Abstract
Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.
Highlights
In plants and nonmammalian animals, polyploidization has conferred some survival advantages, such as tolerance to genome damage, the requirement for fewer cells in organs, and flexibility and strength of tissues [1]
Despite similarities in morphology and the expression of the Embryonic stem cells (ESCs)-positive marker alkaline phosphatase (ALP), the relative proliferation rate of tetraploid embryonic stem cells (TESCs) was significantly lower than that of control ESCs after 3 days of culture (Fig 1E and S1 Fig); cellular apoptosis and necrosis did not account for this difference (S1 Fig). These results indicated that TESC lines were successfully established using an orthodox method and showed that TESCs exhibited similarities in shape, ALP staining, and genome dosage stability, but differences in the proliferation rate compared to ESCs
We found that TESCs maintained intrinsic pluripotency and differentiation potential despite artificial tetraploidization
Summary
In plants and nonmammalian animals, polyploidization has conferred some survival advantages, such as tolerance to genome damage, the requirement for fewer cells in organs, and flexibility and strength of tissues [1]. Despite the prevalence of polyploidy in amphibians and fishes, polyploid animals are often sterile [3,4,5]. Tetraploid mouse embryos can be produced by artificial methods such as electrofusion of two-cell-stage embryos by electrical stimulation [8,9,10]. This electrofusion method shows high efficiency and stably produces embryos consisting entirely of tetraploid cells in the organs of mice [11, 12]. Tetraploid embryos can be developed to the blastocyst stage in standard embryo culture medium in vitro, which implies that tetraploid cells have normal cellular functions in the early embryo before implantation [13,14,15]
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