Name: defining the program of maternal mrna translation during in vitro maturation using a single oocyte reporter assay
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This work was supported by NIH R01 GM097165, GM116926 and Eunice Kennedy Shriver NICHD National Centers for Translational Research in Reproduction and Infertility P50 HD055764 to Marco Conti. Enrico M. Daldello was supported by a fellowship from the Lalor Foundation and Natasja G. J. Costermans was supported by a Rubicon fellowship from the Netherlands Organisation for Scientific Research (NWO).

The experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of California at San Francisco (protocol AN182026).
1. Preparation of media
<ol>
	<li>Add all components, as described in Table 1, to make the basic oocyte collection medium and oocyte maturation medium. For the basic collection medium, set the pH to 7.4. For both collection and maturation medium, add 3 mg/mL of bovine serum albumin (BSA) and 1 &#1...

<ol>
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The presented method describes a strategy to study activation and repression of translation of target mRNA at different transitions during in vitro oocyte meiotic maturation. IL-7, a cytokine released by the oocyte that may be involved in oocyte-cumulus cell communication8,13, was chosen for the purpose of describing this method. IL-7 is known to be increasingly translated during oocyte maturation8 and allows for good visualizatio...

A fully-grown mammalian oocyte undergoes rapid changes in preparation for fertilization and acquisition of totipotency. These changes are essential to sustain embryonic development after fertilization. Although the events associated with nuclear maturation are relatively well described, much less is known about the molecular processes and pathways in the oocyte cytoplasm. During the final stages of oocyte maturation, oocytes are transcriptionally silent, and gene expression is entirely dependent on mRNA translation and degradation1,2. The synthesis of proteins critical for developmental competence, therefore, relies on a program of timed translation of long-lived mRNAs that have been synthesized earlier during oocyte growth1,3. As part of a focus on defining this program of maternal mRNA translation executed during meiotic maturation and at the oocyte-to-zygote transition, this paper presents a strategy to study the activation and repression of the translation of target maternal mRNAs in single oocytes during in vitro meiotic maturation.
In this method, the YPet open reading frame is cloned upstream of the 3&#39; UTR of the transcript of interest. Next, mRNAs encoding this reporter are micro-injected into oocytes together with polyadenylated mRNAs encoding mCherry to control for injected volume. Reporter accumulation is measured during in vitro oocyte meiotic maturation using time-lapse microscopy. The accumulation of yellow fluorescent protein (YFP) and mCherry is recorded in individual oocytes, and YFP signals are corrected by the plateaued level of the co-injected mCherry. After data acquisition, translation rates are calculated for different time intervals during in vitro oocyte meiotic maturation by calculating the slope of the curve obtained by curve-fitting.
This approach provides a tool to experimentally confirm changes in translation of selected endogenous mRNAs. In addition, this method facilitates the characterization of regulatory elements that control translation during oocyte meiotic maturation by manipulating cis-regulatory elements of the 3&#39; UTR of target mRNAs4,5,6. Manipulation of the poly(A) tail length also allows insight into adenylase/deadenylase activity in oocytes5. Mutagenesis of cis-acting elements or RNA immunoprecipitation can be used to study interactions with cognate RNA binding proteins6,7. Additionally, this method can be used to identify essential components of the translation program that are critical for oocyte developmental competence by measuring target 3&#39; UTR translation in models associated with decreased oocyte quality 8,9,10. This method paper presents a representative experiment where denuded oocytes of 21-day-old C57/BL6 mice have been micro-injected with a Ypet reporter fused to the 3&#39; UTR of IL-7. The setup and protocol for oocyte injection, time-lapse recording, and data analysis have been described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Preparation of media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Powder Bioxtra</td>
			<td>Sigma-Aldrich</td>
			<td>SIAL-A3311</td>
			<td></td>
		</tr>
		<tr>
			<td>Cilostamide</td>
			<td>EMD Millipore</td>
			<td>231085</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM alpha</td>
			<td>Gibco</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M2645</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100x Solution, Sterile Filtered</td>
			<td>Genesee Scientific Corporation (GenClone)</td>
			<td>25-512</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate&#160;</td>
			<td>JT-Baker</td>
			<td>3506-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco&#160;</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of mRNA encoding YFP/3&#39; UTR and mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Apex Biomedical&#160;</td>
			<td>20-102QD</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>C1389-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Choo-Choo Cloning Kit</td>
			<td>McLab</td>
			<td>CCK-20</td>
			<td></td>
		</tr>
		<tr>
			<td>CutSmart Buffer (10x)</td>
			<td>New England Biolabs</td>
			<td>B7204</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA loading dye (6x)</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Solution</td>
			<td>New England Biolabs</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs</td>
			<td>R0176</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb DNA ladder</td>
			<td>Thermo Fisher</td>
			<td>SM1333</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar Plates with 100 &#181;g/mL Carbenicillin, Teknova&#160;</td>
			<td>Teknova</td>
			<td>L1010</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium (Capsules)</td>
			<td>MP Biomedicals</td>
			<td>3002-021</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGAclear Transcription Clean-Up Kit</td>
			<td>Life Technologies</td>
			<td>AM1908</td>
			<td></td>
		</tr>
		<tr>
			<td>MfeI-HF restriction enzyme</td>
			<td>New England Biolabs</td>
			<td>R3589</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1344</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High Fidelity DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tailing kit</td>
			<td>Invitrogen</td>
			<td>AM1350</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit&#160;</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C. medium</td>
			<td>Thermo Fisher</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer&#160;</td>
			<td>Apex Biomedical&#160;</td>
			<td>20-193</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Ethidium Bromide Solution</td>
			<td>Life Technologies</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assembly for calibrated micro-pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>A5177-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Calibrated micro-pipettes</td>
			<td>Drummond Scientific Company</td>
			<td>2-000-025&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG- 5000</td>
			<td>Mybiosource</td>
			<td>MBS142665</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 26 G x 1/2</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 ml</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte micro-injection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Dish | No. 0 Coverslip | 20 mm Glass Diameter | Uncoated</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
			<td>For time-lapse microscopy</td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument</td>
			<td>BF100-78-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil for Embryo Culture</td>
			<td>Irvine Scientific</td>
			<td>9305</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Falcon</td>
			<td>351006</td>
			<td>For micro-injection</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>For oocyte incubation</td>
		</tr>
		<tr>
			<td>VacuTip Holding Capillary</td>
			<td>Eppendorf</td>
			<td>5195000036</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biorender</td>
			<td>BioRender</td>
			<td></td>
			<td>Preparation of Figure 1S</td>
		</tr>
		<tr>
			<td>MetaMorph, version 7.8.13.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>&#160;For time-lapse microscopy, analysis of 3&#39; UTR translation&#160;</td>
		</tr>
	</tbody>
</table>

defining the program of maternal mrna translation during in vitro maturation using a single oocyte reporter assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

Denuded prophase I-arrested oocytes of 21-day-old C57/BL6 mice were injected with a reporter mix containing mRNA encoding the Ypet reporter fused to the 3&#39; UTR of IL-7 and mRNA encoding mCherry. YFP and mCherry expression was recorded in 39 oocytes, of which 30 were matured, and 9 were arrested in prophase I as a negative control. Three maturing oocytes were excluded for analysis because they either had a delayed GVBD (N=2) or moved in the dish during the recording (N=1). Figure 3 shows ...

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

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

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.

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