Electroporation of bovine spermatozoa to carry foreign DNA in oocytes.
In the present study, electroporation was used to test the ability of spermatozoa to carry foreign DNA into the bovine oocytes. Frozen-thawed bovine spermatozoa (10(7)/ml) were electroporated using six different combinations of voltage (500, 1,000, or 1,500 V) and capacitance (1 or 25 microFarads) in the presence of 1 mg/ml of plasmid pRGH527. The portions of plasmids retained by sperm cells after three washings (stable for ten washings) were 4.3, 5.5, 5.1, 6.0, 6.8, and 5.8% for 1 microFarad, 500, 1,000, and 1,500 V and 25 microFarads, 500, 1,000, and 1,500 V, respectively. Nonelectroporated cells have retained only 1% of plasmids. In the same experiment, electroporated spermatozoa were acrosome reacted by treatment with ionophore A23187 to evaluate the fraction of marked plasmids joined at the acrosomal membrane. The results show that 3.5, 5.0, 4.4, 5.0, 6.3, and 4.4% remain tied to the ionophore-treated sperm. Only 0.7% of plasmid was retained after removal of the acrosome of nonelectroporated cells. Acrosome reaction was not significantly induced by the electrical field (EF) (P less than 0.005). EF decrease motility significantly for greater than 100 V in 0.3 M mannitol (M) and mannitol-TALP (MT) (1/1) media and greater than or equal to 500 V (P less than 0.05) in TALP medium. The retained plasmid rate was compared between TALP medium M and MT media and resulted in a percentage of 1.0, 2.5, 6.5 at 1 microFarads, 100 V, and 0.9, 3.8, and 3.8 at 25 microFarads, 100 V in TALP, MT, and M medium, respectively. Sperm cells electroporated at 1 microFarad, 500 or 1,000 V, 25 microFarad, 500 V or 1,000 in TALP medium hold plasmids in proportion of 5.2, 5.4, 7.4, and 6.0%. Electroporation above 100 V in M and MT killed the cells. In a part of this experiment, spermatozoa electroporated in the presence of radiolabeled plasmids have been treated with DNase I and results revealed that 35, 28, 54, 58, and 3% of marked DNA remains in sperm cells following digestion after electroporation in TALP (1,000 V, 1 and 25 microFarads), M medium (100 V, 1 and 25 microFarads), and control, respectively. Using in vitro matured bovine oocytes, the electroporation conditions were correlated with the fertilization rate (85% for control and 55% for electroporated spermatozoa). Autoradiography of embryos following fertilization indicated the presence of plasmids in the cytoplasm and in the zona pellucida.(ABSTRACT TRUNCATED AT 400 WORDS)
Gagné
M B
MB
Unité de Recherche en Ontogénie et Reproduction, CHUL Research Center, Québec, Canada.
Pothier
F
F
Sirard
M A
MA
D000818:Animals; D030801:Animals, Genetically Modified / Q000502:physiology; D001345:Autoradiography; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000502:physiology; D003851:Deoxyribonucleases / Q000378:metabolism; D005314:Embryonic and Fetal Development; D005821:Genetic Techniques; D008297:Male; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology*; D010957:Plasmids / Q000502:physiology; D016133:Polymerase Chain Reaction; D013094:Spermatozoa / Q000502:physiology*; D014170:Transformation, Genetic
D000001:Calcimycin; D004247:DNA; D003851:Deoxyribonucleases