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Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT

Published online by Cambridge University Press:  15 January 2018

Esther Sánchez-Villalba ,
María Elena Arias ,
Fabiola Zambrano ,
Pía Loren  and
Ricardo Felmer
Esther Sánchez-Villalba
Affiliation:
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile. Student of Doctoral Program in Sciences in Applied Cellular and Molecular Biology, Universidad de La Frontera, Temuco, Chile.
María Elena Arias
Affiliation:
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile. Department of Animal Production, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile.
Fabiola Zambrano
Affiliation:
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile.
Pía Loren
Affiliation:
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile. Student of Doctoral Program in Sciences in Applied Cellular and Molecular Biology, Universidad de La Frontera, Temuco, Chile.
Ricardo Felmer*
Affiliation:
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Montevideo 0870, P.O. Box 54-D, Temuco, Chile. Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile.
*
All correspondence to: Ricardo Felmer. Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Montevideo 0870, P.O. Box 54-D, Temuco, Chile. Tel: +56 45 2325591. E-mail: rfelmerd@gmail.com
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Summary

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

Keywords

CattleEmbryosICSISMGTSpermatozoaTransgenic

Information

Type
Short Communication
Information
Zygote , Volume 26 , Issue 1 , February 2018 , pp. 104 - 109
Copyright
Copyright © Cambridge University Press 2018 

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References

Anzar, M. & Buhr, M.M. (2006). Spontaneous uptake of exogenous DNA by bull spermatozoa. Theriogenology 65, 683–90.Google Scholar
Arias, M.E., Sanchez, R., Risopatron, J., Perez, L. & Felmer, R. (2014). Effect of sperm pre-treatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection. Reprod. Fertil. Dev. 26, 847–54.Google Scholar
Eghbalsaied, S., Ghaedi, K., Laible, G., Hosseini, S.M., Forouzanfar, M., Hajian, M., Oback, F., Nasr-Esfahani, M.H. & Oback, B. (2013). Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos. Reproduction 145, 97–108.Google Scholar
Feitosa, W.B., Mendes, C.M., Milazzotto, M.P., Rocha, A.M., Martins, L.F., Simoes, R., Paula-Lopes, F.F., Visintin, J.A. & Assumpcao, M.E. (2010). Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation. Theriogenology 74, 563–8.Google Scholar
Garcia-Vazquez, F.A., Garcia-Rosello, E., Gutierrez-Adan, A. & Gadea, J. (2009). Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT. Theriogenology 72, 506–18.Google Scholar
Goodla, L., Morrell, J.M., Yusnizar, Y., Stalhammar, H. & Johannisson, A. (2014). Quality of bull spermatozoa after preparation by single-layer centrifugation. J. Dairy Sci. 97, 2204–12.Google Scholar
Lavitrano, M., Giovannoni, R. & Cerrito, M.G. (2013). Methods for sperm-mediated gene transfer. Methods Mol. Biol. 927, 519–29.Google Scholar
Li, C., Mizutani, E., Ono, T. & Wakayama, T. (2010). An efficient method for generating transgenic mice using NaOH-treated spermatozoa. Biol. Reprod. 82, 331–40.Google Scholar
Moisyadi, S., Kaminski, J.M. & Yanagimachi, R. (2009). Use of intracytoplasmic sperm injection (ICSI) to generate transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 32, 47–60.Google Scholar
Moreira, P.N., Giraldo, P., Cozar, P., Pozueta, J., Jimenez, A., Montoliu, L. & Gutierrez-Adan, A. (2004). Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection. Biol. Reprod. 71, 1943–7.Google Scholar
Perry, A.C., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyoda, Y. & Yanagimachi, R. (1999). Mammalian transgenesis by intracytoplasmic sperm injection. Science 284, 1180–3.Google Scholar
Sim, B.W., Cha, J.J., Song, B.S., Kim, J.S., Yoon, S.B., Choi, S.A., Jeong, K.J., Kim, Y. H., Huh, J. W., Lee, S. R., Kim, S. H., Lee, C. S., Kim, S. U., Chang, K.T. (2013). Efficient production of transgenic mice by intracytoplasmic injection of streptolysin-O-treated spermatozoa. Mol. Reprod. Dev. 80, 233–41.Google Scholar
Szczygiel, M. A., Moisyadi, S., Ward, W.S. (2003). Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol. Reprod. 68, 1903–10.Google Scholar
Zambrano, F., Aguila, L., Arias, M. E., Sanchez, R. & Felmer, R. (2016). Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100. Theriogenology 86, 1489–97.Google Scholar
Zhao, Y., Yu, M., Wang, L., Li, Y., Fan, J., Yang, Q. & Jin, Y. (2012). Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer. Mol. Biol. Rep. 39, 2659–64.Google Scholar