The In Vitro Single Oocyte Reporter Assay: A Technique to Study Target mRNA Translation Regulation During In Vitro Oocyte Maturation

To begin, carefully open the antral follicles by making a small cut in the follicle wall with a 26-gauge needle. Isolate intact COCs with several layers of cumulus cells using a mouth-operated glass pipette. Use a smaller pipette and mechanically denude the COCs by repeated pipetting.

Aspirate the denuded oocytes, and place them in a Petri dish with maturation medium supplemented with 1 micromolar of cilostamide. Place the dish in an incubator for two hours, to let the oocytes recover from the stress induced by their isolation from the follicles.

Place a 10-centimeter long borosilicate glass capillary tube, and a mechanical puller to prepare injection needles. Bend the needle tip at a 45-degree angle using a heated filament for optimal injection. Place 20-microliter droplets of basic oocyte collection medium in a polystyrene dish, and cover the droplets with light mineral oil.

Prepare a larger volume of reporter mix by adding 12.5 micrograms per microliter of Ypet UTR and mCherry each. Store aliquots of these at minus 80 degrees Celsius. Upon thawing, centrifuge the aliquot for two minutes at 20,000 x g. Then, transfer it to a new microcentrifuge tube.

Load the injection needle with approximately 0.5 microliters of reporter mix. Place the holding pipette and the injection needle into the holders, and position them in the droplet of oocyte collection medium.

Open the injection needle by gently tapping it against the holding pipette. Place oocytes in a droplet of basic collection medium, and inject 5 to 10 picoliters of the reporter mix. Incubate the oocytes in the maturation medium with 1 micromolar cilostamide for 16 hours to allow the mCherry signal to plateau.

For time-lapse microscopy, prepare a Petri dish with two 20-microliter droplets of maturation medium for each injected reporter. One droplet with 1 micromolar cilostamide for control prophase-I-arrested oocytes, and one droplet without cilostamide for maturing oocytes. Cover the droplets with light mineral oil, and place them in the incubator.

After pre-incubation, remove the injected oocytes from the incubator, and wash them four times in maturation medium without cilostamide. Keep some oocytes in maturation medium with 1 micromolar cilostamide as a prophase-I oocyte control group.

Transfer the injected oocytes to their respective droplets on the previously prepared time-lapse microscope dish. Cluster the oocytes with a closed glass pipette to prevent their movement during the recording.

Place the dish under the microscope equipped with a light-emitting diode illumination system, and a motorized stage equipped with an environmental chamber maintained at 37 degrees Celsius and 5% carbon dioxide, using the parameters mentioned in the text manuscript.

Enter the appropriate settings for the time-lapse experiment by clicking on "Apps," and then, "Multidimensional acquisition." Select the first tab "Main" and select "Time-lapse," "Multiple stage positions," and "Multiple wavelengths."

Select the tab "Saving" to enter the location where the experiment should be saved. Then, select the tab "Time-lapse" to enter the number of time points, duration, and time interval. Select the tab "Stage." Switch on brightfield and locate the position of the oocytes by opening a new window and selecting "Acquire," "Acquire," and "Show live."

Once the oocytes are located, switch back to the "Multidimensional acquisition window," and press "+" to set the location of the oocytes. Select the tab "Wavelengths," and set three different wavelengths for brightfield, YFP, and mCherry. Adjust the exposure for Ypet and mCherry. Then, start the time-lapse experiment by clicking on "Acquire."

For analysis of Ypet-3' UTR translation, perform two region measurements for each oocyte. The oocyte itself by clipping on "Ellipse region," and a small region surrounding the oocyte to be used for background subtraction by clicking on "Rectangular region." Export the region measurement data to a spreadsheet by clicking "Open log" and then, "Log data."

For each individual oocyte and for all measured time points, subtract the background region measurement from the oocyte region measurement separately for YFP and mCherry wavelengths.