Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro

Abstract

Tetraploid bovine blastocysts were produced experimentally by electrofusion of in vitro matured and fertilized, zona‐enclosed two‐cell embryos (33–35 hr after initiation of sperm–egg incubation) using three fusion protocols. Field strengths of 1.0, 1.4, and 2.4 kV/cm were tested and the rate of fusion, subsequent cleavage, and blastocyst development were measured for each. High rates of fusion (76.5% ± 2.8%), cleavage (72.5% ± 7.4%) and blastocyst development (56.1% ± 6.4%) were achieved with the application of 1.4 kV/cm as a single 100‐μs pulse. Embryos were scored 30 and 60 min after stimulation for fusion. No time effect for fusion, cleavage, or blastocyst development was observed. Chromosome preparations of day 7 blastocysts revealed 12.5% of fused embryos were tetraploid. This is a significant increase from that found in nonfused embryos where spontaneous tetraploidy did not occur. An electrical stimulus of 1.0 kV/cm applied as two 50‐μs pulses produced significantly less one‐cell embryos (64.2% ± 3.0%) compared to 1.4 kV/cm while cleavage (79.9% ± 3.4) and blastocyst development (44.6% ± 4.0%) were not different from that for unexposed control embryos (89.5% ± 2.3% and 57.2% ± 3.2%, respectively). Embryos fused at 2.4 kV/cm applied as a single 30‐μs pulse (69.7% ± 5.7%) showed significantly lower cleavage (72.1% ± 3.7%) and blastocyst rates (40.2% ± 4.6%) compared to the unexposed control. Mol. Reprod. Dev. 56:372–377, 2000. © 2000 Wiley‐Liss, Inc.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres

“…Aneuploidy was increased in 4-to 8-cell stage embryos subjected to the electrical pulse that did not fuse, even though blastocyst development was not affected. Additionally, in accordance with our results, it has been reported in cattle that aneuploidy is a result of asynchronous cleavage between two-cell embryos and asynchronous nuclear status of blastomeres within the embryo suggesting that blastomeres need to be at a certain stage of the cell cycle for consistent production of tetraploids (Curnow et al 2000). In agreement with our results, previous reports showed that higher electric pulse voltage affects efficiency of electrofusion and embryo survival (Meghji & Burnstock 1995, Suo et al 2009, Razza et al 2016.…”

“…Tetraploid (4n-cells) complementation contributes to extra-embryonic tissue and reduces maternalfetal failures during implantation and placentation by improving placenta compatibility (Lin et al 2011, Curnow et al 2000. Tetraploid complementation has been used to study pluripotency and developmental capacity in mice by injecting stem cells or induced pluripotent stem cells (iPS cells) into a tetraploid blastocyst.…”

Development of rat tetraploid and chimeric embryos aggregated with diploid cells

“…In addition, two diploid blastomeres can be fused using polyethylene glycol (Eglitis, 1980; Spindle, 1981), inactivated Sendai virus (O'Neill et al ., 1990) or electrofusion (Berg, 1982; Kubiak & Tarkowski, 1985). Stimulation of 2-cell embryo fusion by electrical pulses appears to be the most efficient method and has been used to produce tetraploid embryos in a number of species including mouse (Kubiak & Tarkowski, 1985), rabbit (Ozil & Modlinski, 1986), cow (Iwasaki et al ., 1989; Curnow et al ., 2000), pig (Prather et al ., 1996; Prochazka et al ., 2004) and rat (Krivokharchenko et al ., 2002).…”

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

“…Until now, no hexaploid mammal was known. Electrofusion of blastomeres at the 2-cell stage was indicated as an effective method to produce tetraploid embryos in mammals (Kubiak et al ., 1985; Prather et al ., 1996; Curnow et al ., 2000; Krivokharchenko et al ., 2002). In the mouse, the aggregation of nuclear transfer embryonic stem cells (nt-ESC) with tetraploid embryos has allowed the birth of cloned offspring completely derived from those terminal differentiated cells like lymphatic or olfactory neurons (Hochedlinger et al ., 2002; Li et al ., 2004).…”