Evaluation of maturation competence of metaphase II
oocytes in mice based on the distance
between pericentriolar materials of meiotic spindle
Distance of PCM during oocyte maturation
Chizuka Sakai & Yumi Hoshino & Yusuke Sato &
Received: 21 July 2010 /Accepted: 13 October 2010 /Published online: 17 November 2010
# Springer Science+Business Media, LLC 2010
Purpose To ascertain whether metaphase II (MII) spindle
shape influences oocyte competence, we examined the
meiotic spindle organization in in vivo ovulated (IVO)
oocytes and in spontaneously matured or follicle stimulat-
ing hormone (FSH)-induced oocytes.
Methods FSH-induced oocytes matured in Waymouth’s
MB752/1 or human tubal fluid (HTF) media and oocytes
matured spontaneously in the basal medium were obtained,
and spindles were detected by immunofluorescence. To
evaluate the fertilization-associated differences in spindle
morphology, we performed in vitro fertilization and
analysed integrin mRNA expression.
Results The distance between the pericentriolar materials
(PCMs) in oocytes matured under all conditions was
initially more, but it reduced gradually and increased again
thereafter. Therefore, oocytes exhibiting a reduction in the
distance between PCMs had the highest development rate
to blastocyst in each condition.
Conclusion These results indicate that the ‘maturation
competence’ of MII oocytes can be evaluated on the basis
of the distance between PCMs.
Keywords In vitro maturation.Maturation competence.
Meiotic spindle.Mouse oocyte.Pericentriolar materials
vesicle breakdown (GVBD), is either a spontaneous or a
gonadotropin-induced response. The former occurs as a
consequence of oocyte removal from the inhibitory environ-
ment of the follicle , while the latter is induced by
gonadotropin action on cumulus cells that produce meiosis-
inducing signals capable of overriding meiotic arrest [2–4]. In
both the processes, a key step is the decrease in intracellular
cyclic adenosine monophosphate (cAMP) levels [5, 6] and the
subsequent activation of the M-phase promoting factor (MPF)
and the mitogen-activated protein kinase (MAPK) pathway
. Follicle stimulating hormone (FSH) induces complete
meiotic maturation, i.e., first polar body (PB1) emission, of
cultured cumulus-oocyte complexes (COCs). Hypoxanthine
maintainsmeiotic arrest inculturedCOCs,while FSH reverses
this inhibitory effect of hypoxanthine .
In vitro maturation (IVM) has been efficiently used to
obtain metaphase II (MII)-arrested mouse oocytes that are
competent to be fertilized and are capable of producing
viable embryos [7, 8]. The developmental competence of in
vitro matured (IVM) oocytes of mammals is likely to be
inferior to that of in vivo matured oocytes [9–12]. After it
was reported that mammalian oocytes can spontaneously
resume and complete meiosis upon removal from the
follicle [1, 13], much effort has been expended to modify
the culture conditions of IVM in order to obtain high-
quality oocytes for embryo production [14, 15]. In some
mammals, such as cattle, IVM represents the industry
standard and is routinely used for in vitro fertilization or
nuclear transfer and embryo production strategies, and this
method affords relatively high rates of blastocyst develop-
ment and implantation [16, 17]. Although live young
Capsule The ‘maturation competence’ of MII oocytes can be
evaluated on the basis of the distance between pericentriolar materials
(PCMs) of spindles.
C. Sakai (*):Y. Hoshino:Y. Sato:E. Sato
Laboratory of Animal Reproduction,
Graduate School of Agricultural Science, Tohoku University,
Sendai 981-8555, Japan
J Assist Reprod Genet (2011) 28:157–166
individuals have been successfully produced from IVM
oocytes of some mammals, including humans, the distin-
guishing features of in vivo ovulated (IVO) oocytes, which
confer a high developmental potential to these oocytes,
remain obscure . From a practical point of view, to
optimize IVM, it is necessary to achieve successful
embryonic development and identify oocyte markers that
predict successful nuclear and cytoplasmic maturation.
In oocytes, the meiotic spindle plays an important role in
chromosome alignment and separation during meiosis.
Typically, meiotic spindles of mouse oocytes are anastral;
however, the degree of spindle pole tapering and minus end
focusing varies widely between species  and with the
conditions under which meiotic maturation occurs .
Significant differences have been observed between IVO
and IVM oocytes of mice with regard to meiotic spindle
shape and size. IVO oocytes exhibit spindles with a focused
pole, whereas IVM oocytes typically display large barrel-
shaped anastral spindles that appear more pronounced after
different treatments [20, 21]. The significance of deviations
in the meiotic spindle shape and size with respect to the
quality of oocytes has not been fully investigated.
We aimed to identify the maturation conditions under
which the shape of the spindle in IVM oocytes was similar
to that of the spindle in IVO MII oocytes and to increase
the maturation competence of IVM oocytes. To this end, we
cultured mouse oocytes in the germinal-vesicle (GV) stage
under different conditions and evaluated the relationship
between spindle shape and ‘maturation competence’.
Materials and methods
ICR mice were purchased from Japan SLC Inc. (Shizuoka)
and bred in our laboratory. Immature 20- to 23-day-old
mice were used for all experiments. The experimental
procedures described in this report were performed in
accordance with the Guide for the Care and Use of
Laboratory Animals published by Tohoku University.
Collection of oocytes matured in vivo and in vitro
The maturation time under each maturation condition was
divided into phase I, II, and III (Fig. 1); phase I was
immediately after PB1 emission, phase II was the common
maturation time, and phase III was the prolonged matura-
tion time after PB1 emission. To obtain in vivo oocytes, we
first primed the mice with 5 IU of pregnant mare’s serum
gonadotropin (PMSG) (Teikoku Hormone MFG, Tokyo)
and then with 5 IU of human chorionic gonadotropin (hCG)
(Teikoku Hormone MFG) after 48 h. At 13 (phase I), 14
(phase II), and 18 h (phase III) after hCG treatment, MII-
Fig. 1 General timeline of mat-
uration in mice. Phase I, II, and
III in in vivo matured oocytes
are 13, 14, and 18 h, respec-
tively. Phase I, II, and III in
spontaneously matured oocytes
are 10, 12, and 18 h, respec-
tively. Phase I, II, and III in
FSH-induced matured oocytes
are 15, 18, and 24 h, respec-
tively. The arrows indicate the
maturation time reached inci-
dence rate to MII oocyte prateau
158 J Assist Reprod Genet (2011) 28:157–166
arrested oocytes released from the oviductal ampullae were
collected in Leibovitz’s L-15 medium (Invitrogen, Grand
Island, NY) containing 0.1% polyvinyl alcohol (PVA)
(Sigma, St. Louis, MO) and 4 mM hypoxanthine (Sigma).
The cumulus cells were removed by treatment with 0.1%
hyaluronidase at room temperature. In vitro oocyte matu-
ration was induced according to previously described
methods . The COCs were isolated from mice at 48 h
after PMSG injection by puncturing the large antral follicles
with a 26-gauge needle and were collected in Leibovitz’s L-
15 medium containing 0.1% PVA and 4 mM hypoxanthine
in order to maintain the oocytes in the GV-stage. For
spontaneous meiotic maturation experiments of COCs, we
used the following culture media: Waymouth’s MB752/1
medium (Invitrogen) or human tubal fluid (HTF) medium
(Irvine Scientific, Santa Ana, CA) containing 5% fetal calf
serum (FCS) (Gemini Bio, CA), 0.23 mM pyruvic acid
(Sigma), 75 mg/l penicillin G (Meiji Seika, Tokyo), and
50 mg/l streptomycin sulphate (Meiji Seika). These media
were designed as the basal media. To evaluate FSH-induced
meiotic maturation, we added 4 mM hypoxanthine and
100 IU/l FSH (Sigma) to the basal media. The COCs were
cultured in 100 μl droplets of the culture medium overlaid
with paraffin liquid (Nacalai Tesque, Kyoto) in a humidi-
fied atmosphere of 5% CO2in air at 37°C for 10 (phase I),
12 (phase II), and 18 h (phase III) (basal medium) or for 15
(phase I), 18 (phase II), and 24 h (phase III) (FSH-induced
medium). At the end of culture, oocytes were removed from
the cumulus cells by treatment with 0.1% hyaluronidase at
Immunodetection of microtubules and pericentrin in mouse
Immunolocalization in oocytes was performed according to
previously described methods . Oocytes were washed
three times in phosphate-buffered saline (PBS, Nissui,
Tokyo) containing 0.1% PVA (PBS-PVA), and denuded
oocytes were fixed with 2% paraformaldehyde (Sigma) in
Dulbecco’s PBS(-) containing 0.1% PVA and 0.2% Triton
X-100 at room temperature for 60 min. Microtubules were
detected by using anti-α-tubulin (Sigma; 1:500) and Alexa
Fluor 488-labelled goat anti-mouse IgG antibodies (Invi-
trogen; 1:200). Pericentriolar materials (PCMs) were
detected by using anti-pericentrin (BD biosciences, NJ;
1:100) and Alexa Fluor 488-labelled goat anti-mouse IgG
antibodies. The chromosomes were labelled with 10 μg/ml
propidium iodide (Sigma), and oocytes were viewed using
a Bio-Rad MRC-1024 confocal scanning laser microscope
mounted on an Axioplan Zeiss microscope. Confocal
images shown in the Results are representative of at least
30 oocytes matured under each maturation condition and
obtained from more than five animals.
Quantification of spindle size
Digital images were obtained from MII oocytes with a Bio-
Rad MRC-1024 confocal scanning laser microscope
mounted on an Axioplan Zeiss microscope, and spatial
measurements were recorded with Motic Images Plus 2.0S
(Shimadzu, Kyoto). The central plane of the spindle was
defined as the region with the largest spindle area in which
the distance between 2 points of PCMs was the maximum.
Only digital images in which spindle boundaries were
clearly defined and the spindles were oriented properly
were used to obtain measurements of spindle area, oocyte
area, distance between PCMs and chromosome width; from
these values, the relative spindle area and distance between
PCMs of meiotic spindle were calculated for each oocyte.
In vitro fertilization and embryo culture
In vitro fertilization and embryo culture were performed
according to the previously described methods .
Spermatozoa were collected from the cauda epididymis
and preincubated for 2–3 h in 400 μl of HTF medium to
allow capacitation before insemination. After capacitation,
the spermatozoa were introduced into 200 μl droplets of the
fertilization medium at a final concentration of 700
spermatozoa/μl. At 4 h after insemination, the penetration
of sperms into the oocytes was confirmed by microscopic
examination; subsequently, the oocytes were washed
thoroughly 5 times and then cultured in the KSOM
medium. All embryos were incubated in 100 μl droplets
of culture medium in a humidified atmosphere of 5% CO2
in air at 37°C.
Reverse transcriptase-polymerase chain reaction
We collected 20 MII oocytes matured under each
maturation condition; total RNA was extracted and cDNA
synthesis was performed by using Cells-to-cDNA™ II
(Ambion, Austin, TX). Polymerase chain reaction (PCR)
was performed using Ex Taq polymerase (TaKaRa, Shiga).
Each primer was designed as described in the previous
reports [22, 23]. For integrin α6, the sense (5′-GAGGAA-
TATTCCAAACTGAACTAC-3′) and antisense (5′-
GGAATGCTGTCATCGTACCTAGAG-3′) primers gener-
ated a 398-bp fragment. For integrin β1, the sense (5′-
GTGACCCATTGCAAGGAGAAGGA-3′) and antisense
TTCCA-3′) generated a 217-bp fragment. For
glyceraldehyde-3-phosphate dehydrogenase (G3PDH),
the sense (5′-CCACTCTTCCACCTTCGATG-3′) and an-
tisense primers (5′-GAGGGAGATGCTCAGTGTTG-3′)
generated a 226-bp fragment. The amplification conditions
were as follows: 94°C for 10 min; 35 cycles of 30 s each
J Assist Reprod Genet (2011) 28:157–166159
of denaturation at 94°C, annealing at 52°C for integrin α6,
52°C for integrin β1, and 57°C for G3PDH, and extension
at 72°C; and a final extension for 10 min at 72°C. The
DNA Data Bank of Japan/European Molecular Biology
Laboratory/GenBank accession numbers for integrin α6,
integrin β1, and G3PDH cDNA sequences are X69902,
Y00769, and M32599, respectively.
There were at least three replicates for each experiment.
Data were expressed as the mean ± SD and analysed using
ANOVA, followed by Bonferroni’s protective least signif-
icant difference test (P<0.05). The statistical significance
was evaluated by using STATVIEW (Abacus Concepts
Inc., Berkeley, CA).
Comparison of spindle size in in vivo- and in vitro-matured
The spindle size in oocytes matured under the three
maturation conditions was analysed by immunostaining
and digital imaging. The representative images of oocytes
from each treatment group are shown in Fig. 2. Oocytes
matured in vivo contained small spindles (Fig. 2A–C). MII
oocytes matured in vitro spontaneously cultured in Way-
mouth’s MB752/1 medium typically contained large and
barrel-shaped spindles (Fig. 2D–F). MII oocytes matured in
FSH-induced Waymouth’s MB752/1 medium exhibited
small spindles, similar to those of in vivo oocytes
To determine whether the culture medium influenced
spindle shape, we next examined the GV-stage oocytes
cultured in FSH-induced Waymouth’s MB752/1 medium
and HTF medium (Fig. 3). Oocytes matured in FSH-
induced HTF medium contained larger spindles than those
matured in Waymouth’s MB752/1 medium. These varia-
tions in the in vitro and in vivo matured oocytes with
respect to spindle size prompted a morphometric analysis of
each experimental group.
We measured the area covered by MII spindle in
individual oocytes from each treatment group using Motic
Images Plus 2.0S. The results are shown in Fig. 4. The
results indicated that the spindle areas of MII oocytes
matured in vitro spontaneously were greater than those of in
vivo oocytes, whereas the areas of MII oocytes matured in
FSH-induced Waymouth’s MB752/1 medium were rela-
tively similar to those of in vivo oocytes. However, the
spindle areas of MII oocytes matured in FSH-induced HTF
Fig. 2 Representative spindle images of in vivo (A–C, a–c),
spontaneous (D–F, d–f), and FSH-induced matured (G–I, g–i) oocytes
probed for microtubules (green) and chromosomes (red). The (A, a),
(B, b), and (C, c) panels illustrate the 13 h (phase I), 14 h (phase II),
and 18 h (phase III) hCG-treated MII oocytes matured in vivo,
respectively. The (D, d), (E, e), and (F, f) panels illustrate
spontaneously matured oocytes cultured for 10 h (phase I), 12 h
(phase II), and 18 h (phase III), respectively. The (G, g), (H, h), and
(I, i) panels illustrate FSH-induced matured oocytes cultured for 15 h
(phase I), 18 h (phase II), and 24 h (phase III), respectively. Small
letters represent morphological models of spindles in oocytes
matured in each maturation condition. In morphological models,
green and red lines represent microtubules and chromosomes,
respectively. Bar, 10 μm
160 J Assist Reprod Genet (2011) 28:157–166
medium were significantly larger than those in in vivo
oocytes and oocytes matured in Waymouth’s MB752/1
medium. These results suggest that although the culture
medium influenced spindle size of mouse MII oocytes, MII
oocytes matured in FSH-induced Waymouth’s MB752/1
medium may exhibit similar spindle size as that observed in
in vivo oocytes. Therefore, we used Waymouth’s MB752/1
medium as the IVM culture medium in all the subsequent
Localization of pericentriolar materials in in vivo- and in
vitro-matured MII oocytes
Next, we determined if PCMs influenced spindle morphol-
ogy. We analysed the PCMs of spindles in oocytes matured
under the three maturation conditions by immunostaining
and digital imaging. The representative images of oocytes
from each treatment group are shown in Fig. 5. The
distance between the 2 points of PCMs in MII oocytes
matured under all maturation conditions was initially more
(phase I); however, this distance reduced with time (phase
II) and increased again thereafter (phase III). These results
prompted a morphometric analysis in each experimental
We measured the distance between the PCMs of spindle in
individual oocytes from each treatment group using Motic
Images Plus 2.0S. The results are shown in Fig. 6. The
distance between the PCMs of spindles in MII oocytes
matured in vitro spontaneously was more and that between
the PCMs of spindles in MII oocytes matured in FSH-
vivo MII oocytes. This result indicates a trend that the distance
conditions was initially more; however, this distance reduced
gradually and increased again thereafter. These results sug-
gested that the distance between the PCMs in MII oocytes may
be affected by maturation conditions and time.
Differences in spindle morphologies of MII oocytes
To determine whether the difference in spindle morphology
in MII oocytes is associated with fertilization, we per-
Fig. 3 Representative spindle images of in vivo oocytes (A, B, a, b)
and FSH-induced matured oocytes cultured in Waymouth’s MB752/1
medium (C, D, c, d) or HTF medium (E, F, e, f) probed for
microtubules (green) and chromosomes (red). The (A, a) and (B, b)
panels illustrate the 14 h (phase II) and 18 h (phase III) hCG-treated
MII oocytes matured in vivo respectively. The (C, E, c, e) and (D, F, d,
f) panels illustrate FSH-induced matured oocytes cultured for 18 h
(phase II) and 24 h (phase III), respectively. Small letters represent
morphological models of spindles in oocytes matured in each
maturation condition. In morphological models, green and red lines
represent microtubules and chromosomes, respectively. Bar, 10 μm.
Way, Waymouth’s MB752/1 medium; HTF, HTF medium
Fig. 4 The effect of different culture conditions on the size of spindle.
Each bar shows in vivo (white), spontaneous (gray), and FSH-induced
oocytes matured in Waymouth’s MB752/1 medium or HTF medium
(black). Relative spindle area indicates the mean value of spindle area/
oocyte area. The total number of oocytes analysed (n) for each
maturation condition are indicated at the bottom of the columns. The
value of spindle size relative to that of in vivo MII oocytes (14 h after
hCG injection) is indicated. Values are expressed as mean ± SD. Bars
with different superscripts (a or b) are significantly different (P<0.05).
Way, Waymouth’s MB752/1 medium; HTF, HTF medium
J Assist Reprod Genet (2011) 28:157–166 161
formed in vitro fertilization using mature MII oocytes that
were matured under all maturation conditions. As illustrated
in Table 1, the sperm penetration rate, pronuclear formation
rate, and development rate to 2-cell embryos did not differ
with the maturation conditions. The rate of development to
blastocyst was the highest (62.7%) in MII IVO oocytes
isolated 14 h after hCG treatment under all maturation
conditions, and it was the second highest (60.7%) in MII
oocytes matured for 18 h in FSH-induced medium.
Moreover, when MII oocytes cultured in each of the
three culture conditions were examined closely, the oocytes
in which the distance between PCMs was reduced (Figs. 5
and 6, phase II) exhibited the highest development rate to
blastocyst in all culture conditions. This result suggests that
reduction in the distance between PCMs in MII oocytes
(phase II) improved their maturation competence.
To investigate this hypothesis, we examined the expres-
sion of integrins α6 and β1 mRNA in MII oocytes under
all maturation conditions. Oocyte integrins are essential for
fertilization. In particular, integrins α6 and β1 on the egg
plasma membrane serve as mammalian sperm receptors and
are related to fertility . As shown in Fig. 7, high levels
of integrins α6 and β1 were detected in MII oocytes
exhibiting high development rate to blastocyst in in vitro
fertilization. These results suggested that integrins α6 and
β1 influence the fertility of MII oocytes, and with reducing
distance between PCMs (phase II), high levels of integrins
α6 and β1 were detected in MII oocytes.
Fig. 6 Morphometric measurement of meiotic spindle under different
culture conditions. Each bar shows in vivo (white), spontaneous
(gray), and FSH-induced (black) groups. Values are individual
distance between PCMs of meiotic spindle (distance between PCMs
(a)/chromosome width (b)). The value of distance between PCMs
relative to that of in vivo MII oocytes (14 h after hCG injection) is
indicated. The total number of oocytes analysed (n) for each
maturation condition are indicated at the bottom of the columns.
Values are expressed as mean ± SD. Bars with different superscripts (a
or b) are significantly different (P<0.05)
Fig. 5 Representative spindle images of in vivo (A–C, a–c),
spontaneous (D–F, d–f), and FSH-induced matured (G–I, g–i) oocytes
probed for PCMs (green) and chromosomes (red). The (A, a), (B, b),
and (C, c) panels illustrate 13 h (phase I), 14 h (phase II), and 18 h
(phase III) hCG-treated MII oocytes matured in vivo, respectively. The
(D, d), (E, e), and (F, f) panels illustrate spontaneous matured oocytes
cultured for 10 h (phase I), 12 h (phase II), and 18 h (phase III),
respectively. The (G, g), (H, h), and (I, i) panels illustrate FSH-
induced matured oocytes cultured for 15 h (phase I), 18 h (phase II),
and 24 h (phase III), respectively. Small letters represent morpholog-
ical models of spindles in oocytes matured in each maturation
condition. In morphological models, red lines and blue dots represent
chromosomes and PCMs, respectively. Bar, 10 μm
162J Assist Reprod Genet (2011) 28:157–166
Oocyte quality profoundly affects monospermic fertiliza-
tion, early embryonic survival, maintenance of pregnancy,
and even fetal development. Therefore, it is important to
identify reliable indicators of oocyte quality for efficient
embryo production and infertility treatment. Determination
of oocyte quality by morphological assessment is a
relatively popular method because it is noninvasive and
convenient. Meiotic spindle morphology provides impor-
tant information for predicting the developmental compe-
tence of oocytes . The length of the MII spindle has
been correlated with the quality of human oocytes [26, 27].
Sanfins et al. (2003) examined the spindle behaviour in
naturally ovulated oocytes, IVO oocytes obtained from
standard superovulation protocols, and COCs undergoing
spontaneous meiotic maturation. They found that while the
MII stage IVO oocytes had normal bipolar spindles with
focused poles, which were characterized by the presence of
distinct γ-tubulin foci, the IVM oocytes exhibited barrel-
shaped spindles with few acetylated microtubules and
diffuse distribution of γ-tubulin in the microtubules.
Similarly, we observed significant differences between the
spindle morphologies of oocytes matured under different
conditions: the spindles in IVO oocytes and oocytes
matured in FSH-induced medium were small and pointed
as compared to the spindles in oocytes that were spontane-
ously matured in vitro, as previously observed by Sanfins et
al. (2003). Interestingly, the spindle morphologies in
spontaneously matured oocytes are similar to those in the
Mos−/−strains of mutant oocytes [4, 28, 29]. The Mos−/−
strains of mutant oocytes have hypertrophied spindles.
Moreover, Mos−/−strains of mutant COCs cannot undergo
cumulus expansion, which occurs during in vivo maturation
and ovulation. Therefore, it is noteworthy that the addition
of FSH to culture medium induces the partial reversion of
spontaneously matured oocytes to the IVO phenotype,
suggesting that COC integrity and hormone-regulated
expansion play pivotal poles in microtubule patterning
during oocyte maturation.
However, we compared spindle morphologies in IVO
oocytes to those in oocytes matured in the 2 FSH-induced
Fig. 7 RT-PCR analysis of integrin α6 (A) and β1 (B) mRNA
expression in different culture conditions. G3PDH was amplified as an
intrinsic control. The experiments were repeated three times and similar
results were obtained. Values are expressed as mean ± SD. Bars with
different superscripts (a or b) are significantly different (P<0.05)
Table 1 Effects of culture conditions on MII oocytes and embryonic development
Maturation conditionsPhase (h) Total number of oocytes Sperm penetration (% ± SD)d
Pronuclei (% ± SD)d
2-cell embryos (% ± SD)e
Blastocyst (% ± SD)e
In vivoI (13)16184.9±7.777.7±7.898.8±1.257.3±6.9a
III (18) 9199.0±1.087.0±6.499.0±1.0
In vitroSpontaneous I (10)13592.1±4.063.9±3.693.1±5.0
II (12) 12088.7±4.8 85.7±3.482.7±4.5
III (18) 78 90.0±3.684.9±4.189.0±6.5
FSH-inducedI (15) 7594.7±0.579.0±13.889.7±5.2
II (18)102 98.0±2.091.5±4.498.9±1.1
III (24) 7793.9±6.1 76.7±12.379.2±10.1
Values are expressed as mean ± SD.a–cValues with different superscripts within each column are significantly different (P<0.05; ANOVA). No
differences were observed in sperm penetration rate, pronuclear formation rate, and the development rate to two-cell embryos.dValues per MII oocyte;
eValues per pronuclei
J Assist Reprod Genet (2011) 28:157–166163
culture media (HTF and Waymouth’s MB752/1 medium);
the spindle areas of MII oocytes matured in FSH-induced
HTF medium were significantly greater than those of IVO
oocytes and oocytes matured in the Waymouth’s MB752/1
medium. This result was consistent with the results of
previous studies [30, 31]. These reports suggested that
spindles in IVM oocytes exhibited a variation in shape from
tapering poles (IVO oocytes) to large barrel shape (oocytes
matured in basal medium); further, the shape of spindles in
IVM oocytes cultured in supplemented medium was similar
to that of IVO oocytes. Therefore, culture conditions may
influence the processes underlying spindle morphogenesis,
and some degree of caution is warranted in attributing
significance to spindle shape variations when comparing in
vivo and in vitro maturation.
In somatic cells, the centrosome is a crucial organelle for
the assembly of mitotic spindles during cell division.
However, centrosomes do not always contain centrioles.
Although MII spindles in mouse oocytes possess centroso-
mal material at both meiotic poles, which is formed by the
assembly of multiple small asters, the centrioles are absent.
Nevertheless, this centrosomal material performs the func-
tions that are typical of microtubule organizing centres
(MTOCs) and can therefore ‘qualify’ as a centrosome .
Vertebrate oocytes contain acentriolar MTOCs in place of
centrosomes, which are involved in spindle assembly
(acentrosomal spindle assembly), and these MTOCs contain
the PCM components, namely, γ-tubulin [33, 34] and
pericentrin . MII oocytes of the pig , sheep ,
and cow  do not contain cytoplasmic MTOCs, whereas
those of mice contain cytoplasmic MTOCs. In mice, the
acentrosomal materials containing PCMs at both meiotic
spindle and cytoplasmic MTOCs are regulated to aid
spindle assembly during meiotic progression [39–42].
However, it has been proposed that the barrel-shaped
spindles observed in Mos−/−strains of mutant and sponta-
neously matured oocytes are formed by the incorporation of
an excessive number of MTOCs in the spindle [20, 40, 43,
44]. In our study, the distance between the PCMs at the
spindle poles in MII oocytes matured in vitro spontaneously
in all phases was more and that in MII oocytes matured in
FSH-induced Waymouth’s MB752/1 medium was less than
that in the IVO oocytes. If spindle-associated MTOCs were
limited in number or mass during spindle morphogenesis,
then smaller and pointed spindles in oocytes would be
expected, as observed in MII oocytes matured in FSH-
induced Waymouth’s MB752/1 medium and in IVO
oocytes. Moreover, in our experiments, the rate of
development to blastocyst was the highest in MII IVO
oocytes isolated at 14 h after hCG treatment and the second
highest in MII oocytes matured for 18 h in FSH-induced
medium; however, the rate of development to blastocyst in
MII oocytes matured spontaneously for 12 h was signifi-
cantly lower than that in the abovementioned two
conditions (Table 1). After fertilization, these MTOCs
function as centres for microtubule nucleation, and they
participate in pronuclear movement and in the formation
of subsequent mitotic spindles [45, 46]. Therefore, an
abnormal reduction of cytoplasmic MTOCs could signif-
icantly reduce the oocyte competence to sustain normal
Our study showed that the distance between PCMs at the
spindle poles in MII oocytes matured under all maturation
conditions was initially more (phase I); however, this
distance reduced gradually (phase II) and increased again
thereafter (phase III) (Fig. 5). Further, MII oocytes with less
distance between PCMs (phase II) exhibited the highest
development rate to blastocyst in each MII oocyte
maturation condition (Table 1). In addition, MII oocytes
with high integrin α6 and β1 mRNA expressions devel-
oped to the blastocyst stage (Fig. 7). We speculate that the
dynamic change in PCMs and the association between the
distance between PCMs and fertility influence spindle
assembly and maintain spindle configuration. The pig
meiotic spindle is formed by bundling of microtubules by
the Nuclear Mitotic Apparatus (NuMA), which is a kind of
centrosomal material, whereas the mouse meiotic spindle is
formed through the gathering of cytoplasmic MTOCs .
First, the perinuclear acentrosomal materials containing
PCMs formed multiple spindle poles. The continued
activity of motor proteins, such as dynein or Ncd, would
promote PCMs clustering, explaining the fusion of several
small poles into large ones containing 2 PCMs clusters, and
this process continued until the formation of two dominant
spindles . In this bipolar configuration, microtubules
are stably aligned due to motor activities and chromosome
biorientation, indicating that microtubule kinetochore con-
tacts have been established . We expected that the
metaphase spindle morphology in phase I (immediately
after PB1 emission) could be on the way of PCMs
clustering at each spindle poles; therefore, the distance
between 2 points of PCMs at each pole was more and the
oocytes could not undergo fertilization. In phase II, this
bipolar configuration could be stabilized, thereby reducing
the distance between 2 points of PCMs and affording
maturation competence to the oocytes. However, it has
been reported that metaphase spindle in over-matured
oocytes exhibit characteristic patterns of centrosome dete-
rioration and frequently display tri- or multipolar spindle
poles; these oocytes could not be fertilized, or alternatively,
the fertilized oocytes exhibited aneuploidy or developmen-
tal abnormalities [48, 49]. It has been shown that MAPK
activities gradually decrease in aging oocytes. MAPK is a
signalling molecule associated with centrosome compo-
nents , and the former is thought to be involved in
centrosome and microtubule stabilization. Therefore, in
164J Assist Reprod Genet (2011) 28:157–166
aging oocytes, the decrease in MAPK activities may
account for centrosome and microtubule destabilization,
thereby influencing fertility.
Meiotic spindle is crucial for accurate chromosomal
alignment and segregation during meiosis. It is important to
coordinate microtubule remodelling with the functions of
cell cycle components during oocyte meiosis to achieve
balanced nuclear and cytoplasmic maturation . There-
fore, the characteristics of the spindle, including its area,
and the distance between PCMs, can be used to evaluate
oocyte quality .
Our results suggest that oocytes exhibiting reduction in
the distance between PCMs have a higher embryo
developmental potential than the oocytes that exhibit
increasing distance between PCMs. Conventionally, the
spindles were imaged mainly by confocal microscopy
analysis, which requires cell fixation and has a fatal effect
on the oocyte. Alternatively, meiotic spindles can be
observed directly  by using a polarization microscope
(Polscope, Cambridge, MA). At present, the results of
morphometric evaluation of the spindle obtained by using a
Polscope are not consistent with those obtained by confocal
analysis ; therefore, the distance between PCMs in
metaphase spindle could not be measured using the
Polscope. However, more advanced polarized light micros-
copy devices (e.g., the Oosight) may provide a better
representation of the spindle constitution. Therefore, in the
future, careful analysis of the distance between PCMs of
spindle in the MII oocytes using these devices may serve as
a noninvasive and reliable method of assessing oocyte
quality and embryonic competence.
The findings of our study draw specific attention to the
determinants of oocyte quality that influences the produc-
tion of superior quality animals, preservation of endangered
animals, and application of assisted-reproductive technolo-
gy, and reinforces the mounting concerns over the wide-
spread use of in vitro maturation for clinical purposes.
for the Promotion of Science Grant to E. Sato (No. 21248032). This
work was also supported in part by Grant-in-Aid for Young Scientists
(B) to Y. Hoshino (No. 21780250) from the Ministry of Education,
Science, and Culture, Japan.
This work was supported by the Japan Society
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