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.

<table><tbody><tr><td>&lt;strong&gt;Preparation of media&lt;/strong&gt;</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&amp;nbsp;</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&amp;nbsp;</td><td>JT-Baker</td><td>3506-1</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Gibco&amp;nbsp;</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>&lt;strong&gt;Preparation of mRNA encoding YFP/3&#039; UTR and mCherry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td>Apex Biomedical&amp;nbsp;</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 &amp;micro;g/mL Carbenicillin, Teknova&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Apex Biomedical&amp;nbsp;</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>&lt;strong&gt;Oocyte collection&lt;/strong&gt;</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&amp;nbsp;</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>&lt;strong&gt;Oocyte micro-injection&lt;/strong&gt;</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>&lt;strong&gt;Software&lt;/strong&gt;</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&amp;nbsp;</td><td>Molecular Devices</td><td></td><td>&amp;nbsp;For time-lapse microscopy, analysis of 3&#039; UTR translation&amp;nbsp;</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 ...

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

Research

JoVE Journal

Developmental Biology

4.3K 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 ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Genetics

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 ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

Cancer Research

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Biology

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

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

Summary

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Chapters in this video

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

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

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

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Xenopus laevis as a Model to Identify Translation Impairment

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers