Isolation and maintenance of calves type A spermatogonia in vitro: in influence of epidermal growth factor on number of cells, colony size and spermetogoia viability

Mammalian spermatogenesis is a complex process of germ cell development within the seminiferous tubules, including the mitotic phase in which type-A spermatogonial stem cells renew themselves. The process of spermatogenesis is regulated not only by gonadotropins, i.e. FSH and LH, but also by the interactions between spermatogenic cells and somatic Sertoli cells (Skinner 1991 and Griswold 1995). Culture systems for recapitulating spermatogenesis in vitro enable us to identify and characterize the factors or genes involved in germ cell proliferation, meiosis and spermatogenesis. In mice, undifferentiated germ cell lines have been established by immortalizing the germ cells with the simian virus 40 large tumor antigens, or with telomerase catalytic component (Fenget al.2002), Type A spermatogonia include a very small number of SSCs and their more numerous differentiating daughter cells. Initial attempts to isolate SSCs started with the isolation of type A spermatogonia and SSC purification. Type A spermatogonia can be obtained in large numbers from young prepubertal bulls, and it is important to note that there are breed differences. Type A spermatogonia isolation can be achieved through mechanical dissociation and enzymatic digestion of the testicular tissue followed by two purification steps, with a final typical bovine type A spermatogonia suspension of 70%. An evaluation for SSC activity using a transplantation assay adapted for bovine SSCs is described. Bovine Type A spermatogonia can be maintained in vitro for short periods (7 to 15 days) with simple culture conditions. However, expansion of SSC can only be achieved under certain conditions such as specially supplemented medium, specific growth factors, and serial sub-culturing for longer periods of time. After expansion, bovine spermatogonia can be cryopreserved while retaining the ability to proliferate and survive (.Aponte&de Rooij 2008). Bovine culture systems require some form of feeder layer to support the germ cells. Autogenous Sertoli cells (Aponte et al. 2006) or embryonic fibroblasts (Oatley etal. 2004) have been used.
Material and Initially, sertoli cells and SSCs were isolated from 3–7-month-old calvesand, and The minced pieces of testis were suspendedin DMEM, which contained 0.5 mgmL−1 collagenase/dispase, 0.5 mgmL−1 trypsin and 0.08 mgmL−1 DNase, for 60 min (with shaking) at 37◦C.). After three washes in DMEM medium and removal of most of the interstitial cells, a second digestion step (45 min at 32◦C) was performed in DMEM by adding fresh enzymes to the seminiferous cord fragments. Separated from the remaining tubule fragments by centrifugation at 30g for 2 min at 37◦C. After filtration through a 70-μm nylon filter, the medium was aspirated, the cells were washed twice and fresh medium was added. Coated plastic dishes were prepared by incubation with a solution of 5μgmL−1 of datura stramonium agglutinin in phosphate-buffered saline (PBS) at 37◦C for 60 min, followed by extensive washing with PBS, supplemented with 0.5% bovine serum albumin The Sertoli cells were isolated using a procedure described by Scarpinoet al. (1998.) with some modifications. The mixed population of the cells obtained byenzymatic digestion was placed on lectin-coated dishes at a concentration of 1.5×105 cells cm−2 and incubated for 1 h at 32◦C in a humidified atmosphere of 5% CO2 in air. After the incubation, the non-adhering cells were collected by being washed twice with medium. After the incubation, the Non-adhering cells were collected by being washed twice with medium. Alternatively, 48 h after being plated on the lectin-coated dishes, the Sertoli cells were detached by ethylenediamine tetraacetic acid (EDTA)–trypsin treatment (0.02% EDTA–0.1% trypsin in Ca2+ and Mg-free PBS) for 5 min at 37◦C, counted and adjusted to desired densities into each well of a 24-well multidish for secondary culture in DMEM at 32◦C in the presence of 10% fetal bovine serum (FBS; Gibco). This method helped isolate the Sertoli cells with more than 95% purity. After the Sertoli cells had been isolated by DSA lectin, the spermatogonia that remained in suspension were collected and kept at 32◦C in a humidified atmosphere and the presence of 10% FBS. More than 95% of the cells were spermatogonial cells. The number of the Sertoli and spermatogonial cells was determined with a hemocytometer. Cell viability was evaluated by means of the dye exclusion test (0.04% trypan blue solution). Two days after the above-mentioned procedure, the Sertoli cells formed a confluent layer, and spermatogonial cells were co-cultured on top of them. Highly purified human FSH and) were added to spermatogonial cell (EGF supplementation: three groups received 4, 12, 20 and 50 ngmL−1of fresh EGF every 3 days for 2 weeks). The diameters and the number of colonies were determined every 3 days after the appearance of the colonies for 2 weeks. An inverted microscope (Zeiss, Germany) was used to determine the number of the colonies, their diameters being measured by ocular grid. The nature of cells in addition to the morphology and the activity of alkaline phosphatase were confirmed through specific markers Oct-4 and vimentin in colonies and Sertoli cells. In the last stage these cells were frozen-melted in the in vitro environment until the purity and strength of biological stem cell is evaluated.
The number and diameter of colonies increased more rapidly in the sertoli coculture, that it agreed with results of Anjamrooz et al., 2006 and Mohamamadi 2010. The numbers of colonies in the EGF-supplemented groups were not significantly different with the control group (P ≤0.05) (table 1-3), that they were Similar to results of Anjamrooz et al.2006 .The diameters of the colonies in the EGF-treated, in4, 12, 20 ngmL−1, had not significantly different with the control group (P ≤0.05) but in 50 ngmL−1 increased the diameters of the colonies slightly (P ≤0.05) that were Similar to results of Anjamrooz et al.2006 and Koruji et al.2009. In the EGF-treated groups, two pathways seem to have affected the spermatogonial cells. In these groups, the number of the colonies was lower than that of the control group.
The results of this study show that spermatogonial stem cells can be separated with high purity from the testicles of calves and also the mutual relationship between spermatogonial stem cells and Sertoli cells and some growth factors can be used to initiate, maintaining the process of spermatogenesis and the enrichment of stem cells during the culture and increasing the purity and viability of them during solidification.