We acknowledge support for this work from the project &#34;Establishment of &#39;capacity building&#39; infrastructures in Biomedical Research (BIOMED-20)&#34; (MIS 5047236), which is implemented under the Action &#34;Reinforcement of the Research and Innovation Infrastructure&#34;, funded by the Operational Program &#34;Competitiveness, Entrepreneurship and Innovation&#34; (NSRF 2014-2020), and co-financed by Greece and the European Union (European Regional Development Fund).

All mice experiments were approved by the local authorities (Region of Ioannina, Greece) and conducted in accordance with the European Communities Council Directives 2010/63/EU. Experiments were conducted with respect to the principles of the 3Rs.&#160;All the CD-1 mice used for the experiments were kept in the animal house facility of the University of Ioannina, Greece, in a room with controlled temperature (22 &#176;C) and humidity (60%) and were fed ad libitum. The animal house has a license to operate a facility for breeding (EL33-BIObr01), supply (EL33-BIOsup01), and experiments (EL33BIO-exp01).
1. Preparation of reagents
<ol>
	<li>Dilute 3-isobutyl-1-methylxanthine (IBMX) powder (see the Table of Materials) in dimethyl sulfoxide (DMSO) (see the Table of Materials) to a final concentration of 200 mM. &#924;ake 10 &#181;L aliquots, and store at &#8722;20 &#176;C. Use the solution within 1 month. 
	NOTE: IBMX powder is kept at &#8722;20 &#176;C.</li>
	<li>Prepare all the immunofluorescence buffers, and store them at 4 &#176;C.
	<ol>
		<li>Prepare sterile phosphate-buffered saline (PBS) by diluting one PBS tablet (see the Table of Materials) in 200 mL of ddH2&#927;.</li>
		<li>Make PHEM buffer by adding in 80 mL of ddH2&#927;, 0.59575 g of HEPES, 1.81422 g of PIPES, 0.38035 g of EGTA, and 0.04066 g of MgCl2 (see the Table of Materials) while agitating with a magnetic stirrer (see the Table of Materials), and simultaneously add NaOH (see the Table of Materials) until the pH reaches 6.9 (check using a pH/ORP meter [see the Table of Materials]). Then, add ddH2&#927; to a final volume of 100 mL.</li>
		<li>Prepare paraformaldehyde-Triton-X-100 (PFA-Tx-100) buffer by diluting PFA powder (see the Table of Materials) in PHEM buffer while agitating with a magnetic stirrer under heating at a final concentration of 4% PFA. Then, filter the buffer using a syringe and a 0.2 &#181;m filter (see the Table of Materials), and add 0.5% Tx-100 (see the Table of Materials). Prepare approximately 10 mL of PFA-Tx-100 (0.4 g of PFA, 50 &#181;L of Tx-100), which is enough for one experiment. Store it at 4 &#176;C for 1 week maximum. 
		&#8203;CAUTION: Wear gloves to handle PFA, and avoid contact with skin and eyes.</li>
		<li>Prepare washing buffer by adding bovine serum albumin (final concentration: 0.5% w/v BSA) (see the Table of Materials) in PBS, and agitate mechanically. Add 10% w/v NaN3 buffer (sodium azide) at a 1:1,000 dilution to minimize the risk of fungal and bacterial contamination. Make a 10% w/v NaN3 buffer by adding 1 g of NaN3 powder (see the Table of Materials) to 10 mL of ddH2O; store the NaN3 buffer at room temperature.</li>
		<li>Prepare blocking buffer by adding BSA (final concentration: 3% w/v) in PBS and agitating mechanically. Add 10% NaN3 buffer at a 1:1,000 dilution.</li>
	</ol>
	</li>
</ol>
2. Collection of GV oocytes from dissected ovaries and induction of DSBs
NOTE: All the tools and solutions should be sterile. Oocyte handling is conducted by using a mouth pipette under a stereo microscope (see the Table of Materials), and all the drops are covered with mineral oil (see the Table of Materials and Figure 1E).
<ol>
	<li>Inject mice intraperitoneally with 7 international units (IU) of pregnant mare&#39;s serum gonadotropin (PMSG) (see the Table of Materials) 46-48 h before culling the mice by cervical dislocation. 
	NOTE: All the mice used should be 8-12 weeks old.</li>
	<li>Filter M2 culture medium (see the Table of Materials) with a syringe and a 0.2 &#181;m filter, and add IBMX 200 mM to a final concentration 200 &#181;M in a 14 mL round-bottom tube (see the Table of Materials) to keep the oocytes arrested at prophase I. Then, prepare drops of M2-IBMX medium in a plastic tissue culture dish (see the Table of Materials), and place it on a hot block (see the Table of Materials) at 37 &#176;C for at least 30 min before oocyte isolation. Store the M2 at 4 &#176;C.</li>
	<li>Sacrifice the mice by cervical dislocation, dissect the ovaries, and place them in a 5 mL round-bottom tube (see the Table of Materials) with M2-IBMX.</li>
	<li>Transfer the ovaries to a plastic lid containing 1.5 mL of M2-IBMX, remove any peri-ovarian adipose tissue or fallopian tube segments, and release the COCs by mechanical perforation of the ovaries with a 27 G needle (see the Table of Materials and Figure 1A-C).</li>
	<li>Transfer the COCs to a culture dish with drops of M2-IBMX (approximately 25-30 &#181;L each), and remove the cumulus cells by repeated pipetting using a narrow-bore glass Pasteur pipette (see the Table of Materials and Figure 1D).</li>
	<li>Select SN GV-stage oocytes, and transfer them in a drop (25 &#181;L) of M2-IBMX medium on a hot block at 37 &#176;C protected from light (Figure 1F).
	<ol>
		<li>Look for SN oocytes based on their bigger size and centrally placed nuclei in contrast with NSN oocytes, in which the nuclei are positioned peripherally21. In any case, observe the DNA configuration under a confocal microscope before taking the final decision about the type of GV oocyte (SN or NSN).</li>
	</ol>
	</li>
	<li>Induce DSBs using etoposide (see the Table of Materials). Place the GV-stage oocytes in drops (25 &#181;L each) of the genotoxic agent for 1 h on the hot block at 37 &#176;C in dark conditions. 
	&#8203;NOTE: Etoposide is a topoisomerase II inhibitor that introduces DSBs to the DNA22. Keep the etoposide in 10 &#181;L aliquots of 20 mg/mL at room temperature protected from light. The concentrations that have been tested are 5 &#181;g/mL, 20 &#181;g/mL, and 50 &#181;g/mL.</li>
	<li>To keep the GV-stage oocytes arrested for a prolonged period, place the oocytes in drops of M16 culture medium (see the Table of Materials) supplemented with 400 &#181;M IBMX in an incubator (see the Table of Materials) at 37 &#176;C and 5% CO2. Store the M16 at 4 &#176;C, filter the medium with a syringe and a 0.2 &#181;m filter, and incubate it for at least 1 h before use.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65494/65494fig01.jpg" /> 
Figure 1: Oocyte isolation process. (A) Removal of peri-ovarian adipose tissue and leftover fallopian tube segments from ovaries in M2 medium with IBMX. Photograph obtained through the stereo microscope eyepieces. Scale bar = 1 mm. (B) Isolated ovaries in M2 medium with IBMX. Image obtained through the stereo microscope eyepieces. Scale bar = 1 mm. (C) Mechanical perforation of ovaries using a 27 G needle in M2 medium with IBMX. Image obtained through the stereo microscope eyepieces. Scale bar = 1 mm. (D) COCs released from ovaries after perforation in M2 medium with IBMX. Image obtained through the stereo microscope eyepieces. Scale bar = 100 &#181;m. (E) Oocyte collection using a mouth pipette. (F) Denuded oocytes, after the removal of the surrounding cumulus cells, in M2 medium with IBMX. Image obtained through the stereo microscope eyepieces. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65494/65494fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Oocyte fixation and immunofluorescence
NOTE: Oocyte handling is conducted by using a mouth pipette under a stereo microscope, and all the drops are covered with mineral oil.
<ol>
	<li>Place the control and etoposide-treated GV oocytes in different plastic tissue culture dishes with PFA-Tx-100 buffer for 40 min at room temperature.</li>
	<li>Wash the oocytes in three different drops of washing buffer (50 &#181;L each) at room temperature. Leave the oocytes for 5 min in each drop.</li>
	<li>Place the oocytes in drops of blocking buffer (25 &#181;L each) for 1 h on a hot block at 37 &#176;C.</li>
	<li>Prepare the primary antibody that recognizes &#947;H2AX (rabbit phospho-&#919;2&#913;&#935;) (Ser139) (see the Table of Materials) (stock solution: 1 mg/mL). Use a 1:200 dilution in blocking buffer, and place the oocytes in drops of primary antibody (15 &#181;L each) at 4 &#176;C overnight. 
	NOTE: Phospho-&#919;2&#913;&#935; (&#947;H2AX) is a common marker for detecting DSBs in both somatic cells and GV oocytes18,23.</li>
	<li>The following day, wash the oocytes in three different drops of washing buffer (50 &#181;L each) at room temperature. Leave the oocytes for 5 min in each drop.</li>
	<li>Prepare the secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit (see the Table of Materials) (stock solution: 2 mg/mL). Use a 1:200 dilution in blocking buffer, and place the oocytes in drops of secondary antibody (15 &#181;L each) for 1 h on a hot block at 37 &#176;C protected from light.</li>
	<li>Transfer the oocytes into drops of DRAQ7 (25 &#181;L each) (stock solution: 0.3 mM; see the Table of Materials), which is a far-red fluorescent DNA dye that only stains DNA in permeabilized cells. Use a 1:250 dilution in washing buffer for 10 min at room temperature in dark conditions.</li>
	<li>Wash the oocytes in three different drops of washing buffer (50 &#181;L each) at room temperature. Leave them for 5 min in each drop, and then transfer them to small drops (approximately 5 &#181;L each) of washing buffer in a 35 mm glass-bottom Petri dish (see the Table of Materials) for confocal microscopy (Figure 2A). 
	&#8203;NOTE: The washing of both the DNA stain and secondary antibody is performed at the same time.</li>
</ol>
4. Confocal microscopy
NOTE: Confocal microscopy should be performed immediately to avoid the reduction in the fluorescence intensity after the placement of the oocytes in glass-bottom dishes. Access to a confocal microscope (see the Table of Materials) with a motorized stage is required.
<ol>
	<li>Microscope setup
	<ol>
		<li>In the confocal system, switch on the laser controller, the lasers, the microscope controller, the lamps for the transmitted light, and the PC (Figure 2B, D).</li>
		<li>Open the confocal software, and choose the 40x oil lens.</li>
		<li>Place the dish into the specimen holder, and try to focus on the oocytes by moving the stage on XY and Z axes using the joystick (Figure 2C).</li>
	</ol>
	</li>
	<li>Scanning of the oocytes
	<ol>
		<li>Set the laser power, the gain, and the pinhole size independently for each experiment in order to minimize any saturation.</li>
		<li>For each oocyte, set the area of interest, specifically in the nucleus at the DNA area. Define the borders of the DNA area, and adjust the z step size to 3 &#181;m. Then, start the scanning.</li>
		<li>Save the images for each cell in the selected folder.</li>
		<li>When the scanning is completed, exit the software, shut down the computer, and turn off the laser controller, the lasers, the microscope controller, and the lamps for the transmitted light.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65494/65494fig02.jpg" /> 
Figure 2: Confocal microscopy. (A) Fixed oocytes after performing the immunofluorescence protocol and DNA staining, which are in separate drops of washing buffer, covered with mineral oil, placed in a glass-bottom dish, and prepared for confocal microscopy imaging. Each drop contains a different experimental category. Image obtained through the stereo microscope eyepieces. Scale bar = 1 mm/100 &#181;m for the zoomed-in portion. (B) Glass-bottom plate placed on the confocal microscope stage. (C) Brightfield image of oocytes obtained through confocal microscopy. Scale bar = 100 &#181;m. (D) The confocal microscopy system. <a href="https://www.jove.com/files/ftp_upload/65494/65494fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Imaging analysis
<ol>
	<li>Download Fiji ImageJ-win64 in the browser (https://imagej.net/software/fiji/downloads), open it, and import the data as TIFF stack files. 
	NOTE: Open each oocyte file separately.</li>
	<li>Click on Image | Color | Split Channels to split all the channels.</li>
	<li>Click on LUT (Look up Table), and choose the preferred colors for each channel.</li>
	<li>Click on Image | Color | Merge Channels to merge the channels for &#947;&#919;2&#913;&#935; and DNA. Leave the brightfield channel unmerged.</li>
	<li>In NSN oocytes and in SN oocytes with low levels of DNA damage, &#947;&#919;2&#913;&#935; is detected as foci in the DNA region. In this case, click on the &#34;Multi-point&#34; or point command, and select every &#947;&#919;2&#913;&#935; focus that coincides with the DNA. Repeat this step for all the stacks.</li>
	<li>In SN oocytes with high levels of DNA damage, the &#947;&#919;2&#913;&#935; signal is distributed throughout the whole DNA region. In this case, click on Image | Stacks | Z project, and with the Freehand selections command, select the entire DNA area.</li>
	<li>To measure the &#947;&#919;2&#913;&#935; fluorescence, click on Analyze | Measure, and copy the measurements into a .xlsx file. Then, calculate the mean fluorescence, normalize the values, and count the number of foci before creating any graphs.</li>
	<li>Click on Analyze | Set Scale to set the scale and then on Analyze | Tools | Scale bar to add a scale bar to the channels.</li>
</ol>

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

<ol>
	<li>Wang, X., Pepling, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+meiotic+prophase+one+in+mammalian+oocytes.">Regulation of meiotic prophase one in mammalian oocytes.</a> Frontiers in Cell and Developmental Biology. 9, 667306 (2021).</li><li>Filatov, M., Khramova, Y., Semenova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+prophase+I+meiotic+arrest+maintenance+and+meiotic+resumption+in+mammalian+oocytes.">Molecular mechanisms of prophase I meiotic arrest maintenance and meiotic resumption in mammalian oocytes.</a> Reproductive Sciences. 26 (11), 1519-1537 (2019).</li><li>Adhikari, D., 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=Inhibitory+phosphorylation+of+Cdk1+mediates+prolonged+prophase+I+arrest+in+female+germ+cells+and+is+essential+for+female+reproductive+lifespan.">Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan.</a> Cell Research. 26 (11), 1212-1225 (2016).</li><li>Sun, S. C., Kim, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+asymmetric+division+in+oocytes.">Molecular mechanisms of asymmetric division in oocytes.</a> Microscopy and Microanalysis. 19 (4), 883-897 (2013).</li><li>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=Mammalian+egg+activation:+From+Ca2++spiking+to+cell+cycle+progression.">Mammalian egg activation: From Ca2+ spiking to cell cycle progression.</a> Reproduction. 130 (6), 813-823 (2005).</li><li>Solc, P., Schultz, R. M., Motlik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prophase+I+arrest+and+progression+to+metaphase+I+in+mouse+oocytes:+comparison+of+resumption+of+meiosis+and+recovery+from+G2-arrest+in+somatic+cells.">Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells.</a> Molecular Human Reproduction. 16 (9), 654-664 (2010).</li><li>Parfenov, V., Potchukalina, G., Dudina, L., Kostyuchek, D., Gruzova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+antral+follicles:+oocyte+nucleus+and+the+karyosphere+formation+(electron+microscopic+and+autoradiographic+data).">Human antral follicles: oocyte nucleus and the karyosphere formation (electron microscopic and autoradiographic data).</a> Gamete Research. 22 (2), 219-231 (1989).</li><li>Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organization+during+mouse+oocyte+growth.">Chromatin organization during mouse oocyte growth.</a> Molecular Reproduction and Development. 41 (4), 479-485 (1995).</li><li>Sun, 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=Comprehensive+analysis+of+nonsurrounded+nucleolus+and+surrounded+nucleolus+oocytes+on+chromatin+accessibility+using+ATAC-seq.">Comprehensive analysis of nonsurrounded nucleolus and surrounded nucleolus oocytes on chromatin accessibility using ATAC-seq.</a> Molecular Reproduction and Development. 90 (2), 87-97 (2023).</li><li>Zuccotti, M., Bellone, M., Longo, F., Redi, C. A., Garagna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully-mature+antral+mouse+oocytes+are+transcriptionally+silent+but+their+heterochromatin+maintains+a+transcriptional+permissive+histone+acetylation+profile.">Fully-mature antral mouse oocytes are transcriptionally silent but their heterochromatin maintains a transcriptional permissive histone acetylation profile.</a> Journal of Assisted Reproduction and Genetics. 28 (12), 1193-1196 (2011).</li><li>Winship, A. L., Stringer, J. M., Liew, S. H., Hutt, K. 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+importance+of+DNA+repair+for+maintaining+oocyte+quality+in+response+to+anti-cancer+treatments,+environmental+toxins+and+maternal+ageing.">The importance of DNA repair for maintaining oocyte quality in response to anti-cancer treatments, environmental toxins and maternal ageing.</a> Human Reproduction Update. 24 (2), 119-134 (2018).</li><li>Lieber, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+human+nonhomologous+DNA+end+joining.">The mechanism of human nonhomologous DNA end joining.</a> The Journal of Biological Chemistry. 283 (1), 1-5 (2008).</li><li>Chiruvella, K. K., Liang, Z., Wilson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+double-strand+breaks+by+end+joining.">Repair of double-strand breaks by end joining.</a> Cold Spring Harbor Perspectives in Biology. 5 (5), 012757 (2013).</li><li>Chang, H. H. Y., Pannunzio, N. R., Adachi, N., Lieber, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-homologous+DNA+end+joining+and+alternative+pathways+to+double-strand+break+repair.">Non-homologous DNA end joining and alternative pathways to double-strand break repair.</a> Nature Reviews. Molecular Cell Biology. 18 (8), 495-506 (2017).</li><li>Shibata, A., Jeggo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+for+the+DNA-PK+complex+and+53BP1+in+protecting+ends+from+resection+during+DNA+double-strand+break+repair.">Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair.</a> Journal of Radiation Research. 61 (5), 718-726 (2020).</li><li>Mohiuddin, I. S., Kang, 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=DNA-PK+as+an+emerging+therapeutic+target+in+cancer.">DNA-PK as an emerging therapeutic target in cancer.</a> Frontiers in Oncology. 9, 635 (2019).</li><li>Marangos, P., Carroll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocytes+progress+beyond+prophase+in+the+presence+of+DNA+damage.">Oocytes progress beyond prophase in the presence of DNA damage.</a> Current Biology. 22 (11), 989-994 (2012).</li><li>Paull, T. T., 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+critical+role+for+histone+H2AX+in+recruitment+of+repair+factors+to+nuclear+foci+after+DNA+damage.">A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.</a> Current Biology. 10 (15), 886-895 (2000).</li><li>Bakkenist, C. J., Kastan, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+activates+ATM+through+intermolecular+autophosphorylation+and+dimer+dissociation.">DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.</a> Nature. 421 (6922), 499-506 (2003).</li><li>Marangos, P., 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=DNA+damage-induced+metaphase+I+arrest+is+mediated+by+the+spindle+assembly+checkpoint+and+maternal+age.">DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age.</a> Nature Communications. 6, 8706 (2015).</li><li>Lavrentyeva, E. A., Shishova, K. V., Zatsepina, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+nuclear+dynamics+in+mouse+GV+oocytes+with+a+diverse+chromatin+configuration.">Differences in nuclear dynamics in mouse GV oocytes with a diverse chromatin configuration.</a> Biology Bulletin Russian Academy of Sciences. 46, 332-341 (2019).</li><li>Montecucco, A., Zanetta, F., Biamonti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+etoposide.">Molecular mechanisms of etoposide.</a> EXCLI Journal. 14, 95-108 (1998).</li><li>Mayer, 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=DNA+damage+response+during+mouse+oocyte+maturation.">DNA damage response during mouse oocyte maturation.</a> Cell Cycle. 15 (4), 546-558 (2016).</li><li>Olive, P., Ban&#225;th, 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+comet+assay:+A+method+to+measure+DNA+damage+in+individual+cells.">The comet assay: A method to measure DNA damage in individual cells.</a> Nature Protocols. 1, 23-29 (2006).</li><li>Wu, D., Dean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EXOSC10+sculpts+the+transcriptome+during+the+growth-to-maturation+transition+in+mouse+oocytes.">EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes.</a> Nucleic Acids Research. 48 (10), 5349-5365 (2020).</li><li>Simon, L., Emery, B., Carrell, D., Agarwal, A., Henkel, R., Majzoub, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage:+COMET+assay.">DNA damage: COMET assay.</a> Manual of Sperm Function Testing in Human Assisted Reproduction. , 202-212 (2021).</li></ol>

The authors have no conflicts of interest.

By using the method described here, we detected DSBs in mammalian oocytes. This method allows for the detection and study of the DNA repair process in oocytes. The same protocol could also be used for analyzing other proteins that participate in physiological processes in mammalian oocytes. It is important to study how oocytes respond to potential DNA damage in order to better understand the cause of female subfertility in humans.
Studying the DNA damage response in mammalian oocytes can be challenging because of the sensitivity of oocytes. Oocyte handling requires specific temperatures and CO2 and O2 concentrations. At the same time, the oocytes must be protected from light. &#913;ll handling should be done by using glass pipettes that are not to&#959; narrow, as this could be harmful for the oocytes, but also not to&#959; wide, as this could cause the dilution of the medium and, thus, negatively affect the fixation procedure. In each step of fixation, several drops of buffers are used to minimize the dilution effect. An alternative way for observing DSBs is the Comet assay24. Even though this technique is more sensitive, it is more complicated. At the same time, by using the Comet assay, it is not possible to detect the exact DNA region where the damage occurs, and in cells with abundant RNA molecules, like GV-stage oocytes25, the background could be increased, leading to a false DNA damage signal26.
By using the immunofluorescence protocol described here, we can detect DSBs with accuracy and estimate the repair progress in GV-stage oocytes, as indicated by the reduction in &#947;H2AX fluorescence over time. Nevertheless, one limitation of this method is that certain antibodies may present nonspecific distribution throughout the ooplasm, thus leading to images with high background fluorescence. The PFA-Tx-100 buffer is used instead of sequential PFA and Tx-100, as we have observed that it improves the fixation process by allowing the detection of less background and non-specific fluorescence. A second limitation of using &#947;H2AX for DSB detection is that damage cannot be estimated after GVBD because of the spontaneous phosphorylation of &#947;H2AX in meiosis23.
In this immunofluorescence protocol, the oocytes remain in a liquid buffer and cannot be stored within slides. This fact makes it difficult to preserve the fixed cells for days after the addition of the secondary antibody. In order to attain good-quality images and not to lose signal, it is preferable to perform the imaging within a few hours after the addition of the secondary antibody. It should also be noted that the scanning of the nuclei through the Z-axis could make the signal become weaker due to overexposure. For that reason, it is preferable to lower the laser power and to increase the speed of the scanning.
Lastly, another limitation of the immunofluorescence protocol is that it can be used only for fixed/non-living cells. Therefore, we can estimate only the presence and absence of factors at specific time points without knowing if there are any fluctuations in their concentration or changes in their behavior through time. This problem could be overcome by using live-cell imaging and fluorescently tagged markers.

The process of meiosis in mammalian female germ cells is initiated in the ovaries before birth. The total number of oocytes is established in the ovaries primarily during embryogenesis. Oocytes enter meiosis and remain arrested at prophase I1. After the onset of puberty and the production and endocrine action of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), oocytes may reinitiate and complete meiosis2. In humans, prophase arrest can last for up to 50 years3. The cell divisions following the entry into meiosis I are asymmetric, resulting in the production of a small polar body and an oocyte that retains its size. Thus, most cytoplasmic components are stored in the ooplasm during early embryogenesis4. Then, the oocytes enter meiosis II, without reforming their nucleus or decondensing their chromosomes, and remain arrested at metaphase II until fertilisation5.
A unique characteristic that distinguishes oocytes from somatic cells is the state of arrest in prophase I, when the oocyte possesses an intact nucleus (germinal vesicle [GV] arrest), referred to as the GV stage6. Based on the chromatin organization, GV-stage oocytes are classified into two categories: non-surrounded nucleolus (NSN) and surrounded nucleolus (SN)7,8. In NSN GV-stage oocytes, the chromatin spreads throughout the whole nuclear region, and transcription is active, while in SN oocytes, the chromatin forms a compact ring that surrounds the nucleolus, and transcription is silent9. Both types of GV-stage oocytes show meiotic competence; they enter meiosis at the same rate, but the NSN oocytes present low developmental capacity and cannot develop beyond the two-cell stage embryo10.
The protracted state of prophase I arrest increases the incidence of DNA damage accumulation11. Therefore, DNA damage response mechanisms in oocytes are essential for allowing the production of gametes with genetic integrity and for ensuring that the resulting embryo has a physiological chromosomal content.
A central aspect of the DNA damage response is DNA repair. The main pathways for DSB repair in eukaryotic cells include non-homologous end joining (NHEJ), homologous recombination (HR), and alternative NHEJ12,13,14,15. NHEJ is a faster but more error-prone mechanism, while HR requires more time to be completed but has high fidelity16.
There is not enough knowledge about the mechanisms that oocytes use for DNA damage repair. Studies have shown that DNA damage induced in fully grown mammalian oocytes by the use of genotoxic agents, such as etoposide, doxorubicin, or UVB or ionizing radiation, does not affect the timing and rates of exit from prophase I arrest17. Oocytes can undergo GV breakdown (GVBD) even in the presence of elevated levels of damage. This damage can be determined by the observation of &#947;H2AX. This phosphorylated form of H2AX (&#947;&#919;2&#913;&#935;) is a DSB marker, which is located at the site of breaks and functions as a scaffold to help repair factors and proteins to accumulate at broken ends18.
The absence of cell cycle arrest following DNA damage is due to an insufficient DNA damage checkpoint that allows oocytes with unrepaired DNA to re-enter meiosis. Following high levels of DNA damage, a checkpoint can maintain prophase arrest through the activation of an ATM/ Chk1-dependent pathway. The limited checkpoint response to DSBs is due to the limited activation of ATM17,19. In the M-phase of meiosis I, research has shown that DNA damage may activate a spindle assembly checkpoint (SAC)-induced meiosis I checkpoint, which prevents the activation of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) and, therefore, M-phase exit. Moreover, the ablation of SAC proteins overcomes the state of M-phase arrest, thus underlining the importance of the SAC in the establishment of the meiosis I chekpoint20.
As previous research clearly shows, DSBs cannot induce a robust prophase checkpoint in mouse oocytes. If such damage is left unrepaired, it could lead to embryos carrying chromosomal abnormalities. Therefore, it is important to study the DNA damage response at different stages of female gametogenesis to better understand the unique pathways that oocytes use to cope with potential genetic insults.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 mL Pasteur pipettes in LDPE, graduated</td>
			<td>APTACA</td>
			<td>1502</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cc syringes</td>
			<td>SoftCare</td>
			<td>114.104.21</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated goat anti-rabbit Secondary Ab</td>
			<td>Biotium</td>
			<td>20012</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-H2A.X (Ser139)</td>
			<td>Merck Millipore</td>
			<td>07-164</td>
			<td></td>
		</tr>
		<tr>
			<td>ARE Heating Magnetic Stirrer</td>
			<td>VELP Scientifica</td>
			<td>F20500162</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FALCON 5 mL Polystyrene Round-Bottom Tubes</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Microlance 3 Needles 27 G - 0.40 x 13 mm</td>
			<td>Becton Dickinson</td>
			<td>300635</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V</td>
			<td>Roche</td>
			<td>10735078001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO Anhydrous</td>
			<td>Biotium</td>
			<td>90082</td>
			<td></td>
		</tr>
		<tr>
			<td>DRAQ7 DNA dye</td>
			<td>BioStatus</td>
			<td>DR71000</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>EMSURE MgCl2. 6H2O</td>
			<td>Merck Millipore</td>
			<td>1058330250</td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>CHEMIPHARM</td>
			<td>L01CB01</td>
			<td></td>
		</tr>
		<tr>
			<td>FALCON 14 mL Polystyrene Round-Bottom Tubes</td>
			<td>Corning Science</td>
			<td>532057</td>
			<td></td>
		</tr>
		<tr>
			<td>FALCON Tissue Culture Dishes, Easy-Grip, 35 x 10 mm Style</td>
			<td>Corning Science</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Culture Dishes (35 mm Petri dish/ 14 mm Microwell, No. 0 coverglass)</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H6147-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACELL 150i CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>50116048</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX powder</td>
			<td>Sigma-Aldrich</td>
			<td>I5879-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica M125 Stereo Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M16 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M7292</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M7167</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Honeywell</td>
			<td>13412H</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck Millipore</td>
			<td>1064981000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon AX ECLIPSE Ti2 Confocal Microscope</td>
			<td>Nikon Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ800N Stereo Microscope</td>
			<td>Nikon Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pippettes, glass, long form 230 mm</td>
			<td>DURAN WHEATON KIMBLE</td>
			<td>357335</td>
			<td></td>
		</tr>
		<tr>
			<td>pH/ORP meter&#160;</td>
			<td>Hanna Instruments Ltd</td>
			<td>HI2211</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline tablets</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P1851</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG Protein Lyophilised</td>
			<td>Genway Biotech (now AVIVA Systems Biology)</td>
			<td>GWB-2AE30A (now OPPA01037)</td>
			<td></td>
		</tr>
		<tr>
			<td>QBD4 Dry block heater</td>
			<td>Grant Instruments (Cambridge) Ltd</td>
			<td>A25218</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Puradisc 25 mm 0.2 &#956;m filters</td>
			<td>GE Healthcare</td>
			<td>6780-2502</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of dna double-stranded breaks in mouse oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Using the procedure demonstrated here, mouse ovaries were dissected, the fat was removed, and fully grown GV-stage oocytes were collected. Then, the cumulus cells were removed by repetitive pipetting using a narrow pipette and were placed in fresh drops of M2-IBMX medium and covered with mineral oil on a hot block (37 &#176;C) (Figure 1A-F). Three different etoposide concentrations were prepared (5 &#181;g/mL, 20 &#181;g/mL, and 50 &#181;g/mL) by using a stock etoposide concentration of 20 mg/mL. The GV-stage oocytes were placed in three distinct etoposide concentrations for 1 h in drops covered with mineral oil and protected from light at 37 &#176;C. The immunofluorescence protocol was then followed, as described in the protocol section in detail, and the oocytes were placed in glass-bottom dishes and observed by confocal microscopy (Figure 2).
In the SN GV-stage oocytes, immediately after DNA damage, the presence of &#947;H2AX increased at all the etoposide concentrations (5 &#181;g/mL, 20 &#181;g/mL, and 50 &#181;g/mL), and the &#947;H2AX was distributed throughout the whole DNA region (Figure 3). DSB quantification and estimation were performed by observing the &#947;H2AX fluorescence intensity at DNA sites. The &#947;H2AX fluorescence intensified proportionally with increasing etoposide concentrations. Moreover, after protracted prophase arrest (20 h after etoposide treatment), the GV-stage oocytes showed the capacity to reduce the &#947;H2AX foci number and intensity, implying the presence of active repair processes in the GV-stage-arrested oocytes (Figure 3E).
Unlike the SN oocytes, in which the &#947;H2AX fluorescence was distributed through the DNA, in the NSN oocytes, &#947;H2AX was shown in foci immediately after treatment with etoposide at 20 &#181;g/mL. We estimated the number of foci that coincided with the DNA area, calculated the fluorescence of every focus, and presented the mean fluorescence of all the oocytes. Both the fluorescence and number of foci showed statistically significant differences between the two oocyte categories (Figure 4).
Confocal microscopy provides information on the number and intensity of foci in different Z stacks, thus helping to identify the presence of DNA damage and the repair dynamics at distinct time points. Galvano scanning provides precision scanning with low background and better analysis of the scanning images.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65494/65494fig03.jpg" /> 
Figure 3: Reduction of &#947;H2AX in SN GV-stage oocytes treated with three different etoposide concentrations after protracted GV arrest. (A) &#947;H2AX fluorescence in SN GV-stage oocytes 0 h after etoposide treatment. The &#947;H2AX increases immediately after exposure at all the etoposide concentrations, and the increase is concentration-dependent (green: &#947;&#919;2&#913;&#935;, magenta: DNA). The images are Z-stack projections, and the brightness/contrast have been adjusted for each channel using Fiji / ImageJ. Scale bar = 10 &#181;m.&#160;(B)&#160;Graph of the &#947;H2AX fluorescence in SN GV-stage oocytes 0 h after treatment with distinct etoposide concentrations. Data represent mean &#177; SEM. Each dot represents one oocyte (the number of oocytes is shown in the graph), (ns = non-significant, ** p &#60; 0.005, **** p &#60; 0.0001, one-way ANOVA with Tukey&#39;s multiple comparisons test).&#160;(C)&#160;&#947;H2AX fluorescence in SN GV-stage oocytes 20 h after etoposide treatment. &#947;H2AX reduces 20 h after exposure at all the etoposide concentrations (green: &#947;&#919;2&#913;&#935;, magenta: DNA). The images are Z-stack projections, and the brightness/contrast have been adjusted for each channel using Fiji/ImageJ. Scale bar = 10 &#181;m. (D)&#160;Graph of the &#947;H2AX fluorescence in SN GV-stage oocytes 20 h after treatment with distinct etoposide concentrations. Data represent mean &#177; SEM. Each dot represents one oocyte (the number of oocytes is shown in the graph), (ns = non-significant, * p &#60; 0.05, ** p &#60; 0.005, *** p &#60; 0.0005, **** p &#60; 0.0001, one-way ANOVA with Tukey&#39;s multiple comparisons test).&#160;(E)&#160;Bar graph of the &#947;H2AX fluorescence reduction in SN GV-stage oocytes after prophase arrest in etoposide-treated oocytes. The number above each column indicates the percentage decline in &#947;H2AX fluorescence. <a href="https://www.jove.com/files/ftp_upload/65494/65494fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65494/65494fig04.jpg" /> 
Figure 4: Phosphorylation of &#919;2&#913;&#935; in NSN GV-stage oocytes after treatment with etoposide at 20 &#181;g/mL. (A) Representative confocal images of one control NSN GV-stage oocyte (green: &#947;&#919;2&#913;&#935;, magenta: DNA). The images are Z-stack projections, and the brightness/contrast have been adjusted for each channel using Fiji/ImageJ. Scale bar = 10 &#181;m. (B)&#160;Representative confocal images of one etoposide-treated NSN GV-stage oocyte (green: &#947;&#919;2&#913;&#935;, magenta: DNA). The oocytes were fixed 0 h after etoposide treatment. The images are Z-stack projections, and brightness/contrast have been adjusted for each channel using Fiji/ImageJ. Scale bar = 10 &#181;m. (C) The normalized &#947;&#919;2&#913;&#935; fluorescence in NSN GV-stage oocytes after 20 &#181;g/mL etoposide treatment. Data represent mean &#177; SEM. Each dot represents one oocyte (the number of oocytes is shown in the graph), taken from two independent experiments (**** p &#60; 0.0001, unpaired non-parametric t-test, Mann-Whitney U-test). (D)&#160;Number of &#947;&#919;2&#913;&#935; foci in NSN GV-stage oocytes after 20 &#181;g/mL etoposide treatment. Data represent mean &#177; SEM. Each dot represents one oocyte (the number of oocytes is shown in the graph), taken from two independent experiments (**** p &#60; 0.0001, unpaired non-parametric t-test, Mann-Whitney U-test). <a href="https://www.jove.com/files/ftp_upload/65494/65494fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos at JoVE.com

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

detection-of-dna-double-stranded-breaks-in-mouse-oocytes

Research

JoVE Journal

Developmental Biology

2.2K Views.  University of Ioannina. Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Video: Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

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 ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...