with 4000 U/ml lipase fromCandida rugose
(Sigma-Aldrich) in 400 mM NaCl, 50 mM Tris
(pH 7.2), 5 mM CaCl 2 , and 0.2% sodium tauro-
cholate supplemented with cOmplete, EDTA-
free Protease Inhibitor Cocktail (Roche) at room
temperature for 20 to 40 min as previously
described ( 31 ).
Peptide preincubation assay
Oocytes were fixed, extracted, and blocked as
for routine immunofluorescence. Mouse mono-
clonal anti-NDC80 (sc-515550; Santa Cruz Bio-
technology), rabbit monoclonal anti-KIFC1-N
(ab172620; Abcam) and polyclonal anti-KIFC1-C
(20790-1-AP; Proteintech), mouse monoclonal
anti-LIS1 (H00005048-M03; Abnova), and rab-
bit polyclonal anti-NUMA (ab97585; Abcam)
were preincubated with recombinant NDC80
(Proteintech), recombinant Strep2-mClover3-
KIFC1 (homemade), recombinant LIS1 (Abnova),
and recombinant His-NUMA-N (homemade),
respectively, before being applied to oocytes as
previously described ( 133 ).
Confocal, super-resolution,
and light-sheet microscopy
For confocal imaging, oocytes were imaged in
2 ml of medium (for live oocytes) or PBS with
1% polyvinylpyrrolidone and 0.5 mg/ml BSA
(for fixed oocytes) under paraffin oil in a 35-mm
dish with a no. 1.0 coverslip. Images were ac-
quired with LSM780, LSM800, LSM880, and
LSM980 confocal laser scanning microscopes
(Zeiss) equipped with an environmental incu-
bator box and a 40× C-Apochromat 1.2 NA
water-immersion objective. For live imaging, a
volume of 50mmby50mm by 37.5mmor35mm
by 35mmby37.5mm centered around the
chromosomes was typically recorded. Auto-
matic three-dimensional (3D) tracking with
a temporal resolution of 5 to 15 min was im-
plemented using a custom-made macro (Zeiss)
on LSM800 and MyPiC ( 137 ) on LSM880.
Super-resolution Airyscan images were ac-
quired using the Airyscan module on LSM880
and LSM980 confocal laser scanning micro-
scopes and processed in ZEN (Zeiss) after ac-
quisition. mClover3, meGFP, mPA-GFP, Alexa
Fluor 488, and Atto 488 were excited with a
488-nm laser line and detected at 493 to 571 nm.
mCherry, mScarlet, SPY555, and Alexa Fluor
568 were excited with a 561-nm laser line and
detected at 571 to 638 nm. Alexa Fluor 594 was
excited with a 594-nm laser line and detected
at605to638nm.miRFP,5-SiR,andAlexa
Fluor 647 were excited with a 633-nm laser line
and detected at 638 to 700 nm.
For light-sheet imaging, oocytes were im-
aged in 2 ml of medium under paraffin oil in
a multiwell sample holder with four compart-
ments (Viventis Microscopy Sàrl). Images
were acquired with LS1 Live (Viventis Micros-
copy Sàrl) equipped with an environmental
incubator box and a 25X 1.1 NA water-dipping
objective. SPY555 was excited with a 561-nm
laser line and detected with a 523/20 nm–610/
25 nm dual band-pass filter. 5-SiR was excited
with a 638-nm laser line and detected with a
Chroma ZET405/488/561/640m emission filter.
Images of the control and experimental
groups were acquired under identical imag-
ing conditions on the same microscope. For
some images, shot noise was reduced with a
Gaussian filter. Care was taken that the imag-
ing conditions (laser power, pixel-dwell time,
detector gain, and exposure time) did not cause
phototoxicity (for live imaging), photobleaching
and saturation.Photoactivation
For analyses of fluorescence dissipation, oocytes
coexpressing mPA-GFP-NUMA, mScarlet-MAP4-
MTBD, and H2B-miRFP were rotated on stage
with an unbroken microinjection needle to
obtain meiotic spindles parallel to the imag-
ing plane. Rectangular regions of interest
(ROIs) were marked and photoactivated using
a 405-nm laser line at the maximum power
after the fifth time point.Fluorescence recovery after photobleaching
For analyses of fluorescence recovery after
photobleaching, oocytes coexpressing mClover3-
KIFC1, mScarlet-MAP4-MTBD, and H2B-miRFP
or oocytes expressing mCherry–a-tubulin were
rotated on stage with an unbroken micro-
injection needle to obtain meiotic spindles
parallel to the imaging plane. Rectangular ROIs
were marked and photobleached using a 488-
and a 561-nm laser line at the maximum power
after the fifth time point.ImmunoÐelectron microscopy and correlative
FIB-SEM
For immune–electron microscopy of NUMA,
mouse MI oocytes expressing mClover3-NUMA
were microinjected with 7 pl of 0.167 mg/ml
2-nm Immunogold (Aurion)–conjugated GFP
VHH (Chromotek) with 0.1% NP-40. Before
fixation in 100 mM HEPES (pH 7.0, titrated
with KOH), 50 mM EGTA (pH 7.0, titrated with
KOH), 10 mM MgSO4, 3% EM-grade glutaral-
dehyde, and 0.5% methanol-free formaldehyde
at 37°C for 1 hour, oocytes were randomly
distributed and attached to a high Grid-500
35 mmm-Dish (iBidi) using Cell-Tak Cell and
Tissue Adhesive (Corning). To preselect oocytes
with optimally oriented spindles, oocytes were
stained with Hoechst 33342 and screened on
a confocal laser scanning microscope. Before
staining for electron microscopy, silver en-
hancement was performed with R-Gent SE-
EM (Aurion).
All of the following processing steps were
performed in a microwave (Ted Pella), and
oocytes were washed three times with water
for 40 s at 250 W in between every staining
step. Oocytes were first stained with 2% osmiumtetroxide–1.5% potassium ferrocyanide in 0.1 M
phosphate buffer (pH 7.4) for 12 min at 100 W
(microwave cycling between on and off every
2 min). Oocytes were then incubated with 1%
thiocarbohydrazide (Sigma-Aldrich) for 12 min
at 100 W (microwave cycling between on and
off every 2 min) and stained with 2% osmium
tetroxide in water for 12 min at 100 W (micro-
wave cycling between on and off every 2 min).
Oocytes were further stained with 1% uranyl
acetate in water for 12 min at 100 W (micro-
wave cycling between on and off every 2 min)
and 0.02 M lead nitrate-0.03 M aspartic acid
(pH 5.5) for 12 min at 100 W (microwave cy-
cling between on and off every 2 min). Oocytes
were subsequently dehydrated in a graded
ethanolseries(10,30,50,75,90,100,and
100%) for 40 s at 250 W and infiltrated in a
graded series (25, 50, 75, 90, 100, and 100%)
of Durcupan resin (Sigma-Aldrich) in ethanol
for 3 min at 250 W.
Infiltrated oocytes were embedded with a
minimal amount of resin as previously de-
scribed ( 138 ). The polymer coverslip with em-
bedded oocytes on top was cut out of the
culture dish using a jigsaw and attached to
a SEM stub (Science Services) using silver-
filled EPO-TEK EE129-4 adhesive (Electron
Microscopy Sciences), and cured overnight
at 60°C. Samples were coated with a 10-nm
gold layer using the high-vacuum sputter
coater EM ACE600 (Leica) at a current of
35 mA. Afterward, samples were placed in
the Crossbeam 540 FIB-SEM (Zeiss). To ensure
even milling and to protect the surface, a
500-nm platinum layer was deposited on
top of the region of interest at a current of
3 nA. Atlas 5 (Atlas 3D, Zeiss) was used to
collect the 3D datasets. Preselected oocytes
were exposed with a 15-nA current, and a 7-nA
current was used to polish the cross-section
surface. Images were acquired at 1.5 kV with
the energy-selective backscattered (ESB) de-
tector at a grid voltage of 450 V (5 nm pixel
size inx-y) using 700 pA as the milling cur-
rent (5 nmz-step).
After acquisition, images were first aligned
using Linear Stack Alignment with SIFT in Fiji
(NIH). Datasets were then cropped, inverted,
and smoothed with a Gaussian filter of 1 sigma in
Fiji. To better visualize microtubules, data-
sets were further subjected to Local contrast
enhancement (CLAHE) in Fiji. The specific
parameters used were 127 for blocksize, 256
for histogram bins, and 1.25 for maximum
slope. To obtain a spindle parallel to the im-
aging plane, the resulting image stacks were
rotated and resliced with Interactive Stack
Rotation in Fiji.Immunoblotting
Ten to 50 mouse and 12 human, bovine, or
porcine MI oocytes (per lane) were exten-
sively washed in protein-free medium andSoet al.,Science 375 , eabj3944 (2022) 11 February 2022 15 of 19
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