The binding of exogenous DNA fragments to bovine spermatozoa

Abstract

Evaluación de la unión espermatozoide-ADN exógeno en espermatozoides porcinos eyaculados y epididimarios # SUMMARY Transgenic biotechnology is a powerful tool for the generation of genetically modified animals with applications in various fields such as veterinary, agriculture and biomedicine. Sperm mediated gene transfer (SMGT) is an interesting tool for animal transgenesis consisting on the intrinsic ability of the spermatic cells to bind and internalize exogenous DNA and allow their transfer into oocytes after fertilization, to become part of the genome of the new embryo. The seminal plasma plays an important role acting as a natural barrier and protecting the spermatozoa from exogenous molecules that could compromise their integrity. So, the epididymal spermatozoa are a valuable model to explore the possible effect of seminal plasma components. The objective of this study was to evaluate the interaction among sperm and transgene using Epididymal (EP) vs Ejaculated (EJ) sperm without seminal plasma. Linealized plasmid (GFP) (5.7 kb) labelled with fluorescein was added (1x10 8 spermatozoa/ml + 5µg DNA/ml) and incubated at 16 ºC. DNA binding and viability were measured simultaneously by flow cytometry during 120 minutes of incubation. The results showed that EP spermatozoa present a similar DNA-binding ability (12.63 ± 1.23% vs 10.94 ± 1.05%, P = 0.31) and viability throughout the incubation (14.64 ± 0.94% vs 13.42 ± 0.61%, P = 0.23) than EJ. We only detected a greater percentage of living DNA-bound spermatozoa in EP compared to EJ (2.10 ± 0.33% vs 1.05 ± 0.14%, P < 0.01). The DNA-binding was associated mainly to dead sperm or with low viability in both groups (EP: 10.53 ± 1.01% vs EJ: 9.89 ± 0.97%, P = 0.98). These results open new ways to explore and use epididymal spermatozoa in diverse applications (artificial insemination, in vitro fertilization and ICSI) associated with SMGT method. Palabras clave: espermatozoides, ADN exógeno, transgénesis, porcino. INTRODUCCIÓN La producción de organismos transgénicos ha sido el mayor avance en el ámbito de la biología en los últimos tiempos, siendo providencial en el estudio de mecanis-mos genéticos de regulación y desarrollo biológico. Se han producido numerosas mejoras en la tecnología de la transgénesis desde su inicio allá por el año 1981 (Gordon y Ruddle 1981), y son varios los sistemas de transferencia de ADN que se realizan hoy en día con una gran eficiencia y con unos costos reducidos. Desde hace casi dos décadas el uso de los espermatozoides como vectores de ADN exógeno (Sperm Mediated Gene Transfer: SMGT) ha centrado la atención de numerosos investigadores en un continuo debate sobre la transgénesis animal. En 1989 se publicó un trabajo donde se demostraba la capacidad que tenían los espermatozoides de transportar ADN exógeno y transferir estas moléculas mediante fecundación dando lugar a animales modificados genéticamente (Lavitrano y col 1989). Esto supuso un importante avance en el campo de la transgénesis, de manera que la SMGT podría ser considerada por ...