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.
Horan
R
R
Department of Microbiology, University College, Galway, Ireland.
Powell
R
R
Gannon
F
F
Houghton
J A
JA
D000177:Acrosome / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D037102:Lectins; D008297:Male; D010063:Ovum / Q000502:physiology*; D037121:Plant Lectins; D013094:Spermatozoa / Q000502:physiology*; D013552:Swine; D015044:Zona Pellucida / Q000502:physiology*
D005456:Fluorescent Dyes; D037102:Lectins; D037121:Plant Lectins; C036292:pea lectin; D000001:Calcimycin; D004247:DNA; D016650:Fluorescein-5-isothiocyanate