Science - USA (2022-02-11)

Soet al.,Science 375 , eabj3944 (2022) 11 February 2022 6 of 19

Microtubules

Dynactin

Prophase

Nuclear

envelope

breakdown

Onset of

microtubule

nucleation

Early bipolar

spindle Prometaphase I

Chromosomes

Metaphase II

Merge

AB

0246810

0.0

0.5

1.0

1.5

2.0

2.5

Enrichment relativeto the cytoplasm

Spindle width (μm)

NUMA

Dynactin

LIS1

Human MI spindle

NUMA

Dynactin

LIS1

Fixed

human

oocytes

( 27 )

Fixed

aMTOC-free

mouse oocytes

( 347 )

0

20

40

60

80

100

Per

ce

nt

age

of

spin

dle

s

Multipolar

With broad poles

Bipolar

I

Live

human

oocytes

( 89 )

Live

aMTOC-free

mouse oocyte

( 100 )

0

20

40

60

80

100

Perc

en

ta

ge

of

oocytes

Multipolar spindle intermediate

(Severe spindle instability)

Apolar spindle intermediate

(Moderate spindle instability)

Bipolar spindle intermediate

F

G

E

BSA

( 7 )

P150-CC1-His

( 12 )

0

20

40

60

80

100

Per

cen

ta

ge

of

sp

in

dl

es

Focused poles

Defocused poles

Nucleus

H

Human

( 16 )

aMTOC-free

mouse ( 50 )

0

20

40

60

80

100

Pe

rc

en

ta

ge

o

f^ defocused s

pi

nd

les

Misaligned microtubules

within the central region

Aligned microtubules within

the central region

NUMA-depleted or

dynein-inhibited

Aligned

microtubules

within the

central region

Misaligned

microtubules

within the

central region

Microtubules

Human Human aMTOC-free mouse

Aligned

microtubules

within the

central region

Directionality

Directionality color-code

+90o -45o 0 o +45o -90o

NUMA-depleted or dynein-inhibited

Chromosomes

C

NEBD Fix

+P150-CC1

15 h

P150-CC1-His

Microtubules Merge

Chromosomes

NUMA

BSA

Metaphase I

D

Fig. 3. Human oocytes focus the spindle poles with dynein-dynactin but lack
an additional mechanism that stabilizes the spindles in aMTOC-free mouse
oocytes.(A) Immunofluorescence images of human oocytes fixed at different
stages of meiosis. Green, dynactin (P150); magenta, microtubules (a-tubulin); blue,
chromosomes (Hoechst). The nucleus is outlined by a yellow dashed-line circle.
(B) Immunofluorescence images of a human MI spindle fixed at 15 hours after NEBD.
Yellow, NUMA; magenta, dynactin (P150); cyan, LIS1. The graph is the fluorescence
profile of NUMA, dynactin, and LIS1 across the spindle pole along the direction
of the yellow arrow. (C) Schematic diagram of the experiment in (D). (D) Immuno-
fluorescence images of MI spindles from human oocytes treated with BSA and
P150-CC1-His, fixed at 15 hours after NEBD. Green, NUMA; magenta, microtubules
(a-tubulin); blue, chromosomes (Hoechst). The inset is the magnification of the region

marked by the white dashed-line box. (E) Manual scoring of spindle pole defocusing in

control and dynein-inhibited human MI oocytes (Fisher’s exact test,P< 0.001). (F) Manual

scoring of spindle polarity in live human oocytes from ( 2 ) and in aMTOC-free mouse

oocytes from this study (Fisher’s exact test,P< 0.0001). (G) Immunofluorescence

images of MI spindles from NUMA-depleted or dynein-inhibited human oocytes fixed at

15 hours after NEBD and aMTOC-free mouse oocytes fixed at 7 hours after release. Gray,

microtubules (a-tubulin); magenta, chromosomes (Hoechst); pseudocolor, directionality.

(H) Manual scoring of the microtubule organization of the central region of the spindle

in NUMA-depleted or dynein-inhibited human and aMTOC-free mouse MI oocytes

(Fisher’s exact test,P< 0.0001). (I) Manual scoring of spindle polarity in fixed human and

aMTOC-free mouse MI oocytes (Fisher’s exact test,P< 0.0001). For (E), (F), (H), and

(I), the number of oocytes analyzed is specified in italics. Scale bars are 5mm.

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