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

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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><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&amp;nbsp;</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 &amp;mu;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

2.3K 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 ...

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Genetics

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 ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

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

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

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

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 ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

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

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

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

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Biology

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

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

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

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

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

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

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 ...

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Summary

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Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

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