The effect of combining vitamin E and C on the viability improvement of transfected ovine spermatogonial stem cells after cryopreservation and thawing
The present study aimed to identify whether vitamin E and C and their combination in basal freezing medium improved the viability of postthaw transfected spermatogonial stem cells (SSCs). For this purpose, SSCs were harvested by enzymatic digestion twice. After enrichment and culture on Sertoli cell feeder layer, SSCs were characterized by using alkaline phosphatase staining and expression of oct-4 and c-kit genes as specific stem cell markers. As to the transfection of SSCs, different concentrations of DNA (0.2, 0.4, and 0.8 μg) and turbofect (0.4 and 0.8 μL) in 100 μL of medium were studied. The results showed that cryopreservation of SSCs in the presence of 25 μg/mL vitamin E and 10 μg/mL vitamin C could increase cell viability and expression of antiapoptotic genes. The best combination of DNA and turbofect for transfection of SSCs in 100 μL of medium was 0.8 μg and 0.4 μL, respectively. However, SSCs indicated lower transfection efficiency compared with Sertoli cells (~30% vs. 70%, respectively). Cryopreservation with the addition of vitamin E and C in the basal freezing medium could increase cell viability of transfected SSCs as well. The findings of this study also suggest the need for further research regarding improvement of the transfection efficiency of SSCs.
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- Zhang Y, Wu S, Luo F, Baiyinbatu, Liu L, Hu T, Yu B, Li G,
Wu Y. CDH1, a novel surface marker of spermatogonial stem
cells in sheep testis. Journal of Integrative Agriculture 2014; 13:
1759-1765.
- Yu X, Riaz H, Dong P, Chong Z, Luo X, Liang A, Yang L.
Identification and IVC of spermatogonial stem cells in
prepubertal buffaloes. Theriogenology 2014; 81: 1312-1322.
- Herrid M, Olejnik J, Jackson M, Suchowerska N, Stockwell S,
Davey R, Hutton K, Hope S, Hill JR. Irradiation enhances the
efficiency of testicular germ cell transplantation in sheep. Biol
Reprod 2009; 81: 898-905.
Herrid M, Vignarajan S, Davey R, Dobrinski I, Hill JR.
Successful transplantation of bovine testicular cells to
heterologous recipients. Reproduction 2006; 132: 617-624.
- Aponte PM, de Rooij DG. Biomanipulation of bovine
spermatogonial stem cells. Animal Reproduction 2008; 5: 16-
22.
- Wang J, Cao H, Xue X, Fan C, Fang F, Zhou J, Zhang Y, Zhang
X. Effect of vitamin C on growth of caprine spermatogonial
stem cells in vitro. Theriogenology 2014; 81: 545-555.
- Oatley JM, Reeves JJ, McLean DJ. Biological activity of
cryopreserved bovine spermatogonial stem cells during in
vitro culture. Biol Reprod 2004; 71: 942-947.
- Aliakbari F, Gilani MAS, Amidi F, Baazm M, Korouji M,
Izadyar F, Yazdekhasti H, Abbasi M. Improving the efficacy of
cryopreservation of spermatogonia stem cells by antioxidant
supplements. Cellular Reprogramming 2016; 18: 87-95.
- Aliakbari F, Gilani MAS, Yazdekhasti H, Koruji M, Asgari
MR, Baazm M, Izadyar F, Nejad EK, Khanezad M, Abbasi
M. Effects of antioxidants, catalase and a-tocopherol on cell
viability and oxidative stress variables in frozen-thawed mice
spermatogonial stem cells. Artificial Cells Nanomedicine and
Biotechnology 2017; 45: 63-68.
- Izadyar F, Matthijs-Rijsenbilt J, Ouden K, Creemers LB,
Woelders H, de Rooij DG. Development of a cryopreservation
protocol for type A spermatogonia. J Androl 2002; 23: 537-545.
- van Pelt A, Morena A, van Dissel-Emiliani F, Boitani C, Gaemers
I, de Rooij D, Stefanini M. Isolation of the synchronized A
spermatogonia from adult vitamin A: Deficient rat testes. Biol
Reprod
1996; 55: 439-444.
- Ebrahimi M, Tavirani MR, Keshel SH, Raeisossadati R, Salavati
BH,
Daneshimehr F. Appraisal of fibroblast viability in different
concentration of glucose as mimicry diabetic condition.
Journal of Paramedical Science 2011; 2: 36-41.
- Kim KJ, Lee YA, Kim BJ, Kim YH, Kim BG, Kang HG, Jung
SE, Choi SH, Schmidt JA, Ryu BY. Cryopreservation of putative
pre-pubertal bovine spermatogonial stem 4 cells by slow
freezing. Cryobiology 2015; 70: 175-183.
- Iannolo G, Conticello C, Memeo L, De Maria R. Apoptosis in
normal and cancer stem cells. Crit Rev Oncol Hematol 2008;
66: 42-51.
- Koruji M, Movahedin M, Mowla SJ, Gourabi H. Colony
formation ability of frozen thawed spermatogonial stem cell
from adult mouse. Iranian Journal of Reproductive Medicine
2007; 5: 109-115.