Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

Abstract

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Living Organisms Author Their Read-Write Genomes in Evolution

“…It is probable that intercellular DNA transfer inside membrane vesicles will turn out to be more ecologically and evolutionarily important than we currently know [ 187 , 188 ]. The use of DNA uptake by sperm cells has proved to be easy, and, like T-DNA, has found broad biotechnology applications in the genetic engineering of animals [ 189 , 190 ]. Sperm-mediated DNA transfer illustrates the principle that horizontal transfer to multicellular eukaryotes encounters fewer obstacles at early stages of development, when genome isolation is less elaborated than in the mature organism [ 191 ].…”

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells

“…Spermatozoa from a variety of species have a spontaneous ability to take up exogenous DNA and internalize it in the nucleus (Anzar & Buhr, 2006). During the fertilization process, this foreign DNA is transferred from the spermatozoa to oocytes and further propagated during the development of transgenic embryos (Arias et al ., 2017). This technique is known as sperm-mediated gene transfer (SMGT) (Smith & Spadafora, 2005).…”

“…6 Laboratório de Vacinologia, Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brasil. transgenic embryos (Arias et al, 2017). This technique is known as sperm-mediated gene transfer (SMGT) (Smith & Spadafora, 2005).…”

A New Toolbox in Experimental Embryology—Alternative Model Organisms for Studying Preimplantation Development

“…Sperm as a vector can be employed during ICSI-mediated gene transfer, where semen is co-incubated with an exogenous transgene before conducting ICSI. This is especially of interest in pig [ 123 , 124 , 125 ], but the vector may also be used in bovine for IVF [ 126 , 127 ] and even AI for both pig and bovine, as well [ 128 , 129 ]. Unfortunately, these techniques come with high variability in success and unprecise modifications (reviewed in [ 130 ]).…”