Production of mouse androgenetic embryos using spindle perturbation

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.The cell cycle in unfertilized vertebrate oocytes is arrested at the metaphase of the second meiosis (Meta-II). Sperm-oocyte fusion triggers the meiotic resumption and onset of the anaphase-II (Ana-II), leading to sister chromatid segregation toward the spindle poles. As a result of highly asymmetric cell division, one set of sister chromatids is extruded to the small second polar body, and the other forms a maternal pronucleus (PN) in the large zygote 1 . Polar body formation is typically a process of cytokinesis with an oocyte-specific feature. The Meta-II spindle is located close to the cortex, and aligned chromosomes induce the formation of a thick cortical actin meshwork called the actin cap, through the small GTPase Ran-mediated signaling cascade that leads to Arp2/3 activation 2,3 . During Ana-II, segregated chromosomes also induce actin cap formation. The actin cap, together with the actomyosin ring that is located in the peripheral region of the actin cap, pushes the plasma membrane to form a membrane protrusion. The actomyosin-based contractile ring is then formed at the neck region of the protrusion, resulting in the formation of a polar body upon completion of cytokinesis 2,4-6 . The formation and function of the contractile ring depends on the numerous proteins accumulated at the overlapping antiparallel microtubules called the central-spindle midzone that forms between the two segregating chromatids 7 . In the anaphase, protein regulator of cytokinesis 1 (PRC1) exhibits strong microtubule bundling activity upon dephosphorylation by PP2A-B55, and plays a critical role in the spindle midzone organization 8-10 , and therefore in contractile ring assembly. During the formation of the second polar body and maternal PN, sperm-derived chromatin forms a paternal PN after extensive c...

Ca2+-driven cytoplasmic backflow secures spindle position in fertilized mouse eggs

“…To clarify the causal relationship between directional changes of cytoplasmic streaming and spindle rotation, we utilized a method of partial spindle disruption using low-dose nocodazole. This approach halted chromosome segregation and spindle rotation without compromising meiotic resumption upon egg activation 28 . The low-dose nocodazole did not affect Ca 2+ oscillations, and the cytoplasmic streaming was oriented outward immediately after each Ca 2+ transient, even in the absence of spindle rotation (Fig.…”

“…Medium drops containing the drugs dissolved in DMSO were prepared as previously reported 28 . For the thapsigargin treatment assay, two Sr 2+ activation medium drops, one containing 10 or 20µm thapsigargin and one without thapsigargin were placed close to each other on a glass-bottom dish and covered with 4 ml liquid paraffin followed by incubation at 37°C under 5% CO 2 for 30 to 60 min before live imaging.…”