Electrical-assisted microinjection for analysis of fertilization and cell division in mammalian oocytes and early embryos

FitzHarris, Greg; Carroll, John; Swann, Karl

doi:10.1016/bs.mcb.2018.03.036

Cited by 15 publications

(13 citation statements)

References 14 publications

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“…Oocytes were isolated from ovaries of 7–10 week old CD-1 mice, cultured in M2 medium under mineral oil and matured to metaphase II in vitro for 16 hr at 37°C. Oocytes were then microinjected with mRNA encoding Cyclin B1-mCherry (Pasternak et al, 2016) using an electric-assisted microinjection system (FitzHarris et al, 2018). A function generator (GW Instek AFG-2005) was used to produce electrical current instead of an intracellular electrometer.…”

Section: Methodsmentioning

confidence: 99%

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

Afonso

Castellani

Cheeseman

et al. 2019

eLife

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According to the prevailing ‘clock’ model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the ‘ruler’ model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular ‘rulers’ and ‘clocks’ licenses mitotic exit only after proper chromosome separation.

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Section: Methodsmentioning

confidence: 99%

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

Afonso

Castellani

Cheeseman

et al. 2019

eLife

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“…Microinjection is one of the most powerful technologies to study reproduction and early embryo development (Wall, 2001;Andras Nagy et al, 2003;FitzHarris et al, 2018). By utilizing it, people learned a lot of knowledge about oocyte maturation and embryo development.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, it should be beneficial to establish a method to obtain a high survival rate for new operators after microinjection. Some monographs and papers have demonstrated the application and methods of microinjection in mouse reproduction (Andras Nagy et al, 2003;Jaffe et al, 2009;FitzHarris et al, 2018), but currently, there is no single protocol that could be used in all the samples from denuded oocyte, cumulus-oocyte complex (COC), and zygote to early embryos (Jaffe et al, 2009;Kline, 2009;Stein and Schindler, 2011). An easy-to-learn method is expected to be applicable to all the mouse oocytes and early embryos at different stages.…”

Section: Introductionmentioning

confidence: 99%

High-Survival Rate After Microinjection of Mouse Oocytes and Early Embryos With mRNA by Combining a Tip Pipette and Piezoelectric-Assisted Micromanipulator

Chen

Fan

Liu

et al. 2021

Front. Cell Dev. Biol.

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Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development in vitro. However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus–oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.

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“…GV-stage oocytes were microinjected in M2 media supplemented with with 200μM IBMX, using a picopump (World Precision Instruments), intracellular electrometer (Warner instruments), and micromanipulators (Narishige), mounted on a Leica DMI4000 inverted microscope (as described previously Fitzharris, 2009; FitzHarris et al, 2018). The mRNAs used for injections were in vitro synthesised using mMessage mMachine kits (T3, T7 and SP6), followed by poly-adenylation using Poly(A)-Tailing kit according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse

Mihajlović

Haverfield

FitzHarris

2021

Preprint

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SUMMARYChromosome segregation errors that cause oocyte aneuploidy increase in frequency with maternal age and are considered a major contributing factor of age-related fertility decline in females. A common age-associated chromosome segregation phenomenon in oocytes is the lagging anaphase chromosome, but whether anaphase laggards actually missegregate and cause aneuploidy is unclear. Here we show unexpectedly that lagging chromosomes in mouse oocytes comprise two mechanistically distinct classes of motion that we refer to as ‘Class-I’ and ‘Class-II’. We use imaging approaches and mechanistic interventions to dissociate the two classes, and find that whereas Class-II laggards are benign, Class-I laggards can directly cause aneuploidy. Most notably, a controlled prolongation of meiosis-I specifically lessens Class-I lagging to prevent aneuploidy. Our data thus reveal lagging chromosomes to be a cause of age-related aneuploidy in mouse oocytes and suggest that manipulating the cell cycle could increase the yield of useful oocytes in some contexts.

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Electrical-assisted microinjection for analysis of fertilization and cell division in mammalian oocytes and early embryos

Cited by 15 publications

References 14 publications

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

High-Survival Rate After Microinjection of Mouse Oocytes and Early Embryos With mRNA by Combining a Tip Pipette and Piezoelectric-Assisted Micromanipulator

Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse

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