We evaluated the effects of BM-MSCs on germinal epithelium of seminiferous tubules from busulfan-induced infertile male rats. Initially, MSCs were isolated from the BM samples of male rats’ tibias. After 10~14 days, each rat received a single dose of busulfan (40 mg/kg BW) for induction of infertility. After approximately 28 days, passages-3 (P3) to 5 (P5) BM-MSCs from each male rat were labeled with PKH26 dye (Sigma, USA). Next, we performed autologous cell transplantations. The rats were divided into three treatment groups. In the first group testicular evaluations were performed 4 weeks after transplantation. In the second group evaluations were performed 6 weeks after transplantation and in the third group evaluations were performed 8 weeks after transplantation.

Male Wistar rats that weighed 250±20 g were used in this study. Animals were kept under controlled temperature (20°C) and a 12-h light/12-h dark cycle with ad libitum access to food and water. All procedures were performed following official permission granted by the Animal Ethics Committee of the Faculty of Veterinary Medicine, Urmia University.

BM sampling and isolation of BM-MSCs were performed according to a new protocol reported in our recently published paper (31). Briefly, after induction of anesthesia in the rats (with an intraperitoneal injection of 60 mg/kg ketamine and 6 mg/kg xylazine) and dorsal recumbency, the knee region was shaved carefully and disinfected using 70% ethanol and povidone iodine. A 22-g needle was slowly inserted through the skin on the anterior face of the proximal end of the tibia bone. The needle was penetrated into the bone lumen by revolving movements. Then, the BM was aspirated gently with a syringe that contained 0.1 ml heparin sulfate. All BM samples were immediately mixed with 1 ml complete culture medium [high-glucose Dulbecco’s modified eagle medium (DMEM; Gibco, Paisley, UK), 15% fetal bovine serum (FBS; Gibco, Paisley, UK), 100 U/ml penicillin G, and 100 U/ml streptomycin (Gibco, Paisley, UK)].BM/medium mixture was centrifuged (1200 rpm for 5 min) and after removal of the supernatant, the cellular pellet was resuspended in fresh warm culture medium in 25 cm² cell culture flasks and transferred to an incubator (37°C and humidified 5% CO₂ atmosphere). The medium was changed every 3~4 days and the cells were sub-cultured prior to complete confluency.

According to criteria suggested by the International Society for Cellular Therapy (32), characterization of rat BM-MSCs was performed based on behavioral characteristics of the cells (adhesion of cells to the bottom of a plastic culture dish), expression of specific CD-markers (flow cytometric analysis) and the trilineage (bone, fat and cartilage) differentiation test. Expressions of CD44, CD90, CD11b and CD34 were assessed in P3 cells with specific phycoerythrin (PE) - or fluorescein isothiocyanate (FITC)-conjugated antibodies (Pharmingen BD, San Diego, CA).

For induction of differentiation into bone, fat and cartilage, P3 BM isolated cells were treated with osteogenic, adipogenic and chondrogenic media which was the same culture media used in our previous study (31) for 21 days. Generation of osteocytes, adipocytes and chondrocytes was confirmed by specific staining (alizarin red for osteogenic, oil red O for adipogenic and toluidine blue for chondrogenic differentiation), reverse transcription polymerase chain reaction (RT-PCR) and detection of the expressions of bone, fat and cartilage specific genes in the cells after 21 days treatment.

RT-PCR was utilized to detect the expressions of bone, cartilage and fat specific genes after treatment of BM-MSCs with osteogenic, chondrogenic, and adipogenic media. The RT-PCR process was performed according to former reports (33, 34). Briefly, TRIzol reagent (Invitrogen, Paisley, UK) was used to extract total RNA from the cells according to the manufacturer’s instructions. The extracted RNA underwent DNase treatment by DNase I (Takara, Shiga, Japan) for elimination of genomic DNA. Using random hexamer primer and the RevertAid^TM First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Roth, Germany), 2 μg of total RNA was reverse transcribed into cDNA. We used 2 μl of produced cDNA together with 1× PCR buffer (AMSTM; CinnaGen Co., Tehran, Iran); 200 μM dNTPs; 0.5 μM each of bone, fat and cartilage tissue specific forward and reverse primers; and 1U Taq DNA polymerase (CinnaGen Co., Tehran, Iran) for the PCR reaction. The primers used for detection of the tissue specific genes in this study are shown in Table 1.

A single intraperitoneal injection of busulfan (40 mg/kg BW, Sigma, USA) was used to induce infertility in the male rats. We confirmed the effects of busulfan on spermatogenesis and induction of infertility in a pilot study before beginning the main study. In this pilot study, we determined the effects of busulfan and its solvent dimethyl sulfoxide (DMSO, Sigma, USA) on testis histology and on sperm count in the vas deferens of the rats. We used 1 cm of the distal end of the vas deferens to determine the sperm count by a hemocytometer.

Previous reports have shown that PKH26 is an efficient and reliable dye for tracking of donor cells in the testis (35, 36), so, here we selected this dye for labeling of the BM-MSCs before transplantation into the testes. Labeling with PKH26 (Sigma, USA) was performed according to the manufacturer’s instructions. Briefly, after detachment of the BM-MSCs by trypsin/EDTA, the cells were counted and washed with medium and no serum. The supernatant was completely removed and immediately we added 1 ml of fresh dye solution that consisted of 2 μmol of PKH26 in 1 ml of PBS to each of the 10×10⁶ cells, after which the cells were resuspended by pipetting. The cell/dye mixture was incubated for 4 min. An equal volume of FBS (1 ml) was added to the suspension to block the staining process. After 1 min, the suspension was centrifuged at 1200 rpm for 10 min and the supernatant was cautiously removed. We washed the cells three times with 5 ml complete culture medium and after checking for viability, they were resuspended in 1 ml complete culture medium and placed on ice until transplantation.

PKH26-labeled cells were transplanted into the testes of infertile recipient rats by a method suggested by Ogawa and colleagues (37). There were three study groups in this research- 4 weeks, 6 weeks and 8 weeks. Each treatment group consisted of 6 male Wistar rats. In 5 rats, the left testis was considered as the non-injected control group whereas the right testis received BM-MSCs. In one rat, the left testis was not manipulated, while the other testis received culture medium without cells (medium only or sham group). For transplantation, the recipient rats were anesthetized according to the same method used for BM sampling. The testis was removed from the body cavity through an incision perpendicular to the midline at the upper region of the scrotum. The cell suspension at a concentration of 8–10×10⁶ cells/ml was transplanted into the rete testis via a sharp thin pipette (each testis received about 2.5×10⁵ labeled cells). For better monitoring of the transplantation procedure and observing movement of the cell suspension, a small amount of trypan blue was added to the injection medium. At the end of transplantation, the testis was returned to the body and the muscular layer and skin of the incised region were separately sutured.

The recipient testes were evaluated at 4, 6 and 8 weeks after transplantation of BM-MSCs. For this purpose the rats were euthanatized by CO₂ inhalation. The testes were removed and some of the seminiferous tubules were observed directly under fluorescent microscope (Olympus, Japan) in order to check the existence of PKH-positive transplanted cells. Then, the testes were placed in Bouin’s solution followed by 4% paraformaldehyde and processed to produce paraffin sections by standard procedures. The 5 μm sections were immunostained for Dazl and Stella (markers of GCs) and vimentin (a marker of SCs) and observed for the existence of PKH-positive cells (red fluorescent) that expressed other stained markers. After treatment of the sections with 3% H₂O₂ in distilled water for 30 min to eliminate endogenous peroxidase, the sections were washed twice in PBS for 5 min each time. Antigen retrieval was performed by boiling the sections in citrate buffer for 8~10 min in a microwave followed by washing twice with PBS/Tween (10 min each time). Next, the sections were placed in PBS with 5% goat serum (PBS-GS) for 1 hour at 37°C for blocking and then washed twice with PBS/Tween (5 min each time). Primary antibodies were rabbit polyclonal to Dazl (unconjugated, Abcam, Cambridge, MA, USA, 1/200 in PBS-GS), anti-Stella rabbit polyclonal IgG (unconjugated, Santa Cruz, CA, USA, 1/200 in PBS-GS) and mouse monoclonal to vimentin (FITC-conjugated, Abcam, Cambridge, MA, USA, 1/200 in PBS-GS). The slides were exposed overnight to primary antibodies at 2~8°C. Secondary antibodies were goat anti-rabbit IgG (Alexa Fluor^® 488-conjugated, Abcam, Cambridge, MA, USA, 1/200 in PBS-GS) for Dazl and goat anti-rabbit IgG (FITC-conjugated, Santa Cruz, CA, USA, 1/100 in PBS-GS) for Stella, which were incubated with the slides for 1 hour at room temperature. After washing three times with PBS/Tween (5 min each time), the cells’ nuclei were stained with DAPI (Vector Laboratories, Burlington, ON, Canada) and slides were assessed under a fluorescent microscope (Olympus, Japan). For each group, we determined the following items: existence of PKH-positive cells; percentage of tubules that contained transplanted cells; percentage of tubules positive for donor cells in their germinal epithelium (homed cells); numbers of transplanted BM-MSC-derived colonies and their ratio to the numbers of transplanted cells; percentage of tubules that contained homed transplanted BM-MSCs in their germinal epithelium that also expressed GC- or SC-specific markers; differentiation of transplanted cells into spermatogonia (any type), spermatocytes, spermatids or spermatozoa; calculation of the Tubular Differentiation Index (TDI) which is defined as the percentage of tubules that reach a particular stage of development (having at least three GCs at a specific stage) (38); existence of any transplanted cell-derived sperm (PKH-positive sperm) in the epididymal contents; and finally the existence of transplanted cells in the lungs, BM, spleen and liver (probable migration from the testes after transplantation).

One-way ANOVA and Tukey’s HSD were used for data analysis. Version 18 SPSS software (SPSS Inc., Chicago, IL, USA) was used for analysis. p<0.05 was considered to be statistically significant.

Three main characteristics of MSCs- adhesion onto the bottom of culture flasks, expression of specific CD markers and potency of differentiation into osteocytes, adipocytes and chondrocytes were checked in the isolated cells obtained from the BM samples. The isolated cells were adhesive cells that had fibroblast-like morphology in the culture. Flow cytometric analysis showed that isolated cells were highly positive for CD44 and CD90 and negative for CD11b and CD34 (Fig. 1). The trilineage differentiation test showed that after 21 days treatment of BM-MSCs with osteogenic, adipogenic and chondrogenic media, the cells differentiated into osteocytes, adipocytes and chondrocytes, respectively. Differentiation was confirmed by specific staining, RT-PCR, and detection of expressions of bone, fat and cartilage-specific genes in the treated cells (Fig. 2).

We performed a pilot study to confirm the efficacy of busulfan for induction of infertility in male rats. Histological evaluations indicated that a single injection of 40 mg/kg BW busulfan in the rats completely destroyed the germinal epithelium of the seminiferous tubules. We observed that DMSO (busulfan solvent) had no negative impact on the germinal layer. Busulfan effectively disturbed spermatogenesis and significantly decreased the sperm count (Fig. 3).

PKH-labeled BM-MSCs were transplanted into each testis through the rete testis by a thin pipette. The addition of trypan blue enabled us to monitor the medium flow in the seminiferous tubules (Fig. 4D).

We removed the testes at 4, 6 and 8 weeks after transplantation in order to determine the fate of transplanted BM-MSCs in the testes at different times after transplantation. Any sign of tumor formation or infection was recorded in the testes of the different study groups.

Direct observation of seminiferous tubules from all three study groups showed the presence of transplanted BM-MSCs in the testes at 4, 6 and 8 weeks post-transplantation (Fig. 4A and B).

Histological evaluations showed that although the transplanted cells were alive in the testes of all three treatment groups, the mean percentage of seminiferous tubules that contained donor cells differed among the three groups. This percentage was approximately 12.2% for 4-week, 4.8% for 6-week and 1.2% for 8-week testes (Table 2). Comparison of the results showed that the percentage of seminiferous tubules that contained transplanted cells decreased significantly with increasing time (Fig. 4C).

Evaluation of histological sections from the testes of the three treatment groups revealed that some of the transplanted PKH-positive BM-MSCs located or homed at the germinal epithelium on the basement membrane of the seminiferous tubules. The mean percentage of tubules that contained these homed cells was 0.16% for 4-week, 0.072% for 6-week and 0.008% for 8-week testes (Table 2). Immunohistochemistry showed that most of these homed cells expressed GC-specific markers, Stella and Dazl (Figs. 5 and 6). Fig. 5E shows a comparison of the mean percentages of homed cell-containing tubules in the three treatment groups.

Among all study group testes, only one testis from the 4-week group contained a cell colony-like compartment that originated from the transplanted PKH-positive cells (Table 2). Fig. 6 shows atransplanted PKH-positive cell-derived cell mass together with a number of homed cells that expressed the GC marker Dazl.

Testes tissue sections were immunostained for two GC markers (Dazl and Stella) and a SC marker (vimentin). Evaluations indicated that most transplanted cells that homed at the germinal epithelium of the seminiferous tubules expressed Dazl and/or Stella (Figs. 5 and 6). This finding confirmed the differentiation of BM-MSCs into male spermatogonia-like GCs. In addition, we measured TDI for different stages of GC development (spermatogonia, spermatocytes, spermatids and sperm). TDI for spermatogonia was 0.14% in 4-week, 0.05% in 6-week and 0.0068% in 8-week testes. TDI for spermatocytes, spermatids and sperm was 0 in all study groups. We observed no PKH-positive sperm in the epididymal (vas deferens) contents of all three groups. In addition, our results showed that transplanted MSCs did not express vimentin (Fig. 6E and F). They did not differentiate into SCs in any of the study groups (Table 2).

We assessed the lungs, BM, spleen and liver in order to determine if any labeled BM-MSCs migrated into other organs after transplantation into the testis. No PKH-labeled cells were observed in these organs in any of the treatment groups. Therefore no migration occurred after injection of the cells into the testes.