the mRNA for 12 hours before release. For live
imaging of the spindle assembly with Trim-
Away, bovine oocytes were microinjected with
3.5 pl ofH2B-mScarlet,mClover3-MAP4-MTBD,
andbTRIM21mRNAs at a needle concentra-
tion of 0.16, 2.5, and 210.5 ng/ml, respectively.
Oocytes were allowed to express the mRNAs
for 12 hours before release. For live imaging
of porcine spindle assembly, oocytes were
microinjected with 2 pl ofH2B-mCherryand
meGFP-MAP4mRNAs at a needle concentra-
tion of 3 and 30 ng/ml, respectively. Oocytes
were allowed to express the mRNAs for 3 hours
before release.
Microinjection of proteins
For inhibition with RanT24N, immature mouse
oocytes were microinjected with 12 pl of recom-
binant His-RanT24N (Cytoskeleton) at a needle
concentration of 2 mg/ml with 0.05% NP-40.
Corresponding amount of BSA was micro-
injected as a control.
For acute inhibition with P150-CC1 and
NUMA-N in mouse meiosis I (MI) oocytes,
oocytes were microinjected at 7 hours after
release with 7 pl of recombinant P150-CC1-His
and His-NUMA-N at a needle concentration
of 50 and 15 mg/ml, respectively, with 0.1%
NP-40. A corresponding amount of BSA was
microinjected as a control.
For inhibition with P150-CC1 in human
oocytes, oocytes were microinjected immedi-
ately after NEBD with 10 to 15 pl of recombi-
nant P150-CC1-His at a needle concentration
of 47.85 mg/ml with 0.03% NP-40. A corre-
sponding amount of BSA was microinjected
as a control.
For introduction of exogenous KIFC1, re-
combinant KIFC1 protein was used instead of
KIFC1mRNA for better control over the final
KIFC1 protein level in the oocytes. Immature
human oocytes were microinjected with 11 pl
of recombinant Strep2-mClover3-KIFC1 at a
needle concentration of 0.875 mg/ml with
0.05% NP-40.
Trim-Away in mouse, bovine,
and human oocytes
Only affinity-purified antibodies were used
in this study for Trim-Away–mediated pro-
tein depletion ( 56 ). Mouse monoclonal anti-
NDC80 (sc-515550; Santa Cruz Biotechnology),
rabbit polyclonal anti-KIFC1-C (20790-1-AP;
Proteintech), mouse monoclonal anti-LIS1
(H00005048-M03; Abnova), rabbit polyclonal
anti-NUMA (ab97585; Abcam), and mouse
monoclonal anti-PCNT (611814; BD Biosciences)
were purified as previously described ( 133 ). The
control immunoglobulin Gs (IgGs) used were
normal mouse IgG (12-371; Millipore) and nor-
mal rabbit IgG (12-370; Millipore).
For constitutive Trim-Away in immature
mouse oocytes, 3.5 pl of mRNAs and 3.5 pl of
antibodies were coinjected as previously de-
scribed ( 133 ). All antibodies were microinjected
at a needle concentration of 1 to 2 mg/ml with
0.1% NP-40. Target proteins were allowed to
be depleted for 3 to 4 hours before the oocytes
were released. For acute Trim-Away in mouse
MI oocytes, immature oocytes were first mi-
croinjected with 3.5 pl of mRNAs and then
released after 3 hours. At around 7 hours after
release, oocytes were further microinjected
with 3.5 pl of antibodies to acutely deplete the
target proteins.
For constitutive Trim-Away in immature
bovine oocytes, partially stripped oocytes were
first microinjected with 3.5 pl of mRNAs and
allowed to express the mRNAs for 12 hours.
Before release, oocytes were fully stripped and
further microinjected with 7 pl of antibodies
at a needle concentration of 2.5 mg/ml with
0.05% NP-40.
For constitutive Trim-Away in immature hu-
man oocytes, oocytes were first microinjected
with 10 to 15 pl of recombinant hTRIM21 at
a needle concentration of 4.125 mg/ml with
0.0375% NP-40. After 3 to 4 hours, oocytes
were subsequently injected with 12 pl of anti-
body at a needle concentration of 2.5 mg/ml
with 0.03% NP-40.Cold treatment
To selectively depolymerize non–kinetochore-
bound microtubules, mouse MI oocytes were
incubated on ice for 15 min and immedi-
ately fixed as for routine immunofluorescence.
To depolymerize all spindle microtubules,
mouse MI oocytes were incubated on ice for
1 hour and immediately fixed as for routine
immunofluorescence.Nocodazole treatment
Nocodazole (Sigma-Aldrich) was reconstituted
in dimethyl sulfoxide (DMSO) (Sigma-Aldrich)
as 10 mM stock. To acutely depolymerize spin-
dle microtubules, nocodazole was added to a
final concentration of 10mM at around 7 hours
after release.Staining of human oocytes for live imaging of
spindle assembly
5-SiR-CTX ( 135 )and5-SiR-Hoechst( 135 ) were
reconstituted in DMSO as 0.9 and 1 mM stock,
respectively. SPY555-DNA (Spirochrome) and
SPY555-tubulin (Spirochrome) were reconsti-
tuted in DMSO following the manufacturer’s
instructions. 5-SiR-CTX and SPY555-DNA
were used in most experiments and present
throughout imaging at 15 nM and 1:2000
dilution, respectively. 5-SiR-Hoechst and SPY555-
tubulin were also used in some experiments
and present throughout imaging at 50 nM
and 1:5000 dilution, respectively.Immunofluorescence
To obtain mouse metaphase I spindles, oocytes
were incubated at 37°C for around 7 hours afterreleased into dbcAMP-free medium. To obtain
porcine metaphase I and bovine metaphase I
and II spindles, oocytes were incubated at 39°C
and 5% CO 2 for around 11, 12, and 24 hours,
respectively, after being released into RO-3306-
free medium. To obtain human metaphase I
and II spindles, oocytes were incubated at
37°C for around 15 and 24 hours after NEBD,
respectively. Except for meiosis II oocytes in
Figs. 6G and 7D, none of the oocytes used for
immunofluorescence analyses were subjected
to live imaging before fixation.
Oocytes were fixed in 100 mM HEPES (pH 7.0,
titrated with KOH), 50 mM EGTA (pH 7.0, ti-
trated with KOH), 10 mM MgSO 4 , 2% methanol-
free formaldehyde, and 0.5% Triton X-100 at
37°C for 15 to 60 min. Fixed oocytes were ex-
tracted in phosphate-buffered saline (PBS) with
0.5% triton X-100 (PBT) overnight at 4°C and
blocked in PBT with 5% BSA (PBT-BSA) over-
night at 4°C. All antibody incubations were
performed in PBT-BSA at 10mg/ml overnight at
4°C (for primary antibodies) and at 20mg/ml
for 1 hour at room temperature (for second-
ary antibodies). Primary antibodies used were
human anti-centromere antibody (15-234-
0001; Antibodies Incorporated), rabbit anti-
ASPM (NB100-2278; Novus Biological), rat
anti–a-tubulin (MCA78G; Bio-Rad), rabbit
anti-CAMSAP3 ( 136 ), rabbit anti-CDK5RAP2
(ABE236; Merck Millipore), rabbit anti-CEP192
(18832-1-AP; Proteintech), rabbit anti-DHC (12345-
1-AP; Proteintech), rabbit anti-KIF11 (NB500-181;
Novus Biologicals), goat anti-GFP (600-101-215;
Rockland Immunochemicals), rabbit anti–g-
tubulin (T3559; Sigma-Aldrich), mouse anti-
NDC80 (sc-515550; Santa Cruz Biotechnology),
rabbit anti-KANSL3 (HPA035018; Sigma-
Aldrich), rabbit anti-KIFC1-N (ab172620;
Abcam), rabbit anti-KIFC1-C (20790-1-AP;
Proteintech), mouse anti-LIS1 (H00005048-
M03; Abnova), rabbit anti-MCRS1 (HPA039057;
Sigma-Aldrich), rabbit anti-NUMA (ab97585;
Abcam), rabbit anti-NUMA (ab84680; Abcam),
mouse anti-P150 (612708; BD Biosciences), mouse
anti-PCNT (611814; BD Biosciences), and rabbit
anti-TPX2 (NB500-179; Novus Biological). Sec-
ondary antibodies used were Alexa Fluor 488-,
594-, or 647-conjugated AffiniPure Fab Fragment
anti-goat, anti-rabbit, and anti-rat (Jackson
ImmunoResearch Europe), Alexa Fluor 568-
conjugated Nano-Secondary anti-mouse IgG1
and anti-mouse IgG2b (ChromoTek), Atto 488-
conjugated FluoTag-X2 anti-mouse IgG2a/b
(NanoTag Biotechnologies), and Alexa Fluor
488-conjugated anti-human IgG (Molecular
Probes). DNA was stained with Hoechst 33342
(Molecular Probes).Optical clearing of bovine and porcine oocytes
Oocytes were fixed, extracted, and blocked as
for routine immunofluorescence. Before incu-
bation with primary antibodies, lipid droplets
in bovine and porcine oocytes were clearedSoet al.,Science 375 , eabj3944 (2022) 11 February 2022 14 of 19
RESEARCH | RESEARCH ARTICLE