BIOLOGY OF REPRODUCTION (2013) 89(3):57, 1–11
Published online before print 24 July 2013.
DOI 10.1095/biolreprod.113.109439
Production of Fertile Offspring from Oocytes Grown In Vitro by Nuclear Transfer in
Cattle1
Yuji Hirao,2,5 Kenji Naruse,6 Masahiro Kaneda,3,5 Tamas Somfai,5 Kosuke Iga,6 Manabu Shimizu,6 Satoshi
Akagi,5 Feng Cao,7 Tomohiro Kono,7 Takashi Nagai,5 and Naoki Takenouchi4,6
5
Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, Tsukuba,
Ibaraki, Japan
6
Livestock and Forage Research Division, NARO Tohoku Agricultural Research Center, Morioka, Iwate, Japan
7
Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, Japan
INTRODUCTION
Because of recent advancements in reproductive technology,
oocytes have attained an increasingly enriched value as a unique
cell population in the production of offspring. The growing
oocytes in the ovary are an immediate potential source that
serve this need; however, complete oocyte growth before use is
crucial. Our research objective was to create in vitro-grown
(IVG) oocytes that would have the ability to perform specialized
activities, including nuclear reprogramming, as an alternative to
in vivo-grown oocytes. Bovine oocyte–granulosa cell complexes
with a mean oocyte diameter of approximately 100 lm were
cultured on Millicell membrane inserts, with culture medium
supplemented with 4% polyvinylpyrrolidone (molecular weight,
360 000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5
ng/ml bone morphogenetic protein 7. Oocyte viability after the
14-day culture period was 95%, and there was a 71% increase
in oocyte volume. Upon induction of oocyte maturation, 61% of
the IVG oocytes extruded a polar body. Eighty-four percent of
the reconstructed IVG oocytes that used cumulus cells as donor
cells underwent cleavage, and half of them became blastocysts.
DNA methylation analyses of the satellite I and II regions of the
blastocysts revealed a similar highly methylated status in the
cloned embryos derived from in vivo-grown and IVG oocytes.
Finally, one of the nine embryos reconstructed from the IVG
oocytes developed into a living calf following embryo transfer.
Fertility of the offspring was confirmed. In conclusion, the
potential of a proportion of the IVG oocytes was comparable to
that of in vivo-grown oocytes.
Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a
unique cell population that is able to produce offspring. In
particular, promising oocytes are of an inestimable value in the
field of fertility treatment and preservation [1]. An immediate
potential source of oocytes is ovarian follicles, which are
continuously growing in the ovary. Culturing such oocytes to
their full size requires a straightforward approach, and several
culture systems have been developed for that purpose, as
reviewed in reference [2]. The one requirement is that oocyte
growth should be completed so that the resulting embryos can
develop to term and the offspring can develop to fertile adults.
In large mammals, the fulfillment of this requirement is
extremely challenging and time consuming.
The oocyte is characterized by cytoplasm (ooplasm) that is
specialized in quantity and quality, which is necessary for
events until zygotic transcription begins to control embryogenesis [3]. Oocyte growth in mammals takes several weeks or
even months. In a large nucleus called the germinal vesicle
(GV), the molecules are progressively expressed that provide
the structural and functional bases for the oocyte [4]. One of
the best characterized processes is the acquisition of meiotic
competence. Meiotic competence is gradually acquired when
oocytes reach 80%–90% of their maximum diameter (mice [5];
pig [6, 7]; cattle [8]) as a result of the expression and
accumulation of specific kinases and phosphatases (mice [9–
13]; pig [7, 14–16]; goat [17]). Developmental competence is
acquired subsequently, although there appears to be a slight
overlap in mice in the timing of acquisitions of meiotic
competence to the progression to metaphase II and the
developmental competence to the blastocyst stage [18]. In
contrast, the completion of growth does not ensure that bovine
oocytes become viable embryos with the competence to
develop to term; rather, adequate oocyte chromatin status,
which is achieved during final follicular growth, has been
suggested to represent the readiness of the oocytes [19].
However, most follicles and oocytes degenerate without
growing to mature stages in the ovary. Oocyte-specific
genomic imprinting in growing oocytes has also been indicated
in mice [20, 21] and cattle [22].
The first successful culture was achieved in the work of
Eppig and Schroeder [23], who obtained mouse offspring from
the oocyte–granulosa cell complexes prepared from preantral
follicles. Later, the team developed oocytes from primordial
follicles [24] by using the tandem combination of ovarian
organ culture and isolated complex culture. The culture system
for the oocyte–granulosa cell complexes has been modified and
applied to bovine oocytes around the late mid-growth phase
cattle, cell culture, oocyte development, oocyte growth,
reprogramming, somatic cell nuclear transfer
1
Supported by grants from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, 22380151 (Y.H.) and the National
Agriculture and Food Research Organization (NARO), Japan (Y.H.,
M.K., M.S., T.K.). Presented in part at the 2nd World Congress on
Reproductive Biology, 9–11 October, 2011, Cairns, Queensland,
Australia.
2
Correspondence: Yuji Hirao, Animal Breeding and Reproduction Research
Division, NARO Institute of Livestock and Grassland Science, 2 Ikenodai,
Tsukuba, Ibaraki 305-0901, Japan. E-mail: [email protected]
3
Current address: Division of Animal Life Science, Tokyo University of
Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.
4
Current address: NARO Kyusyu Okinawa Agricultural Research
Center, Koshi, Kumamoto 861-1192, Japan.
Received: 18 March 2013.
First decision: 9 April 2013.
Accepted: 12 July 2013.
Ó 2013 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
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ABSTRACT
HIRAO ET AL.
[25]. A bovine oocyte has a size (125 lm) that is comparable
with those of human [26] and pig oocytes [6]. Technically,
oocytes that are 100 lm in diameter are approximately 50% of
their maximum volume. They are small and cannot mature
[25]. With the use of the bovine 2-week culture system, a
proportion of oocytes could develop the capabilities of meiotic
maturation, fertilization, and embryonic development [25].
However, competence of the oocytes grown in vitro still has
not matched that of oocytes grown in vivo. Bovine oocytes
provide a unique opportunity to refine the technology, and the
use of bovine oocytes as a model system is potentially
applicable to other large mammal species in which fully grown
oocytes may be difficult to obtain.
In addition, a large amount of research now indicates that
oocytes can possess the uncanny ability to reprogram
transferred somatic cell nuclei [27]. Despite remarkable
advances in nuclear transfer (NT) technology [28–30], animal
cloning continues to be difficult, mainly because of insufficient
reprogramming of the transferred nucleus [27]. The preparation
of highly qualified cytoplasts is an essential task undertaken for
successful cloning. In mice, ovulated ova are used exclusively
as the source of cytoplasts to create live offspring from NT
embryos [31]. To the best of our knowledge, no mice offspring
have yet been produced from NT embryos prepared from in
vitro-matured (IVM) oocytes, despite there being efficient
culture systems for mouse oocyte maturation. However, in
cattle and pigs, IVM oocytes as well as ovulated ova have been
used to prepare cytoplasts, and many of the offspring have been
obtained from the resulting NT embryos (reviewed in reference
[27]), although with reduced developmental frequencies
compared with those from ovulated ova [32]. A question that
remains is whether in vitro-grown (IVG) oocytes can make
recipient cytoplasts that are competent in supporting the
development of NT embryos. However, this question is more
difficult to answer than it appears because the investigation has
been restricted to mice and cattle, in which, so far, the offspring
have been obtained from IVG oocytes (cultured from the state
of meiotic incompetence) even through in vitro fertilization [2,
25, 33]. Of these species, IVG oocytes in mice are apparently
incompatible with NT technology, considering that even IVM
oocytes have not been used for this purpose. Thus, only bovine
IVG oocytes at this stage can provide an opportunity to
examine their quality and potential as the recipient cytoplasts.
Therefore, this study was undertaken to test the hypothesis that
bovine IVG oocytes produce recipient cytoplasts with
sufficient competence to support the complete development
of NT embryos. To this end, we adapted and improved a
method of producing IVG oocytes that has been described
previously [25], and aimed to establish a sequential system that
incorporated in vitro growth and maturation of bovine oocytes
to produce oocytes that were as capable as oocytes grown in
vivo.
cell complexes isolated from antral follicles 3–6 mm in diameter were used as
the in vivo-grown control oocytes. Details of the collection procedure are
described in the Supplemental Materials and Methods (all Supplemental Data
are available online at www.biolreprod.org). Only healthy looking oocytes that
were 90–106 lm in diameter (1227 oocytes in 35 experiments) were introduced
to the culture.
In Vitro Growth of Oocytes
In Vitro Maturation of Oocytes
Oocyte maturation was induced by maintaining meiotic arrest (16 h) and
facilitating oocyte maturation (20 h). The basal medium was a modified TCM199 solution with essentially the same composition as that used for oocyte
growth, except that it was free of PVP and the StemPro nutrient supplement.
For the arresting period (16 h), the basal medium was supplemented with 12.5
lM (s)-roscovitine (Enzo Life Sciences, Inc., Plymouth Meeting, PA), an
inhibitor of cyclin-dependent kinases [37], and 100 lM milrinone, an inhibitor
of phosphodiesterase [38, 39]. After the arresting period, the oocyte–cumulus
cell complexes were washed five times to remove the inhibitors and cultured for
20 h in the basal medium to facilitate oocyte maturation. The IVG oocytes and
in vivo-grown oocytes that extruded the first polar body are referred to as the
IVG-IVM oocytes and in vivo-IVM oocytes, respectively. More details about
culture conditions are provided in Supplemental Materials and Methods.
MATERIALS AND METHODS
Chemicals
Oocyte Enucleation
Unless otherwise specified, chemicals were purchased from Sigma-Aldrich
Corporation (St. Louis, MO). The concentration of supplemental fluid in the
medium was expressed as a volume/volume percentage.
Oocyte enucleation and reconstruction with the donor cell was performed as
described previously [40] in TCM-199 medium containing 20 mM HEPES, 5
mM sodium bicarbonate, 1 mM sodium pyruvate, and 0.08 mg/ml kanamycin
sulfate (hereafter referred to as NT medium). NT medium was used with
supplementations suitable for each procedure as described in Supplemental
Materials and Methods; the final medium was called NT medium ( þ
supplemented substance). Enucleation was performed in NT medium ( þ 20%
FBS and 2.5 lg/ml cytochalasin D). Successfully enucleated oocytes were
washed five times with NT medium ( þ 10% FBS and 50 lM 2mercaptoethanol) prior to coupling with the donor cell.
Collection of Oocyte–Granulosa Cell Complexes
From a total of 310 ovaries collected from Japanese Black or F1 (Japanese
Black 3 Holstein) cows at an abattoir, follicles 0.5–0.8 mm in diameter were
dissected and placed in minimum essential medium (MEM; pH 7.2, Nissui
Pharmaceutical Co., Ltd., Tokyo, Japan). Oocyte–granulosa cell complexes
were collected from the follicles as described previously [25]. Oocyte–cumulus
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The general experimental design is shown in Supplemental Figure S1. The
culture was started on Day 0 and continued until Day 14. All cultures were
maintained at 38.58C in incubators. The medium used for oocyte growth was a
modified TCM-199 solution described previously [34] with modifications.
Briefly, TCM-199 was supplemented with 1 mM sodium pyruvate, 40 mg/ml
polyvinylpyrrolidone (PVP; molecular weight [MW], 360 000), 0.08 mg/ml
kanamycin sulfate, 0.3 mM cysteine, 1 lM vitamin B12, 0.4 lM biotin, 150 lM
ascorbic acid 2-glucoside (AA2G; Hayashibara Co., Ltd., Okayama, Japan),
5% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Logan, UT), 0.685
mM GlutaMAX (Life Technologies, Carlsbad, CA), 1% MEM vitamin mixture
(Life Technologies), and 0.5% StemPro nutrient supplements (Life Technologies). The medium was also supplemented with 20 ng/ml androstenedione
[35] and 2 mM hypoxanthine [36]. In some initial experiments, the effects on
promotion of oocyte growth with the supplementation of bone morphogenetic
protein 7 (BMP7; R&D Systems, Inc., Minneapolis, MN) in culture medium
were examined.
Unless specifically stated otherwise, the oocyte–granulosa cell complexes
were placed in groups on Millicell inserts (30-mm diameter, 0.4-lm pore size,
product no. PICMORG50; EMD Millipore, Billerica, MA), which were set in
Petri dishes (Falcon no. 351007; Becton Dickinson and Co., Franklin Lakes,
NJ). In total, 1 and 5 ml of medium were placed on the membrane (top) and in
the dish (bottom), respectively. In some experiments, complexes were also
cultured on Biocoat cell culture inserts (product no. 35442; Becton Dickinson),
which were coated with collagen I, and that fit in a 6-well culture plate. The
culture atmosphere was 5% O2, 5% CO2, and 90% N2 from Day 0 to Day 3 and
5% CO2 in air from Day 3 to Day 14 [34]. Half the medium was replaced with
fresh medium on Days 3, 6, 9, and 12.
Oocyte diameters were remeasured on Day 1 to exclude the possibility that
oocytes that were out of the desired range (90–106 lm) or those showing
degenerated morphology. In total, 92.4% of the oocytes (1134/1227) met the
requirements (mean 6 SD of 16.2 6 4.5 oocytes per insert). After the 14-day
culture period, the oocyte–cumulus cell complexes were extracted from the
complexes with a fine pipette. Oocytes were considered live when they showed
no signs of degeneration, such as insubstantial cytoplasm or membrane
disruption, and were covered with cumulus cells over at least half of their
surface. Oocyte/embryos were manipulated on a plate set at 38.08C, except for
the procedures performed at room temperature (i.e., donor cell–cytoplast fusion
and calcium ionophore treatment).
FERTILITY OF BOVINE OOCYTES AFTER GROWTH IN VITRO
Preparation of Donor Cells
Total RNA Extraction from Oocytes and Microarray
Analyses
Cumulus cells obtained after maturation of the control oocytecumulus cell
complexes were used as nuclei donors [41, 42]. The cumulus cells dispersed
with the help of hyaluronidase were cultured to confluency as described
previously [40]. Details about preparation of donor cells are provided in
Supplemental Materials and Methods.
From RNA extraction to microarray analyses, a system optimized for small
quantities of total RNA from samples [49] was used. Fragmented cRNA
samples (10 lg) were hybridized to the GeneChip bovine genome array
(Affymetrix, Inc., Santa Clara, CA) for 12 oocytes: the IVG-IVM oocytes (n ¼
6) and in vivo-IVM oocytes (n ¼ 6). The oocytes were sampled individually in
order to examine whether there was possible (extensive) heterogeneity in the
oocyte population after growth in vitro. Details of RNA extraction and array
analyses are shown in Supplemental Materials and Methods.
Donor Cell-Cytoplast Fusion and Activation
The donor cell was inserted into the perivitelline space in NT medium ( þ
20% FBS, 2.5 lg/ml cytochalasin D, 50 lg/ml phytohemagglutinin, and 50 lM
2-mercaptoethanol), and the couples were centrifuged for 1 min at 390 3 g
before being transferred into Zimmermann fusion medium [43]. The fusion
medium was prepared by excluding the calcium chloride in the original
composition to avoid the simultaneous occurrence of fusion and parthenogenetic activation [44]; the composition of the medium was 280 mM sucrose, 0.5
mM magnesium acetate tetrahydrate, 1 mM potassium phosphate dibasic, 0.1
mM glutathione, and 3 mg/ml bovine serum albumin. A single direct-current
pulse of 30 V was applied to the couples for 10 lsec, using an electrocell-fusion
generator (LF101; Nepa Gene Co., Ltd., Chiba, Japan). The couples were then
incubated in modified calcium-free MEM. The fusion took place within 10–30
min in most cases. Only couples that fused within 1 h were cultured for 3 h in
NT medium ( þ10% FBS) until chemical activation treatment. This and all
subsequent cultures were conducted in an atmosphere of 5% O2, 5% CO2, and
90% N2. The reconstructed oocytes were then exposed to 5 lM calcium
ionophore A23187 for 5 min at room temperature [45] in NT medium ( þ 4 mg/
ml PVP [MW, 40 000]), in conjunction with subsequent 5-h treatment with 10
lg/ml cycloheximide and 2.5 lg/ml cytochalasin D [45] in NT medium ( þ 3
mg/ml bovine serum albumin). The NT oocytes (or embryos) from the IVGIVM oocytes are referred to as the IVG-NT oocytes or IVG-NT embryos,
depending on the developmental stages. Likewise, NT oocytes (or embryos)
from the in vivo-IVM oocytes are referred to as the in vivo-NT oocytes or in
vivo-NT embryos.
Oocyte Fixation and Examination
Two types of oocyte fixation methods were used according to the purposes
of this study: for nuclear status examinations, acetic alcohol was used on the
oocytes mounted on glass slides, which were then stained with orcein. For
immunofluorescence microscopy observations, oocytes/reconstructed embryos
were fixed with a microtubule stabilization buffer [50] as described by Ju et al.
[51] with modification. Morphologies of DNA, microtubules, and microfilaments were evaluated according to the method described by Somfai et al. [52].
Details of the method are described in Supplemental Materials and Methods.
Data Presentation and Statistical Analyses
RESULTS
Embryo Culture
Oocyte Survival and Growth In Vitro
After being treated with cycloheximide, the reconstructed oocytes were
cultured with a synthetic oviduct fluid medium (SOFaaci), previously described
by Holm et al. [46] with modification. During the first 4 days, the embryos
were cultured in microdrops of serum-free medium (2 ll/embryo) overlaid with
paraffin oil. Following this, half of the medium was replaced with fresh
medium containing 10% FBS. Development to the blastocyst stage was
determined 8 days post-NT. Blastocysts were either transferred to foster
mothers or vitrified and preserved with a Cryotop device (Kitazato BioPharma
Co., Ltd., Shizuoka, Japan) for later use or sampled for genomic DNA
methylation analysis. When appropriate, the blastocysts were subjected to one
of the treatments before 8 days.
Figure 1A shows the early antral follicles (0.5–0.8 mm in
diameter) from which the oocyte–granulosa cell complexes
were collected. The morphological appearance of the complex
after overnight culture is shown in Figure 1B. Oocytes at this
stage (Day 1) were incompetent to resume meiosis; 98.2% 6
3.6% of the oocytes (n ¼ 67, 100.2 6 2.6 lm in diameter)
remained in the GV stage after 24-h culture with folliclestimulating hormone (FSH), luteinizing hormone (LH), and
epidermal growth factor (EGF) (five experiments). One
exceptional oocyte ended up in the diakinesis stage.
Two series of experiments were preliminarily conducted in
order to determine the detailed culture conditions that
maximized the oocyte volume after 14 days of culture. In the
first series, the effects of two kinds of cell culture membrane
inserts were compared: Biocoat (BD) inserts with collagencoated membranes, which were used in our previous study
[25], and Millicell inserts for organ culture. Both of the
membrane inserts allowed the complexes to form a welldeveloped dome-like structure (Fig. 1, C and D). However, a
distinct difference was found in terms of the degree of
granulosa cell migration and proliferation over to the marginal
areas between the domes; vigorous cell migration and
proliferation was observed on Biocoat inserts (Fig. 1E) but
not on Millicell inserts (Fig. 1F). No appreciable differences
were observed between the two groups in regard to the
morphology of the oocyte–cumulus cell complexes that were
recovered from the domes. However, the volume of the oocytes
on Millicell inserts was significantly larger than those on
Biocoat inserts (P , 0.01) (Fig. 2A). In the second series of
experiments, we examined the effects of BMP7 supplementation (0–50 ng/ml) on the growth of the oocytes cultured for 14
days. As shown in Figure 2B, 5 and 50 ng/ml BMP7 resulted in
a positive effect on oocyte growth (P , 0.05). Based on these
Embryo Transfer
All animal care and embryo transfers were approved by the Committee on
Animal Experimentation of the Tohoku Agricultural Research Center and
conducted according to its guidelines. Recipient cows were synchronized for
estrus with the insertion of a controlled intravaginal drug-releasing device
(EAZI-Breed; InterAg, Hamilton, New Zealand) for administration of 0.5 mg of
cloprostenol, a prostaglandin F2a analog (Estrumate; Nagase Medicals Co.,
Ltd., Itami, Japan) for 4–8 days. Six fresh blastocysts and three vitrifiedwarmed blastocysts were nonsurgically transferred into nine recipient cows.
Genomic DNA Methylation Analysis
Genomic DNA was prepared from the oocytes (collected on Days 0 and
14), in vivo-grown oocytes, blastocysts, and cumulus cells. Twenty oocytes that
were denuded completely were pooled for each sample. The blastocysts were
individually sampled, and cumulus cells were collected from the remaining
donor cell populations prepared for NT. All the cells were washed with PBS
containing 1 mg/ml polyvinyl alcohol prior to digestion in DNA lysis buffer
[21]. Genomic DNA was extracted with standard procedures, and the bisulfite
treatment of DNA was performed as described previously [21]. Regions of
interest, including the satellite-I, satellite-II, a-satellite, and art2 regions, were
amplified by polymerase chain reactions. The primers used were previously
described by Kang et al. [47, 48]. Primer sequences are listed in Supplemental
Table S1.
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Unless specified, the results of percentage data are given as mean
percentages of at least three independent experimental replicates; the variations
between experiments are described by SD. For statistical analyses of the
percentages, the data were transformed with arcsine transformation. Comparisons between two groups were conducted using Welch t-tests. For multiple
comparisons, Tukey tests were used. To compare with the control, Dunnett test
was applied. For nonparametric multiple comparisons, Steel-Dwass tests were
used. P values ,0.05 were considered significant.
HIRAO ET AL.
FIG. 1. Morphology of the follicles that were dissected from the ovaries
and the oocyte–granulosa cell complexes in vitro. A) Early antral follicles,
0.5–0.8 mm in diameter, are shown in comparison with larger follicles (a,
2 mm; b, 3 mm). B) Oocyte–granulosa cell complex that was placed on a
Millicell insert and cultured for a day. oo, oocyte; gc, granulosa cells. C–
F) Oocyte–granulosa cell complexes cultured for 14 days. Complexes
cultured on a Biocoat membrane insert (C) and a Millicell membrane
insert (D). E and F) Enlarged images of the complexes marked by
rectangles in C and D, respectively. E) Arrowheads indicate a cell-dense
area between the domes. Bars ¼ 2 mm (A), 100 lm (B), 1 mm (C and D),
and 500 lm (E and F).
results, it was decided that Millicell inserts and 5 ng/ml BMP7
would be used in the culture system for further study.
In 35 replicated experiments, 95.0% 6 5.9% of the oocytes
(1075/1134) were recovered alive after 14 days. Most of the
oocyte–granulosa cell complexes developed into dome-like
structures between Days 6 and 14 (Supplemental Fig. S2, A
FIG. 2. Comparison of the volumes of oocytes cultured for 14 days in the oocyte–granulosa cell complexes isolated from early antral follicles. A)
Complexes were cultured on either Biocoat inserts or Millicell inserts; total numbers of oocytes were 40 and 44, respectively. Data are pooled from three
experiments. *Significant differences (P , 0.01; Welch t-test). B) Effects of BMP7 (0, 0.5, 5, and 50 ng/ml) on oocyte growth; total numbers of oocytes were
38, 37, 32, and 33, respectively. Data are pooled from three experiments. Numbers above the box plots indicate the mean diameter (lm) of the oocytes in
that group. *Significant differences (P , 0.05; Dunnett test).
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FIG. 3. Morphology of the oocyte–granulosa cell complexes cultured for
14 days. A) An intact dome of granulosa cells on Day 14. Bar ¼ 500 lm. B)
Dome after mechanical removal of the oocyte–cumulus cell complex
(arrow) using a glass pipette; arrowhead points to the hole left in the
dome. C–E) Confocal microscopy images of an oocyte–cumulus cell
complex fixed on Day 14. C) Microfilaments are visualized by Alexa Fluor
488 phalloidin. oc, oocyte cytoplasm; zp, zona pellucida; cc, cumulus
cells. Bar ¼ 50 lm. D) Hoechst 33342 staining of DNA; arrow points to
one of the nuclei of cumulus cells arranged along with the oocyte. E)
Merged image. Note the numerous transzonal projections traversing the
zona pellucida.
FERTILITY OF BOVINE OOCYTES AFTER GROWTH IN VITRO
FIG. 5. Morphologies of IVG oocytes and cumulus cells during and after
in vitro maturation. A) Oocytes with compact cumulus cell mass after
recovery from the membrane. The complexes slightly loosened (arrowheads) after 16 h (B) of the arresting period and 20 h (C) after transfer to the
maturation medium without inhibitors. The morphology of expanded
cumulus cells (ec) was similar to that of control oocytes (D) grown in vivo
and matured in vitro. Bar ¼ 500 lm. E and F) Confocal images of
metaphase-II oocytes visualized by the combined staining of DNA (blue),
microtubules (green), and microfilaments (red): IVG-IVM oocyte (E) and
in vivo-IVM oocyte (F). Metaphase chromosomes and spindles are shown
with arrows. E) Arrowheads indicate abnormal filamentous actin clusters.
and B). Figure 3, A and B, shows the morphologies of a
complex before and after the mechanical removal of the
oocyte–cumulus cell complex, respectively. Confocal microscopic examination of the recovered complexes revealed
numerous transzonal projections traversing the zona pellucida
(Fig. 3C) from cumulus cells to the oocyte (Fig. 3, D and E).
The oocytes recovered on Day 14 were in the GV stage (n ¼
37, 3 experiments).
The mean volume of the oocytes increased significantly
from approximately 522 600 lm3 (99.8-lm diameter) to
892 100 lm3 (119.2-lm diameter) from Day 1 to Day 14 (P
, 0.01) (Fig. 4A). This was a 71% increase, but the IVG
oocytes did not achieve the volume of the in vivo-grown
oocytes (Fig. 4A). The relative volumes of the oocytes on Days
1 and 14 were 48% and 82%, respectively, compared with that
of the oocytes grown in vivo (Fig. 4A).
cortical microfilaments in the IVG-IVM oocytes (n ¼ 21).
However, as shown in Figure 5, E and F, abnormal filamentous
actin clusters in the cortical area were observed more
frequently in the IVG-IVM oocytes (62%) than in the in
vivo-IVM oocytes (9%).
In Vitro Maturation of IVG Oocytes
Oocyte Enucleation and Reconstruction
During the first 16 h, nuclear progression was transiently
inhibited by a combination of roscovitine and milrinone
(Supplemental Materials and Methods: In Vitro Maturation
of Oocytes). The cumulus cell mass, which was compact when
collected from the domes (Fig. 5A), became slightly loose after
the arrest (Fig. 5B). In the following 20 h of culture (Fig. 5C),
cumulus expansion reached a level comparable with that found
around the oocytes grown in vivo (Fig. 5D). The kinetics of
meiotic arrest and the maturation of the IVG oocytes are
described briefly in Supplemental Materials and Methods.
The first polar body was extruded in 61.1% 6 12.2% of the
IVG oocytes (n ¼ 830, 29 experiments). The volume of these
IVG-IVM oocytes was 81% compared with that of the in vivoIVM oocytes (Fig. 4B). Confocal image analyses indicated the
formation of microtubule spindles and the organization of the
Enucleation was successful in 60%–70% of the oocytes
(Fig. 6A). Reductions in the cytoplasm volume caused by
enucleation were 21% and 14% for the IVG-IVM oocytes and
in vivo-IVM oocytes, respectively. The resulting volume of the
IVG-IVM cytoplasts was 74% the volume of the in vivo-IVM
cytoplasts (Fig. 4C).
Compared with the couples before centrifugation (Fig. 6, B
and C), centrifuged cytoplasts were characterized by cytoplasmic lipids on one side of a semisphere (Fig. 6, D–G). The
intimate contact between the couple was improved by
centrifugation (Fig. 6, F and G), as suggested by the high
fusion rate, which was approximately 95% (Fig. 6A). The rate
was 73% 6 14% when couples were fused without
centrifugation (n ¼ 53, five preliminary experiments).
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FIG. 4. Box plots indicate the volume of oocytes or ooplasm at various
time points from in vitro growth to enucleation. A) Immature oocytes; B)
mature oocytes (with the first polar body); and C) cytoplasts (enucleated
oocytes). Numbers above the box plots indicate the mean diameter (lm)
of the oocytes in that group. A) Total numbers of oocytes on Days 1 and 14
of culture and those grown in vivo were 1134, 1075, and 347,
respectively. B) IVG, in vivo, oocytes only matured in vitro; total oocytes
were 77 and 78, respectively. C) IVG and in vivo represent the values after
enucleation of the IVG-IVM oocytes and in vivo-IVM oocytes, respectively; total cytoplasts were 43 and 48, respectively. a–cDifferent letters
indicate significant differences (P , 0.01, Steel-Dwass test). B and C)
*Significant differences (P , 0.01, Welch t-test). Volume calculation was
based on the oocyte diameter measured with an ocular micrometer at
3150 magnification (A) or photographs taken at 3200 magnification (B
and C). Relative volumes of the oocytes and cytoplasts are also indicated,
with the mean value of in vivo-grown oocytes shown in A designated
100%.
HIRAO ET AL.
FIG. 6. Oocyte enucleation and fusion with donor cells. A) Percentage of
successful enucleation and fusion of oocytes. Enucleation: total numbers
of IVG-IVM oocytes and in vivo-IVM oocytes were 424 (22 experiments)
and 258 (12 experiments), respectively. Fusion: total numbers of IVG-IVM
oocytes and in vivo-IVM oocytes were 224 (17 experiments) and 160 (12
experiments), respectively. Data are means 6 SD. B–G) Morphologies of
the donor cell–cytoplast couples before and after centrifugation. B, D, and
F) Couples were made of IVG-IVM cytoplasts; (C, E, and G) couples were
made of in vivo-IVM cytoplasts. B and C) Before centrifugation. D–G)
After centrifugation at 390 3 g for 1 minute. F and G) Enlarged images of
the couples shown in the rectangles in D and E, respectively; arrows point
to the donor cell attached on the cytoplast surface. Note the cytoplasmic
lipids deflected to one side. Bars ¼ 200 lm (B–E) and 50 lm (F and G).
ionophore and cycloheximide) (Fig. 7E). In both groups, more
than 60% of the embryos developed to the blastocyst stage
after cleavage (Fig. 7, C–E). The cleavage of the IVG-NT
embryos was very rare after cycloheximide treatment alone,
whereas the same treatment led to the cleavage of two-thirds of
the in vivo-NT embryos (Fig. 7E).
DNA Methylation Profiles
Preimplantation Development of IVG-NT Embryos
Figure 8A shows the typical DNA methylation patterns of
the satellite-I region of the GV stage oocytes on Days 0 and 14
and those grown in vivo. Methylation levels were approximately 50% in all groups. Figure 8B further indicates that in
three (satellite-I, satellite-II, and art2) of the four regions,
methylation levels did not differ significantly. Only the asatellite region exhibited lower levels in the growing oocytes
than in the oocytes grown in vivo; however, the difference was
not significant in IVG oocytes.
Details of the nuclei and cytoskeleton of the IVG-NT
oocytes are described in Supplemental Tables S2 and S3.
Figure 7, A and B, shows the cleaved embryos on the next day
of activation. The cytoplasm of the IVG-NT embryos was
partly transparent (Fig. 7A) compared with the dense-looking
cytoplasm of the in vivo-NT embryos (Fig. 7B). The cleavage
rates of the IVG-NT embryos and in vivo-NT embryos were
both high after the combined activation treatment (calcium
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FIG. 7. In vitro preimplantation development of NT embryos. A and C)
IVG-NT embryos and (B and D) in vivo-NT embryos. A and B) Cleaved
embryos on the day after calcium ionophore-cycloheximide treatment.
Note that the overlapping cells (arrows) are visible in IVG-NT embryos
and the lipid-rich impenetrable cytoplasm (arrowheads) of in vivo-NT
embryos. C and D) Blastocysts developed from these embryos are shown
in A and B, respectively. All bars ¼ 100 lm. E) Percentage of embryos that
cleaved and developed to the blastocyst stage. Data from six experiments
are means 6 SD. Values with different superscripts (a, b) in the same
frame differ significantly (P , 0.05, Tukey test). The column with
superscript c represents one blastocyst of two cleaved embryos; this value
was excluded from statistical analysis. Total numbers of fused oocytes
included were 58, 52, 47, and 47 (from the left column to the right).
FERTILITY OF BOVINE OOCYTES AFTER GROWTH IN VITRO
FIG. 9. The DNA methylation profile of the satellite-I and satellite-II
regions of blastocysts produced by either NT or in vitro fertilization. A)
Digestion by Aci I (satellite I) or AccII (satellite II). B) Examples of
methylation profiles of the satellite-I region in the blastocysts of four
different origins. C) Methylation levels in NT blastocysts and in vitro
fertilization blastocysts derived either from IVG oocytes or in vivo-grown
oocytes. Total numbers of blastocysts included were 23, 17, 10, and 15
(from the left column to the right). Data are means 6 SD. a,bDifferent
superscripts indicate significant differences (P , 0.05, Tukey test).
The electrophoresis patterns shown in Figure 9A suggest no
apparent differences between the IVG-NT embryos and in
vivo-NT embryos in terms of DNA methylation levels of the
satellite-I and -II regions. Hypermethylation status was obvious
in both NT embryos (Fig. 9B). The differences between the
groups of NT embryos and those derived from in vitro
fertilization were significant (Fig. 9C). The methylation level
of the donor (cumulus) cells was approximately 57% (Fig. 9C).
IVM oocytes were spread out along the X-axis. The oocytes of
both these groups contributed to the variation associated with
the Y-axis.
Figure 10C shows a hierarchical clustering dendrogram of
the oocytes. One of the IVG-IVM oocyte groups (Fig. 10D)
and one of the in vivo-IVM oocyte groups (Fig. 10B) were
more closely related to each other than to the other IVG-IVM
oocyte group (Fig. 10C). The other in vivo-IVM oocytes (Fig.
10A) had a greater clustering distance than the other three
groups.
Gene Transcript Profiles of Metaphase II Oocytes
Figure 10A shows a similarity matrix that was constructed
with the Pearson correlation coefficients for the pair-wise
comparisons of the 12 oocytes. According to the similarities,
data from the in vivo-IVM oocytes and IVG-IVM oocytes were
named vivo-1 to vivo-6 and vitro-1 to vitro-6, respectively. In
the in vivo-IVM oocyte groups, intense correlations were
found between the pairs of vivo-1, vivo-2, and vivo-3 and the
pairs of vivo-4, vivo-5, and vivo-6; these groups of three
oocytes were classified as B and A, respectively. Similarly, the
IVG-IVM oocytes were divided into two groups: two oocytes
(vitro-1 and vitro-2), and the other four oocytes were grouped
as C and D, respectively.
The transcript profiles were analyzed with principal
component analysis (PCA). Figure 10B is representative of
the first three components (X-axis ¼ 19.2%, Y-axis ¼ 13.6%,
Z-axis ¼ 10.9%). It appeared that the IVG-IVM oocytes were
located along the Z-axis but made a minor contribution to the
variation associated with the X-axis. However, the in vivo-
Birth of the Calf and Growth to a Fertile Adult
From one of the freshly transferred embryos, a female calf
weighing 45 kg was delivered (Day 279) by induced
parturition. The calf was born without any obvious abnormalities, except for a high birth weight. Genetic identities of the
calf (Japanese Black) and cumulus cells used as the donor were
confirmed. At 12 months of age (Supplemental Fig. S3), the
first ovulation occurred. After artificial insemination was
conducted 576 days after birth, its pregnancy was confirmed,
which resulted in the birth of a female calf (Japanese Black)
with a normal weight of 27 kg (Day 289).
7
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FIG. 8. DNA methylation profile at satellite regions of the three types of
GV stage oocytes before and after growth. A) Examples of bisulfite
methylation studies of the satellite-I region. Open circles, unmethylated
cytosines; filled circles, methylated cytosines. B) Methylation levels of the
various regions in the oocytes. Data are means 6 SD of four independent
experiments. a,bDifferent superscripts indicate significant differences (P ,
0.05, Tukey test).
HIRAO ET AL.
DISCUSSION
FIG. 10. Analysis of accumulated gene transcript profiles of metaphase-II
oocytes after in vitro or in vivo growth. Data were based on 9661 probe
sets. A) Similarity matrix of gene transcript profiles for each pairwise
comparison of individual IVG-IVM oocytes (vitro) and in vivo-IVM
oocytes (vivo). B) PCA analysis: three-dimensional map comparing
individual IVG-IVM oocytes and in vivo-IVM oocytes. The first, second,
and third principal components are represented by X, Y, and Z axes,
respectively. C) Condition tree constructed by hierarchical clustering
analysis using a Pearson centered distance metric with a centroid linkage
rule. Red, yellow, and green represent high, median, and low expression,
respectively.
8
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Here, we described the production of highly competent IVG
oocytes and the first cloned animal from a cytoplast that was
prepared from the IVG oocytes. Our primary findings are that
1) IVG-IVM oocytes were fully potent and withstood
manipulations, such as NT; 2) approximately half of the
embryos that were reconstructed with the IVG-IVM ooplasm
and donor cumulus cells developed into blastocysts; and 3)
restoration of a new totipotent state of the transferred nucleus
occurred in the IVG-IVM ooplasm.
In our previous bovine study, several medium supplements
were found to be beneficial for sustaining oocyte growth (see
reference [53] for review). Of these, PVP [25], androstenedione
(or estradiol) [35], and hypoxanthine [36] were critically
influential. In particular, steroid hormone supplementation was
essential to compensate for the absence of products from theca
cells [35]. Under appropriate conditions, oocytes and granulosa
cells collectively develop a bidirectional communication loop,
which stimulates various important events such as antral cavity
formation [54], the preparation of cumulus cell expansion [55],
and the acquisition of developmental competence by the
oocytes [56]. All these features were realized in the present
study, suggesting that bidirectional communication was
reestablished in vitro.
Considering the various activities of theca cells and their
factors that are involved in granulosa cell regulation [57], it is
unlikely that androstenedione supplementation alone could
fully substitute for the theca cell-derived factors, which may
potentially benefit cultured oocytes. Among such factors,
BMP7, which is expressed by theca cells [58] and which uses
ActRII of granulosa cells as a receptor [59], has been shown to
promote granulosa cell proliferation in rats [60] and reduce
cumulus cell apoptosis in cattle [61]. Although not as definite
as the influence of androstenedione which has been shown in a
previous study [35], the results of the present study suggested
that BMP7 treatment can increase the growth of the oocytes
that were cultured in the oocyte–granulosa cell complexes (Fig.
2B). The observed increase in oocyte size probably reflected
improved granulosa cell activities induced by BMP7 treatment.
This result represented another example of compensating, in
part, for the absence of theca cells by adding factors to the
culture medium. When some other potential theca cell-derived
factors that can benefit oocyte growth are characterized, the
same approach should be able to add size and perhaps quality
to the oocytes grown in vitro.
Another modification that was made to the culture system
from the previous study [25] was the use of Millicell inserts in
place of Biocoat inserts because Millicell inserts resulted in a
more positive effect on the growth of the oocytes than Biocoat
inserts. However, it should be noted that the characteristic
dome-like structures were developed by the oocyte–granulosa
cell complexes on both types of inserts. Within the complexes,
the oocytes were separated from the membrane inserts by
layers of granulosa cells. Thus, the reason for the difference
that emerged between the oocytes due to different inserts was
not certain. A manifest difference between the two groups was
the more active outgrowth of granulosa cells radiating away
from the complexes on Biocoat inserts than on Millicell inserts.
Because the collagen coating can facilitate the adherence of the
complexes, the different outcomes may be attributable to the
collagen coating on the membrane of Biocoat inserts. The
effects of the collagen-coated membranes, when combined
with the culture medium that was used in the present study,
may have been exaggerated because a proportion of the
complexes on Biocoat inserts had become almost flat within a
FERTILITY OF BOVINE OOCYTES AFTER GROWTH IN VITRO
to be smaller than the in vivo-grown oocytes. Second, a
proportion of the IVG oocytes exhibited abnormalities in the
cytoskeleton. Third, additional defined culture conditions, such
as the use of serum-free medium and further simplified
supplementation, are required. Nevertheless, the present study
indicated the IVG oocytes can be as capable as the in vivogrown oocytes, including in the ability to perform highly
specialized activities such as nuclear reprograming. These
findings have added significance and reliability to the IVG
oocytes and the technology that has been developed to create
the IVG oocytes.
day after seeding (Supplemental Fig. S4). This initial
deformation of the complexes might have adversely affected
the physical association between the oocytes and the
accompanying granulosa cells.
Only 5% of the oocytes degenerated during the 14-day
culture period. In the bovine ovary, approximately 14 days are
required for follicular growth to reach 8.56 mm in diameter
from 0.68 mm [62]. However, the vast majority of such
follicles have been shown to regress in vivo as subordinate
follicles during the final stages of follicular development [62,
63]. Thus, had the IVG oocytes in the present study remained
in the ovary, most of them would have degenerated. The IVG
oocytes survived because of the absence of the mechanisms
involved in follicular selection in the ovary. First, the oocyte–
granulosa cell complexes were nourished by culture medium
and not by the vascular network, because deprivation of the
vascular system is a major cause of oocyte death in the ovary
[64]. Second, the oocytes nearly exposed to culture medium
have a better chance of escaping detrimental influences than
the oocytes in the follicle that cannot escape when most
neighboring cells are dying. In fact, small groups of granulosa
cells were sufficient to reexpand the population around the
oocyte in the present study, probably because of the promotion
of mitosis [65] and prevention of apoptosis [61] in the
cumulus/granulosa cells by the factor(s) from oocytes.
The favorable frequency of preimplantation development by
the IVG-NT embryos may imply that only highly qualified
IVG-IVM oocytes had been selected through the manipulations
of NT. The birth of a calf further indicated the potential of the
IVG-IVM ooplasm. However, as for the DNA methylation
patterns in the satellite regions, the typical hypermethylation
profiles, which were consistently observed in the cloned
embryos [66], have now been extended to the IVG-NT
embryos. This observation seemed to be reasonable because
the IVG oocytes would, at best, recapitulate the problem if the
in vivo-grown oocytes had a problem.
DNA methylation profiles of the GV stage oocytes were
also analyzed. According to the study by O’Doherty et al. [22],
the methylation profiles of some maternal imprinting genes still
altered the change in bovine oocytes that .100 lm in diameter.
Interestingly, however, the genomic contents of 100-lm
oocytes can support fetal development to term when they are
combined with the appropriate ooplasm and paternal genome
[67]. Our observations of 100-lm oocytes suggested that the
methylation patterns had almost been established in three of the
four regions. The difference from the in vivo-grown oocytes,
however, became insignificant when the oocytes were grown in
vitro.
Microarray analyses suggested that a proportion of the IVGIVM oocytes had gene transcript profiles similar to those of the
in vivo-IVM oocytes, although the differences derived as a
result of different oocyte sources were also revealed. When a
PCA was performed, the first component indicated a large
variability between the in vivo-IVM oocytes and a limited
variability between IVG-IVM oocytes. In contrast, the third
component indicated variability between the IVG-IVM oocytes. The Z-axis could represent a component associated with
the stress caused by culture. Remarkably, the hierarchical
clustering analysis suggested that one group of the in vivoIVM oocytes resembled a group of the IVG-IVM oocytes more
than the other group of in vivo-IVM oocytes. This similarity
found in the gene transcript profiles was encouraging for the
use of technology to produce IVG oocytes in research and
reproductive biology.
Certainly, the culture system used in the present study
requires further improvement. First, the IVG oocytes continued
ACKNOWLEDGMENT
The authors thank Drs. J.J. Eppig, A. Ogura, F. Matsuda, Y. Obata, S.
Watanabe, and K. Sawai for their valuable suggestions. The authors thank
Dr. A.F. Parlow and the National Hormone and Pituitary Program of the
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
for providing FSH and LH.
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