Sequential enzymatic digestion of testicular tubules was performed essentially according to the method of Dym et al (16) with minor modifications. Briefly, testes of ten C57BL/6 3 to 6 days old mice were isolated, placed into a Petri dish, and covered with sterile Phosphate Buffered Saline (PBS).

Testes were decapsulated under a dissection microscope and were suspended in Dulbecco's Minimum Eagle's Medium (DMEM; Sigma, St Louis, MO, USA) containing 0.5 mg/ml collagenase/dispase, 0.5 mg/ml trypsin and 0.08 mg/ml DNase (all from Sigma) supplemented with 14 mM NaHCO₃ (Sigma), single-strength non-essential amino acids, 100 IU/ml penicillin, 100 µg/ml streptomycin and 40 µg/ml gentamycin (all from Invitrogen, Carlsbad, Calif, USA). They were incubated for 30 min at 37 °C on a shaker with occasional gentle pipetting to dissociate the interstitial Leydig cells from the seminiferous tubes. After this incubation step, the tubules were allowed to settle down and the supernatant containing the Leydig cells was pipetted out. The cells were then incubated in DMEM medium containing collagenase (1.5 mg/ml), hyaluronidase (1.5 mg/ml), trypsin (0.5 mg/ml), and DNase (1 µg/ml) (all from Sigma) for 20–30 min using the conditions described above.

The dispersed cells were then washed twice with DMEM medium and filtered through 70 µm nylon mesh (BD Falcon, Becton-Dickinson, USA). Isolated cells were cultured at 32 °C in 5% CO₂ in air on 5 cm plastic dishes at a concentration of 10×10⁶ cells/dish. The cells were expanded by several passages of selected cultures by digesting with trypsin and were seeded onto new dishes. They were then harvested after trypsin digestion, washed in DMEM medium by centrifugation, and resuspended in DMEM for transplantation to recipient testes. Total cell count was determined before transplantation.

Isolated cells were centrifuged at 30×g for 5 min onto glass slides in a Cytospin centrifuge (Shandon, Cheshire, UK). The cells were fixed and permeabilized with ice-cold acetone for 3 min and then washed 3 times with PBS containing 0.5% Triton X-100 and 10% rabbit serum. Endogenous peroxidase activity was blocked with 0.1 – 1% H₂O₂ for 5 – 10 min at room temperature. Blocking of nonspecific antibody binding was performed using 10% nonimmune rabbit serum. Cells were then incubated at 37 °C for 1 hr with 1:500 dilution of mouse monoclonal anti c-kit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing in PBS, the cells were incubated with 1:50 dilution of Horse Radish Peroxidase (HRP)-conjugated anti mouse IgG as secondary antibody (Sigma) for 45 min. After washing with rinsing buffer, 10% 3, 3’-Diaminobenzidine (DAB) solution (Sigma) was applied for 10 min. For the negative controls, the experiments were repeated but without using the primary antibodies. For cytokeratin immunocytochemistry, cells were fixed for 10 min in ethanol-acetone (1:1) at −20 °C rinsed with PBS and incubated for 10 min with 3% H₂O₂ in PBS to quench endogenous peroxidase activity. Cells were washed for 30 min with PBS containing 0.2% bovine serum albumin (BSA), followed by 30 min incubation in 1% BSA in PBS and then incubated with the primary antibody for 1 hr. Mouse monoclonal anticytokeratin Pan (Boeringer-Mannheim, Germany) antibody was used as primary anti-body. The secondary antibody was a peroxidase conjugated anti mouse IgG (Sigma) which was incubated for 90 min. After washing with rinsing buffer, 10% DAB solution was applied for 10 min. For the negative controls, the experiments were repeated but without using the primary antibody.

Cells were removed from the seminiferous tubules by sequential enzymatic digestion and testis's tissue cryosections (positive control) were evaluated for their expression of alkaline phosphatase after 3 days in culture.

Briefly, the cells were fixed in situ with ice-cold acetone for 2 min and washed twice with 0.2 M Tris buffer (pH= 8.74). The sub-strate reagent, prepared fresh, consisted of 0.01% naphtol-AS-MX phosphate and 0.06% Fast Violet salt (both from Sigma) in 0.1 M Tris buffer (pH= 8.74). This solution was immediately filtered and incubated with the fixed cells for 30 min at 37 °C. After incubation, the cells were washed with distilled water and observed under light microscope for a red bright color indicating the expression of alkaline phosphatase against a yellow background.

The mixed population of the cells which were obtained by enzymatic digestion of testis's samples was placed on microscopic cover slip in 12-well tissue culture test plate and DMEM medium containing 10% fetal bovine serum (FBS) was added. When the cells grew to about 70% confluence (proliferation phase), BrdU incorporation was performed by adding 0.1 mM BrdU to the culture medium for 24 hr. The BrdU incorporated cells were centrifuged at 30×g for 5 min onto glass slides in a cytospin centrifuge and fixed in ice-cold acetone for 2 min. The cells were washed in PBS containing 1% BSA, 0.05% Tween 20, and 0.1% sodium azide. DNA denaturation was done by 2N HCl for 30 minutes at 37 °C. After rinsing, the cells were placed in 0.1 M borate buffer (pH = 8.5) for 10 min for acid neutralization. Endogenous peroxidase activity was blocked with 1% H₂O₂ and 10% goat serum for 15 min at 37 °C. Monoclonal anti BrdU antibody (Sigma) was diluted at 1:500 and applied to the cells for 2 hr at 37 °C. Subsequently, the cells were incubated for 45 min with Horse Radish Peroxidase (HRP)-conjugated anti mouse IgG as second antibody diluted at 1:50.

After extensive washing, 10% DAB solution was applied for 10 min. For the negative controls, the experiments were repeated but without using the primary antibodies.

In the experiments with busulfan-induced degeneration of testicular spermatogonial stem cells, different amounts of busulfan (Sigma) were dissolved in Dimethyl Sulfoxide (DMSO; Sigma), and an equal volume of sterile distilled water was added to provide a final concentration of 15, 30, and 45 mg/kg of mice body weight. Adult mice (>6 wks of age) received a single intraperitoneal injection of busulfan. In each experiment, two to three animals received busulfan at each dose.

In the experiments with transplantation, busulfan (30 mg/kg) was used to deplete endogenous germ cells to prepare recipients. They were used as recipient mice for transplantation at least 6 wks after busulfan treatment. The donor cells were suspended in a volume of approximately 15 µl of DMEM/FCS (fetal calf serum) and transplanted into left Rete testis of recipient with a microinjecttion needle. Viability of cells was greater than 95% as determined by trypan blue (Sigma) dye exclusion. For testicular injections, the cells (approximately 10⁶) were maintained on ice and then microinjected into the seminiferous tubules using injection needle in one of the testes of each of the recipient mice; the other testis served as an internal control.

The testes of busulfan-injected animals were recovered at 35 and 70 days after injection. The testes were fixed in 10% formalin (Merck, Darmstadt, Germany) and processed for paraffin sectioning. All sections were stained with hematoxylin and eosin. Two histological sections were made from the testes of each animal, and each slide was studied to determine the extent of spermatogenesis. The values for each time point were determined in at least three experiments and represented data from more than six testes. The number of tubule cross-sections showing spermatogenesis (defined as the presence of multiple layers of germ cells in entire circumference of the seminiferous tubule) or not showing spermatogenesis were recorded for one section from each testis. For evaluation of spermatogonial stem cell colonization, recipient mouse testes were recovered 2 months after donor cell transplantation and analyzed by BrdU staining. This method allowed the specific identification of donor germ cells that had previously been incorporated with BrdU, because endogenous host testis cells did not stain with anti BrdU monoclonal antibody.

Transplanted testes were washed in PBS and then were placed in tissue frozen medium. This medium was frozen in liquid nitrogen for 2 min. Serial sectioning with 5 µm thickness were prepared. The sections were then immunostained with a primary anti-BrdU in order to visualize the donor-derived spermatogenesis.

Twenty four mice (C57BL/6) aged 15 days old were selected and divided into 4 equal groups (one control and three case groups). Animals derived from original stocks were purchased from Pasture Laboratory (Tehran, Iran). The groups 2, 3, and 4 received 2 subcutaneous injections of three different doses of hCG including 5, 10 and 50 IU respectively, on the days 15 and 25 of the mouse life. The level of serum testosterone was determined on days 28 and 65 of the mouse life. The mice were then killed and the different testicular cell populations were determined according to DNA contents by flow cytometric analysis.

Serum testosterone levels were measured using a radioimmunoassay kit (Immunotech, Marseille, France). For DNA flow cytometry, freshly harvested testicular tissues were separated from the tunica albuginea and minced in PBS. The resultant single-cell suspensions were washed twice in PBS, and 100 µl aliquots of the suspensions were fixed with ethanol in PBS. After centrifugation, the pellets were resuspended in propidium iodide after subjection to RNA digestion by RNase (Sigma). DNA histograms were then obtained on a flow cytometer (Becton-Dickinson FAC scan, San Jose, Calif, USA). The data thus obtained were analyzed by Cell Quest software (Becton-Dickinson).

After spermatogonial stem cell transplantation, a group of transplanted mice received 50 IU hCG and 2 months later were compared with uninjected group by evaluating cyclin B1 expression.

For examination of alkaline phosphatase activity the cells were fixed for 10 min in ethanol–acetone (1:1) and then incubated for 30 min in a solution containing 0.5 mg/ml Fast Blue RR (Sigma) and 40 µl/ml α-naphtol phosphate (0.25% solution, Sigma). After washing in water, the samples were mounted and examined.

Fifty round testis tubules were randomly selected for each testis and the diameters of tubules and epithelium thickness were measured under light microscopy.

RNA was extracted from the freshly isolated testis tissues by the AGPC (Acid Guanidium Thiocyanate-Phenol-Chloroform) method (17). In brief, 400 µl RNA-Bee (Biosite, Stockholm, Sweden) solution was added per 0.5 mg fresh tissue, minced on ice using sterile scalpels and scissors and homogenized with a few strokes in a glass-Teflon homogenizer. Two hundred µl ice-cold chloroform (Merck) was added and the mixture was shaken strongly for 10 sec followed by centrifugation at 18000×g for 10 min at 0 °C.

Upper aqueous phase was transferred very carefully, using a pipette to a new micro centrifuge tube. Equal volume of ice-cold isopropanol (Merck) was added to precipitate the RNA, followed by overnight incubation at −20 °C.

The tubes were centrifuged at 29000×g for 15 min at 0 °C and the supernatants were discarded. The RNA precipitate was then washed by adding 500 µl of ice-cold 75% ethanol, and then centrifuged at 41000×g for 10 min at 0 °C. The pellet was dried in air and resuspended in distilled water.

A semi-quantitative Reverse Transcription (RT)-PCR method was used to measure gene expression (c-kit, cyclin B1, and oct-4) in the testis tissue. Total RNA (1 µg) was heat-denatured at 65 °C for 5 min, and reverse-transcribed in the reaction mixture (20 µl) containing 5x Buffer (pH = 8.3; 50 mM Tris-HCl, 40 mM KCl, 6 mM MgCl₂; Roche, Mannheim, Germany), 10 mM dNTP (Roche), N6 random hexamers (20 pmol/µl; Cyber Gene, Stockholm, Sweden), 200 U/µl M-MULV Reverse Transcriptase (Roche), and 2 µl distilled water at 42 °C for 1 hr. PCR amplification was done in a reaction mixture consisting of 10x PCR reaction buffer (pH = 8.3; 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl₂; Roche), 10 mM dNTP (Roche), the primers (10 pmol/µl), and Taq DNA Polymerase (5 U/µl; Roche). All primers were purchased from Cyber Gene and are listed in table 1.

Each cycle consisted of denaturation for 1 min at 94 °C, annealing (according to table 1) for 30 sec and extension for 30 sec at 72 °C. Extension during the last cycle was prolonged to 7 min. After PCR amplification, 10 µl of PCR products were applied to 2% ethidium bromide stained agarose gels for electrophoresis. To compare the expression levels of c-kit and cyclin B1 genes, PCR products of the house keeping gene glyceraldehyde 3-phosphat dehydrogenase (GAPDH) and the specific genes (c-kit or cyclin B1) were loaded in the same well synchronously. Due to difference in the amplicon size, the PCR bands were separated and the strengths of the bands were analyzed by the Lab Works software (UVP, Upland, CA, USA).

Protein extracts of testis's cells collected from 6 weeks old (control samples) and busulfan-treated mice were used for western blot analysis. Total protein lysates were prepared by homogenizing the testes in ice cold RIPA buffer (PBS with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF (Phenyl Methyl Sulfonyl Fluoride), 10 mg/ml of leupeptin, 10 mg/ml of aprotinin, 2 mM Na₃VO₄, and 10 mM NaF; Sigma). Lysates were cleared by centrifugation for 30 min at 20000×g. Protein levels were measured using Bradford assay. Proteins were separated using SDS-PAGE and blotted onto a nitrocellulose membrane (Sigma). After transfer was completed, the membrane was stained with Ponceau S (Merck) to estimate transfer efficiency.

Western blots were blocked using PBS containing 2% BSA. The membrane was incubated in 1% BSA solution containing 1:500 dilution of primary antibody (c-kit monoclonal antibody, Santa Cruz) for 90 min at room temperature with gentle shaking. After 3×10 min washing in PBS Tween, incubation with 1:10,000 diluted HRP conjugated rabbit anti mouse immunoglobulins (Sigma) was performed for 30 min. For band detection, DAB solution was added and incubated for 10 min with gentle shaking.

Statistical analysis was done using SPSS, version 11.5, statistical software program. The Kruskal-Wallis test was applied for comparison of control and treatment groups and Mann-Whitney Rank Sum test was applied for comparison of treatment groups with the control and p values less than 0.05 were considered statistically significant.

Morphological analysis of mouse testis structure following busulfan treatment showed that mortality was dose dependent and 90 mg/kg was the lethal dose for mice. In agreement with the results reported previously (18), injection of busulfan decreased the testis size and weight and depleted spermatogenesis in a dose-dependent manner (Figures 1a and b). Morphological evaluation at 35 days after treatment revealed that, whereas only a few of seminiferous tubules showed spermatogenesis in animals treated with busulfan at doses of 45 and 90 mg/kg, spermatogenesis was observed in 15% and 53% of the tubules of animals given busulfan at doses of 30 and 15 mg/kg, respectively (Figures 1c-e). These observations indicated that the effects of busulfan were dose dependent.

Seventy days after busulfan treatment, the increase in weight of the testis was most apparent in animals treated at a dose of 15 mg/kg, suggesting extensive regeneration of spermatogenesis at this dose. Histological analysis showed that most testes contained no germ cells in animals treated with busulfan at 45 mg/kg, and that regeneration occurred in a few tubules.

On the other hand, regeneration of spermatogenesis was more significant in animals given the drug at 15 and 30 mg/kg, with averages of 80% and 35% of tubules showing spermatogenesis, respectively. Particularly in animals treated with busulfan at 15 mg/kg, virtually all seminiferous tubules showed apparently normal spermatogenesis with mature spermatozoa, and the size of the testis was almost comparable to that in uninjected controls. These results indicated that a significant proportion of spermatogonial stem cells were eliminated at 45 mg/kg, and that surviving stem cells could regenerate to show spermatogenesis following injection of 15 and 30 mg/kg of busulfan. We selected a dose of 30 mg/kg for further analysis, because the stem cell regeneration, as suggested by the testis weight and the number of repopulated tubules, occurred extensively at this dose, but there are enough spaces for cell injection.

Spermatogonial stem cells express c-kit and we used it for confirmation of stem cells presence in colonies (Figure 2a). Therefore c-kit immunoreactivity as a molecular marker for spermatogonial cells was positive for the cells (Figure 2b). Figure 2c shows positive staining of Sertoli cells with anti-cytokeratin antibody throughout the cytoplasm. BrdU was added to the donor cells 2 days before transplantation and staining was examined just before transplantation. More than 80% of all the cells were labeled with BrdU before transplantation (Figure 2d).

Two months after transplantation, the cells showing nuclear BrdU staining were considered as transplanted cells. Transplanted spermatogonial cells resulted in full spermatogenesis in the recipient mouse testes. The right testis served as control group.

Alkaline phosphatase is highly expressed in mouse intestine (brush border of villous), as well as in adult and neonatal testes (Figures 3a-c) that served as positive control group in our study, but it is not expressed in spermatogonial stem cells (Figure 3d).

Testis capsular thickness was evaluated by optical microscopy and was increased after busulfan treatment. Capsular thickness increase and tubular diameter reduction depended on busulfan dosage. There was no significant difference in capsular thickness and tubular diameter between doses 15, 30, and 45 mg/kg, but there was a significant difference in these parameters when the control group was compared with the experimental groups regardless of dosage of busulfan (Figure 4a-d).

Two months after transplantation of cells onto seminiferous tubules of left testes, spermatogenic cells, as detected by tracing BrdU incorporated cells, were found in the tubule cross-sections of recipient mice left testes (Figure 5a). No BrdU positive cells were found in the non-transplanted group (right testis which was considered as control group), confirming the specificity of this identification method (Figure 5b).

Day 28 testosterone was shown to have increased as compared to the control group and the highest testosterone values were detected in group 4 (Table 2). In contrast to day 28, different results were found for day 65. In this regard, the testosterone levels were declined with increasing dose of hCG in different groups in comparison to the control group. Accordingly, group 4 had the lowest level of testosterone (Table 3). There was also no statistically significant difference detected between the case groups and the control group. Additionally, DNA flow cytometric analysis revealed that the germ cell numbers had remarkably declined in the case groups in comparison with the control group on day 65 and this reduction was statistically significant for groups 3 and 4 (Table 4).

Our results revealed that Oct-4 was exclusively expressed in testis cells of 3–6 days old mice and adult mouse testis and testicular cells from busulfan treated mice did not express Oct-4 (Figure 6a). In adult mouse testis, spermatogonial stem cell differentiation resulted in down regulation of Oct-4 expression and its transcript was not detected in RT-PCR gel electrophoresis.

We evaluated the expression of these genes in adult (control group), 3–6 days old, and busulfan treated (experimental groups) mouse testes by semi-quantitative RT-PCR. During proliferation of testis cells, expression rates of c-kit and cyclin B1 was increased and thus these expressions in adult mice was more than that in neonatal testis. Busulfan eliminates most germ cells from the testis and destroys seminiferous epithelial leading to Sertoli cell disappearance. Germ cell reduction results in c-kit and cyclin B1 expression decrease in adult busulfan treated mouse testis (Figures 6a and b). Using semi-quantitative RT-PCR analysis, expression rate of each gene was compared with GAPDH expression and cyclin B1/ GAPDH and c-kit/ GAPDH ratios were evaluated.

After hCG injection in stem cell transplanted mice, cyclin B1 expression was evaluated by semi quantitative RT-PCR. Results revealed that cyclin B1 expression increased after hCG injection. High quantity of gene expression showed that spermatogonial stem cell colonization and endogenous stem cells number increased during hCG injection (Figure 6c).

The presence of c-kit protein in the testis was determined by SDS-PAGE/ immunoblot analysis. Total proteins (10 µg) from testis lysate of control and busulfan treated mice were separated by SDS-PAGE, transferred to Nitrocellulose membrane (NC) and probed with anti-c-kit antibody. The antibody recognized a single protein band of ∼150 kDa in normal mice. No detectable c-kit protein was seen in busulfan treated testes, busulfan treatment caused complete depletion of germ cells, and thus these animals were used as recipients for germ cell transplantation (Figures 7a and b).