes in human seminiferous tubules during development and degeneration. Am J Pathol, 150, 1421–1431.

52.

Wheeler

M.B.

, and Walters

E.M.

(2001). Transgenic technology and applications in swine. Theriogenology, 56, 1345–1369.

53.

Wheeler

M.B.

, Walters

E.M.

, and Clark

S.G.

(2003). Transgenic animals in biomedicine and agriculture: outlook for the future. Anim Reprod Sci, 79, 265–289.

54.

(2009). Anchoring stem cells in the niche by cell adhesion molecules. Cell Adh Migr, 3, 396–401.

55.

, and Mosher

(2011). Fibronectin and other adhesive glycoproteins. In The Extracellular Matrix: An Overview. Mecham

R.P.

, ed. (Springer Berlin Heidelberg, New York), pp. 41–75.

56.

Zagzag

, Shiff

, Jallo

G.I.

, Greco

M.A.

, Blanco

, Cohen

, et al. (2002). Tenascin-C promotes microvascular cell migration and phosphorylation of focal adhesion kinase. Cancer Res, 62, 2660–2668.