Effect of electrofusion pulse in either electrolyte or nonelectrolyte fusion medium on subsequent murine embryonic development

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Abstract
This study was designed to determine what effect electropulse parameters would have on rate of fusion, lysis, and embryo viability when embryos were subjected to electrofusion treatment in nonelectrolyte or electrolyte pulse media. Previous experiments have shown electrolyte medium (i.e., phosphate-buffered saline; PBS) to have a positive effect on electric pulse-induced murine oocyte activation. In addition, these results also indicated that pulse media containing 0.9 mM Ca2+ induced a dramatic increase in the rate of murine oocyte activation compared with oocytes pulsed in media containing 0.0 or 0.05 mM Ca2+. Pronuclear or two-cell-stage embryos were obtained from superovulated prepubertal randomly bred Swiss (albino) female mice. Embryos were randomly assigned to three nonelectrolyte and three electrolyte treatment media. Nonelectrolyte media consisted of 0.3 M mannitol (T1), 0.3 M mannitol + 0.05 mM CaCl2 (T2), and 0.3 M mannitol + 0.9 mM CaCl2 (T3). Electrolyte media consisted of Ca2+ -free PBS (T4), PBS containing 0.05 mM CaCl2 (T5), and PBS containing 0.9 mM CaCl2 (T6). Three experiments were carried out; the objective of the first was to determine the rate of fusion and rate of lysis in murine two-cell embryos placed in the two types of (0.3 M mannitol, T1-T3; and PBS, T4-T6) fusion media and subjected to a fusion procedure (3 V, 5 sec AC alignment pulse, followed by a 1.56 kV · cm−1, 99 μsec DC fusion pulse). Control two-cell embryos were placed in T1 for 2 min and did not receive a fusion pulse. The objective of experiment 2 was to evaluate the development of pronuclear stage embryos to the blastocyst stage in vitro after receiving the fusion pulse in T1, T3, and T6. Control embryos were not subjected to fusion treatment. In experiment 3, T3 and T6 were used to test the rate of fusion and rate of development for pronuclear-stage karyoplasts fused to enucleated pronuclear-stage cytoplasts. Micromanipulations were carried out, and all pronuclear embryos were placed into culture and the number developing to the four-cell stage and subsequently to the blastocyst stage was assessed. Two-cell-stage embryos pulsed in T5 and T6 exhibited significantly higher rates of fusion (96.9 and 92.9%, respectively) compared with T1 (83.7%), T2 (84.7%), or T3 (77.6%) ( P > 0.05). There was no significant difference (P > 0.05) in rate of lysis between any of the treatment groups. Pronuclear-stage embryos placed in T1, T3, and T6 and subjected to the electrofusion procedure resulted in 55.5%, 52.3%, 50.0%, and 54.2% of T1, T3, T6, and control embryos developing to the blastocyst stage, respectively. There was no difference (P > 0.05) between treatment groups in development to the blastocyst stage after 98 hr in culture. Finally, nuclear transplant results indicated no difference in the rate of pronuclear karyoplast-cytoplast fusion between T3 (79.7%) and electrolyte T6 (85.5%) media (P > 0.05). There was also no difference in the rate of development to the four-cell stage (78% vs. 72.9%) or blastocyst stage (59.3% vs. 54.2%, T3 and T6, respectively) (P > 0.05). However, a difference was observed in rate of development to the four-cell stage and blastocyst stage between nonpulsed control pronuclear stage embryos and T6-treated karyoplast-cytoplast constructs (86.3% vs. 72.9% and 68.5% vs. 54.2%, respectively) (P < 0.05). In addition, there was no difference in the rate of lysis observed between T3, T6, and control embryos. These data indicate that the application of a 3 V, 5 sec AC alignment pulse prior to a single 1.56 kV · cm−1 DC fusion pulse to electrolyte PBS results in successful fusion of murine two-cell blastomeres at a rate equal to that of nonelectrolyte 0.3 M mannitol. In vitro development of pronuclear-stage or fused pronuclear transferred karyoplast-cytoplasts after an AC alignment pulse followed by a DC fusion pulse in either 0.3 M mannitol containing 0.9 mM Ca2+ or PB1 does not adversely effect early murine embryonic development in vitro.