Identification of exogenous factors affecting spermatogonial stem cells (SSCs) proliferation in vitro, provides worthy ways to study the basic biology of the cells. The aim of this study was to investigate the effects of a GnRH analogue (alareline acetate) on SSCs colonization in short-term co-culture with sertoli cells. Five, three-month old Holstein male calves were used to isolate spermatogonial and sertoli cells. Testicular germ cell collection was made by enzymatic digestion methods. The cells were co-cultured in a 15 day period and in vitro effects of various doses (0.5, 1, 2 and 4 µg/ml) of GnRHa on SSCs colonization were assessed. Effects of GnRHa on SSCs proliferation were dose dependent. In conclusion, it was demonstrated that 1 µg/ml GnRHa was the optimum dose for SSCs colonization in comparison with control group. The highest treatment dose (4 µg/ml GnRHa), negatively affected SSCs colonization in comparison with control group.

This study presents a simple method for isolation, expansion and purification of neonatal mouse spermatogonial stem cells. We used enzymatic digestion to isolate a cell suspension of spermatogonia and Sertoli cells from neonatal 2-day-old mice. The cells were cultured in DMEM/F12 that contained 10% serum for two weeks. Sertoli and spermatogonia cell characteristics were confirmed by examining for the presence of vimentin and PLZF proteins, respectively. To assess the rate of spermatogonia stem cell expansion, the area and number of colonies were measured during the two weeks of culture. At the end of the second week, we detected spermatogonia cell-specific expressions of the Stra8, Piwill2, DAZL, and Mvh genes. Current results indicated that isolated Sertoli and spermatogonia cells were immunopositive for specific markers. During the culture period, a significant difference was seen in the number and area of spermatogonial stem cell colonies (P<0.05) at four time points. In addition, spermatogonial specific gene expression demonstrated that these cells were undifferentiated after two weeks of culture. Our study showed that co-culture of spermatogonia and Sertoli cells from same source provides a convenient and efficient environment. This co-culture, without the addition of external growth factors and chemical manipulations, can be used for proliferation of spermatogonia stem cells.

Stem cells are generally defined as clonogenic cells capable of both self-renewal and differentiation. Probably the best method for long-term preservation of spermatogonial stem cells is cryopreservation. In this study, effects of Follicle Stimulating Hormone andTestosterone on viability rate of cryopreserved spermatogonial stem cell after Thawingwere investigated. Sertoli and spermatogonial cells were isolated from 3-5 months old calves. Cocultured sertoli and spermatogonial cells were treated with Follicle Stimulating Hormone and Testosterone in treatment groups before cryopreservation. Results indicated that Follicle Stimulating Hormone increased viability rate of cryopreserved spermatogonial cells in comparison with Testosterone and control group. In conclusion, using Follicle Stimulating Hormone provided proper bovine spermatogonial stem cell culture medium for in vitro culture and cryopreservation of these cells.

Spermatogenesis is a complex developmental process that originates from spermatogonia stem cell. This process consists of sequential, highly organized steps of cell proliferation and differentiation resulting in the generation of functional spermatozoa. Therefore, the aim of the present study was to determine the effects of human chorionic gonadotropin (hCG) on spermatogonial stem cell colony formation and differentiation after in vitro Coculture with Sertoli cells. In this experimental study, Sertoli and spermatogonial cells were isolated from 3- to 5-month-old calves. Co-cultured Sertoli and spermatogonial cells were treated with hCG in treatment groups before colony assay with 2, 5 and 10 IU/ml culture medium. Vimentin and oct-4 immunohistochemical staining were used for Sertoli and spermatogonial stem cell colony detection, respectively. The present study showed that hCG increase both colony diameter and colony number. hCG can induce both proliferative pathway and differentiation process in SSCs Coculture with Sertoli cells.

Spermatogenesis is a highly complex and regulated process in which germ stem cells differentiate into spermatozoa. These stem cells، called spermatogonial stem cells (SSCs)، are in the base of seminiferous tubules and have the ability of self-renewal and differentiation into functional germ cells. Due to this ability، SSCs can restore spermatogenesis after testicular damage caused by cytotoxic materials or following transplantation into an infertile recipient. Therefore، self-renewal of these cells is critical for the preservation of SSC populations and restoration of fertility. While previous studies have shown that the SSCs of mice and other species can survive and proliferate for long periods of time، little information is available about suitable culture media for the growth of human SSCs. Identification of SSC markers allows for the isolation of these populations of cells. The isolated cell can be expanded in culture and transplanted into infertile recipients. Consequently، the recognition of markers and the establishment of long-term culture systems for human SSCs will be essential for using the potential of these cells in a clinical setting. In this article، we focus on the markers that have been identified for human SSCs and in vitro culture techniques used for human SSCs proliferation.

Recently، attention has been diverted to the pluripotency characteristic of Spermatogonia Stem Cells (SSCs) in in-vitro culture. The aim of this study was to evaluate the morphological and molecular genetics changes of differentiated SSCs to Embryonic-like Stem cell (ESC). The SSCs were collected from neonatal mouse testis via a two-step mechanical and enzymatic digestion and cultured in DMED containing 15% FBS. ES-like cells colonies was appeared in the fifth passage. The expression of pluripotency and spermatogonia specific genes; Nanog، SOX2، Klf4، Stra8، DAZL، Plzf as well as the expression of puripotency markers Oct4 and Alkaline Phosphatase Activity (ALP) of ESC respectively was investigated using RT-PCR and immunocytochemical techniques and compared with ESCs. The pluripotent characteristic of ES-like cells derived from SSCs was confirmed based on morphological investigation and high expression of ALP as well as pluripotency marker Oct4. The expression of specific spermatogonia genes like Stra8، DAZL، Plzf was decreased and the expression of pluripotency genes such as Nanog، SOX2، Klf4 was increased during the differentiation of SSCs to ES-like cells. In vitro culture of SSCs was conformed to induced ES-like cells. This process was accompanied by wide alteration in molecular profile expression of specific spermatogonia and pluripotency genes. In over all، SSCs can be considered as an opening window to generate pluripotent stem cell.

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.
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.
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 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.

The goal of this paper was to study the effect of ginger extracts and vitamin E on the performance of ovine Spermatogonial Stem Cells (SSCs). For this purpose, different concentrations of hydro and hydroethanolic extracts of ginger and vitamin E on viability, colony formation and expression of inhibiting (bcl2ll and bcl2)and inducing (bax) apoptosisgenes were studied. Spermatogonial Stem Cells (SSCs) were extracted from lamb testes with slaughterhouse origin using two steps enzymatic digestion method and enrichment by differential plating method. The characterization of SSCs was carried by alkaline phosphatase staining and expression of c-kit and oct-4 genes. Results have shown that the viability of SSCs was decreased significantly by using more than 800 µg/mL of hydroethanolic extract of ginger, in comparision with control group (P

This study was aimed to investigate the effect of Calligonum extract on the number of colonies, ROS production and apoptosis related genes expression in sheep’s spermatogonial stem cells (SSCs). In this study, SSCs at the basal membrane of seminiferous tubules were isolated from testes of Afshari sheep using enzymatic digestion steps. The samples assigned into four groups. The control group received basic medium and the other groups contained H2O2 (30 µM), Calligonum (10 µg/ml) and Calligonum+H2O2 (30 µM H2O2 along with 10 µg/ml Calligonum), respectively. The cells were cultured for 3 weeks and the colonizing abilities were evaluated on the 5th, 14th and 21th days of culture. At the end of culturing period, ROS production and apoptosis related genes expression (BAX and BCL2) were evaluated. On the 5th and 14th days of culture, the number of colonies were higher (P≤0.05) in the control and Calligonum groups compared to the other groups. On the 14th day of culture, the highest and the least (P≤0.05) number of colonies were observed in Calligonum and H2O2 groups, respectively. The least and the highest (P≤0.05) rate of ROS contained SSCs, BAX expression and BAX/BCL2 ratio were observed in Calligonum and H2O2 groups, respectively. Moreover, the highest (P≤0.05) expression of BCL2 gene was found in Calligonum group. In conclusion, using Calligonum extract in culture medium could be a helpful strategy to improve the quality and decrease apoptosis status in sheep’s SSCs during research studies.