The aim of this project was to establish if DNA molecules bind to porcine spermatozoa after incubation in vitro. In an initial experiment, the DNA was mixed with porcine spermatozoa to test for the presence of DNases. From bacterial transformation and gel electrophoresis studies, it was established that the presence of DNases within the sperm or in the sperm suspension was negligible. By the addition of radiolabeled DNA, spanning a large size range, it was demonstrated that the DNA molecules interacted with the sperm during a 15 to 20 min period of incubation. These samples were centrifuged and washed extensively to confirm that the DNA was closely associated with the sperm. Under the experimental conditions used, it was calculated that approximately 3.8 x lo2 DNA molecules were associated with each spermatozoon. Percoll gradient experiments, which allowed differentiation between motile and nonmotile sperm, showed that motile sperm were more efficient at capturing DNA molecules than nonmotile sperm. In situ hybridization studies revealed that approximately 30% of the motile sperm carried the DNA on their surface, and the associated DNA was attached only to the sperm head with no attachment to the sperm tails. Heterologous foreign DNA can become closely associated in a nonrandom manner with porcine spermatozoa.

This study was conducted to evaluate the effects of an electric pulse (electroporation/ electropermeabilization) on the binding of foreign DNA molecules to porcine spermatozoa. We previously examined various parameters involved in the association of foreign DNA with sperm after a simple incubation procedure and now report the effects of electroporation on this association. Using end-labeled and random primer labeled X HindIII DNA fragments (23-0.125 kb), it was demonstrated that the DNA fragments interacted with the sperm after electroporation. These samples were then centrifuged and washed extensively to establish if any of the labeled DNA was associated with the spermatozoa. It was determined that approximately lo8 molecules of DNA were associated with 1.5 x lo7 mL-' motile spermatozoa after five medium washes. After each wash, samples were withdrawn for gel analysis and scintillation counting. Gel analysis followed by autoradiography revealed the distinctive band pattern of X HindIII DNA associating with sperm. In situ visualization studies with biotin-labeled DNA revealed that approximately 75 % of motile sperm carried DNA bound to the post-acrosomal region. However, the intensity of the binding varied, with some sperm being more strongly stained than others. Using [3H]dCTP-labeled DNA followed by light microscope autoradiography, approximately 70% of the sperm were strongly stained in the post-acrosomal region. There was a 5-10% increase in the amount of DNA bound by sperm when the samples were electroporated.

The aim of this study was to determine the fate of foreign DNA molecules bound to porcine sperm that had been capacitated and acrosome reacted in vitro using calcium ionophore and then used in the in vitro fertilization of zona-free hamster ova and zona-intact pig ova. Fluoresceinated Pisum sativum agglutinin (PSA) labeling was used to differentiate between acrosome-intact and acrosome-reacted sperm. This revealed that up to 80% of the sperm treated with calcium ionophore were acrosome reacted. Up to 70% of these acrosome-reacted sperm were labeled with the foreign DNA at the post-acrosomal region. Following association of DNA with the acrosome-reacted sperm, insemination droplets were prepared and zona-free hamster oocytes or zona-intact pig oocytes were added. The gametes were allowed to interact and then fixed and stained to visualize decondensed sperm heads that had penetrated into the oocytes. The sperm were stained with streptavidin peroxidases to detect the biotinylated foreign DNA bound to the decondensed heads. These studies revealed that 54% of fertilized hamster and pig oocytes contained decondensed sperm that had retained the post-acrosomal pattern of bound foreign DNA. After incubation with DNA-associated sperm, the oocytes were washed and cultured for 15-17 h. After fixation, up to 30% of hamster oocytes and 10% of porcine oocytes were found to contain sperm pronuclei. However, using the streptavidin peroxidase detection system, it was not possible to determine if any of these pronuclei contained the foreign DNA.

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 ...