Introduction

Proliferation and self-renewal of spermatogonial stem cells is based on the presence of hormonal support and growth factors (1). Hormonal support is very important for proliferation of germ cells (2) and can be used for increase in transplantation efficiency. It is postulated that germ cell development is controlled by the hypothalamic-pituitary-gonadal axis (3) and established that spermatogenesis is gonadotropin-dependent, and optimal function of the testis is supported by FSH and by intratesticular androgens induced by luteinizing hormone (4). Human chorionic gonadotropin (hCG) has more than 90% similarity with Luteinizing Hormone (LH) in structure and function (5) and therefore can be used alternatively. Androgen production is induced by LH and promotes germ cell proliferation and maturation. Hypophysectomy or neutralization of circulating gonadotropins increases degeneration of spermatogenic cells (6 - 8).
Spermatogonial cell transplantation is an efficient technique that can help infertility treatment and leads to restoration of spermatogenesis (9). It may be proposed to preserve a fraction of the testicular stem cell of the patient that awaits cancer chemo or radiotherapy for use as auto-transplant after treatment (9). This may be especially effective for prepubertal boys in whom spermatogenesis has not started and thus are lacking sperm.
Combining germ cell transplantation with culture and germ cell population enrichment of spermatogonia and hormonal support opens new pathways for conservation of livestock and plays important roles in medical sciences and reproduction. Events that cause an excessive loss of germ cells such as exposure to high doses of irradiation stimulate their proliferation (10, 11). Self-renewal and differentiation of the testis stem cells is dependent on their close association with Sertoli cells in the seminiferous epithelium (12). These processes are mediated by growth factors produced by the Sertoli cells, which induce or inhibit self-renewal, differentiation and further development of all germ cells (13, 14).
Hormonal environment following transplantation is an essential factor for effective proliferation of transplanted stem cells and absence of such hormonal support may lead to failure in stem cell function and transplantation efficacy. Previously the effects of hCG injection on postpubertal germ cell maturation and androgen production in rat testis was evaluated (15). However, to our knowledge, the effects of hCG treatment on germ cell status and their proliferation before and after spermatogonial stem cell transplantation has not been studied. Therefore, the aim of this study is to evaluate the effect of different doses of hCG on postnatal testicular germ cell proliferation in normal and following stem cell transplantation.

Materials and Methods

Isolation and culture of mouse type A spermatogonia: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 day 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.
Immunocytochemical localization of c-kit receptor and cytokeratin: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 antibody. 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.
Cytochemical detection of alkaline phosphatase: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, th

Result

Regeneration of spermatogenesis after busulfan injection: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.
Immunostaining: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).
Capsular thickness examination and spermatogenesis regeneration of the control and busulfan treated mice testes: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 (Figures 4a-d).
Transplantation efficiency: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 tu

Discussion

Biomedical researches have been boosted after stem cell discovery. The testis is among the organs that contain adult stem cells and proliferation and differentiation of these cells play an important role in continuation of spermatogenesis (19). Research on spermatogonial stem cells can provide the scientific community with valuable information about the process of spermatogenesis. Using these cells may enlighten the future of infertility treatment (20). By time, some of the unaffected stem cells undergo spontaneous regeneration which may have important application in spermatogonial stem cell transplantation (21). This study revealed that increase in germ cell population leads to stem cell expansion that appears following busulfan treatment. Previous morphological studies have contributed to form the basic concept of stem cell self-renewal (22). Our study revealed that regeneration of spermatogonial stem cells after busulfan treatment increased the efficiency of transplantation.
The results of busulfan injection confirmed that regeneration after cytotoxic treatment is based on stem cell expansion. Removal of endogenous germ cells by busulfan treatment of recipient testes had a significant impact on donor stem cell colonization efficiency. As busulfan destroys primitive germ cells that account for less than 1% of the total number of testis cells (23, 24), most of the differentiated progenitor cells remain and continue differentiation with normal kinetics. However, the latter gradually mature and disappear by 35 days, owing to the absence of self-renewal activity (23, 24).
Therefore, as the stem cell numbers continue to recover, the number of differentiated germ cells decreases, and the ratio of stem cells to differentiated germ cells changes markedly during regeneration. Eventually, all progenitor cells must be derived from the stem cells that initially survived busulfan injection. These processes are reflected in the changes in the weight and cell recovery observed during regeneration. The renewed and the re-induction of spermatogenesis in the left testis can be used as a measure of evaluating regeneration of spermatogonial division.
In this regard spontaneous recovery of spermatogonial stem cells in the right testis makes this evaluation difficult as there is no specific markers for discrimination of the donor stem cells for the endogenous recipient stem cells which is usually performed using transgenic animals.
Since, in our study, long term analysis of spermatogonial regeneration was not necessary and the objective was to survey the effect of hormone administration as well as stem cell transplantation on cell proliferation, we chose to administer the simple and reliable technique of BrdU incorporation in spermatogonial stem cells instead of using transgenic animals.
The present study used a mouse model and demonstrated that hCG injection following stem cell transplantation and evaluation of cyclin B1 expression, has increased the efficiency of transplantation. Our results showed that hCG leads to increase in the proliferation of stem cells that may be due to increase testosterone levels shortly after injection. These results are in agreement with previous studies in rat (15).
However, hCG administration to neonate mice may have adverse effects on germ cell population, androgen secretion and probably germ cell apoptosis. Furthermore, the effects of hCG administration on the testis were time dependent, i.e. early administration of hCG in neonate mice increased testosterone levels and the number of germ cells. However, late administration of hCG showed absolutely opposite effects. The terms early and late refer to the evaluation of cell number and testosterone levels in a short and long time after hCG administration, respectively. The adverse effect of hCG on germ cells is probably due to the androgen withdrawal. Administration of hCG stimulates androgen production by the prepubertal testis (25).
Following withdra

Acknowledgement

We greatly appreciate the collaboration of Dr. A. S. Tabatabaei Panah, Dr. E. Javadi and Dr. H. Soltanghoraee for their helpful discussions. We would also like to thank Dr. H. Zeraati for the statistical analyses and G. E. Kazemisefat, G. Ghasemi, M. Mobaraki, and M. Rashedan for their excellent technical assistance.