Science - USA (2022-02-11)

45% of KIFC1-depleted bovine oocytes under-

went tridirectional division (Fig. 6C), which

was likely because fewer oocytes restored

spindle bipolarity at anaphase onset (Fig. 6D).

Depletion of KIFC1 also resulted in misaligned

chromosomes at anaphase onset and severe-

ly lagging chromosomes during anaphase in

more than 75% of bovine oocytes (Fig. 6, E

and F). In addition, chromosome counts at

metaphase II revealed that only 6.5% of KIFC1-

depleted bovine oocytes were euploid (Fig. 6G).

Thus, depletion of KIFC1 results in spindle

instability and promotes aneuploidy in mam-

malian oocytes that lack aMTOCs.

Introducing exogenous KIFC1 rescues spindle

instability in human oocytes

To determine if the spindle instability in human

oocytes is indeed caused by the deficiency of

KIFC1, we purified recombinant mClover3-

KIFC1 protein from human embryonic kidney

(HEK) 293 cells, injected it into human im-

mature oocytes, and performed live imaging

of spindle assembly (Fig. 7, A and B; and

movies S16 and S17). Successful delivery of

exogenous KIFC1 was confirmed by mClover3

detection in live oocytes and immunofluores-

cence in fixed oocytes (Fig. 7, C and D). The

duration of spindle instability was signifi-

cantly reduced from around 10.5 hours in

noninjected oocytes to around 3.5 hours in

mClover3-KIFC1Ðinjected oocytes (Fig. 7E). By

contrast, introduction of mClover3-KIFC1 did

not significantly alter the time of early spindle

bipolarization and anaphase onset (Fig. 7, F

and G), implying that KIFC1 is specifically

required for spindle stability. Furthermore,

introduction of mClover3-KIFC1 increased the

frequency of oocytes with aligned chromo-

somes at anaphase onset from 44 to 75% (Fig.

7H) and the frequency of oocytes with no

lagging chromosomes during anaphase from

33 to 63% (Fig. 7I). Thus, introducing ex-

ogenous KIFC1 rescues spindle instability

and reduces the risk of aneuploidy in human

oocytes.

KIFC1Õs cross-linking and sliding activities are

required for proper bipolar spindle assembly in

the absence of aMTOCs

KIFC1 is dispensable for spindle stability in

wild-type mouse oocytes during meiosis I

( 103 , 104 ). To understand how KIFC1 stabilizes

aMTOC-free spindles, we first compared the

localization of mClover3-KIFC1 in wild-type

and in aMTOC-free mouse oocytes. As expected

( 103 ), mClover3-KIFC1 was uniformly localized

throughout the spindle in wild-type mouse

oocytes (Fig. 8, A to C; and movie S18). By

contrast, mClover3-KIFC1 was enriched at

the forming spindle before bipolarization, and

subsequently at the poles, in aMTOC-free

mouse oocytes (Fig. 8, A to C; and movie S18).

Fluorescence recovery after photobleaching

Soet al.,Science 375 , eabj3944 (2022) 11 February 2022 8 of 19

siControl

( 46 )

siKIFC1

( 45 )

0

1

2

3

4

5

6

No. o

f NUMA clusters

****

C

siControl

( 46 )

siKIFC1

( 45 )

0

20

40

60

80

100

P

erce

ntag

e of

s

pin

dl

es

Bipolar

With broad poles

Multipolar

siControl

( 46 )

siKIFC1

( 42 )

0

10

20

30

40

50

Total vol

ume

o

f N

U

MA

clusters (μm

3 )

N.S.

D

Control

IgG ( 41 )

anti-

KIFC1-C ( 42 )

0

20

40

60

80

100

Per

centa

ge

of

spin

dle

s

Multipolar

With broad poles

Bipolar

H

E

A

+siPNCT Fix

+siKIFC1

9 - 12 days 6 h

Harvest oocyte NEBD

~1 h

dbcAMP washout

siKIFC1

siPCNT

siControl

NUMA

Chromosomes

Microtubules

Merge

siKIFC1

Metaphase I

B

F

anti-KIFC1-C

Merge

Chromosomes

Microtubules

bTRIM21

Control IgG anti-KIFC1-C

Metaphase I

+bTRIM21 mRNA Fix

~12 h 11 h

RO3306 washout

+anti-KIFC1-C

NEBD

~1 h

G

Fig. 5. Depletion of KIFC1 in aMTOC-free mouse oocytes and in bovine oocytes recapitulates the spindle
instability of human oocytes.(A) Schematic diagram of the experiment in (B). (B) Immunofluorescence
images of MI spindles from control and KIFC1-depleted aMTOC-free mouse oocytes, fixed at 7 hours after
release. Green, NUMA; magenta, microtubules (a-tubulin); blue, chromosomes (Hoechst). (CandD) Manual
scoring (Fisher’s exact test,P< 0.0001) and automated quantification of spindle polarity in control and
KIFC1-depleted aMTOC-free mouse MI oocytes. ****P< 0.0001. (E) Quantification of the total volume of NUMA
clusters in control and KIFC1-depleted aMTOC-free mouse MI oocytes. N.S., not significant. (F) Schematic
diagram of the experiment in (G). (G) Immunofluorescence images of MI spindles from control and KIFC1-
depleted bovine oocytes, fixed at 12 hours after release. Green, microtubules (a-tubulin); magenta,
chromosomes (Hoechst). (H) Manual scoring of spindle polarity in control and KIFC1-depleted bovine oocytes
(Fisher’s exact test,P< 0.0001). Yellow arrowheads highlight well defined spindle poles. Yellow dashed
lines highlight poorly defined spindle poles. The number of oocytes analyzed is specified in italics. See Materials
and methods for box plot specifications. Scale bars are 5mm.

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