Merck

Production of mouse androgenetic embryos using spindle perturbation.

Scientific reports (2020-04-18)
Takaya Totsuka, Miho Ohsugi
ABSTRACT

To study the functional differences between maternal and paternal genomes in mammalian development, embryos with only one parental genome are often used. Androgenetic embryos are produced by the removal of maternal chromosomes before or after fertilization by techniques that require specialized skills and are associated with high risk of cellular damage. Here, we developed a novel method for producing androgenetic mouse embryos without the invasive enucleation process. We found that during in vitro fertilization in the presence of low-dose nocodazole, a microtubule destabilizing drug, whole oocyte chromosomes were extruded into the second polar body resulting in the production of androgenetic embryos. We further demonstrated that low-dose nocodazole decreased the spindle size and prevented chromosome segregation but did not compromise oocyte meiotic resumption. This led to the formation of a protrusion around the chromosomes, accumulation of protein regulator of cytokinesis 1 (PRC1) to the microtubules around the chromosomes, and assembly of a contractile ring at the neck region of the protrusion. Our method uses the intrinsic cytokinetic mechanism to exclude maternal chromatin from zygotes and may be applicable to other mammals.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
M16 Medium, With sodium bicarbonate and lactic acid, without penicillin and streptomycin, liquid, sterile-filtered
Sigma-Aldrich
Nocodazole, ≥99% (TLC), powder
Sigma-Aldrich
Mineral oil, light oil, (neat)
Sigma-Aldrich
M2 medium, With HEPES, without penicillin and streptomycin, liquid, sterile-filtered, suitable for mouse embryo cell culture