Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Abstract

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (nZ96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (nZ751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68Z56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (nZ48) or GFP fluorescence (nZ94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

Evaluating bovine sperm transfection using a high-performance polymer reagent and assessing the fertilizing capacity of transfected spermatozoa using an in vitro fertilization technique

“…Analysis of IVF-derived bovine embryos revealed no EGFP expression, indicating that bovine spermatozoa incubated with exogenous DNA and X-tremeGENE HP could not fertilize oocytes. A similar result was reported by Eghbalsaied et al (2013), Canovas et al (2010) and Pereyra-Bonnet et al 2011, who did not detect any EGFP fluorescence signal among fertilized embryos. Inasmuch as in vitro fertilization of bovine oocytes using transfected sperm cells did not result in producing transgenic embryos, we sought another approach by which the fertilizing potential of spermatozoa could be kept after transfection.…”

“…In comparison with the control group, X-tremeGENE HP could remarkably increase DNA uptake which was congruent to the study of Hoelker et al (2007). In contrast, the percentage of DNA-bound spermatozoa in this study was lower than the report of Eghbalsaied et al (2013), who used Lipofectamine for transfection of bovine sperm cells, and higher than the results of Hoseini Pajooh et al (2016) and Teymoornejad et al (2017), who transfected the bulls' spermatozoa with the same reagent after 60 and 30 min incubation, respectively. This variability in transfection efficiency could be due to the kind of construct and the concentration of DNA (Sperandio et al, 1996;Lavitrano et al, 2003), the characteristics of the liposome (Yonezawa et al, 2001) and gene transfer method (Teymoornejad et al, 2017).…”

“…Sasaki et al (2000) revealed a considerable reduction in sperm motility and fertility rate in murine sperm cells, and Jurkiewicz et al (2010) indicated a decrease in sperm cells' motility and percentage of live spermatozoa of boars. Eghbalsaied et al (2013) reported that bovine spermatozoa transfected with Lipofectamine were impotent to fertilize the oocytes. Another study by Hoseini Pajooh et al (2016) identified that Lipofectamine was unable to improve the transfection rate and motility of ovine sperm cells.…”

Transgenic bovine as bioreactors: Challenges and perspectives

“…34 Initially, SMGT was reported to be inefficient with bovine sperm for the production of blastocysts in vitro. 35 A modification of this method using spermatogonial stem cells now offers a new method for producing transgenic animals. These cells can be isolated from the testis, modified genetically and transplanted into the seminiferous tubules of males, where they start the production of transgenic sperm to be used in in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) techniques.…”

Functionalization of Mesoporous Silica Nanoparticles with a Cell-Penetrating Peptide to Target Mammalian Sperm in vitro

“…These techniques include, but are not limited to, gene transfer into reproductive tissues [3,4], sperm-mediated gene transfer (SMGT) [5], an investigative tool based on the physiological property of sperm to bind, incorporate and deliver exogenous DNA into the oocyte at the time of fertilization resulting in the production of transgenic/mosaic embryos, and the sorting of sperm into subpopulations driven by the detection of a specific intracellular marker, for example, sex sorting [6]. Although these experimental techniques have been extensively studied over the last few decades, their efficacy remains suboptimal [3,[6][7][8], and their relative application is thus compromised.…”