Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Abstract

Sperm mediated gene transfer (SMGT) could provide the opportunity to carry out transgenesis on a mass scale using spermatozoa as vectors for exogenous DNA. However, the efficiency of sperm-mediated DNA transfer is still questionable, and the mode of transmission to the egg has not yet been well understood. Our aim was to investigate the capacity of bovine spermatozoa to carry exogenous DNA and its relationship to sperm functionality. We studied these parameters using flow cytometry to measure viability (necrosis and apoptosis) and capacitation status, computer-assisted semen analysis (CASA) to measure motility parameters and in vitro fertilization (IVF) to assess fertilizing capacity. Furthermore, we studied the effect of capacitation status on interaction with exogenous DNA, and the role of heparin supplementation in this process. Bull spermatozoa showed a high capacity to bind DNA quickly and reached a maximum after 30 min, with approximately half of the DNA-bound spermatozoa being viable. Incubation with exogenous DNA induced a decrease in sperm viability and motility and increased the proportion of apoptotic cells, but did not affect the cleavage rate in IVF assay. Heparin increased high-lipid disorder and the number of sperm with DNA bound (viable and dead). In conclusion, this study shows that live spermatozoa can bind exogenous DNA with a slight negative effect in some parameters of sperm function that in our opinion, would not drastically compromise fertility.

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

“…A number of studies have employed indirect methods to investigate this phenomenon. These include liquid scintillation counting of radioactively labelled DNA co-precipitating with sperm (Lavitrano et al 2002, Kang et al 2008, Kim et al 2010, flow cytometry of sperm incubated with fluorescently labelled plasmids (Canovas et al 2010) and genomic PCR assays after incubating sperm with DNase to destroy any non-internalised exogenous DNA (Hoelker et al 2007, Kang et al 2008. While these assays demonstrated association between exogenous DNA and sperm, they provide no direct evidence for DNA uptake across the plasma membrane into the sperm cytoplasm or nucleus.…”

Transfección de espermatozoides bovinos para su posterior utilización en transgénesis mediada por espermatzoides (SMGT)

“…Los resultados demuestran que los espermatozoides poseen la capacidad intrínseca de unir ADN exógeno, ya que la totalidad de los espermatozoides unieron ADN luego de simple la co-incubación con ADN exógeno, observándose una alta proporción de ellos vivos (>75%). Estos resultados difieren con lo observado por Canovas et al, (2010), quien observaron alrededor de un 13% de espermatozoides vivos con ADN unido luego de incubar espermatozoides con ADN. En relación a la motilidad total y progresiva, los resultados observados son levemente superiores a lo reportado en el estudio de Canovas et al, (2010).…”

Nanobiotechnology in reproduction – pros and cons. A review

“…This method is designed to breed transgenic animals for use as preclinical models of human diseases, bioreactors for pharmaceutical products and in xenotransplantation experiments (Canovas et al, 2010;Parrington et al, 2011). Traditional and forced transfection methods for SMGT are electroporation, lipofection, DNA/ DMSO complexes, and restriction-enzyme-mediated integration.…”