Biomedical subjects

F Gannon

Publications and source records attributed to F Gannon.

129 records · Page 8Linked to original sources

PubMedDate not supplied

Association of foreign DNA with porcine spermatozoa.

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 10(2) 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.

Animals↗

PubMedDate not supplied

Effects of electropermeabilization on the association of foreign DNA with pig sperm.

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 lambda 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 10(8) molecules of DNA were associated with 1.5 x 10(7) mL-1 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 lambda 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.

Animals↗

PubMedDate not supplied

The fate of foreign DNA associated with pig sperm following the in vitro fertilization of zona-free hamster ova and zona-intact pig ova.

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.

Acrosome↗