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 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.
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, γ-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.
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
2. Oocyte microinjection
3. Oocyte maturation
4. Oocyte preparation for ablation and imaging
5. Microscope preparation for ablation
6. Ablation of mcMTOCs
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).
Figure 1: Oocyte microinjection. Microinjection of a prophase I-arrested mouse oocyte with mCh-Cep192 cRNA. Scale bar: 10 µm. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
Supplementary File 1: Compositions of Chatot, Ziomek, and Bavister (CZB) and bicarbonate-free minimal essential medium (MEM/PVP). Please click here to download this File.
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 (>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 µm15, 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 γ-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.
The authors have nothing to disclose.
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 the mCherry-Cep192 construct. This study was supported by R35GM142537 (NIGMS, NIH) to AZB.
4 IN thinwall GL 1.0 OD/.75 ID | World precision instrument | TW100F-4 | Injection needles |
Borosilicate glass | Fisherbrand | Cat# 13-678-20D | |
Borosilicate glass capillarities | World Precision Instrument | Cat# TW100-6 | Holding needles |
Bovine serum albumine | MilliporeSigma | Cat# A4503 | |
Cage incubator for Leica DMI6000 B microscope | Life Imaging Services GmbH | ||
Calcium chlrode dihydrate | MilliporeSigma | Cat# C7902 | |
CO2 controller | Pecon | # 0506.000 | |
CO2 Cover HP | Pecon | # 0506.020 | |
DL-Lactic acid | MilliporeSigma | Cat# L7900 | |
DMi8 | Leica | N/A | Microscope |
EDTA | MilliporeSigma | Cat# E5134 | |
Femtojet 4i | Eppendorf | N/A | Microinjector |
Femtotips Microloader | Fisher scientific | E5242956003 | |
Gentamicin | MilliporeSigma | Cat# G1272 | |
Gentamycin | MilliporeSigma | Cat# 1272 | |
Glass bottom dish | Mat Tek Corporation | Cat# P35G-1.0-20-C | |
Hepes | MilliporeSigma | Cat# H3784 | |
Hepes Sodium Salt | MilliporeSigma | Cat # H3784 | |
Hera Cell vios 160i | Thermo | N/A | CO2 incubator |
Leica TCP SP8 spectral laser scanning confocal micorscope with inverted stand DMI6000 B | Leica Microsystems, Inc | N/A | |
L-Glutamine | MilliporeSigma | Cat#G8540 | |
Magnesium sulfate dihydrate | MilliporeSigma | Cat# M7774 | |
MaiTai DeepSee Ti-Sapphire femtosecond laser | Spectra-Physics | N/A | |
mCH-Cep192 cRNA | N/A | ||
Medium Essential Medium Eagle – MEM | MilliporeSigma | Cat #M0258 | |
Milrinone | MilliporeSigma | Cat# M4659 | |
Mineral oil | MilliporeSigma | Cat# M5310 | |
Minimum essential medium eagle (MEM) | MilliporeSigma | Cat# M0268 – 1L | |
Mouse: Cep192-eGFP reporter CF-1 | N/A | ||
Petri dish (100 mm) | Fisherbrand | Cat# FB0875712 | |
Petri dish (35 mm) | Corning | Cat# 430165 | |
Petri dish (60 mm) | Falcon | Cat# 351007 | |
Phenol Red | MilliporeSigma | Cat# P5530 | |
Polyvinylpyrolidone | MilliporeSigma | Cat# P2307 | |
Polyvinylpyrolidone (PVP) | MilliporeSigma | Cat# P2307 | |
Potassium chloride | MilliporeSigma | Cat# P5405 | |
Potassium phosphate monobasic | MilliporeSigma | Cat# P5655 | |
Pregnant mare´s serum gonadotropin | Lee BioSolutions | Cat# 493-10-10 | |
Pyruvic acid | MilliporeSigma | Cat# P4562 | |
Pyruvic acid | MilliporeSigma | Cat# P4562 | |
Sewing needles | D.M.C | N/A | N° 5 * 16 Needles |
Sodium bicarbonate | MilliporeSigma | Cat# S5761 | |
Sodium chloride | MilliporeSigma | Cat# S5886 | |
Stage-top heating insert P | Pecon | # 0426.300 | |
Sterezoom S9i | Leica | N/A | Stereomicroscope |
Syringe 1 mL | BD company | Cat# 309597 | |
Taurine | MilliporeSigma | Cat# T0625 | |
Temperature controller Tempcontrol 37-2 digital | Pecon | # 0503.000 | |
The Cube temperature controller for the cage incubator | Life Imaging Services GmbH |