Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development

Abstract

Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two-cell embryos were fused and cultured in vitro in Chatot-Ziomek-Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8-10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12-14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm.

Altered Gene Expression Profiles in Mouse Tetraploid Blastocysts

“…In mid-blastocyst stages, Oct4-positive cells randomly appear within the ICM, whereas Cdx2-positive cells are localized to the outer surface of the ICM. As we previously reported [20], Oct4 expression from the 2-cell to the morula stage was similar in tetraploid and diploid embryos. Interestingly, we discovered that at the expanded blastocyst stage, Oct4 expression had disappeared in most of the tetraploid-derived embryonic cells, though some embryonic cells at the ICM site did remain Oct4-positive.…”

“…Interestingly, we discovered that at the expanded blastocyst stage, Oct4 expression had disappeared in most of the tetraploid-derived embryonic cells, though some embryonic cells at the ICM site did remain Oct4-positive. Previously, we reported that 83% of the tetraploid-derived blastocysts had 80 chromosomes, and 17% exhibited mixploidy [20], indicating a close relationship between the numbers of tetraploid-derived blastocysts with Oct4-positive cells and aberrant chromosome numbers. We also demonstrated that the transcription levels of ICM-related genes such as Oct4, Sox2 and Klf4 were 0.1-fold lower in tetraploid-derived blastocysts compared with diploid blastocysts (P<0.05).…”

“…However, at the expanded blastocyst stage, Oct4 was localized only in the ICM nuclei and was largely absent from the nuclei of the TE. In tetraploid embryos, Cdx2-positive nuclei were first detected at the late compaction stage, with greatly increased Cdx2 expression at the blastocyst and expanded blastocyst stages, whereas Oct4 expression had disappeared by the time tetraploid embryos reached the expanded blastocyst stage [20].…”

Efficient Production of Bovine Tetraploid Embryos by Electrofusion of In Vitro Produced Two-Cell Stage Embryos

“…For fusion 2-cell stage mammalian embryos, different electrofusion parameters were used [23,40] according to the type of fusion buffer, electrofusion instrument and animal species [38]. In mice, the highest tetraploid blastocyst formation rate (93.0%) of fused embryos was achieved when electrofusion was performed using 20-volt AC and 100-volt DC [41]. In rabbits, tetraploid embryos were obtained with a single pulse of 1.4 kV/ cm for 100 μs and 74.5% of them developed to morula and blastocyst stages, with 72.7% of the morulae/blastocysts being tetraploid [40].…”

Tetraploidy Induced by Electrofusion - A Review

“…May 2016 | Volume 4 | Issue 5 | Page 268 Park et al (2011) reported that most favourable electrofusion voltages were 100 volts DC pulse and 20 volts alternating current in mouse. Tetraploid embryos were further developed and 2-cell stage tetraploid embryos (93%) were achieved after 16 hours of electrofusion, whereas 4-cell stage tetraploid embryos (80%) were achieved after 24 hours.…”