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

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