Abstract
The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase (AP) staining and expression of vasa and thy1 genes as specific bovine SSC markers. To evaluate the effect of antioxidants on vitality, colony formation, and the expression of pro- and anti-apoptotic genes of bovine SSCs, various concentrations of vitamin C (5, 10, 25 and 50 μg/mL) and Trolox (a water soluble α-tocopherol analogue) (12.5, 25, 50 and 100 μg/mL) were added to the SSC culture medium. The results showed that SSCs treated with 50 μg/mL of vitamin C or 25 μg/mL of Trolox individually could increase cell viability and colony formation significantly in comparison with other concentrations and the control group. Additionally, the expressions of bax (as a pro-apoptotic gene) and bcl2 (as an anti-apoptotic gene) were significantly lower and higher than the control group, respectively. To optimize the transfection condition, the effective dosages of vitamin C or Trolox, with various concentrations of two transfection reagents (X-tremeGENE HP and Turbofect) and DNA, at day 8 of culture, were studied. Results showed that 1 μl X-tremeGENE HP or 0.5 μl Turbofect and 2 μg of DNA are the best concentrations for transfecting SSCs. However, X-tremeGENE HP expressed more potential for transfecting SSCs in comparison with Turbofect. Besides, no difference was observed between the use of defined doses of vitamin C or Trolox.Keywords: Apoptosis, gene transfer, primary cells, viability, vitamin C, Trolox
Highlights
Since the 1980s, when transgenic mice were first generated (Gordon et al, 1980), transgenesis has been used widely to produce genetically modified animals (Niu & Liang, 2008)
The main purpose of the first experiment was to evaluate the effectiveness of various concentrations of vitamin C and Trolox alone or in combination on the vitality of bovine spermatogonial stem cells (SSCs), colony formation and the expression of pro- and anti-apoptotic gens
The results from the MTT assay demonstrated that the viability of SSCs in cultures that were separately supplemented with 50 μg/mL of vitamin C or 25 μg/mL of Trolox was higher (P
Summary
Since the 1980s, when transgenic mice were first generated (Gordon et al, 1980), transgenesis has been used widely to produce genetically modified animals (Niu & Liang, 2008). Along with other gene transfer techniques, there has been cumulative interest in the possibilities of using the male germline to create transgenic animals (Aponte, 2009). Spermatogonial stem cell-based methods of transferring genes to new generations provide a promising future in the field of animal science to accelerate the generation of transgenic animals more rapidly and effectively (Wang et al, 2014). To gain these desirable outcomes, some difficulties need to be surmounted (Kim et al, 1997)
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