The authors would like to thank all members of the Balboula laboratory for their valuable help and discussions. The authors thank Melina Schuh for kindly sharing&#160;the mCherry-Cep192 construct. This study was supported by R35GM142537 (NIGMS, NIH) to AZB.

All the methods described here were approved by the University of Missouri (Animal Care Quality Assurance Ref. Number 9695). Cep192-eGFP reporter female mice aged 6-8 weeks old were used in the present study. To generate Cep192-eGFP reporter mice, CRISPR/Cas9-mediated homology-directed repair was used to integrate the EGFP reporter gene into the CF-1 mouse genome. The EGFP reporter was fused at the C-terminus of Cep192 (an integral component of MTOCs)15. To maintain the mouse colony, homozygous Cep192-eGfp reporter mice were used. All the animals were maintained in cages (up to four animals/cage) at 21 &#176;C and 55% humidity, with a 12 h light/dark cycle and ad libitum access to food and water.
1. Mouse oocyte collection
<ol>
	<li>Prepare the culture medium (Chatot, Ziomek, and Bavister, CZB19, see the Table of Materials), and incubate it at 37 &#176;C and 5% CO2 overnight. 
	NOTE: The CZB medium can be stored at 4 &#176;C for 1 month (see Supplementary File 1 for the media composition).</li>
	<li>Supplement the CZB medium with glutamine (1 mM) and milrinone (2.5 mM) (CZB + M) (see Table of Materials), and place it in the incubator. 
	&#8203;NOTE: Milrinone is a phosphodiesterase inhibitor that maintains the oocytes arrested at prophase I and prevents meiotic resumption20.</li>
	<li>Prepare the collection medium (bicarbonate-free minimal essential medium, MEM) containing 3 mg/mL polyvinylpyrolidone (PVP), 25 mM HEPES (pH 7.3) (Supplementary File 1), and milrinone (2.5 mM) (MEM/PVP + M, see the Table of Materials).</li>
	<li>Prepare the collection and culture dishes, make four microdrops (100 &#181;L) of collection medium (MEM/PVP + M) and two microdrops (100 &#181;L) of culture (CZB + M) medium in 60 mm and 35 mm Petri dishes, respectively, and cover them with mineral oil (see Table of Materials). Keep the collection dish on the slide warmer, and put the culture dish into the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Inject intraperitoneally 5 IU of pregnant mare&#39;s serum gonadotropin (PMSG, see the Table of Materials) into sexually mature (6-8 week-old) Cep192-eGFP reporter female mice 44-48 h before oocyte collection.</li>
	<li>Sacrifice the mice by cervical dislocation, identify and remove the ovaries21, and transfer them into a watch glass containing prewarmed collection medium (MEM/PVP + M) at 37 &#176;C.</li>
	<li>Fix the ovary by touching a 1 mL syringe to the bottom of the watch glass and puncturing it several times (~40 times per ovary) using sewing needles bundled together to release the oocytes into the medium.</li>
	<li>Using a plastic transfer pipet, transfer all the medium containing the cumulus-oocyte complexes (COCs) from step 1.7 into an empty 100 mm plastic Petri dish.</li>
	<li>Under&#160;a stereomicroscope, collect the COCs using a Pasteur glass pipet, and transfer them to the collection dish that contains MEM/PVP + M.</li>
	<li>Using a narrow Pasteur glass pipet (approximately 100 &#181;m in diameter), denude the oocytes mechanically by gentle repetitive pipetting, followed by transferring and washing them in four microdrops of (100 &#181;L) MEM/PVP + M (the collection dish) prior to their transfer into the CZB + M culture dish.</li>
	<li>Incubate the denuded oocytes for 1 h at 37 &#176;C with 5% of CO2 in the air.</li>
</ol>
2. Oocyte microinjection
<ol>
	<li>Place a 250 &#181;L drop of MEM/PVP + M into a 100 mm plastic Petri dish lid, and cover it with mineral oil. 
	NOTE: The Petri dish lid has a lower rim that provides more space for adjusting the micromanipulator.</li>
	<li>Turn on the microinjection system (see the Table of Materials).</li>
	<li>Place the microinjection dish on the warming stage (37 &#176;C) of the microscope.</li>
	<li>Load the injection needle with 0.5 &#181;L of mCh-Cep192cRNA using microloader pipette tips (see Table of Materials), and secure the injection needle to the micromanipulator.</li>
	<li>Transfer the denuded oocytes (step 1.11) to the 250 &#181;L microdrop (step 2.1).</li>
	<li>Under a 20x or 40x objective lens, adjust the position and the focus of the injection and holding needles according to the oocyte position (Figure 1).</li>
	<li>Set up the microinjection unit, and set the injection pressure (pi), compensation pressure (pc), and injection time (ti) to be able to inject 5-10 pl of mCh-Cep192 cRNA (to exogenously label the MTOCs).</li>
	<li>Carefully inject the oocytes without touching the nucleus.</li>
	<li>Once all the oocytes are injected, wash them in three microdrops of CZB + M, transfer them to the culture dish (CZB + M), and incubate them for 3 h at 37 &#176;C to allow mCh-Cep192 expression.</li>
</ol>
3. Oocyte maturation
<ol>
	<li>Prepare the maturation medium by adding glutamine (1 mM) to CZB medium pre-equilibrated in an incubator with 5% CO2 in air at 37 &#176;C for at least 3 h.</li>
	<li>Make two microdrops (100 &#181;L) of the maturation medium in a 35 mm Petri dish, and cover them with mineral oil (maturation dish).</li>
	<li>Wash prophase I-arrested oocytes at least three times in 100 &#181;L milrinone-free CZB microdrops to completely remove the milrinone and allow meiotic resumption.</li>
	<li>Transfer the oocytes to the maturation dish, and incubate them for 5 h (prometaphase I stage) in a humidified incubator with 5% CO2 in air at 37 &#176;C.</li>
</ol>
4. Oocyte preparation for ablation and imaging
<ol>
	<li>Transfer the prometaphase I oocytes (step 3.4) to a glass-bottom culture dish containing 100 &#181;L of maturation medium (step 3.1) covered with mineral oil.</li>
</ol>
5. Microscope preparation for ablation
<ol>
	<li>At least 30 min before ablation, turn on the temperature controller for the stage incubator (see Table of Materials) and set it at 37 &#176;C.</li>
	<li>Turn on the CO2 controller and set it at 5% CO2.</li>
	<li>Select a 40x oil immersion apochromatic objective, and apply a small drop of the immersion oil.</li>
	<li>Mount the glass-bottom culture dish with the oocytes on a stage-top incubator, and cover the incubator with a gas lid.</li>
	<li>In the image acquisition software (see the Table of Materials), select an option enabling the saving of multiple stage positions (e.g., &#34;Define mark and find experiment&#34;). Using transmitted light brightfield illumination, center on the individual oocytes and save their positions.</li>
	<li>In the image acquisition software, select the XYZ scanning mode, set the image format to 256 x 256 pixels, set the zoom factor to 2.5x - 3.0x, and select a scanning frequency of 600 Hz. This corresponds to a pixel dwell time of approximately 1.6 &#181;s.</li>
	<li>Set a 488 nm excitation laser line at approximately 10% of the laser power (which corresponds to 4.0 &#181;W at the sample level) (see Table of Materials), and use a spectral bandwidth of 500-550 nm to observe the GFP signal from the MTOCs. For the simultaneous observation of fluorescence from the mCherry-tagged Cep192, set a 585 nm excitation laser line at approximately 8% of the laser power (which corresponds to 10.7 &#181;W at the sample level), and use another detector set to a spectral bandwidth of 595-645 nm. 
	NOTE: It is helpful to use the confocal microscope&#39;s transmitted light detector (TLD) for concurrently detecting oocyte boundaries.</li>
	<li>Using a live scanning mode and the manual control of the z-drive, screen the oocyte for MTOCs.</li>
	<li>Once an mcMTOC is detected, draw a square region of interest (ROI) around it.</li>
</ol>
6. Ablation of mcMTOCs
<ol>
	<li>Set a femtosecond laser to a 740 nm wavelength. 
	NOTE: The laser wavelength can be changed according to the microscope and the laser conditions.</li>
	<li>Use the electro-optic modulator of the multi-photon microscope (see Table of Materials) to set the laser power to 70%-80%, corresponding to 60-70 mW power at the sample plane. 
	NOTE: If the laser is equipped with a femtosecond pulse compensator, use it to correct for group delay dispersion (GDD). GDD correction reduces the amount of laser power required for efficient mcMTOC ablation and minimizes photodamage to the oocyte. The GDD control is usually integrated with the image acquisition software of commercial multi-photon microscopes.</li>
	<li>Use the same image format, zoom factor, and scanning frequency as in step 5.6. Set the line and frame averaging parameters to 1.</li>
	<li>Click on the Scan button in the software to perform the ablation of the mcMTOC by conducting a single laser scan of the selected ROI.</li>
	<li>Use the channel settings from step 5.7 to review the results of the ablation by comparing the images of the GFP-labeled structures taken before and after the multi-photon laser exposure. If the ablation is successful, the intensity of the GFP fluorescence in the targeted mcMTOC decreases to the levels observed in the background.</li>
	<li>If some fragments of a GFP-labeling structure remain, repeat step 6.4 one or more times by focusing on various z planes of the mcMTOC.</li>
	<li>Use the channel settings from step 5.7 to verify the ablation efficiency by the complete loss of mcMTOC-associated mCherry signals (mCh-Cep192).</li>
	<li>Repeat step 5.8 to step 6.7 until all the MTOCs of an oocyte (at various focal planes) are ablated. 
	NOTE: This protocol uses mCh-Cep192 microinjection as a strategy to confirm mcMTOC depletion following multi-photon laser exposure and exclude the possibility that the loss of GFP fluorescence is caused by GFP photobleaching. However, the microinjection of this probe is optional and not required for the ablation procedure.</li>
</ol>

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The authors declare no competing interests.

Different methods exist to disturb the cytoskeleton-related structures within cells22,23,24,25. However, finding efficient techniques to selectively perturb the targeted structure without compromising the cell viability is challenging. The multi-photon laser ablation method presented here is an efficient strategy to induce a selective mechanical perturbation to mcMTOCs within the oocyte without altering the oocyte viability.
Laser ablation has been extensively used to understand the molecular mechanisms controlling chromosome segregation during mitosis and meiosis22,23,26,27. Due to the relatively large size (&#62;80 mm in diameter) of mammalian oocytes compared to somatic cells28, the ablation of their intracellular structures represents a challenge. Moreover, the average mcMTOC volume in metaphase I oocytes is ~20 &#181;m15, representing an additional challenge. An efficient method that offers deeper tissue penetration must be adopted to overcome these challenges. The main advantage of using the multi-photon laser for ablation is its ability to reach deeper into the cell while minimizing off-target effects29.
To verify the efficacy and efficiency of the laser ablation method to deplete the targeted structure, it is recommended to use a fluorescently labeled protein to identify the targeted structure over time (before and after ablation)23. It is important to notice that laser ablation depletes the whole mcMTOC as a structure, and although smaller mcMTOCs require only a single laser exposure to be depleted, larger mcMTOCs may require more than one laser exposure at different focal planes. It is also recommended to fix and immunolabel a subset of control and mcMTOC-ablated oocytes with an MTOC marker (such as &#947;-tubulin, pericentrin, or Cep192) to confirm the efficiency of the mcMTOC ablation further. In control oocytes, areas of the cytoplasm just adjacent to but not overlapping with mcMTOCs will be exposed to the laser.
This experiment requires several mcMTOC ablations while moving among different focal planes within the oocytes. Therefore, it is highly recommended to practice this technique several times before executing the experiment to minimize the experiment time, thereby increasing oocyte viability. Moreover, it is important to use the minimum laser power that is sufficient to deplete the mcMTOCs without affecting oocyte viability.
This technique has some limitations. First, multi-photon confocal microscopes are relatively more expensive than regular confocal microscopes. Second, perturbing all mcMTOCs at different focal planes is more time-consuming than chemical or genetic perturbations. Third, this protocol requires technical skills to ablate all mcMTOCs in the shortest possible time. However, once mastered, the use of the multi-photon laser provides an excellent strategy to perturb several intracellular structures in mouse oocytes, including mcMTOCs, contributing to the understanding of molecular mechanisms regulating spindle positioning and its timely migration in mammalian oocytes.

Haploid gametes (sperm and oocyte) are produced through meiosis, which entails one round of DNA replication followed by two consecutive divisions that are necessary for chromosome number reduction prior to fertilization. In mammals, during early fetal life, the oocyte undergoes a lengthy arrest (until puberty) at the diplotene stage of prophase I of the first meiotic division, a stage called the germinal vesicle (GV) stage. Following meiotic resumption, the GV oocyte undergoes nuclear envelope breakdown (NEBD), and the spindle is assembled mainly at the oocyte center1,2,3. Later, driven by F-actin, the spindle migrates in a timely manner from the oocyte center to the cortex to ensure highly asymmetrical division, resulting in an egg with a tiny polar body (PB)4,5,6.
In mitotic cells, the centrosomes consist of a pair of centrioles surrounded by peri-centriolar material components (PMC), such as pericentrin, &#947;-tubulin, Cep152, and Cep1927. These centriole-containing centrosomes contribute to the fidelity of bipolar spindle formation8. However, centrioles are lost during early oogenesis in various species, including rodents9. Therefore, mouse oocytes adopt a centriole-independent spindle assembly pathway using numerous acentriolar microtubule (MT) organizing centers (MTOCs)9,10. Upon meiotic resumption, the perinuclear MTOCs undergo three distinct steps of recondensation, stretching, and fragmentation into a large number of smaller MTOCs11,12. The fragmented MTOCs are then clustered and sorted to organize a bipolar spindle10,13,14. Another pool of MTOCs is located in the cytoplasm during NEBD. Some of these cytoplasmic MTOCs migrate and form spindle poles (polar MTOCs, pMTOCs)10,11. Recently, another subset of cytoplasmic MTOCs was discovered, termed metaphase cytoplasmic MTOCs (mcMTOCs), that do not contribute to spindle pole formation but remain in the oocyte cytoplasm during metaphase I (Met I)15. Depleting mcMTOCs by multi-photon laser ablation or abnormally increasing their numbers by autophagy inhibition perturbs spindle positioning and migration and increases the incidence of aneuploidy in metaphase II oocytes15.
Interestingly, mcMTOCs differ from pMTOCs in many aspects15. For example, in contrast to pMTOCs, which mainly originate from perinuclear MTOCs, mcMTOCs originate from the oocyte cortex. When the spindle is still at the oocyte center, the mcMTOCs are localized asymmetrically opposite to the side to which the spindle migrates for PB extrusion15. Astral-like MTs cannot reach the cortex in the relatively large oocyte cell. Therefore, these mcMTOCs nucleate MTs to anchor the spindle (via astral-like MTOCs) to the cortex. These findings suggest a model in which the mcMTOC-nucleated MT force counteracts the F-actin-mediated force that drives spindle migration toward the cortex. The balance between these two opposing forces is essential to regulate the central spindle positioning and timely spindle migration15.
To date, all the examined PMC proteins (pericentrin, g-tubulin, Cep192, and Aurora kinase A) localize to both MTOC pools: mcMTOCs and pMTOCs15. Therefore, there is no chemical or genetic approach to selectively perturb mcMTOCs without perturbing pMTOCs. These limitations can be circumvented by selectively targeting the mcMTOCs with laser ablation. Among the laser-based technologies developed for microablation, pulsed multi-photon femtosecond lasers show great potential due to their precision impact limited to the focal plane, the high penetration depth of near-infrared light, and the reduced phototoxicity and thermal damage to the cell16,17,18. This work describes a selective approach to ablate mcMTOCs in mouse oocytes using a multi-photon laser coupled to an inverted microscope.

<table><tbody><tr><td>4 IN thinwall GL 1.0 OD/.75 ID</td><td>World precision instrument&amp;nbsp;</td><td>TW100F-4</td><td>Injection needles&amp;nbsp;</td></tr><tr><td>Borosilicate glass&amp;nbsp;</td><td>Fisherbrand&amp;nbsp;</td><td>Cat# 13-678-20D</td><td></td></tr><tr><td>Borosilicate glass capillarities&amp;nbsp;</td><td>World Precision Instrument&amp;nbsp;</td><td>Cat# TW100-6</td><td>Holding needles&amp;nbsp;</td></tr><tr><td>Bovine serum albumine&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# A4503</td><td></td></tr><tr><td>Cage incubator for Leica DMI6000 B microscope</td><td>Life Imaging Services GmbH</td><td></td><td></td></tr><tr><td>Calcium chlrode dihydrate</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# C7902</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; controller&amp;nbsp;</td><td>Pecon</td><td># 0506.000</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Cover HP</td><td>Pecon</td><td># 0506.020</td><td></td></tr><tr><td>DL-Lactic acid&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# L7900</td><td></td></tr><tr><td>DMi8</td><td>Leica&amp;nbsp;</td><td>N/A</td><td>Microscope&amp;nbsp;</td></tr><tr><td>EDTA</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# E5134</td><td></td></tr><tr><td>Femtojet 4i</td><td>Eppendorf&amp;nbsp;</td><td>N/A</td><td>Microinjector&amp;nbsp;</td></tr><tr><td>Femtotips Microloader</td><td>Fisher scientific&amp;nbsp;</td><td>E5242956003</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# G1272</td><td></td></tr><tr><td>Gentamycin&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# 1272</td><td></td></tr><tr><td>Glass bottom dish&amp;nbsp;</td><td>Mat Tek Corporation</td><td>Cat# P35G-1.0-20-C&amp;nbsp;</td><td></td></tr><tr><td>Hepes&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# H3784</td><td></td></tr><tr><td>Hepes Sodium Salt</td><td>MilliporeSigma</td><td>Cat # H3784</td><td></td></tr><tr><td>Hera Cell vios 160i</td><td>Thermo&amp;nbsp;</td><td>N/A</td><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator&amp;nbsp;</td></tr><tr><td>Leica TCP SP8 spectral laser scanning confocal micorscope with inverted stand DMI6000 B</td><td>Leica Microsystems, Inc</td><td>N/A&amp;nbsp;</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>MilliporeSigma</td><td>Cat#G8540</td><td></td></tr><tr><td>Magnesium sulfate dihydrate</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# M7774</td><td></td></tr><tr><td>MaiTai DeepSee Ti-Sapphire femtosecond laser</td><td>Spectra-Physics</td><td>N/A</td><td></td></tr><tr><td>mCH-Cep192 cRNA</td><td></td><td>N/A</td><td></td></tr><tr><td>Medium Essential Medium Eagle - MEM</td><td>MilliporeSigma</td><td>Cat #M0258</td><td></td></tr><tr><td>Milrinone</td><td>MilliporeSigma</td><td>Cat# M4659</td><td></td></tr><tr><td>Mineral oil</td><td>MilliporeSigma</td><td>Cat# M5310</td><td></td></tr><tr><td>Minimum essential medium eagle (MEM)</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# M0268 - 1L</td><td></td></tr><tr><td>Mouse: Cep192-eGFP reporter CF-1</td><td></td><td>N/A</td><td></td></tr><tr><td>Petri dish (100 mm)</td><td>Fisherbrand&amp;nbsp;</td><td>Cat# FB0875712</td><td></td></tr><tr><td>Petri dish (35 mm)</td><td>Corning&amp;nbsp;</td><td>Cat# 430165</td><td></td></tr><tr><td>Petri dish (60 mm)</td><td>Falcon&amp;nbsp;</td><td>Cat# 351007</td><td></td></tr><tr><td>Phenol Red&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P5530</td><td></td></tr><tr><td>Polyvinylpyrolidone</td><td>MilliporeSigma</td><td>Cat# P2307</td><td></td></tr><tr><td>Polyvinylpyrolidone (PVP)</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P2307</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P5405</td><td></td></tr><tr><td>Potassium phosphate monobasic&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P5655</td><td></td></tr><tr><td>Pregnant mare&amp;acute;s serum gonadotropin&amp;nbsp;</td><td>Lee BioSolutions</td><td>Cat# 493-10-10&amp;nbsp;</td><td></td></tr><tr><td>Pyruvic acid&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P4562</td><td></td></tr><tr><td>Pyruvic acid&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P4562</td><td></td></tr><tr><td>Sewing needles&amp;nbsp;</td><td>D.M.C</td><td>N/A</td><td>N&amp;deg; 5 * 16 Needles&amp;nbsp;</td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# S5761</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# S5886</td><td></td></tr><tr><td>Stage-top heating insert P</td><td>Pecon</td><td># 0426.300</td><td></td></tr><tr><td>Sterezoom S9i</td><td>Leica&amp;nbsp;</td><td>N/A</td><td>Stereomicroscope&amp;nbsp;</td></tr><tr><td>Syringe 1 mL</td><td>BD company&amp;nbsp;</td><td>Cat# 309597</td><td></td></tr><tr><td>Taurine&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# T0625</td><td></td></tr><tr><td>Temperature controller Tempcontrol 37-2 &lt;em&gt;digital&lt;/em&gt;</td><td>Pecon</td><td># 0503.000</td><td></td></tr><tr><td>The Cube temperature controller for the cage incubator</td><td>Life Imaging Services GmbH</td><td></td><td></td></tr></tbody></table>

multi-photon laser ablation of cytoplasmic microtubule organizing centers in mouse oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

Multi-photon laser ablation provides an efficient method to selectively ablate intracellular structures. The current study employed multi-photon laser ablation to deplete mcMTOCs in mouse oocytes selectively. Laser ablation depleted the mcMTOCs efficiently, as shown by the reduction in the endogenous GFP fluorescence in the targeted mcMTOCs to a level comparable to the background. Exogenous mCh-Cep192 in the targeted mcMTOCs was also abolished following laser ablation. The laser ablation of mcMTOCs should be performed at different focal planes within the oocyte (where mcMTOCs are located, Figure 2) to ensure that all the mcMTOCs within the oocyte are depleted (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64439/64439fig01.jpg" /> 
Figure 1: Oocyte microinjection. Microinjection of a prophase I-arrested mouse oocyte with mCh-Cep192 cRNA. Scale bar: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64439/64439fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64439/64439fig02.jpg" /> 
Figure 2: Depletion of mcMTOCs in mouse oocytes. Representative images of single focal planes. The white squares indicate an mcMTOC before and after multi-photon laser ablation. Scale bar: 40 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64439/64439fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64439/64439fig03.jpg" /> 
Figure 3: Depletion of mcMTOCs at different focal planes in a mouse oocyte. Representative maximum projection images of an mcMTOC-ablated mouse oocyte. Scale bar: 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64439/64439fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Compositions of Chatot, Ziomek, and Bavister (CZB) and bicarbonate-free minimal essential medium (MEM/PVP). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64439/64439_Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes at JoVE.com

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

multi-photon-laser-ablation-cytoplasmic-microtubule-organizing

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.6K Views.  University of Missouri. An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser. 

Video: Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

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 ...

Genetics

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

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of &#946;-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.

Typical microtubule inhibitors, used widely in basic and applied research, have far-reaching effects on cells. Recently, photostatins emerged as a class of photoswitchable microtubule inhibitors, capable of instantaneous, reversible, spatiotemporally precise manipulation of microtubules. This step-by-step protocol details the application of photostatins in a 3D live preimplantation mouse embryo.

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. ...

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 ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

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.

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.

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 ...

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 ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

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Chapters in this video

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

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

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

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes