Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

This method may be used to identify and characterize regulatory elements that control translation during oocyte maturation. We applied time-lapse microscopy to assess reporter accumulation throughout in-vitro oocyte maturation. This accurately pinpoints the time when translational activation or repression takes place during 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 one 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 in 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 times 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 five to 10 picoliters of the reporter mix.

Incubate the oocytes and the maturation medium with one 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 one 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 one 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 elimination 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 multi-dimensional acquisition window and press plus 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 three prime UTR translation, perform two region measurements for each oocyte, the oocyte itself by clicking 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. The expression of mCherry and YFP in prophase I and maturing oocytes was recorded in 39 oocytes, of which 30 were matured, and nine were arrested in prophase I as a negative control. Individual YFP to mCherry ratios for signals of prophase I-arrested and maturing oocytes were corrected for injected volume by dividing the YFP signal by the average mCherry signal of the last 10 time points for prophase I-arrested and maturing oocytes.

Translation rates of the reporter YFP were measured by curve fitting the YFP to mCherry ratios in prophase I and in maturing oocytes during the first zero to two hours, or eight to 10 hours after cilostamide release using linear regression. The accumulation of the reporter does not follow a linear pattern, as indicated by a significant difference in translation rates between zero to two hours and eight to 10 hours after cilostamide release, indicating activation of interleukin 7 translation during oocyte myotic maturation. When attempting this protocol, make sure that the exposure time is adequate at the beginning of the recording.

Keep in mind that the signal might saturate during the time course if translation is activated during oocyte meiotic resumption. Stability of the MRMA may be confirmed by PCR, using primers for YFP. To confirm changes in translation, you can use Western blotting with an antibody against a V5 tech.