Colony formation ability of frozen thawed spermatogonial stem cell from adult mouse

Background: The basis of spermatogenesis is the spermatogonial stem cells (SSCs). The concentration of SSCs is very small. However, a system that supports the proliferation and maintenance of SSCs in vitro could be used to preserve and expand SSCs numbers as well as increase success in transplantation. It is a new avenue to restore spermatogenesis in azoospermia subjects. Objective: Proliferation and enhancement of frozen-thawed SSCs numbers during in vitro culture. Materials and Methods: Both Sertoli and spermatogonial cells were isolated from adult mouse testes. Frozen-thawed spermatogonial cells were cultured in two groups: simple culture (Experimental 1) and co culture with Sertoli cells (Experimental 2). Also, Fresh cells were considered as control groups: simple culture (control1) and co culture with Sertoli cells (control 2).Assay of the spermatogonial-cell-derived colonies was carried out at the end of each week. Results: Results indicated that the viability rate of the frozen cells after thawing (68.4±10.2%) was influenced by cryopreservation procedure significantly (p =0.001). In addition, the number of the colonies and their diameters in the co-culture system with fresh cells (25.1±5.2 and 205.8±50 μm, respectively) were more than other groups and the differences were significant (p<0.001). Number of the colonies and their diameters in experimental 1(9.5±4.3 and 124±35.9 μm, respectively), experimental 2 (15.6±3.5 and 157.6±41.9μm, respectively) groups were better than control 1 group (3.1±2.2 and 87.5±30.6μm, respectively) and the differences were significant (p<0.001). Conclusion: We demonstrated that co-culture system with Sertoli cells can increase in vitro colony formation of adult fresh and frozen-thawed spermatogonial cells in mouse

The roles of Sertoli cells in fate determinations of spermatogonial stem cells

Background: Spermatogenesis is a complex and highly organized process of proliferation and differentiation of spermatogonial stem cells. Spermatogonial stem cells (SSCs) as a unique stem cell have the potential to self-renewal, differentiation and transmit genetic information to the next generation and play a vital role in maintaining fertility. Sertoli cells as the only somatic cells within the seminiferous epithelium play central roles in the formation of niche and balance between self-renewal and differentiation by secrete many growth factors. Given the importance and widespread use of SSCs, particularly in the treatment of infertility, the aim of this study was to create an optimal environment for the proliferation of SSCs. So we decided to study of undifferentiated (ID4) and differentiated (c-Kit) gene expression in SSCs followed by co-culture with Sertoli cells for a one-month. Methods: This experimental study was conducted from November 2013 to December 2014 in Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, on immature NMRI mouse (6-3 days old). Initially, Sertoli cells and SSCs were isolated from neonates mouse testes during the two-step enzymatic digestion characteristics Sertoli cells with vimentin marker and SSCs with promyelocytic leukemia zinc-finger (PLZF) marker were confirmed. Then SSCs were cultured in two groups: co-culture with Sertoli and without co-culture (control). Undifferentiated (ID4) and differentiation (c-Kit) gene expression were evaluated by Real-time PCR technique. Results: Spermatogonial stem cells purity was obtained 66.91% by flow cytometry. The relative expression levels of gene ID4 in co-culture group at the end of each week, compared to the control group showed a significant increase (P<0.05). While the expression of this gene significantly decreased in each group over time (P<0.05). The results of the comparison of the relative expression of c-Kit gene in co-culture group are indicated significant decrease than the control group at the end of each week (P<0.05). In addition, this gene expression was showed significant increase in each group individually over time (P<0.05) ID4 gene expression showed a significant (P<0.05) increase toward the control group, while in the expression of c-Kit was observed a significant (P<0.05) decrease compared with the control group at the end of each week. Conclusion: According to the results of this study, co-culture with Sertoli cells maintains SSCs in the prolifration stage for long-term, so can be used to optimize the culture medium at the clinic

Autologous Transplantation of Adult Mice Spermatogonial Stem Cells into Gamma Irradiated Testes

Objective: We evaluated structural and functional changes of fresh and frozen-thawed adult mouse spermatogonial stem cells following auto-transplantation into gamma-irradiated testes.Materials and Methods: In this experimental research, the right testes from adult mice (n=25) were collected, then Sertoli and spermatogonial cells were isolated using two-step enzymatic digestion, lectin immobilization and differential plating. Three weeks after cultivation, the Bromodeoxyuridine (BrdU)-labeled spermatogonial cells were transplanted, via rete testis, into the other testis of the same mouse, which had been irradiated with 14Gy. The mice were transplanted with: fresh cells (control 1), fresh cells co-cultured with Sertoli cells (control 2), the frozen-thawed cells (experimental 1) and frozen-thawed cells co-cultured with Sertoli cells (experimental 2). The morphological changes between different transplanted testes groups were compared in 8 weeks after transplantation. The statistical significance between mean values was determined by Kruskal Wallis and one-way analysis of variance in efficiency of transplantation.Results: The statistical analysis revealed significant increases in the mean percentage of testis weight and normal seminiferous tubules following spermatogonial stem cells transplantation in the recipient’s testes. The normal seminiferous tubules percentage in the co-culture system with fresh cells and frozen-thawed groups were more than those in non-transplanted and fresh cell transplanted groups (p≤0.001).Conclusion: Our results demonstrated that spermatogonial stem cells in the colonies could result sperm production in the recipient’s testes after autologous transplantation

The effect of aqueous extract of Phoenix Dactylifera Pollen on In vitro viability and proliferation rate of neonatal mouse spermatogonial stem cells

Introduction: There is a fast growing tendency in the consumption of herbal remedies in the developing countries. One of the traditional medicines used for male infertility is Date palm (Phoenix dactylifera) pollen (DPP). The goal of the present study was to investigate the effect of aqueous extract of DPP on In vitro viability and proliferation rate of neonate mouse spermatogonial stem cells (SSCs). Methods: cell suspension includes sertoli cells and SSCs were isolated from neonatal 6 day-old mice testes by 2 steps enzymatic digestion. The cell suspension was cultured in DMEM and FCS 4% in the absence or presence of 0.06, 0.25 and 0.62 mg/ml of aqueous extract of DPP for 2 weeks. In order to evaluate the rate of SSCs expansion at the end of culture, the mean number of whole cells and living cells were considered as proliferation and survival rates respectively. Data analysis was done with ANOVA test. The significancy of the data was analyzed using ANOVA and Tukey post test. Results: The results showed that there were no significant differences between the mean percent of viability and proliferation rate between control and 0.06, 0.25 and 0.62 mg/ml of DPP-treated groups (P> 0.05). Conclusion: Our study showed that treatment of neonatal mouse testicular cell suspension with DPP had no toxic effects on viability percent and proliferation rate of these cells. Thus, we can use DPP for evaluate the in vitro pattern of SSCs colonization in the future studies