Electroporation of bovine spermatozoa to carry foreign DNA in oocytes

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Abstract
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 (107/ml) were electroporated using six different combinations of voltge (500, 1,000, or 1,500 V) and capacitance (1 or 25 μ Farads) 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 μFarad, 500, 1,000, and 1,500 V and 25 μFarads, 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 < 0.005). EF decrease motility significantly for > 100 V in 0.3 M mannitol (M) and mannitol‐TALP (MT) (1/1) media and ≥500 V (P<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 μFarads, 100 V, and 0.9, 3.8, and 3.8 at 25 μFarads, 100 V in TALP, MT, and M medium, respectively. Sperm cells electroporated at 1 μFarad, 500 or 1,000 V, 25 μFarad, 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 μFarads), M medium (100 V, 1 and 25 μFarads), 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. Finally, the use of Polymerase Chain Reaction (PCR) revealed the presence of plasmids in blastocyst cells in 12% for control (n=67) and 22, 17, and 19% for electroporated spermatozoa in TALP (n=188,25μFarads, 1,000 V), MT (n=29, 25 μFarads, 100 V) and M (n=21, 25 μFarads, 100 V), respectively. In conclusion, our results indicate A) that electroporation results in an increase absorption of DNA by the spermatozoa, B) that the male gametes can carry foreing DNA in oocytes at fertilisation, and C) that an increased fraction of day 5 embryos is showing a positive response for the inserted gene with the polymerase chain reaction.