Isolation and in vitro Culture of Mouse Oocytes

Greaney, Jessica; Subramanian, Goutham Narayanan; Ye, Yunan; Homer, Hayden

doi:10.21769/bioprotoc.4104

Cited by 4 publications

(5 citation statements)

References 22 publications

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“…Embryos were fixed and stained similar to described previously for oocytes (Wei et al , 2018, 2020; Zhou et al , 2019; Iljas et al , 2020; Subramanian et al , 2020; Greaney et al , 2021). Briefly, 2‐cell embryos were quickly washed in PHEM buffer, pre‐permeabilised in 0.25% Triton‐X in PHEM before being fixed in 3.7% paraformaldehyde solution in PHEM for 20 min.…”

Section: Methodsmentioning

confidence: 99%

“…Microinjection of zygotes was performed similar to previously described for prophase‐arrested oocytes (Wei et al , 2018, 2020; Zhou et al , 2019; Iljas et al , 2020; Subramanian et al , 2020; Greaney et al , 2021). Briefly, cRNA was produced using the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit (Ambion) using linearised DNA template, as previously verified.…”

Section: Methodsmentioning

confidence: 99%

“…To induce follicular development, female mice were given an intraperitoneal injection of 7.5 international units (IU) pregnant mare serum gonadotrophin (PMSG, Pacific vet) dissolved in saline, as previously described (Wei et al , 2018, 2020; Zhou et al , 2019; Iljas et al , 2020; Subramanian et al , 2020; Greaney et al , 2021). After 48 h, 5 IU of human chorionic gonadotropin (hCG, ProSpec, HOR‐250) was administered in the same manner to induce ovulation.…”

Section: Methodsmentioning

confidence: 99%

“…To collect oocytes, female mice were given a single PMSG injection and sacrificed 48 h later. Oocytes were collected from punctured ovaries and denuded in handling medium supplemented with 100 μM 3‐isobutyl‐1‐methylxanthine (IBMX) (Greaney et al , 2021). Oocytes were then transferred into IBMX‐containing M16 medium (Fig 4C) or washed into IBMX‐free M16 medium to resume meiosis (Appendix Fig S4B).…”

Section: Methodsmentioning

confidence: 99%

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Two‐step nuclear centring by competing microtubule‐ and actin‐based mechanisms in 2‐cell mouse embryos

Homer

2022

EMBO Reports

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Microtubules typically promote nuclear centring during early embryonic divisions in centrosome-containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time-lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubuledependent manner. High cytoplasmic viscosity in 2-cell embryos is associated with non-diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro-centring actin-dependent mechanisms work against microtubule-dependent de-centring forces. Disrupting the equilibrium of this tug-of-war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule-dependent de-centring forces.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Two‐step nuclear centring by competing microtubule‐ and actin‐based mechanisms in 2‐cell mouse embryos

Homer

2022

EMBO Reports

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“…6. Sacrifice the mice by cervical dislocation, identify and remove the ovaries 21 , and transfer them into a watch glass containing prewarmed collection medium (MEM/ PVP + M) at 37 °C.…”

Section: Inject Intraperitoneally 5 Iu Of Pregnant Mare's Serum Gonad...mentioning

confidence: 99%

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

Londoño-Vásquez

Jurkevich

Balboula

2022

JoVE

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The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy.The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes. ProtocolAll the methods described here were approved by the University of Missouri (Animal Care Quality Assurance Ref. Number 9695). Cep192-eGFP reporter female mice aged 6-8 weeks old were used in the present study.To generate Cep192-eGFP reporter mice, CRISPR/Cas9-

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Isolation and in vitro Culture of Mouse Oocytes

Cited by 4 publications

References 22 publications

Two‐step nuclear centring by competing microtubule‐ and actin‐based mechanisms in 2‐cell mouse embryos

Two‐step nuclear centring by competing microtubule‐ and actin‐based mechanisms in 2‐cell mouse embryos

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

A surge in cytoplasmic viscosity triggers nuclear remodeling required for Dux silencing and pre-implantation embryo development

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