Discussion
Our data elucidate a cause of spindle instability
in human oocytes: Human oocytes are deficient
in the molecular motor KIFC1, a key spindle-
stabilizing factor present in oocytes of other
mammalian species. By contrast, most other
mammalian and nonmammalian oocytes ex-
press KIFC1 to promote proper spindle as-
sembly ( 22 , 23 , 103 , 108 – 110 ). We report that
KIFC1 ensures the coalescence of clustered
microtubule minus ends into two spindle poles
in mammalian oocytes that lack aMTOCs (Fig.
8K). Spindle stabilization is likely achieved
through the formation of static cross-links
along parallel microtubules at the poles and
the alignment of antiparallel microtubules in
the central region of the spindle. Because hu-
man oocytes are deficient in KIFC1, we propose
that the deficiency of these activities renders
their spindles unstable.
Our data also reveal notable differences in
spindle pole organization in different mam-
malian oocytes. Whereas aMTOCs dominate
the organization of spindle poles in mouse
oocytes ( 30 , 32 – 35 ), an adaptation in NUMA
dynamics can compensate for the absence of
aMTOCs in mammalian oocytes. At aMTOC-
free spindle poles, NUMA becomes strongly
enriched at and stably associated with micro-
tubule minus ends. Owing to its multimodular
nature, NUMA can cross-link microtubules at
the minus ends and also engage minus end–
directed dynein complexes. All of these fea-
tures allow stably associated NUMA to cluster
microtubule minus ends at the spindle poles,
replacing the microtubule anchoring function
of aMTOCs. In addition to anchoring micro-
tubules, aMTOCs promote microtubule nucle-
ation ( 30 , 32 , 35 ). However, NUMA neither
nucleates microtubules nor interacts with
microtubule nucleators, in line with the crucial
requirement of the Ran pathway for micro-
tubule nucleation in nonrodent mammalian
oocytes ( 2 , 36 ).
Dynein and KIFC1 appear to have similar
roles in pole focusing inXenopusegg extracts,
DrosophilaS2 cells, and nonmammalian
oocytes ( 22 – 25 , 29 , 109 – 114 ). By contrast,
perturbation of NUMA-dynein and of KIFC1
result in distinct phenotypes in mammalian
oocytes that lack aMTOCs, suggesting that
they have nonredundant roles at the spindle
poles. Although both dynein and KIFC1 are
minus end–directed motors with microtubule
cross-linking and sliding activities, their dif-
ferential roles may be explained by their differ-
ent distribution on the aMTOC-free spindles in
mammalian oocytes. NUMA-dynein is confined
at microtubule minus ends and hence acts
more locally, whereas KIFC1 localizes through-
out the spindle with an enrichment at the
polar regions and hence acts more globally.
In normal mitotic cells with two centro-
somes, KIFC1 is largely dispensable for bipolar
spindle assembly ( 107 , 115 – 117 ). By contrast,
mitotic cells with supernumerary centrosomes
require KIFC1 to cluster extra centrosomes into
two spindle poles, preventing spindle multi-
polarity ( 115 , 116 , 118 , 119 ). Hence, KIFC1 has
been proposed as a selective target for treating
cancers, where supernumerary centrosomes
are frequently observed ( 120 ). Here, we dem-
onstrate that KIFC1 also prevents spindle
multipolarity in mammalian oocytes that lack
aMTOCs. Spindle pole organization in these
oocytes may be intrinsically error-prone owing
to the absence of additional mechanisms that
enforce spindle bipolarity such as those that
regulate the number of centrosomes ( 121 ) and
aMTOCs ( 34 ). Thus, KIFC1 is also required to
cluster extra spindle poles formed in cancer cells
and in mammalian oocytes that lack aMTOCs.
ThedeficiencyofKIFC1mayalsorender
zygotic spindles unstable. Human zygotes have
low levels ofKIFC1mRNA and a high incidence
of multipolar spindles ( 9 – 13 ). Spindles are,
however, mostly bipolar in human two-cell–
stage embryos ( 13 ). This sudden reduction in
multipolar spindles coincides with a rise in
KIFC1mRNA from the two-cell stage onward.
Indeed, bovine oocytes express KIFC1, and
multipolar spindles are rare in bovine zygotes
( 122 – 124 ). Thus, differences in maternal KIFC1
expression may also explain why human zygotes
are more prone to assembling multipolar spin-
dles than other mammalian zygotes.
If spindle instability is caused by a general
deficiency of KIFC1 in human oocytes, why are
spindles in 20% of human oocytes stable ( 2 )
and why aren’t 80% of human eggs aneuploid?
One contributing factor could be heterogeneous
KIFC1 expression between individual oocytes.
Our immunoblot data show that the average
level of KIFC1 in human oocytes is very low.
However, as revealed by single-cell RNA-seq of
human meiosis I oocytes, there might be in-
dividual oocytes expressing a higher level of
KIFC1 that could be sufficient to assemble
stable spindles. Moreover, human oocytes
might have developed additional mechanisms
to compensate for the deficiency of KIFC1.
Indeed, many human oocytes succeed in form-
ing bipolar spindles shortly before anaphase
onset, despite progressing through long phases
of spindle instability ( 2 , 3 ). Furthermore, al-
though most human oocytes with unstable
spindles show lagging chromosomes during
anaphase ( 2 ), only a fraction of lagging chro-
mosomes will eventually lead to aneuploidy
( 125 ). Spindle instability also appears to occur
more frequently in meiosis I than in meiosis II
and the first mitotic division ( 2 , 11 – 13 , 126 ).
This could be because of additional differences
in the spindle architecture and the process of
spindle assembly. For instance, the kinetics of
spindle assembly in meiosis II and the first
mitotic division (2 to 4 hours) is much faster
than in meiosis I (12 to 16 hours) ( 2 , 12 , 13 ).Nevertheless, we do not exclude the possibility
that in vitro matured oocytes, which are the
main source of immature human oocytes avail-
able for research, are more prone to spindle
instability during meiosis I than oocytes ovu-
lated in vivo.
Our data reveal a cause of the differences
in spindle stability between human oocytes
and other mammalian oocytes and provide a
potential method for reducing chromosome
segregation errors in human oocytes. By quan-
titatively delivering a defined amount of KIFC1
protein into human oocytes, we were able to
rescue spindle instability and reduce the risk
of aneuploidy in human oocytes. Thus, the
deficiency of KIFC1 contributes to spindle
instability in human oocytes and the titrated
administration of KIFC1 protein has the poten-
tial to increase the fidelity of spindle assembly
and chromosome segregation in human oocytes.Materials and methods
Preparation and culture of mouse oocytes
and follicles
All mice were maintained in a specific pathogen-
free environment at the Animal Facility of the
Max Planck Institute for Multidisciplinary Sci-
ences according to The Federation of European
Laboratory Animal Science Associations guide-
lines and recommendations.
Oocytes were isolated from ovaries of 8- to
12-week-old FVB/N female mice. Fully grown
oocytes of around 75mm in diameter with a
centered nucleus were arrested at prophase in
homemade phenol red-free M2 supplemented
with 250mM dibutyryl cyclic AMP (dbcAMP)
(Sigma-Aldrich) under paraffin oil (ACROS
Organics) at 37°C. To induce resumption of
meiosis, oocytes were released into dbcAMP-
free M2 at 37°C.
Follicles were mechanically isolated from
10- to 12-day-old (C57BL/6J × CBA) F 1 female
mice in HEPES-buffered minimal essential
medium (MEM) with GlutaMAX (Gibco) sup-
plemented with 5% fetal bovine serum (FBS)
(Gibco) and 0.1× penicillin G/streptomycin
(Sigma-Aldrich). Compact follicles of around
100 mm in diameter with a centered oocyte
were cultured in MEM alpha with GlutaMAX
and nucleosides (Gibco) supplemented with
5% FBS, 0.03mg/ml ovine follicle stimulat-
ing hormone (National Hormone and Peptide
Program), 1× insulin/transferrin/sodium sel-
enite (Sigma-Aldrich), and 0.1× penicillin
G/streptomycin on a 12-mm Transwell-COL
collagen-coated 0.4-mm pore polytetrafluoro-
ethylene (PTFE) membrane insert (Corning) at
37°C and 5% CO 2 as previously described ( 55 ).
Half of the medium surrounding the insert
was replaced every 3 days. After 10 to 12 days
of culture, in vitro grown oocytes were denuded
and matured in modified M2 supplemented
with 10% FBS instead of 4 mg/ml bovine serum
albumin (BSA).Soet al.,Science 375 , eabj3944 (2022) 11 February 2022 12 of 19
RESEARCH | RESEARCH ARTICLE