A.A.J. and M.A. co-wrote the manuscript and all co-authors commented on the manuscript. M. A. is supported by CNRS and &#34;Projet Fondation ARC&#34; (PJA2022070005322).A.A.J. is supported by Fondation des Treilles, Fonds Saint-Michel, and Fondation du Coll&#232;ge de France.

All animal experiments were performed in accordance with the guidelines of the European Community and were approved by the French Ministry of Agriculture (authorization No. 75-1170) and by the Direction G&#233;n&#233;rale de la Recherche et de l&#39;Innovation (DGRI; GMO agreement number DUO-5291). Mice were housed in the animal facility on a 12 h light/dark cycle, with an ambient temperature of 22-24 &#176;C and humidity of 40%-50%. Mice used here include female OF1 (Oncins France 1, 8 to 12 weeks old) and female C57BL/...

Impact of Cytoskeleton on Nuclear Shape and Condensate Dynamics in Mouse Oocytes

<ol>
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E., Verlhac, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+nucleus+positioning+in+mouse+oocytes.">Control of nucleus positioning in mouse oocytes.</a> Semin Cell Develop Biol. 82, 34-40 (2018).</li><li>Brunet, S., Maro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germinal+vesicle+position+and+meiotic+maturation+in+mouse+oocyte.">Germinal vesicle position and meiotic maturation in mouse oocyte.</a> Reproduction. 133 (6), 1069-1072 (2007).</li><li>Levi, M., Ghetler, Y., Shulman, A., Shalgi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+molecular+markers+are+correlated+with+maturation-competence+of+human+oocytes.">Morphological and molecular markers are correlated with maturation-competence of human oocytes.</a> Hum Reprod. 28 (9), 2482-2489 (2013).</li><li>Almonacid, 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=Active+diffusion+positions+the+nucleus+in+mouse+oocytes.">Active diffusion positions the nucleus in mouse oocytes.</a> Nat Cell Biol. 17 (4), 470-479 (2015).</li><li>Almonacid, 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=Active+fluctuations+of+the+nuclear+envelope+shape+the+transcriptional+dynamics+in+oocytes.">Active fluctuations of the nuclear envelope shape the transcriptional dynamics in oocytes.</a> Dev Cell. 51 (2), 145-157 (2019).</li><li>Al Jord, A., 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=Cytoplasmic+forces+functionally+reorganize+nuclear+condensates+in+oocytes.">Cytoplasmic forces functionally reorganize nuclear condensates in oocytes.</a> Nat Commun. 13 (1), 5070 (2022).</li><li>Shin, Y., Brangwynne, C. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+mapping+of+nuclear+domain-associated+transcripts+reveals+speckles+and+lamina+as+hubs+of+functionally+distinct+retained+introns.">Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns.</a> Mol Cell. 82 (5), 1035-1052.e9 (2022).</li><li>Guo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+stochastic,+motor-driven+properties+of+the+cytoplasm+using+force+spectrum+microscopy.">Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy.</a> Cell. 158 (4), 822-832 (2014).</li><li>Verlhac, M. H., Kubiak, J. Z., Clarke, H. J., Maro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+and+chromatin+behavior+follow+MAP+kinase+activity+but+not+MPF+activity+during+meiosis+in+mouse+oocytes.">Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes.</a> Development. 120 (4), 1017-1025 (1994).</li><li>Reis, A., Chang, H. Y., Levasseur, M., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APCcdh1+activity+in+mouse+oocytes+prevents+entry+into+the+first+meiotic+division.">APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division.</a> Nat Cell Biol. 8 (5), 539-540 (2006).</li><li>El-Hayek, S., 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=Control+of+oocyte+growth+and+development+by+intercellular+communication+within+the+follicular+niche.">Control of oocyte growth and development by intercellular communication within the follicular niche.</a> Results Probl Cell Differ. 58, 191-224 (2016).</li><li>Dumont, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+centriole-+and+RanGTP-independent+spindle+assembly+pathway+in+meiosis+I+of+vertebrate+oocytes.">A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes.</a> J Cell Biol. 176 (3), 295-305 (2007).</li><li>Kal&#225;b, P., Pralle, A., Isacoff, E. Y., Heald, R., Weis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+a+RanGTP-regulated+gradient+in+mitotic+somatic+cells.">Analysis of a RanGTP-regulated gradient in mitotic somatic cells.</a> Nature. 440 (7084), 697-701 (2006).</li><li>Lafontaine, D. L. J., Riback, J. A., Bascetin, R., Brangwynne, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleolus+as+a+multiphase+liquid+condensate.">The nucleolus as a multiphase liquid condensate.</a> Nat Rev. Mol Cell Biol. 22 (3), 165-182 (2021).</li><li>Terret, M. E., 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=DOC1R:+a+MAP+kinase+substrate+that+control+microtubule+organization+of+metaphase+II+mouse+oocytes.">DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes.</a> Development. 130 (21), 5169-5177 (2003).</li><li>Dumont, J., Verlhac, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+FRET+to+study+RanGTP+gradients+in+live+mouse+oocytes.">Using FRET to study RanGTP gradients in live mouse oocytes.</a> Methods Mol Biol. 957, 107-120 (2013).</li><li>McSwiggen, D. 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ImageJ Available from: <a target="_blank" href="https://imagej.github.io/update-sites/big-epfl">https://imagej.github.io/update-sites/big-epfl</a> (2023)</li><li>Hebert, B., Costantino, S., Wiseman, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+image+correlation+spectroscopy+(STICS)+theory,+verification,+and+application+to+protein+velocity+mapping+in+living+CHO+cells.">Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.</a> Biophys J. 88 (5), 3601-3614 (2005).</li><li>Yi, K., 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=Dynamic+maintenance+of+asymmetric+meiotic+spindle+position+through+Arp2/3-complex-driven+cytoplasmic+streaming+in+mouse+oocytes.">Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes.</a> Nat Cell Biol. 13 (10), 1252-1258 (2011).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9, 676-682 (2012).</li><li>. Stowers ImageJ Plugins Available from: <a target="_blank" href="https://research.stowers.org/imagejplugins/">https://research.stowers.org/imagejplugins/</a> (2023)</li><li>Luisier, F., Vonesch, C., Blu, T., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+interscale+wavelet+denoising+of+Poisson-corrupted+images.">Fast interscale wavelet denoising of Poisson-corrupted images.</a> Signal Process. 90 (2), 415-427 (2010).</li><li>. Ovocyte_Nucleus Available from: <a target="_blank" href="https://github.com/orion-cirb/Ovocyte_Nucleus">https://github.com/orion-cirb/Ovocyte_Nucleus</a> (2023)</li><li>Letort, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> gletort/ImageJFiles: GLijplugs_v0.2.0. , (2022).</li><li>. ImageJFiles Available from: <a target="_blank" href="https://github.com/gletort/ImageJFiles/tree/master/radioak">https://github.com/gletort/ImageJFiles/tree/master/radioak</a> (2023)</li><li>Chu, F. Y., Haley, S. C., Zidovska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+shape+fluctuations+of+the+cell+nucleus.">On the origin of shape fluctuations of the cell nucleus.</a> Proc Natl Acad Sci U S A. 114 (39), 10338-10343 (2017).</li><li>Caragine, C. M., Haley, S. C., Zidovska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+fluctuations+and+coalescence+of+nucleolar+droplets+in+the+human+cell+nucleus.">Surface fluctuations and coalescence of nucleolar droplets in the human cell nucleus.</a> Phys Rev Lett. 121 (14), 148101 (2018).</li><li>Introini, V., Kidiyoor, G. R., Porcella, G., Cicuta, P., Cosentino Lagomarsino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centripetal+nuclear+shape+fluctuations+associate+with+chromatin+condensation+in+early+prophase.">Centripetal nuclear shape fluctuations associate with chromatin condensation in early prophase.</a> Commun Biol. 6 (1), 1-11 (2023).</li><li>Boija, A., Klein, I. A., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+condensates+and+cancer.">Biomolecular condensates and cancer.</a> Cancer Cell. 39 (2), 174-192 (2021).</li></ol>

Key steps in this protocol include proper microinjection of oocytes without affecting their survival or normal function9,10,11, as well as microinjecting predefined amounts of cRNA that would allow correct visualization of relevant structures, like nuclear speckles.
Establishing the link between cytoplasmic and (intra)-nuclear dynamics is essential when studying how the cytoskeleton agitates the nucle...

Nuclear positioning is essential for multiple cellular and developmental functions1,2,3,4,5. Mammalian female germ cells named oocytes remodel their cytoplasm to position the nucleus at the cell center despite undergoing an asymmetric division in size, which relies on subsequent chromosome off-centering6 (Figure 1). This centering of the nucleus predicts successful oocyte development in mice and humans7, 8, and thus, their embryogenic potential (Figure 1).
Cytoplasmic remodeling in mouse oocytes is driven primarily by the actomyosin cytoskeleton9 (Figure 2). Its activity generates mechanical forces that agitate, reposition, and penetrate the nucleus10 (Figure 2). Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear messenger RNA-processing organelles named nuclear speckles11, one of several membrane-less organelles in the nucleus known as biomolecular condensates12,13,14,15,16 (Figure 2).
Live imaging has been decisive in deciphering the functional implications of nuclear agitation. Movies of nuclear migration over hours, as well as high-temporal resolution movies of the actin mesh and the bulk cytoplasm, largely contributed to the elaboration of a theoretical model for nuclear positioning, linking different timescales9. Also, high temporal resolution movies of the cytoplasm, nuclear outline and nuclear components such as chromatin and nuclear condensates, highlighted the role of cytoskeleton-based agitation of the nucleus on RNA-processing and gene expression in mouse oocytes, bridging different spatiotemporal scales within the cell10,11. Altogether, such a scale-crossing approach based on live imaging provided the first rationale linking cytoskeletal agitation of the nucleus to the developmental success of oocytes.
The protocol provides the imaging and image analysis pipeline used to study the transmission of cytoplasmic forces (generated primarily by F-actin and partly by microtubules) to the nucleus and its internal components in mouse oocytes. The outcome of these experiments is to capture the continuum of forces across spatial scales, from the cytoskeleton in the cytoplasm to the nuclear interior via high temporal resolution movies as shown in two recent studies10,11, that established the link between cytoplasmic active movements, fluctuations of the nuclear outline, as well as movement and surface fluctuations of a single type of nuclear biomolecular condensates: nuclear speckles. The same approach may be applied to other model systems where cytoplasmic forces are expected to change, such as in the context of malignant cancer cells17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma&#160;</td>
			<td>A3311</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-X1-M1 spinning disk</td>
			<td>Yokogawa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMI6000B microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet microinjector</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter wheel</td>
			<td>Sutter Instruments Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorodish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Metamorph software&#160;</td>
			<td>Universal Imaging,&#160;</td>
			<td></td>
			<td>version 7.7.9.0</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M8410-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OF1 and C57BL/6 mice&#160;</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tailing kit&#160;</td>
			<td>Thermo Fisher</td>
			<td>AM1350</td>
			<td></td>
		</tr>
		<tr>
			<td>Retiga 3 CCD camera&#160;</td>
			<td>QImaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNAeasy kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>SC35 antibody</td>
			<td>Abcam</td>
			<td>ab11826</td>
			<td>Nuclear speckle antibody; mouse IgG1 anti-SRSF2/SC35 (1:400)</td>
		</tr>
		<tr>
			<td>SRSF2-GFP plasmid&#160;</td>
			<td>&#160;OriGene Technologies</td>
			<td>MG202528</td>
			<td>NM_011358</td>
		</tr>
		<tr>
			<td>Stripper Micropipette</td>
			<td>&#160;XLAB Solutions</td>
			<td></td>
			<td>specialized for oocyte collection</td>
		</tr>
		<tr>
			<td>T3 mMessage mMachine</td>
			<td>Thermo Fisher</td>
			<td>AM1384&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 mMessage mMachine&#160;</td>
			<td>Thermo Fisher</td>
			<td>AM13344</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic chamber</td>
			<td>Life Imaging Service</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Windows Excel</td>
			<td>Windows</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing cytoskeleton-based agitation of the mouse oocyte nucleus across spatial scales

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

The lab aims to better understand the mechanisms involved in the acquisition of female gamut quantity, which is essential for the reproduction of sexual species using the mouse oocytes model system. Recently, we identified a novel mechanical transduction process that promotes agitation of the nucleus and its content, leading to the fine regulation of maternal RNAs in mouse oocytes. The imaging and image analysis pipeline presented in this protocol allows us to highlight the transmission of cytoskeletal forces as a continuum across scales from the cytoplasm to the nucleus and its components, including nuclear RNA processing bodies in mouse oocytes.This protocol provides simple tools that allow us to address first transmission across multiple cellular scales in a single pipeline for the first time. Complemented with biophysical modeling, this protocol constitutes a non-invasive technique to address changes in nuclear mechanics and the dissipation of energy across cellular compartments.

Image panels in Figure 3 show examples of a typical fully grown oocyte (Figure 3A), the nucleoplasm in a fully grown oocyte expressing YFP-Rango (Figure 3B), the nucleoplasm in a fully grown oocyte expressing a correct (left panel; Figure 3C) or an excessive (right panel; Figure 3C) dose of SRSF2-GFP cRNA, and an immunostaining of nuclear speckles in a fully grown oocyte us...

Watch this Scientific Journal Video about Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes at JoVE.com

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...

capturing-cytoskeleton-based-agitation-mouse-oocyte-nucleus-across

Research

JoVE Journal

Biology

Oocyte Collection, Preparation, and Microinjection in Mouse for Live Imaging Studies

Live Cell Imaging and Analysis of Nuclear Dynamics in Mouse Oocytes

465 Views.  Collège de France, CNRS, INSERM, Université PSL.  This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

Impact of Cytoskeleton on Nuclear Shape and Condensate Dynamics in Mouse Oocytes

Summary