title: Cell viability of bovine spermatozoa subjected to DNA electroporation and DNAse I treatment.
abstract: Many mechanisms involved in sperm-mediated gene transfer (SMGT) are still unknown. It is still a matter of debate whether exogenous DNA fragments incorporated by the embryo are originated from those bound to the sperm membrane or by those that penetrated the intracellular compartment. In an attempt to elucidate the transmission mechanism of exogenous DNA molecules by sperm, some authors suggested a treatment with DNAse I to remove DNA molecules outside the sperm. But little is known regarding the effects of DNAse I treatment on sperm viability and its impact on sperm organelles. An important aspect of the SMGT technique is the amount of exogenous DNA incubated with sperm, which may influence the internalization rate. Due to the inconsistencies found in literature, this work aimed to contribute to bovine sperm physiology knowledge evaluating the effects of different DNA concentrations, electroporation, and DNAse I treatments on sperm viability characteristics, DNA uptake, and IVF. For that, the effects of different concentrations of exogenous DNA (250, 500 and 1000 ng/10(6) cells) and incubation or electroporation were tested on sperm functional characteristics and in vitro embryo production. No effect of DNA concentration was observed on uptake, plasma membrane integrity, and mitochondrial membrane potential. The addition of exogenous DNA induced a decrease on acrosomal lesion in the 500-ng group when compared to the control. Cells incubated with DNA, electroporated, and treated with DNAse I presented a deleterious influence on mitochondrial membrane potential. In vitro fertilization was made with 1000 ng of DNA, sperm cells incubated or electroporated followed by DNAse I treatment. No significant difference was found in cleavage rate. Blastocyst rates were 24.36% for the control; 19.65% for incubated; 3.5% for electroporated control; and 17.40% for electroporated. There is a significant difference in blastocyst rate between the control and electroporated control groups. The incubated group yielded five and electroporated two positive blastocysts evaluated by epifluorescence microscopy. Polymerase chain reaction screening shows 17% of positive embryos for incubation and 11% for electroporation. Fluorescence in situ hybridization showed the presence of exogenous gene in embryos. These results show that exogenous DNA molecules can be conducted by an intracellular mechanism. The SMGT protocol using electroporation and DNAse I treatment reduces sperm mitochondrial function, in vitro embryo production and increases sperm DNA fragmentation.
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Paulo Varoni
authors.initials: PV
authors.lastname: Cavalcanti
authors.affiliation: Natural Sciences and Humanities Center (CCNH), Federal University of ABC (UFABC), Santo André, São Paulo, Brazil.
authors.forename: Marcella Pecora
authors.initials: MP
authors.lastname: Milazzotto
authors.affiliation: Natural Sciences and Humanities Center (CCNH), Federal University of ABC (UFABC), Santo André, São Paulo, Brazil.
authors.forename: Renata
authors.initials: R
authors.lastname: Simões
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Marcilio
authors.initials: M
authors.lastname: Nichi
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Flavia Regina
authors.initials: FR
authors.lastname: de Oliveira Barros
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Visintin
authors.affiliation: Department of Animal Reproduction, School of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, São Paulo, Brazil. Electronic address: meoaa@usp.br.
authors.forename: Mayra Elena Ortiz D'Avila
authors.initials: ME
authors.lastname: Assumpção
mesh_terms: D000818:Animals; D002417:Cattle / Q000502:physiology*; D002470:Cell Survival; D003850:Deoxyribonuclease I / Q000378:metabolism*; D018274:Electroporation / Q000662:veterinary*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D008297:Male; D010957:Plasmids; D013094:Spermatozoa / Q000502:physiology*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003850:Deoxyribonuclease I
keywords: Cattle; In situ hybridization; Recombinant farm animals; Reproduction; Spermatozoa; Transgenic