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c Cambridge University Press 2016


doi:10.1017/S0967199416000290

Early germinal vesicle breakdown is a predictor of high


preimplantation developmental competent oocytes in mice

Shogo Higaki1 , Masao Kishi3 , Keisuke Koyama4 , Masashi Nagano3 , Seiji Katagiri3 , Tatsuyuki Takada2
and Yoshiyuki Takahashi3
Laboratory of Cell Engineering, Department of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan;
Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine,
Hokkaido University, Sapporo, Hokkaido, Japan; amd Dairy Cattle Group, Konsen Agricultural Experiment Station,
Hokkaido Research Organization, Nakashibetsu, Hokkaido, Japan

Date submitted: 30.05.2016. Date revised: 01.08.2016. Date accepted: 31.08.2016

Summary
The preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is
crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte
quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break
down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been
reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD
timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration
during IVM may affect the nuclear kinetics, all experiments were performed under two distinct
O2 concentrations: 20% and 5% O2 . First, we determined the time course of changes in nuclear
maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate
GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent
GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we
compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage
and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2 , respectively, and
67.6% and 47.3% under 5% O2 , respectively) were almost double those of late GVBD oocytes (44.8% and
26.0% under 20% O2 , respectively, and 40.4% and 17.9% under 5% O2 , respectively). With no observable
alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte
evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method
for high developmental competent oocytes.

Keywords: Developmental competence, Germinal vesicle breakdown, Mouse, Oocyte

Introduction
1
All correspondence to: Shogo Higaki. Laboratory of Cell
Engineering, Department of Pharmaceutical Sciences, In vitro maturation (IVM) of oocytes is increasingly
Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu, demanded in both human-assisted reproductive tech-
Shiga 525 8577, Japan. Tel: +81 77 561 2266. E-mail: nology and animal in vitro embryo production (IVP).
shogohigaki@gmail.com However, in vitro-matured oocytes have been shown
2
Laboratory of Cell Engineering, Department of Pharmaceut-
ical Sciences, Ritsumeikan University, Kusatsu, Shiga 525 to have lower fertilization and blastocyst formation
8577, Japan. rates than their in vivo-matured counterparts (Banwell
3
Laboratory of Theriogenology, Department of Veterinary & Thompson, 2008). This low IVP efficiency using
Clinical Sciences, Graduate School of Veterinary Medicine, in vitro-matured oocytes may be related to a hetero-
Hokkaido University, Sapporo, Hokkaido 0600818, Japan. geneous oocyte population at the beginning of IVM
4
Dairy Cattle Group, Konsen Agricultural Experiment
Station, Hokkaido Research Organization, Nakashibetsu, (Sirard et al., 2006; Fair, 2009). Thus, preselection of
Hokkaido 0861135, Japan. the immature oocytes that are most likely to develop

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2 Shogo Higaki et al.

is crucial for improving the IVP efficiency when using Collection of oocytes
IVM oocytes.
Immature (19 to 23 days old) female mice were
Many morphological, cellular, and molecular pre-
treated intraperitoneally with 5 IU of equine chorionic
dictors of oocyte developmental competence have
gonadotropin (eCG; Aska Pharmaceutical. Co. Ltd,
been reported (Wang & Sun, 2006; Bukowska et al.,
Tokyo, Japan) and euthanized 48 h later by cervical
2012). Several intrinsic markers of oocyte quality are
dislocation. Mouse ovaries were collected in a 35-
also known to be closely related to the onset of
mm plastic dish containing 2.5 ml of Leibovitzs L-15
the nuclear maturation (germinal vesicle breakdown,
medium (Gibco Life Technologies, Grand Island, NY,
GVBD), such as maturation-promoting factor (MPF)
USA) supplemented with 4 mg/ml of bovine serum
activity (Naito et al., 1992; Christmann et al., 1994;
albumin (BSA; A-8022, Sigma-Aldrich, St. Louis, MO,
Chesnel & Eppig, 1995; Ledda et al., 2001; Catal
USA) and 50 g/ml of gentamicin sulfate (G-3632,
et al., 2011). However, although some reports refer
Sigma-Aldrich). Cumulusoocyte complexes (COCs)
to the relationship between the timing of completion
were released from the large antral follicles using a 25-
of nuclear maturation (first polar body extrusion or
G needle. Only oocytes with a diameter >70 m (ex-
reaching metaphase II: MII stage) and subsequent
cluding the zona pellucida thickness) and completely
developmental competence of in vitro-matured oocytes
enclosed by cumulus cells were selected. COCs with
in human and bovine (Dominko & First, 1997; Son
dark, shrunken, or irregularly shaped oocytes were
et al., 2005), a direct comparison between GVBD
discarded.
timing and immature oocyte quality has never been
reported.
Most procedures used to evaluate intrinsic pre-
dictors can have adverse effects on oocyte viability In vitro maturation (IVM)
and/or subsequent developmental competence with
varying degrees. Therefore, the objective of this study IVM was performed as described previously (Adam
was to establish a non-invasive oocyte evaluation et al., 2004) with a slight modification in the number of
method based on GVBD timing for preselecting more COCs cultured in one droplet. In short, approximately
developmental competent oocytes in a commonly 10 COCs were transferred to a 50-l droplet of
used mouse oocyte model. First, we investigated Waymouths MB 752/1 (Gibco Life Technologies)
the time course of changes in nuclear maturation supplemented with 5% fetal calf serum (FCS; Gibco
and preimplantation developmental competence of Life Technologies), 0.23 mM sodium pyruvate (P-4562,
in vitro-matured oocytes to estimate GVBD timing Sigma-Aldrich), 1 IU/ml of porcine follicle stimulating
in high developmental competent oocytes. Second, hormone (FSH; Antrin R, Kawasakimitaka Co. Ltd,
we compared the preimplantation developmental Kawasaki, Japan), 10 ng/ml of human recombinant
competence of early and late GVBD oocytes. Because epidermal growth factor (hrEGF; Sigma-Aldrich), and
the O2 concentration during IVM may affect the 50 g/ml of gentamicin sulfate; then, the entire droplet
nuclear kinetics (Zeilmaker et al., 1972; Mingoti et al., was covered with paraffin oil. COCs were incubated
2011), all experiments were performed under two for varying times at 37C in a humidified atmosphere
distinct oxygen concentrations generally used for of 5% CO2 in air (20% O2 ) or 5% CO2 , 5% O2 , and 90%
mammalian oocyte culture: 5% CO2 in air (20% O2 ) or N2 (5% O2 ).
5% CO2 , 5% O2 , and 90% N2 (5% O2 ).

In vitro fertilization (IVF)


Materials and methods
Following IVM, oocytes surrounded by the cumulus
cells were subjected to IVF by adapting the procedure
Experimental animals
of Toyoda (1971). In brief, spermatozoa collected from
Male and female ICR mice purchased from Japan the caudal epididymis of matured (3 to 6 months old)
SLC Inc. (Shizuoka, Japan) were housed in the animal male mice were preincubated for 2 h in a 0.4-ml droplet
housing facility at the Graduate School of Veterinary of KrebsRinger bicarbonate medium under paraffin
Medicine, Hokkaido University. Mice were kept under oil at 37C in a humidified atmosphere of 20% O2 .
light- and temperature-controlled conditions (12-h Preincubated sperm was added to a 0.4-ml droplet of
light/12-h dark cycle; 23 2C) and given chow pellets KrebsRinger bicarbonate medium containing approx-
and water ad libitum. All procedures described herein imately 10 COCs to give a final sperm concentration
were conducted in accordance with the Hokkaido of 2.0 105 cells/ml. COCs were coincubated with
University guidelines for the care and use of animals sperm for 5 h at 37C in a humidified atmosphere of
(approved protocol: 17023). 20% O2 .

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Timing of GVBD is a predictor of oocyte quality 3

In vitro culture (IVC) and evaluation of embryonic Experiment 2: Relationship between GVBD timing and
development preimplantation developmental competence of oocytes
In vitro maturing oocytes were separated into two
After IVF, presumptive zygotes freed from cumulus
groups according to the presence (+) or absence ()
cells were transferred to a 25-l droplet of potassium
of a clear germinal vesicle (GV) (Fig. 1) examined
simplex optimized medium (KSOM) (Erbach et al.,
at 400 magnification under an inverted microscope
1994) and cultured at 37C in a humidified atmosphere
3 h and 4 h after the initiation of IVM under 20%
of 5% O2 in air. Cleavage and development to the
and 5% O2 , respectively. Both GV+ and GV oocytes
blastocyst stage were determined at 24 and 120 h
were separately transferred to new droplets in groups
after IVF, respectively. Cell numbers of the blastocysts
of approximately 10, cultured until 15 h from the
obtained after 120 h of IVC were determined using an
initiation of IVM under distinct O2 tension, and then
air-dry procedure described elsewhere (Takahashi &
subjected to IVF and IVC; cleavage and blastocyst
First, 1992).
formation rates and the cell number of blastocysts
were examined as described above. This experiment
was repeated seven times using 21 to 56 oocytes for
Experimental design
each replicate. Oocytes with an unknown GV status
Experiment 1: Time course of changes in nuclear stage and (without checking GV status) that were not transferred
preimplantation developmental competence of oocytes to new droplets were used as controls. In another five
To examine the time course of changes in nuclear replicates (24 to 51 oocytes per replicate), cleavage
stage, the nuclear status of the oocytes was first rates and nuclear status of uncleaved oocytes were
examined for every 2 h interval from 0 to 12 h examined by whole-mount preparation 24 h after
of IVM in either 20% O2 or 5% O2 . Thereafter, initiation of IVC. Cleaved and uncleaved oocytes with
nuclear status was also examined at two additional two pronuclei were considered fertilized.
time points; 7 h (the expected time of first oocyte
reached MII stage) and 15 h (the time used for Statistical analysis
routine IVM work in our laboratory (Adam et al.,
2004)) of IVM, to more fully characterize the time Data are expressed as mean standard error of
course of changes in nuclear stage of IVM oocytes. the mean (SEM). Percentage data were transformed
The nuclear status of oocytes was assessed by using the arcsine transformation (arcsin %) before
whole-mount preparation according to a previous analysis to normalize the variance. The effects of
report (Bedford, 1971) with a slight modification. the maturation period and O2 concentration during
Briefly, COCs were transferred to a 50-l droplet IVM on the percentage of nuclear stage, cleavage,
of Leibovitzs L-15 medium supplemented with and blastocyst formation were analysed by two-
300 U/ml hyaluronidase (H-3506, Type 1-S from way analysis of variance (ANOVA) followed by
bovine testis; Sigma-Aldrich). The surrounding a Bonferroni post-hoc test. The effects of GVBD
granulosa cells were removed by pipetting at timing on cleavage and blastocyst formation rates
room temperature (20 to 25C) for 5 min. The and the cell number of blastocysts were analysed
cumulus-free oocytes were mounted on a glass by one-way ANOVA followed by a Bonferroni post-
slide and gently pressed down with coverslips hoc test. Differences in the frequency of fertilized,
supported by four beads of paraffin-vaseline unfertilized, and fragmented GV+ and GV oocytes
wax until trapped. Oocytes were then fixed with were evaluated by chi-squared analysis with the Yates
10% neutral formalin for over 6 h, stained with correction. Data analysis was performed using SPSS
1% (w/v) aceto-orcein, decolored with aceto- software (ver. 13.0, SPSS Inc., Chicago, IL, USA).
glycerol, and examined under a phase-contrast
microscope. This experiment was repeated four times
at each time point using 16 to 48 oocytes for each
Results
replicate.
To examine the time course of changes in
Time course of changes in nuclear stage and
preimplantation developmental competence,
preimplantation developmental competence of
oocytes cultured for 10, 12, 15, and 17 h in IVM
oocytes
medium under 20% and 5% O2 were subjected
to IVF and IVC, and cleavage and blastocyst Under 20% O2 , the first oocytes that underwent
formation rates were examined as described GVBD were found at 2 h after initiation of IVM,
above. This experiment was repeated four times and the percentage of oocytes undergoing GVBD
at each time point using 22 to 60 oocytes for each linearly increased to a maximum level after 4 h of
replicate. IVM (Fig. 2A). The percentage of oocytes that reached

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4 Shogo Higaki et al.

Figure 1 Morphology of cumulus-enclosed mouse oocytes with (+) or without () intact germinal vesicle (GV). Representative
images of cumulus-enclosed GV+ or GV oocytes 3 h after the initiation of in vitro maturation (IVM) under 20% O2 at low
magnification (A). High magnification images of GV+ (B) and GV (C) oocytes. Arrowheads indicate GV. Bar = 100 m (A),
50 m (B, C).

MI and MII stages was almost parallel to the time IVM (P < 0.05). The rate of blastocyst formation from
course of changes in GVBD with 2 h and 6 h the 2-cell stage in GV oocytes cultured under 5%
delays, respectively. Under 5% O2 , the percentage of O2 was higher than that of GV+ oocytes (P < 0.05),
oocytes undergoing GVBD increased in a biphasic but not when cultured under 20% O2 . There was
manner; namely, approximately two-thirds of oocytes no difference between the cell number of blastocysts
underwent GVBD during the first 4 h of IVM, derived from GV+ and GV oocytes, regardless of
gradually followed by the remaining third (Fig. 2B). A the O2 concentration during IVM. Mean percentages of
similar biphasic pattern was also observed for oocytes cleavage, 2-cell to blastocyst, and blastocyst formation
that reached MI and MII stages with 2 h and 6 h delays, as well as the cell number of blastocysts in GV+ and
respectively. GV oocytes were not different from control oocytes,
There were no significant differences between regardless of the O2 concentration during IVM.
cleavage or blastocysts formation rates of oocytes As shown in Table 2, the frequencies of GV+ and
cultured under 20% (Fig. 3A) or 5% O2 (Fig. 3B) at GV fertilized oocytes stopped at the pronucleus
each time point. Cleavage rates tended to increase stage, unfertilized oocytes stopped at the MI stage, and
with prolonged periods of IVM, whereas blastocyst fragmented oocytes were not different, regardless of
formation rates reached a maximum after 12 h, and the O2 concentration during IVM. The frequency of
these rates were not affected by prolonging the IVM GV+ unfertilized MII-stage oocytes was higher than
period up to 17 h. that of GV oocytes, regardless of the O2 concentration
during IVM (P < 0.05).
Relationship between GVBD timing and
preimplantation developmental competence of
oocytes Discussion
As shown in Table 1, GV oocytes showed higher Although there were no comparative data on the
cleavage and blastocyst formation rates than GV+ nuclear progression of mouse oocytes cultured under
oocytes, regardless of the O2 concentration during 5% O2 , the time course of changes observed in the

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Timing of GVBD is a predictor of oocyte quality 5

Table 1 Preimplantation developmental competence of early and late germinal vesicle (GV) breakdown mouse oocytes
Oocyte groupa

Oxygen tension Developmental stage GV+ GV Total Controlb

20% O2 No. of oocytes (replicates) 97 (7) 210 (7) 307 (7) 160 (4)
Cleavage (%) 44.8 7.1c 80.2 3.3d 68.8 2.9d 69.0 1.8d
2-cell to blastocyst (%) 56.9 5.6 66.8 4.0 63.8 2.4 55.6 6.3
Blastocyst (%) 26.0 5.4c 52.7 4.5d 44.1 3.0d 38.3 6.9d
Cell number of blastocyst 34.5 3.1 38.4 3.0 37.4 3.1 39.2 2.7
5% O2 No. of oocytes (replicates) 82 (7) 173 (7) 255 (7) 162 (4)
Cleavage (%) 40.4 4.5c 67.6 2.7d 58.9 2.2d 64.8 4.1d
2-cell to blastocyst (%) 45.3 7.6c 66.1 2.5d 65.2 2.5c,d 60.5 3.9c,d
Blastocyst (%) 17.9 3.4c 47.3 2.0d 38.1 1.0d 39.3 3.2d
Cell number of blastocyst 37.2 3.0 38.7 3.3 38.2 3.2 37.9 2.6
a
Oocytes with (GV+) or without (GV) clear GV 3 and 4 h after initiation of in vitro maturation under 20% and 5% O2 ,
respectively.
b
Oocytes with an unknown GV status (without checking GV status) not transferred to new droplets.
c,d
Values with different superscripts within the same oocyte developmental stage and O2 concentration differ significantly
(P < 0.05).

Table 2 Fertilization and nuclear status of early and late germinal vesicle (GV) breakdown in mouse oocytes 24 h after in
vitro fertilization
Oocyte groupa

Oxygen tension Fertilization/nuclear status GV+ GV P-value

20% O2 No. of oocytes (replicates) 70 (5) 149 (5)


No. (%) of fertilized oocytes Total 36 (51.4) 117 (78.5) <0.05
2PN 1 (1.4) 2 (1.3) 1
Cleavage 35 (50.0) 115 (77.2) <0.05
No. (%) of unfertilized oocytes Total 31 (44.3) 28 (18.8) <0.05
MI stage 8 (11.4) 7 (4.7) 0.121
MII stage 23 (32.9) 21 (14.1) <0.05
No. (%) of fragmented oocytes 3 (4.3) 4 (2.7) 0.829
5% O2 No. of oocytes (replicates) 62 (5) 128 (5)
No. (%) of fertilized oocytes Total 27 (43.5) 95 (74.2) <0.05
2PN 2 (3.2) 4 (4.2) 1
Cleavage 25 (40.3) 91 (71.1) <0.05
No. (%) of unfertilized oocytes Total 33 (53.2) 29 (22.7) <0.05
MI stage 6 (9.7) 4 (3.1) 0.121
MII stage 27 (43.5) 25 (19.5) <0.05
No. (%) of fragmented oocytes 2 (3.2) 4 (3.1) 1
a
Oocytes with (GV+) or without (GV) clear GV 3 and 4 h after initiation of in vitro maturation under 20% and 5% O2 ,
respectively.
2PN, two pronuclei formation; MI, metaphase I; MII, metaphase II.

nuclear stage under 20% O2 in this study fitted well 4 h of IVM under 5% O2 (early nuclear maturation
with the previous studies (Donahue, 1968; Messinger group), showed similar nuclear kinetics to the oocytes
& Albertini, 1991; Liu et al., 2005). Biphasic time course cultured under 20% O2 . Higher levels of intracellular
of changes in the percentage of oocytes that reached reactive oxygen species in oocytes matured under
GVBD, MI, and MII stages under 5% O2 indicated 20% O2 compared 5% O2 was reported for bovine
that heterogeneous oocyte populations contained early (Hashimoto et al., 2000), and oxidative stress is known
and late nuclear maturation oocytes, even if they were to stimulate meiotic resumption in mouse oocytes
obtained solely from large antral follicles of eCG- (LaRosa & Downs, 2006).
primed immature mice. Interestingly, two-thirds of Contrary to nuclear maturation, there were no de-
the oocytes that underwent GVBD during the first tectable differences in cleavage or blastocyst formation

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6 Shogo Higaki et al.

Figure 3 Time course of changes in preimplantation devel-


Figure 2 Time course of changes in nuclear maturation of
opmental competence of in vitro-matured mouse oocytes
in vitro-matured mouse oocytes cultured under 20% O2
cultured under 20% O2 (A) and 5% O2 (B). Percentages
(A) and 5% O2 (B). Cumulative percentages of oocytes
of oocytes developed to 2-cell (white circle) and blastocyst
reached different stages of nuclear maturation [germinal
(triangle) stages. a,b,c Denotes statistical significance within
vesicle breakdown (GVBD), black circle; metaphase I (MI),
the same developmental stage among oocytes cultured
triangle; metaphase II (MII), square]. a,b,c,d,e Denotes statistical
under the same O2 concentration (P < 0.05). This experiment
significance within the same nuclear stage among oocytes
was repeated four times. n represents the total number of
cultured under the same O2 concentration (P < 0.05).
oocytes examined at each time point.
Asterisks () represent significantly lower values from
oocytes at the same nuclear stage and time point cultured
under 20% O2 (P < 0.05). This experiment was repeated four
times. n represents the total number of oocytes examined at
each nuclear stage at each time point.
To confirm this, we compared the preimplantation
developmental competencies of early and late nuclear
rates for oocytes cultured under 20% or 5% O2 as maturation oocytes selected based on the presence
reported previously (Eppig & Wigglesworth, 1995; (+) or absence () of clear GV 3 h and 4 h after
Banwell et al., 2007; Preis et al., 2007). Furthermore, IVM initiation under 20% and 5% O2 , respectively.
the blastocyst formation rate did not increase after 12 These times were chosen because about two-thirds
h of IVM even though one-third of oocytes cultured of oocytes were found to have undergone GVBD
with 5% O2 underwent GVBD after that time. These under the distinct O2 concentrations. GV oocyte
results may indicate that early nuclear maturation cleavage and blastocyst formation rates were almost
oocytes seemed mainly to contribute to the blastocyst double that of GV+ oocytes, regardless of the O2
yield, and such high quality oocytes may acquire concentration during IVM. This might be consistent
the developmental competence even under highly with previous reports in humans and bovine; oocy-
oxidative conditions. tes completing nuclear maturation early showed

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Timing of GVBD is a predictor of oocyte quality 7

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bovine oocytes were improved, their developmental
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This study was supported in part by a Grant-Aid for Kubiak, J.Z., Ciemerych, M.A., Hupalowska, A., Sikora-
the 21st Century COE Program from the Ministry of Polaczek, M. & Polanski, Z. (2008). On the transition

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