Oocyte and egg collection and culture
Sexually mature CF-1 female mice (6–10 weeks of age) were used for all experiments (Envigo, Indianapolis, IN, USA). All animals were maintained in accordance with the guidelines and policies from the Institutional Animal Use and Care Committee at Rutgers University (Protocol# 201702497) and the Animal Care Quality Assurance at the University of Missouri (Reference# 9695). Experimental procedures involving animals were approved by these regulatory bodies. Mice were housed in a room programmed for a 12-hour dark/light cycle and constant temperature, and with food and water provided ad libitum. Females were injected intraperitoneally with 5 I.U. of pregnant mare serum gonadotropin 48 hours prior to oocyte collection (Lee Biosolutions, Cat# 493 − 10). Prophase I-arrested oocytes were harvested as previously described 56. Briefly, cells were collected in minimal essential medium (MEM) containing 2.5 µM milrinone (Sigma-Aldrich, M4659) to prevent meiotic resumption, and cultured in Chatot, Ziomek, and Bavister (CZB) media 57 without milrinone in a humidified incubator programmed to 5% CO2 and 370 C for 11–12 hours for cytokinesis at meiosis I, or overnight for certain drug treatments.
For evaluating midbodies in meiosis II, ovulated eggs were activated with 10 mM strontium chloride (Sigma Aldrich, Cat# 25521) to induce Anaphase II onset. To collect ovulated eggs, mice were injected with human chorionic gonadotropin (hCG) (Sigma Aldrich, Cat# CG5) 48 hours after PMSG injection to stimulate ovulation of Metaphase II-arrested eggs. 14–16 hours following hCG injection, eggs were harvested from the ampulla region of the oviducts in MEM containing 3 mg/ml of hyaluronidase (Sigma Aldrich, Cat# H3506) to aid detachment of cumulus cells. Eggs were then transferred to center-well organ culture dish (Becton Dickinson, Cat# 353037) with activation media, consisting of Ca2+/Mg2+-free CZB with 10 mM of strontium chloride, and cultured in a humidified incubator programmed to 5% CO2 and 370 C. After 3 hours, activated eggs were cultured for 3 additional hours in KSOM + amino acids media (Sigma Aldrich, Cat# MR-106-D). For parthenogenetic activation of eggs, the activation and KSOM media were supplemented with 5 µg/ml cytochalasin D (Sigma Aldrich, Cat# C2743). Parthenogenetically activated eggs were incubated for 48 hours in KSOM + amino acids media to assess embryo cleavage rate.
For microinjection, collected oocytes were maintained arrested at Prophase I with milrinone before injection to prevent nuclear disruption and after injection to allow translation of cRNAs. To induce symmetric division of oocytes, cells were compressed at Metaphase I 29. Briefly, after culturing for 8 hours (Metaphase I time point), cells were transferred to a 5–7 µl drop of CZB covered with mineral oil (Sigma Aldrich, Cat# M5310). A glass cover slip was placed on top of the media drop and pressed down on the edges to spread the media to cover the entire surface of the cover slip. The cover slip was then pressed down until oocytes flattened and the zona pellucida became indistinguishable from the cell membrane. Cells were then cultured for additional 3 hours to observe cytokinesis.
Inhibition and disruption of mMB
To depolymerize microtubules and actin during mMB formation in early Telophase I, oocytes were cultured in CZB for 11 hours and then transferred to media containing nocodazole (Sigma Aldrich, Cat# M1404) (0, 10, 25, and 50 µM) or latrunculin A (Cayman Chemical Company, Cat# 10010630) (0, 5, and 10 µM) in a center-well dish for 30 additional minutes.
For translation inhibition, oocytes were cultured for 9 hours prior to overnight in center-well organ dishes with CZB media supplemented with glutamine, containing either cycloheximide at 50 µg/ml (Sigma-Aldrich, Cat# C7698) or puromycin at 1 µg/ml (Sigma-Aldrich, Cat# P7255).
Ablation of mMB cap by laser ablation
Prophase I-arrested oocytes were cultured in vitro in milrinone-free CZB medium supplemented with 100 nM SiR-tubulin (Cytoskeleton #NC0958386) in a humidified, microenvironmental chamber (5% CO2 and 370 C) equipped to a Leica TCP SP8 inverted microscope. After culturing cells for 11 hours, mMB caps were partially ablated using a multi-photon laser as previously described 58. In brief, a 4µm2 square region of interest within the mMB cap was exposed to a 780 nm wavelength and 60–70 mW power laser beam at the sample plane. For control-ablated oocytes, the cytoplasmic region adjacent to the mMB (cytoplasmic ablated), the egg side of the spindle (egg MT-ablated) or the PB side of the spindle (PB MT-ablated) were exposed to the same protocol. A subset of cap-ablated, control-ablated and non-ablated oocytes were fixed and immunostained with MKLP1 antibody to assess the efficiency of laser ablation and mMB cap disruption.
Immunofluorescence
Following meiotic maturation, oocytes or activated eggs were fixed in various conditions to detect localization of proteins. For detection of PRC1 (Proteintech, 15617-1-AP, 1:100), CIT-K (BD Biosciences, 611376, 1:100), RACGAP1 (Santa Cruz, sc-271110, 1:50), MKLP1 (Novus Biologicals, NBP2-56923, 1:100), and MKLP2 (Proteintech, 67190-1, 1:100), oocytes were fixed in 2% PFA in phosphate-buffered saline (PBS) for 20 minutes at room temperature. For detection of ECT2 (Fortis Life Sciences, A302-348A, 1:100), oocytes were fixed in 2%PFA with 0.1% Triton-X in PBS for 20 minutes at room temperature. For detection of RPS3 (Cell Signaling Technology, 2579S, 1:30), RPS6 (Santa Cruz, sc-74459, 1:30), RPS14 (Proteintech, 16683-1-AP, 1:30), RPL24 (ThermoFisher, PA5-62450, 1:30), and CEP55 (Proteintech, 23891-1-AP, 1:50), oocytes were fixed in cold methanol (Sigma Aldrich, Cat# A452-4) for 10 minutes. For detection of CHMP4B (Proteintech, 13683-1-AP, 1:30), zona pellucida were removed from oocytes by brief treatment with acidic Tyrode’s solution (Sigma Aldrich, Cat# MR-004-D) and fixed with 2% PFA in PBS for 20 minutes at room temperature. After fixation, oocytes were incubated in blocking buffer (0.3% BSA and 0.01% Tween in PBS) for at least 10 minutes before proceeding. For permeabilization, oocytes were incubated in PBS containing 0.2% Triton-X for 20 minutes and blocked with blocking buffer for 10 minutes. For ECT2, RPS3, RPS6, RPS14, RPL24, CEP55, RACGAP1, and CHMP4B, cells were incubated overnight at 40 C with primary antibody. For all other proteins, primary incubation was performed for 1 hour at room temperature. For secondary antibody incubation, oocytes were incubated for 1 hour in a dark, humidified chamber at room temperature. Both antibody incubations were followed by three washes in blocking solution, 10 minutes each. After the last wash, oocytes were mounted in 10 µl of Vectashield (Vector Laboratories, Cat# H-1000) containing 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Life Technologies, Cat# D1306; 1:170) for confocal microscopy.
For super-resolution microscopy using the tau-3X STED module from Leica Biosystems, the same steps as the ones described above for confocal microscopy were followed except for the following changes: 1) antibody concentrations were doubled for primary antibodies and 2) after the third wash following secondary antibody incubation, cells were mounted in 10 µl of EMS glycerol mounting medium with DABCO (EMS, Cat# 17989-10).
RNA in situ hybridization
To detect RNA molecules, fluorescence in situ hybridization (FISH) against the poly-A tail of transcripts was performed using an oligo-dT probe that consists of 21 thymine nucleotides with a 3’ modification of a fluorophore as described 59. Briefly, oocytes were fixed in increasing volumes of methanol-free 4% formaldehyde diluted in RNase-free 1X PBS at 370 C for 45 minutes. Oocytes were then dehydrated in increasing concentrations of methanol and stored at -200 C until further processing. Oocytes were prepared for hybridization by washing through 1X PBS with 0.1% Tween-20 (PBT), followed by 10% formamide and 2X SSC in nuclease-free water (Wash A). For the hybridization reaction, oocytes were incubated in a 10% formamide, 2X SSC and 10% dextran sulfate solution in nuclease-free water with 12.5 µM of the probe overnight at 370C. After hybridization, samples were rinsed through several volumes of fresh, pre-warmed Wash A and PBT before mounting on 10 µl of Vectashield with DAPI for imaging.
Nascent protein detection assay
Translation activity at the midbody was assessed by detecting protein synthesis level using an L-HPG-translation kit (ThermoFisher, Cat# C10429) as previously described 37. In summary, oocytes were collected and matured for 11.5 hours, then transferred to DMEM medium lacking methionine (ThermoFisher, Cat# 42-360-032) and containing HPG diluted 1:50 for 30–45 minutes, followed by fixation with 2% PFA in PBS for 20 minutes at room temperature and subsequent detection of HPG signal by immunofluorescence.
Control and ablated oocytes (mMB cap, egg MT-ablated and PB MT-ablated) were treated as previously described. Laser ablation was performed in CZB medium followed by oocyte incubation in DMEM + HPG for 30 min. Oocytes were fixed and processed for immunostaining with MLKP-1 antibody as described.
Image acquisition and live-cell imaging
Confocal and super-resolution images were acquired using a Leica SP8 confocal microscope with Lightning module equipped with a 40X, 1.30NA oil-immersion objective. Super-resolution STED images were acquired using a Leica SP8 confocal microscope with τ-STED module equipped with a 93X, 1.30NA glycerol-immersion objective. For each image, optical z-sections were obtained using 0.5-1 µm step-size with a zoom factor of 2.5-6. Z-series imaging was used to determine the PB/egg sides. Regardless of spindle orientation, the PB is extruded beyond the egg's confines. Therefore, using z-series imaging, the DNA of the PB always appears outward, whereas the DNA of the egg appears inward, regardless of oocyte orientation. Oocytes from experiments involving comparison of intensities or stages were processed on the same day and imaged maintaining laser settings equal across samples.
Live-cell confocal image acquisition was performed using a Leica SP8 confocal microscope system with a 40X, 1.30NA oil-immersion objective, equipped with a heated, humidified stage top incubator with 5% CO2 and 370 C (Tokai Hit, STX stage top incubator). To observe progression through cytokinesis, images of oocytes were acquired every 20 minutes with 15 optical sections across the entire thickness of each oocyte at 1024x1024-pixel image resolution and 600 Hz acquisition speed. For EB3-GFP tracking during cytokinesis, images were taken every 0.5 second at a single plane at 1024x512-pixel image resolution and 1000 Hz acquisition speed.
Cloning and cRNA preparation
Full-length clone of mouse Mklp1 (MGC: 54492) was purchased from Open Biosystems and amplified for insertion into pIVT-eGFP or pIVT-mCherry 60. Gap43-eGFP was a gift from Dr. Greg FitzHarris (U. of Montreal). To generate cRNA of Eb3-egfp61, Mklp1-eGfp, Mklp1-mCherry, and Gap43-eGfp, plasmids were linearized and transcribed in vitro using mMessage mMachine T7 or SP6 kit (Ambion, Cat# AM1344) according to manufacturer’s protocol.
cRNA was purified using SeraMag Speedbead (Sigma Aldrich, Cat# GE65152105050250) nucleotide purification method previously described 62. Briefly, in vitro transcription reaction solution was brought up to 150 µl and mixed with 100 µl of magnetic beads and let stand for 5 minutes. Beads were then pelleted using a magnetic stand and washed with 80% ethanol. cRNA was eluted using 20 µl nuclease-free water and stored at -800 C.
Image analysis and quantification
All images and videos were analyzed and quantified using Imaris software (Bitplane, Oxford Instrument Company) and Fiji 63. Quantification of volume and intensity were performed by creating a region of interest (ROI) with the “surfaces” tool in Imaris. To determine ROI, threshold of signal was determined from control groups and applied in treatment groups. For co-localization analyses of MKLP1-CITK and MKLP1-RACGAP1, the “co-localization analysis” tool in Imaris was used to determine the Manders overlap coefficient, which quantifies the co-incidence of two pixels in different channels within a set threshold64. Briefly, each channel was used to determine an ROI, which was used for the “co-localization analysis”, and the remaining channel’s intensity to be measured was set to determine the Manders overlap coefficient.
For EB3-GFP speed tracking, only cells oriented parallel to the imaging plane were imaged to prevent differences due to angular orientations. The imaging plane was selected based on distinct visualization of the dark zone. Videos were processed by Gaussian filter blend and background subtraction. Individual puncta were then determined using the “spots” tool and filtering for spots that could be tracked in at least 3 continuous frames. For mapping the directionality of the EB3-GFP comets, “track path displacement” tool was used on Imaris. For EB3-GFP intensity measurements, the first frame of each video was used to compare the intensity of the egg side to the PB side. The dark zone was used as a reference to distinguish the egg and the PB and mark ROIs.
Statistical analysis
As indicated in the figure legends, one-way ANOVA and unpaired Student’s t-test analyses were performed to examine statistical differences between groups using GraphPad Prism software. p < 0.05 was considered statistically significant. All error bars shown reflect standard errors of means.
Data Availability
No sequence or proteomic data has been generated in this study. All data supporting the findings of this study are available from the corresponding author upon request. An excel file of image analysis source data used for calculations is available on figshare 10.6084/m9.figshare.24221440.