Hosein SHABANI, Mohammad ZANDI, Hamideh OFOGHI, Mohammad Reza SANJABI, Khosro HOSEINI PAJOOH

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.

Keywords:

Spermatogonial stem cells cryopreservation, transfection, vitamin E, vitamin C, sheep,

PDF

___

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.