SPINDLE MORPHOGENESIS AND THE MORPHOLOGY OF CHROMOSOME IN
MOUSE NUCLEAR TRANSFER: AN ABNORMAL START IN CLONING OF MICE
VanThuan Nguyen, Sayaka Wakayama, Satoshi Kishigami and Teruhiko Wakayama
[email protected]
Laboratory for Genomic Reprogramming, RIKEN-Center for Developmental Biology, Kobe, JAPAN
Introduction
Results
Although both the spindle and nuclear mitotic apparatus Initial spindle assembly (α-tubulin) in mouse somatic cell nuclear
protein (NuMA) are thought to play important roles in the transfer
DNA
α tubulin Merged
cell cycle and early reprogramming after somatic nuclear A
A’’
A’
Fig. 1. The initial spindle morphologies detected in the
first mitosis of mouse somatic nuclear transfer. Oocytes
transfer, the spindle and NuMA morphogenesis during the
were fixed within 30 minutes after NT and stained by
early stage after NT is not well understood.
anti-α-tubulin-conjugated FITC (green). DNA were
B
B’
B’’
In this study we systematically characterized the
stained with propidium iodide (red). A-A’’) Bipolar
spindle phenotype. B-B’’) Monopolar spindle phenotype.
spindle morphogenesis, the correlation between the
C-C’’) Multipolar spindle phenotype.
donor nuclear membrane and spindle formation, and C
C’
C’’
chromosome configuration in the early stage of the first
mitosis in mouse NT.
Conclusion
The morphological change of NuMA before and after activation in
the first mitosis of mouse NT oocytes/embryos
Centrosomal morphologies at the first mitotic metaphase of
mouse NT
DNA
A
B
C
NuMA
A’
B’
C’
Merged
A’’
B’’
C’’
Fig. 5. Centrosomal morphologies at
the first mitotic metaphase of mouse
NT. Nuclear transferred oocytes were
fixed 60 minutes after NT. NuMA
was stained with Anti-NuMA and
Alexa 488 (green); DNA was stained
with propidium iodide (Red). A-A’’)
A bipolar spindle morphology at the
first mitosis of mouse NT with two
mitotic NuMA foci always
col o ca li z ed w ith mi cr otu bu l eorganizing centers (NuMA-MTOCs)
was detected. B-B’’) A monastral
bipolar spindle morphology at the
first mitosis of mouse NT with only
one NuMA-MTOC was detected. CC’’) A Bipolar spindle morphology of
the metaphase II oocytes with two
NuMA-MTOCs was detected
(Control).
Effects of intact or disrupted nuclear membrane on the
spindle morphogenesis in mouse somatic nuclear transfer
%
80
73
60
40
29
0
20
16
7
4
6-8 µm
Multipolar
Not determined
57
53
20
Bipolar
Monopolar
21
19
8
3
5-6 µm
2-3 µm
Fig. 8. The frequencies of initial spindle
morphologies after injection of somatic nuclei
into enucleated oocytes by micropipettes of
different diameters. This experiment was
repeated four times to obtain 160 nuclear
transfer oocytes per treatment.
Full term development
Group
of Exp.
Live
2-cell
embryo offspring
120 (92%) 0 (0.0%)
The spindle morphogenesis was independent of the DNA NuMA Merged
6-8 µm
A’
A’’
Fig. 2. The initial centrosomal morphologies detected in
presence or absence of a metaphase II spindle and it was A
the first mitosis of mouse somatic NT. Oocytes were
5-6 µm 116 (94%) 2 (1.6%)
initiated by a variety of spindle defects, including a high
fixed within 30 minutes after NT and stained by Antiproportion of the monopolar spindle phenotype.
NuMA and Alexa 488 (green). DNA were stained with Transformation from monopolar to bipolar spindle in mouse 2-3 µm 118 (97%) 0 (0.0%)
Hello, my name is Belief
B’’
B’
propidium iodide (Red). A-A’’) Bipolar centrosomal
The phenotypes of spindle morphogenesis were B
somatic
nuclear
transfer
Table 1. Only two cloned mice (1.6%) were obtained after we transferred 116
phenotype.
B-B’’)
Monopolar
centrosomal
phenotype.
correlated with intact or disruptive donor nuclear
of the 2-cell embryos from the 5-6 µm pipette to the recipient mouse.
C-C’’) Multipolar centrosomal phenotype.
membrane.
C
C’
D
E
C’’
The model of the first mitotic spindle morphogenesis in
Our study also uncovered a novel model for
Localization of NuMA in mouse NT
mouse nuclear transfer
(D) and in ICSI (E) 6 h after
incomplete spindle remodeling by NT in the mouse, which
activation or sperm injection.
occurs when a monopolar spindle is converted to a
A. Bipolar pathway
B. Monopolar pathway
Fig. 9. A) Bipolar pathway, in
bipolar spindle or a monastral bipolar spindle lacks a Effect of intact and enucleated oocytes on the spindle morphogenesis
which donor nuclear membrane
after
nuclear
transfer
10
pole and is therefore an incomplete spindle remodeling %
Fig. 6. More than 600 nuclear transferred oocytes at 5-minute intervals from 10 to
was broken down and α-tubulin
30
Figure
3:
The
spindle
morphogenesis
in
40 minutes after NT and stained them with α-tubulin and NuMA to observe the min
(Fig. 9).
was assembled at the poles of the
C
D
80
Materials and Methods
Oocytes collection and nuclear transfer:
Oocytes were collected from mature female B6D2F1 mice approximately 16 hr after hCG injection. The
donor nuclei using in this study is cumulus cells. MII oocyte enucleation was carried out using a piezoactuated micromanipulator system (Prime Tech, Tsuchiura, Japan) by enucleation pipette in a droplet of
HEPES-CZB containing 5 µg/ml cytochalasin B under mineral oil. Donor nucleus was injected into intact
oocytes or enucleated oocytes by using piezo pulses system.
76
71
60
40
20
15
13
11
8
4
3
0
Enucleated MII oocyte
Intact MII oocyte
Fixation and differential staining:
Bipolar
Monopolar
Oocytes were fixed at 10, 20, 30, 40, 60 and 360 minutes after somatic cell injection in PBS containing
3.5% paraformadehyde and then keep overnight at 4 degree C in PBS-BSA containing 1% Triton X-100.
For double labeling, DNA was stained with PI (red), α tubulin NuMA, and lamin A/C were stained with
FITC, Alexa 488 (green) and Alexa 568 (red). All samples were visualized with a BioRad Radiance 2100
confocal scanning laser microscope.
Multipolar
Not determined
Disruption of nuclear membrane using a small micropipette
To disrupt the nuclear membrane, we drew cumulus cells in and out of the injection pipette while
decreasing the diameter of the injection pipette from 8 to 2 µm. To confirm whether the cumulus cell nuclear
membrane was disrupted, we fixed nuclear transferred oocytes immediately after injection and stained them
with anti-lamin-A/C and Alexa 568.
Embryo transfer
We transferred 10-15 two-cell cloned embryos, 24 to 30 hours after activation, to oviducts of surrogate
mothers (ICR) on day 1 of pseudopregnancy following mating with ICR vasectomized males.
mouse nuclear transfer after injection of
cumulus cell in to an intact or enucleated
oocyte. A total of 920 oocytes (420 intact
MII oocytes and 500 enucleated oocytes)
were injected with cumulus cells and fixed at
10, 20, 30 min after NT. The initial spindle
morphology of donor nuclear injected into
intact oocytes and enucleated oocytes could
be determined in 365 and 425 oocytes,
respectively.
Chromosome morphology in the first mitosis of mouse somatic
nuclear transfer
Fig. 4. The first mitotic metaphase after
injection of a somatic nucleus into an intact (A)
or enucleated oocyte (B). C) A metaphase II
oocyte with chromosomes located at the
equatorial region of the spindle. D) During the
first mitotic metaphase of mouse NT, chromosomes were not located in the equatorial
region of the metaphase spindle, they tended to be located in a polar region of the
spindle.
A
B
C
Metaphase II
Donnor cell
Donnor cell
D
transformation of monopolar to bipolar spindle in NT of mice. The conversion of
a monopolar spindle to a bipolar spindle by bipolarization of a centrosome
Disruption of nuclear membrane using a small micropipette
A
Fig. 7. To disrupt the nuclear membrane,
we drew cumulus cells in and out of the
injection pipette while decreasing the
B diameter of the injection pipette from 8 to
6-8 µm
2 µm (A, A’, A’’).To confirm whether the
A’
cumulus cell nuclear membrane was
disrupted, we fixed nuclear transferred
oocytes immediately after injection and
C
5-6 µm
stained them with anti-lamin-A/C and
A’’
Alexa 568. B) An intact cumulus nuclear
membrane (control), lamin A/C was
detected in the nuclear membrane. C) An
D intact cumulus nuclear membrane by a
2-3 µm
6-8 µm micropipette. D) A disruption of cumulus nuclear membrane by 2-3 µm
micropipette.
A cumulus cell
spindle. The α-tubulin was
prolonged, there was chromosome
condensation, and chromosomes
were ultimately located at the
60
equatorial region of the spindle.
B) Monopolar pathway, in which
120
min
donor nuclear membrane was
Bipolar spindle
Bipolar spindle
Monastral
intact and α-tubulin was
bipolar spindle
assembled at the single pole of the
Chromosome
α tubulin
NuMA
spindle C) Two poles were
formed. from monopolar centrosome and migrated to two poles of the spindle at the
metaphase stage. Chromosomes were located at the equatorial region of the spindle
(bipolar spindle). D) Monopolar centrosome was not duplicated; however, a
monastral bipolar spindle was formed in which one of the poles lacked a
centrosome.
30
50
min
Contact address: VanThuan Nguyen, PhD.
RIKEN KOBE INSTITUTE
CENTER FOR DEVELOPMENTAL BIOLOGY
Phone: +81-78-306-3094 Fax: +81-78-304-3095
E-Mail: [email protected]