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 &#181;M cilostamide on the day of use.</li>
</ol>
2. Preparation of mRNA encoding for Ypet-3&#39; UTR and mCherry
<ol>
	<li>Obtain the 3&#8242; UTR sequences of the mRNAs of interest. 
	NOTE: For this study, sequences were previously obtained from mouse oocyte cDNA.</li>
	<li>Design primers to amplify the target 3&#39; UTRs from oocyte cDNA and portions of the pcDNA 3.1 vector containing a Ypet coding sequence, V5-epitope tag, and a T7 promoter.</li>
	<li>Amplify using a high-fidelity DNA polymerase kit. Run the polymerase chain reaction (PCR) products on a gel to check if the fragments have the correct size, cut out the bands, and extract the DNA from the gel using a gel extraction kit according to the manufacturer&#39;s instructions.</li>
	<li>Fuse the PCR fragments to a vector using a PCR cloning kit. 
	NOTE: PCR fragments were incubated on ice for 4 h to facilitate a more efficient recombination process. This is contrary to the manufacturer&#39;s instructions, which recommend an incubation time of only 45 min.</li>
	<li>Transfect PCR fragments into competent 5-&#945; Escherichia coli bacteria.
	<ol>
		<li>Add the mix and plasmid to the bacteria, and incubate on ice for 30 min.</li>
		<li>Heat shock the mix and plasmid by placing the mixture in a water bath at 42 &#176;C for 45 s, and immediately cool on ice for 3 min.</li>
		<li>Add 500 &#181;L of Super Optimal broth with Catabolite repression (SOC)&#160;medium and incubate for 1 h at 37 &#176;C.</li>
		<li>Spin down at 7000 &#215; g for 2 min, and remove most of the supernatant. Resuspend and plate on a Luria Broth (LB) agar plate with the appropriate selection antibiotic. 
		NOTE: Carbenicillin was used in this study.</li>
		<li>Incubate overnight at 37 &#176;C. Isolate the colonies by lightly pressing a pipette tip on one of the colonies and placing it in 3 mL of LB medium with 100 &#181;g/mL of carbenicillin. Incubate for 12-24 h at 37 &#176;C.</li>
		<li>Extract the DNA of the plasmids using the plasmid DNA isolation kit according to the manufacturer&#39;s instructions and confirm the sequences via DNA sequencing.</li>
	</ol>
	</li>
	<li>For Ypet/3&#39; UTR:
	<ol>
		<li>Produce a linear PCR template for in vitro transcription. Use a forward primer upstream of the Ypet sequence and a reverse primer with 20 additional thymine residues. See Table 2 for the sequence of Ypet/IL-7 3&#39; UTR and the sequences of the forward and reverse primers. 
		NOTE: These additional thymine residues will add oligo(A) to the linear mRNA after in vitro&#160;transcription.</li>
		<li>In vitro transcribe the PCR product using a T7 transcription kit according to the manufacturer&#39;s instructions.</li>
		<li>Purify the resulting complementary RNA (cRNA) using a transcription clean-up kit according to the manufacturer&#39;s instructions.</li>
		<li>Elute the purified cRNA in RNAse-free water, measure&#160;concentration&#160;and evaluate&#160;integrity by agarose electrophoresis. Store at &#8722;80 &#176;C.</li>
	</ol>
	</li>
	<li>For mCherry:
	<ol>
		<li>Produce a linear PCR template for in vitro transcription by using a high-fidelity restriction enzyme; use the restriction enzyme mfEI-HF if replicating this example. Digest in a digestion buffer overnight at 37 &#176;C.</li>
		<li>Run a gel to purify the sample, and extract the linear DNA using a gel extraction kit according to the manufacturer&#39;s instructions.</li>
		<li>In vitro transcribe the PCR product using a T7 transcription kit according to the manufacturer&#39;s instructions.</li>
		<li>Polyadenylate the cRNA (150-200 nucleotides) using a poly(A) tailing kit according to the manufacturer&#39;s instructions.</li>
		<li>Purify the resulting cRNA using a transcription clean-up kit according to the manufacturer&#39;s instructions.</li>
		<li>Elute the purified cRNA in RNAse-free water, measure mRNA concentrations, and evaluate the message integrity by gel electrophoresis. Store at &#8722;80 &#176;C.</li>
	</ol>
	</li>
</ol>
3.&#160;Experimental procedure
NOTE: A schematic overview of oocyte micro-injection and subsequent time-lapse microscopy is given in Figure 1.
<ol>
	<li>Day 1
	<ol>
		<li>Intraperitoneally inject 21-day-old mice with 5 IU of pregnant mare&#39;s serum gonadotropin to promote follicle growth to the antral stage11.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Oocyte collection
		<ol>
			<li>Sacrifice mice 44-48 h after priming to collect the ovaries, and place them in a plastic Petri dish with basic oocyte collection medium.</li>
			<li>Carefully open the antral follicles by making a small cut in the follicle wall with a 26 G needle. Isolate intact cumulus-enclosed oocytes (COCs) with several layers of cumulus cells using a mouth-operated glass pipette.</li>
			<li>Use a smaller pipette (slightly larger than the diameter of the oocyte), and mechanically denude the COCs by repeated pipetting. 
			NOTE: Alternatively, perform micro-injection on intact COCs.</li>
			<li>Using a larger pipette, aspirate the denuded oocytes and place them in a Petri dish with maturation medium supplemented with 1 &#181;M of the phosphodiesterase inhibitor, cilostamide, to prevent resumption of meiotic maturation12. Place the dish in the incubator for 2 h to let the oocytes recover from the stress induced by isolation of the oocytes from the follicles.</li>
		</ol>
		</li>
		<li>Oocyte micro-injection
		<ol>
			<li>Prepare the injection needles by placing a 10 cm long borosilicate glass capillary tube in a mechanical puller. For optimal injection, bend the needle tip in a 45&#176; angle using a heated filament.</li>
			<li>Prepare polystyrene dishes with 20 &#181;L droplets of basic oocyte collection medium, and cover the droplets with light mineral oil.</li>
			<li>Prepare a reporter mix by adding 12.5 &#181;g/&#181;L Ypet-3&#39; UTR and 12.5 &#181;g/&#181;L mCherry. Prepare a larger volume and make aliquots for future experiments to ensure similar reporter concentrations. Store these aliquots at -80 &#176;C. Upon thawing, first centrifuge the aliquot for 2 min at 20,000 x g, and transfer to a new microcentrifuge tube. 
			NOTE: This centrifugation will prevent the injection needle from getting clogged by potential aggregates in the reporter mix.</li>
			<li>Load the injection needle by capillarity with approximately 0.5 &#181;L of reporter mix.</li>
			<li>Place the holding pipette and loaded injection needle into the holders, and position in the droplet of oocyte collection medium. Aspirate some of the medium into the holding pipette.</li>
			<li>Open the injection needle by gently tapping it against the holding pipette.</li>
			<li>Place oocytes in a droplet of basic collection medium, and inject 5-10 pL of the reporter mix.</li>
			<li>Incubate the oocytes in the maturation medium with 1 &#181;M cilostamide for 16 h to allow the mCherry signal to plateau.</li>
			<li>Prepare a Petri dish that will be used for time-lapse microscopy with at least two 20 &#181;L droplets of maturation medium for each injected reporter: one droplet with 1 &#181;M cilostamide for control prophase I-arrested oocytes and one droplet without cilostamide for maturing oocytes. Cover the droplets with light mineral oil and place in incubator.</li>
		</ol>
		</li>
		<li>Time-lapse microscopy
		<ol>
			<li>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 &#181;M cilostamide as a prophase I-arrested oocyte control group.</li>
			<li>Transfer the injected oocytes to their respective droplets on the previously prepared time-lapse microscope dish. Cluster the oocytes by using a closed glass pipette (a closed pipette can be prepared by holding the tip in a flame for a few seconds). 
			NOTE: Clustering the oocytes helps to prevent their movement during the recording.</li>
			<li>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 &#176;C and 5% CO2. To replicate this study, use the following parameters: filter set: dichroic mirror YFP/CFP/mCherry 69008BS; YFP channel (Excitation (Ex): S500/20 &#215; 49057; Emission (EM): D535/30 m 47281), mCherry channel (Ex: 580/25 &#215; 49829; Em: 632/60 m).</li>
			<li>Enter the appropriate settings for the time-lapse experiment (see Table of Materials for the software used in this study): Click on Apps | Multi Dimensional Acquisition. Select the first tab Main and select timelapse, multiple stage positions, and multiple wavelengths.</li>
			<li>Select the tab Saving to enter the location where the experiment should be saved.</li>
			<li>Select the tab Timelapse to enter the number of time points, duration, and time interval. 
			NOTE: The duration of the time-lapse experiment depends on the animal species studied, as timing of oocyte meiotic maturation differs among species. In this experiment with mouse oocytes, oocytes were recorded every 15 min for 16 h.</li>
			<li>Select the tab Stage. Switch on brightfield and locate the position of the oocytes by opening a new window by selecting Acquire | Acquire | Show Live. Once the oocytes are located, switch back to the Multi Dimensional Acquisition window, and press + to set the location of the oocytes.</li>
			<li>Select the tab Wavelengths, and set 3 different wavelengths for brightfield (exposure 15 ms), YFP (exposure 150 ms for Ypet/IL7 3&#39; UTR), and mCherry (exposure 75 ms for Ypet/IL7 3&#39; UTR). 
			NOTE: Adjust the exposure for each Ypet/3&#39; UTR reporter based on the level of reporter accumulation and the injected volume. Ensure that the YFP signal falls in the center of the range of detection at the start of the experiment to prevent underestimation or saturation in case of activation of translation. For mCherry, adjust the exposure for each batch of mCherry as the signal depends on the number of adenine nucleotides that were added during the polyadenylation procedure. Because of the variation in polyadenylation efficiency, use the same batch of mCherry in different experiments for a better comparison between experiments.</li>
			<li>Start the time lapse experiment by clicking on Acquire. See Figure 2 and the Supplemental File for an example of a time-lapse recording in a single oocyte.</li>
		</ol>
		</li>
		<li>Analysis of Ypet-3&#39; UTR translation
		<ol>
			<li>For this analysis (see Table of Materials for the software used in this study), perform two region measurements for each oocyte: the oocyte itself-click on ellipse region and surround the oocyte-and a small region close to the oocyte to be used for background subtraction-click on rectangular region. 
			NOTE: Ensure that the oocytes do not move out of the selected region during the time-lapse recording. Pay special attention to the placement of the region measurement around polar body extrusion, as this causes movement in the oocyte and may distort the recording.</li>
			<li>Export the region measurement data to a spreadsheet by clicking first on Open Log and then on Log Data to export the data analysis software.</li>
			<li>For each individual oocyte and for all measured time-points, subtract the background region measurement from the oocyte region measurement. Do this for YFP and mCherry wavelengths separately.</li>
			<li>Record the timing of germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) for later reference. 
			NOTE: Exclude maturing mouse oocytes when GVBD exceeds 2 h.</li>
			<li>Plot YFP and mCherry expression over time for each oocyte to check for outliers. 
			NOTE: This should be a smooth curve, if it is not this may indicate that the oocyte moved during the recording.</li>
			<li>For each individual oocyte, calculate average mCherry expression for the last ten time-points of the recording. For each time-point, divide the YFP expression by the averaged mCherry expression to correct for injected volume to obtain a YFP/mCherry ratio at every time-point for each individual oocyte.</li>
			<li>Calculate translation rates for a specific time-interval by fitting the slope of the curve by linear regression. Assess the differences in translation rates using statistical inference.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

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.

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	<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 ...

Watch this Scientific Journal Video about Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay at JoVE.com

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 ...

defining-program-maternal-mrna-translation-during-vitro-maturation

Research

JoVE Journal

Developmental Biology

4.2K Views.  University of California at San Francisco. This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...