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>
	<li>Clarke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+control+of+gene+expression+during+mouse+oogenesis.">Post-transcriptional control of gene expression during mouse oogenesis.</a> Results and Problems in Cell Differentiation. 55, 1-21 (2012).</li><li>Gosden, R., Lee, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portrait+of+an+oocyte:+our+obscure+origin.">Portrait of an oocyte: our obscure origin.</a> Journal of Clinical Investigation. 120 (4), 973-983 (2010).</li><li>Conti, M., Sousa Martins, J. P., Han, S. J., Franciosi, F., Menon, K. M. J., Goldstrohm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+the+germ+line.">Translational control in the germ line.</a> Posttranscriptional Mechanisms in Endocrine Regulation. , 129-156 (2015).</li><li>Dai, X. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combinational+code+for+mRNA+3'UTR-mediated+translational+control+in+the+mouse+oocyte.">A combinational code for mRNA 3'UTR-mediated translational control in the mouse oocyte.</a> Nucleic Acids Research. 47 (1), 328-340 (2019).</li><li>Luong, X. G., Daldello, E. M., Rajkovic, G., Yang, C. R., Conti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+a+switch+in+the+translational+program+upon+oocyte+meiotic+resumption.">Genome-wide analysis reveals a switch in the translational program upon oocyte meiotic resumption.</a> Nucleic Acids Research. 48 (6), 3257-3276 (2020).</li><li>Yang, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA-binding+protein+DAZL+functions+as+repressor+and+activator+of+mRNA+translation+during+oocyte+maturation.">The RNA-binding protein DAZL functions as repressor and activator of mRNA translation during oocyte maturation.</a> Nature Communications. 11, 1399 (2020).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+translation+reveals+a+critical+role+for+deleted+in+azoospermia-like+(Dazl)+at+the+oocyte-to-zygote+transition.">Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition.</a> Genes &amp; Development. 25 (7), 755-766 (2011).</li><li>Cakmak, H., Franciosi, F., Musa Zamah, A., Cedars, M. I., Conti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+secretion+during+meiotic+reentry+integrates+the+function+of+the+oocyte+and+cumulus+cells.">Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells.</a> Proceedings of the National Academy of Sciences. 113 (9), 2424-2429 (2016).</li><li>Daldello, E. M., Luong, X. G., Yang, C. R., Kuhn, J., Conti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B2+is+required+for+progression+through+meiosis+in+mouse+oocytes.">Cyclin B2 is required for progression through meiosis in mouse oocytes.</a> Development. 146 (8), (2019).</li><li>Franciosi, F., Manandhar, S., Conti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FSH+regulates+mRNA+translation+in+mouse+oocytes+and+promotes+developmental+competence.">FSH regulates mRNA translation in mouse oocytes and promotes developmental competence.</a> Endocrinology. 157 (2), 872-882 (2016).</li><li>Peters, H., Byskov, A. G., Himelstein-braw, R., Faber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+growth:+the+basic+event+in+the+mouse+and+human+ovary.">Follicular growth: the basic event in the mouse and human ovary.</a> Reproduction. 45 (3), 559-566 (1975).</li><li>Shu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cilostamide+and+forskolin+on+the+meiotic+resumption+and+embryonic+development+of+immature+human+oocytes.">Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes.</a> Human Reproduction. 23 (3), 504-513 (2008).</li><li>Cheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-expressed+Interleukin+7+suppresses+granulosa+cell+apoptosis+and+promotes+oocyte+maturation+in+rats.">Oocyte-expressed Interleukin 7 suppresses granulosa cell apoptosis and promotes oocyte maturation in rats.</a> Biology of Reproduction. 84 (4), 707-714 (2011).</li><li>Ma, J., Fukuda, Y., Schultz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+dormant+Cnot7+mRNA+promotes+deadenylation+of+maternal+transcripts+during+mouse+oocyte+maturation.">Mobilization of dormant Cnot7 mRNA promotes deadenylation of maternal transcripts during mouse oocyte maturation.</a> Biology of Reproduction. 93 (2), 1-12 (2015).</li><li>Sha, Q. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNOT+6L+couples+the+selective+degradation+of+maternal+transcripts+to+meiotic+cell+cycle+progression+in+mouse+oocyte.">CNOT 6L couples the selective degradation of maternal transcripts to meiotic cell cycle progression in mouse oocyte.</a> The EMBO journal. 37 (24), 99333 (2018).</li><li>Yartseva, V., Giraldez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maternal-to-zygotic+transition+during+vertebrate+development:+A+model+for+reprogramming.">The maternal-to-zygotic transition during vertebrate development: A model for reprogramming.</a> Current Topics in Developmental Biology. 113, 191-232 (2015).</li><li>Ma&#353;ek, T., Val&#225;&#353;ek, L., Posp&#237;&#353;ek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+and+RNA+purification+from+sucrose+gradients.">Polysome analysis and RNA purification from sucrose gradients.</a> Methods in Molecular Biology. 703, 293-309 (2011).</li><li>Larsson, O., Tian, B., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+genome-wide+landscape+of+translational+control.">Toward a genome-wide landscape of translational control.</a> Cold Spring Harbor Perspectives in Biology. 5 (1), 012302 (2013).</li><li>Martins, J. P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAZL+and+CPEB1+regulate+mRNA+translation+synergistically+during+oocyte+maturation.">DAZL and CPEB1 regulate mRNA translation synergistically during oocyte maturation.</a> Journal of Cell Science. 129 (6), 1271-1282 (2016).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+mRNAs+with+distinct+3'+UTRs+define+the+temporal+pattern+of+Ccnb1+synthesis+during+mouse+oocyte+meiotic+maturation.">Maternal mRNAs with distinct 3' UTRs define the temporal pattern of Ccnb1 synthesis during mouse oocyte meiotic maturation.</a> Genes &amp; development. 31, 1302-1307 (2017).</li><li>Cheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive,+coordinated+protein-level+changes+driven+by+transcript+isoform+switching+during+meiosis.">Pervasive, coordinated protein-level changes driven by transcript isoform switching during meiosis.</a> Cell. 172 (5), 910-923 (2018).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324 (5924), 218-223 (2009).</li><li>Piqu&#233;, M., L&#243;pez, J. M., Foissac, S., Guig&#243;, R., M&#233;ndez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combinatorial+code+for+CPE-mediated+translational+control.">A combinatorial code for CPE-mediated translational control.</a> Cell. 132 (3), 434-448 (2008).</li><li>Morgan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+3'+uridylation+and+poly(A)+tail+length+sculpt+the+mammalian+maternal+transcriptome.">mRNA 3' uridylation and poly(A) tail length sculpt the mammalian maternal transcriptome.</a> Nature. 548 (7667), 347-351 (2017).</li><li>Yang, F., Wang, W., Cetinbas, M., Sadreyev, R. I., Blower, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+identifies+cis-acting+elements+regulating+mRNA+polyadenylation+and+translation+during+vertebrate+oocyte+maturation.">Genome-wide analysis identifies cis-acting elements regulating mRNA polyadenylation and translation during vertebrate oocyte maturation.</a> RNA. 26 (3), 324-344 (2020).</li><li>Magidson, V., Khodjakov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumventing+photodamage+in+live-cell+microscopy.">Circumventing photodamage in live-cell microscopy.</a> Methods in Cell Biology. 114, 545-560 (2013).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+cells+regulate+maternal+mRNA+translation+and+developmental+competence+of+mouse+oocytes.">Somatic cells regulate maternal mRNA translation and developmental competence of mouse oocytes.</a> Nature Cell Biology. 15 (12), 1415-1423 (2013).</li></ol>

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>
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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 ...

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