International Journal of

Introduction
 The spermatogonial stem cells (SSCs) are the foundation of spermatogenesis. They can self-renew and produce a large number of differentiated germ cells in vivo (1). Sometimes, male fertility is related to spermatogenic arrest. In these cases, interruption of germ cell differentiation leads to azoospermia or oligozoospermia (2). In vitro maturation of germ cells and promoting their differentiation may be helpful for these patients (3). Because of the rare number of SSCs in the testis, proliferation of these cells is required. It has been previously demonstrated that the incubation of mouse SSCs in presence of feeder cells or growth factors enables their long-term proliferation in vitro (4). In this study, it was attempted to use Sertoli cells as feeder layer not only for proliferation, for differentiation of SSCs.
Sertoli cells are the somatic cells of the testis and play pivotal role in testis formation in the embryo and spermatogenesis in the adult males also, provide a structural and nutritional support for germ cells (1, 5). There is a specialized interaction between Sertoli cells and germ cells (6, 7).
The molecular control of germ cell fate is precisely regulated by intrinsic gene expression in the SSCs and extrinsic factors from testis niche (5, 8, 9). Sertoli cells thigh junctions, the main structural component of the seminiferous tubule, provide a restricted tissue environment for SSCs (10). The paracerine regulations and cell-cell adhesion through gap junctions facilitate interactions between SSCs and Sertoli cells. Sertoli cells produce different growth factors involving in proliferation and differentiation of SSCs (11).
The important factor that is necessary for SSCs proliferation and maintenance in vitro and in vivo is Glial derived neurotrophic factor (GDNF) (12). GDNF is a Sertoli-cell-derived factor that controls the balance between self-renewal and differentiation of the spermatogonial stem cells (13). Basic fibroblast growth factor and epidermal growth factor (EGF) are the other necessary factors, secreted by Sertoli cells, for SSCs self-renewal (14).
In addition, Sertoli cells express follicle stimulating factor (FSH) and testosterone receptors (5). Both of these hormones are necessary for spermeiogenesis. The high concentration of FSH could stimulate germ cell meiosis in vitro (15). On the other hand, FSH could regulate SSCs proliferation by GDNF/FSH pathway (16). Therefore, Sertoli cells regulate SSCs proliferation and differentiation by different mechanisms. LIF is the other growth factor that its role in survival of embryonic stem cells has been identified (17). In testis, LIF is secreted by peritubular cells of seminiferous tubules and acts via its receptors. SSCs express LIF receptors and are able to bind to LIF, whereas these receptors are absent on the meiotic cells (18). Therefore, in this study, this cytokine was used for proliferation of SSCs.
Some data demonstrate an improvement in culture system over time through changes in the culture medium composition and in the germ cell microenvironment (4). Today, spermatogonial and testicular somatic cells culture has become important and innovative tools for applied research in reproductive biology (19). Development of in vitro differentiation assays would help in introducing culturing condition to induce spermatogenesis process in SSCs (20).
In this study, it was co-cultured adult SSCs with primary culture of Sertoli cells to replicate conditions somewhat similar to the natural niche applicable in clinical approach. By using this condition, it was investigated the potential role of Sertoli cells for increasing the number of SSCs and following efficient differentiation of SSCs to spermatid-like cells.
 
Materials and methods
 
Experimental animals
In this experimental study, four to six week-old national medical research institute (NMRI) male mice were maintained under standard conditions with free access to food and water. The ethics committee of Arak University of Medical Sciences approved the animal experiments (91-133-5), in accordance with University guidelines.
 
SSC enrichment
For obtaining testicular cells, a modified method published by Guan was used (21). Briefly, decapsulated testis was cut into small pieces and seminiferous tubules were transferred to collagenase type I (1 mg/ml) (Sigma, Germany), Deoxyribonuclease I (10 μg/ml) (DNase; Sigma, Germany) solution. Cells were incubated at 37˚C in a CO₂ incubator and dispersed by pipetting every 2–5 min until the tubules separated after about 20 min. Cells were washed twice with 10 ml of phosphate buffered saline (PBS; Sigma, Germany) as reported previously. The dissociated testis cell suspension was overlaid on 30% (v/v) Percoll (Sigma, Germany) prepared in PBS containing 1% fetal bovine serum (FBS; Gibco, USA) and centrifuged at 600×g for 8 min at 4˚C. For magnetic-activated cell sorting (MACS), Miltenyi Biotec protocol was used. Sedimented cells (bottom fraction) of the Percoll gradient (3–8×10⁶ cells in 90 μl of PBS) were incubated with 10 μl of thymus cell antigen 1 (Thy-1) antibody (30-H12; Miltenyi Biotec, Germany) microbeads for 20 min at 4˚C. After rinsing with PBS containing 0.5% bovine serum albumin (BSA; Sigma, Germany), Thy-1⁺ cells were selected by passing them through a large separating column (Miltenyi Biotec, Germany) that was placed in a magnetic field.
 
Flow cytometry
Flow cytometric analysis was performed on populations of testis cells before and after enrichment. Briefly, 10⁶ cells were suspended in 0.1 ml of PBS/1% FBS and 10μl: fluorescence isothiocyanate conjugated hamster anti-rat β1-integrin (CD29) (R&D, USA). Antibody was added to the cells for 20 min at 4^°C. The cells were washed twice in 1 ml of PBS/FBS. Then, 0.1 ml of PBS/1% FBS and 10μl phycoerythrin (PE) conjugated rat anti-human a6-integrin (CD49f) (R&D, USA) was added to the cells for 20 min at 4^°C. The cells were washed twice with 1 ml of PBS/FBS. Control cells were not treated with antibody (22).
Sertoli cells isolation and culture
Adult Sertoli cells were extracted from the testis of adult mice as previously described (23). Briefly, culture dishes were coated with 5 μg/ml of lectin Datura stramonium agglutinin (DSA; Sigma, Germany) in PBS at 37^°C for 1 h.
Then, the coated plastic dishes were washed three times with 0.5% BSA. The mixed population of the cells obtained by enzymatic digestion was placed on the lectin-coated dishes and incubated for 1 h at 32^°C in a humidified atmosphere of 5% CO₂. After incubation, the non-adhering cells were collected and washed twice with Dulbecco's modified eagle (DMEM; Gibco, USA). Then, DMEM and 10% FBS were added. After 7 days the Sertoli cells formed a confluent layer.
 
Immunocytochemistry for characterization of adult Sertoli cells
Vimentin was detected in cultured Sertoli cells by immunocytochemistry. After fixation with 4% paraformaldehyde (Sigma, Germany), permeabilization by 0.4% Triton X100 (Sigma, Germany) and blocking with 10% goat serum (Sigma, Germany), the cells were incubated for 2 h at 37^°C with mouse monoclonal anti-Vimentin antibody diluted 1:100 (Sigma, Germany).
After washing with PBS, the secondary antibody, goat anti-mouse labeled with fluorescence isothiocyanate (Sigma, Germany) diluted 1:100, was applied for 3 h. Control cells were treated under similar conditions, while instead of the primary antibody, horse serum was added. Nuclei were stained (5μg/ml Propidium iodide; PI; Sigma, Germany) (24).
 
SSCs proliferation and differentiation
Enriched SSCs were cultured (6–10 ×10⁴ cells/cm²) in Minimum essential medium alpha (MEMα; Gibco, USA) containing 10% FBS, 1 x non-essential amino acids (Gibco, USA), 0.1 mM 2-mercaptoethanol )Sigma, Germany), 103U/ml LIF (B&D, USA), 0.4mM Pyruvate (Sigma, Germany), 1x Glutamine (Gibco, USA), 100u/ml Penicillin and 100 μg/ml Streptomycin (Sigma, Germany), on Sertoli cells or gelatin (Sigma, Germany) coated dishes for 7 days. In another groups the cells were cultured in a MEMα medium containing the above-mentioned without LIF, co-culturing with gelatin or Sertoli cells. The culture medium was changed every 3 day. All culture media were maintained at 32˚C in an atmosphere humidified with 5% CO₂ (21). SSCs colonies were then stained with Alkaline Phosphatase (25). The SSCs number was determined with a hemacytometer. Trypan blue (0.4%) (Sigma, Germany) exclusion assays were used to determine the percentage of the surviving cells following the isolation and culture (26).
 
Reverse transcriptase polymerase chain reaction
After 7 days, the expression of specific genes such as Stimulated by retinoic acid gene 8 (Stra8) and Detected in azoospermia- like (Dazl) expressed in SSCs, H2A, Synaptonemal complex protein 3 (Scp3), and TH2B expressed in spermatocytes, and Ubiquitin-activated enzyme (Ube1y), Transition protein 1(TP1), TP2 and Protamine 1 (PRM1) expressed in spermatids were studied by RT-PCR. Table I gives primer sequences. Total RNA was extracted by Trizol reagent (Invitrogen, CA) according to the manufacturer's protocol.
RT-PCR using a Complementary DNA (cDNA) synthesis kit (Bioneer, South Korea) using 1 μl of total RNA was performed according to the manufacturer's protocol. RT-PCR was performed using Tag DNA polymerase (Cinagene, Iran) in a Gene Amp PCR system 9600 (PerkinElmer Life and Analytical Sciences, MA). After an initial denaturation at 94^°C for 5 min, cDNA was subjected to 33 cycles of PCR.
Testicular cells from adult testis were used as positive control and liver cells were used as negative control. To normalize RT-PCR samples, expression of β-actin was examined. The PCR products on 2% agarose gels was observed, and the intensity of bands by UVI doc gel documentation system was assayed (Cambridge, CB4 1QB-UK).
 
Statistical analysis
The results were expressed as Mean ± standard deviation (SD). The statistical significance between the mean values was determined by one-way analysis of variance (ANOVA), Tukey and Duncan post-test. A level of p≤0.05 was considered as the level of significance.
Results
 
SSCs enrichment
In the current study, SSCs were isolated using MACS method and anti-Thy-1 antibody. The purity of the isolated cells was determined by flow cytometery using α6 and β1-integrin antibodies. The flow cytometric analysis revealed enriched Thy-1 purified cells, 67.2 ± 2.2% (n=6) expressed β1 integrin, 37.7 ± 1.8% (n=6) expressed α6 integrin, and 30.8 ± 1.9% (n=6) of Thy-1-enriched cells expressed both integrins (Figure 1), consistent with previous data (51.3 ± 11.9) for β1 integrin (22).
 
Sertoli cell isolation and characterization
Cells cultured on lectin DSA-coated plates were initially rounded and adhered firmly to the bottom of the dish. After 1 day,the cells started to flatten; they gradually spread and took on an epithelioid appearance (Figure 2A). The presence of Vimentin protein on Sertoli cells (24) was confirmed by immunocytochemistry (Figure 2B/C).
 
SSCs proliferation and differentiation
Isolated Thy1+ SSCs were cultured in the presence or absence of LIF co-culturing Sertoli cells. In presence of LIF, on the first day of culture, SSCs were single and attached to Sertoli cells or gelatin coated dishes (Figure 3A).
After two days, the majority of round cells aggregated into clumps in which no cytoplasmic bridges could be discerned (Figure 3B), and then colonies were formed on days 2–3 (Figure 3C). These cell colonies were positive for alkaline phosphatase activity (Figure 3D). The cells exhibited expression levels of Stra8 and Dazl, and they lost detectable postmeiotic transcripts, but the expression level of this genes was significantly (p<0.05) higher in the group SSCs co-cultivated with Sertoli cells (Figure 4). Isolated Thy1+ SSCs were cultured in the absence of LIF and co-cultured with gelatin or Sertoli cells. The SSC colonies developed in a limited fashion and lost detectable Stra8 and Dazl expression. In both groups, the cells exhibited expression levels of primary spermatocyte-specific genes, H2A and Scp3 and spermatocyte gene; Th2b. But, Sertoli cells increased significantly the expression level of these genes. It was also detected only one of the spermatid-specific transcripts (Ube1y) in the group SSCs cultured on gelatin. A significant rise of TP1 (p=0.01), TP2 (p=0.005), and PRM1 (p=0.01) cDNA levels were only detectable in the groups co-cultured with Sertoli cells (Figure 4). All genes were expressed in the testis as positive control, but were absent in the liver as the negative control.