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

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 °C and 55% humidity, with a 12 h light/dark cycle and ad libitum access to food and water.

1. Mouse oocyte collection

1. Prepare the culture medium (Chatot, Ziomek, and Bavister, CZB¹⁹, see the Table of Materials), and incubate it at 37 °C and 5% CO₂ overnight.
   NOTE: The CZB medium can be stored at 4 °C for 1 month (see Supplementary File 1 for the media composition).
2. 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.
   ​NOTE: Milrinone is a phosphodiesterase inhibitor that maintains the oocytes arrested at prophase I and prevents meiotic resumption²⁰.
3. 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).
4. Prepare the collection and culture dishes, make four microdrops (100 µL) of collection medium (MEM/PVP + M) and two microdrops (100 µL) 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 °C and 5% CO₂.
5. Inject intraperitoneally 5 IU of pregnant mare'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.
6. 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 °C.
7. 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.
8. 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.
9. Under a stereomicroscope, collect the COCs using a Pasteur glass pipet, and transfer them to the collection dish that contains MEM/PVP + M.
10. Using a narrow Pasteur glass pipet (approximately 100 µm in diameter), denude the oocytes mechanically by gentle repetitive pipetting, followed by transferring and washing them in four microdrops of (100 µL) MEM/PVP + M (the collection dish) prior to their transfer into the CZB + M culture dish.
11. Incubate the denuded oocytes for 1 h at 37 °C with 5% of CO₂ in the air.

2. Oocyte microinjection

1. Place a 250 µL 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.
2. Turn on the microinjection system (see the Table of Materials).
3. Place the microinjection dish on the warming stage (37 °C) of the microscope.
4. Load the injection needle with 0.5 µL of mCh-Cep192cRNA using microloader pipette tips (see Table of Materials), and secure the injection needle to the micromanipulator.
5. Transfer the denuded oocytes (step 1.11) to the 250 µL microdrop (step 2.1).
6. 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).
7. Set up the microinjection unit, and set the injection pressure (p_i), compensation pressure (p_c), and injection time (t_i) to be able to inject 5-10 pl of mCh-Cep192 cRNA (to exogenously label the MTOCs).
8. Carefully inject the oocytes without touching the nucleus.
9. 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 °C to allow mCh-Cep192 expression.

3. Oocyte maturation

1. Prepare the maturation medium by adding glutamine (1 mM) to CZB medium pre-equilibrated in an incubator with 5% CO₂ in air at 37 °C for at least 3 h.
2. Make two microdrops (100 µL) of the maturation medium in a 35 mm Petri dish, and cover them with mineral oil (maturation dish).
3. Wash prophase I-arrested oocytes at least three times in 100 µL milrinone-free CZB microdrops to completely remove the milrinone and allow meiotic resumption.
4. 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 °C.

4. Oocyte preparation for ablation and imaging

1. Transfer the prometaphase I oocytes (step 3.4) to a glass-bottom culture dish containing 100 µL of maturation medium (step 3.1) covered with mineral oil.

5. Microscope preparation for ablation

1. At least 30 min before ablation, turn on the temperature controller for the stage incubator (see Table of Materials) and set it at 37 °C.
2. Turn on the CO₂ controller and set it at 5% CO₂.
3. Select a 40x oil immersion apochromatic objective, and apply a small drop of the immersion oil.
4. Mount the glass-bottom culture dish with the oocytes on a stage-top incubator, and cover the incubator with a gas lid.
5. In the image acquisition software (see the Table of Materials), select an option enabling the saving of multiple stage positions (e.g., "Define mark and find experiment"). Using transmitted light brightfield illumination, center on the individual oocytes and save their positions.
6. 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 µs.
7. Set a 488 nm excitation laser line at approximately 10% of the laser power (which corresponds to 4.0 µW 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 µW 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's transmitted light detector (TLD) for concurrently detecting oocyte boundaries.
8. Using a live scanning mode and the manual control of the z-drive, screen the oocyte for MTOCs.
9. Once an mcMTOC is detected, draw a square region of interest (ROI) around it.

6. Ablation of mcMTOCs

1. Set a femtosecond laser to a 740 nm wavelength.
   NOTE: The laser wavelength can be changed according to the microscope and the laser conditions.
2. 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.
3. Use the same image format, zoom factor, and scanning frequency as in step 5.6. Set the line and frame averaging parameters to 1.
4. 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.
5. 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.
6. 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.
7. Use the channel settings from step 5.7 to verify the ablation efficiency by the complete loss of mcMTOC-associated mCherry signals (mCh-Cep192).
8. 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.