Birth of Healthy Offspring following ICSI in In Vitro-Matured Common Marmoset (Callithrix jacchus) Oocytes
et al. (2014) Birth of Healthy Offspring following ICSI in In Vitro-Matured Common Marmoset
(Callithrix jacchus) Oocytes. PLoS ONE 9(4): e95560. doi:10.1371/journal.pone.0095560
Birth of Healthy Offspring following ICSI in In Vitro- Matured Common Marmoset (Callithrix jacchus ) Oocytes
Tsukasa Takahashi 0
Kisaburo Hanazawa 0
Takashi Inoue 0
Kenya Sato 0
Ayako Sedohara 0
Junko Okahara 0
Hiroshi Suemizu 0
Chie Yagihashi 0
Masafumi Yamamoto 0
Tomoo Eto 0
Yusuke Konno 0
Hideyuki Okano 0
Makoto Suematsu 0
Erika Sasaki 0
W. Steven Ward, University of Hawaii at Manoa, John A. Burns School of Medicine, United States of America
0 1 Department of Applied Developmental Biology, Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki, Kanagawa, Japan, 2 Department of Biochemistry, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan, 3 Department of Oncology, Juntendo University Nerima Hospital, Nerima-ku, Tokyo, Japan, 4 Marmoset Research Department, Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki, Kanagawa, Japan, 5 Department of Biomedical Research, Central Institute for Experimental Animals , Kawasaki-ku, Kawasaki, Kanagawa, Japan, 6 Altair Corporation, Kohoku-ku, Yokohama-shi, Kanagawa , Japan , 7 Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan, 8 Keio Advanced Research Center, Keio University , Shinjuku-ku, Tokyo , Japan
Intracytoplasmic sperm injection (ICSI), an important method used to treat male subfertility, is applied in the transgenic technology of sperm-mediated gene transfer. However, no study has described successful generation of offspring using ICSI in the common marmoset, a small non-human primate used as a model for biomedical translational research. In this study, we investigated blastocyst development and the subsequent live offspring stages of marmoset oocytes matured in vitro and fertilized by ICSI. To investigate the optimal timing of performing ICSI, corrected immature oocytes were matured in vitro and ICSI was performed at various time points (1-2 h, 2-4 h, 4-6 h, 6-8 h, and 8-10 h after extrusion of the first polar body (PB)). Matured oocytes were then divided randomly into two groups: one was used for in vitro fertilization (IVF) and the other for ICSI. To investigate in vivo development of embryos followed by ICSI, 6-cell- to 8-cell-stage embryos and blastocysts were nonsurgically transferred into recipient marmosets. Although no significant differences were observed in the fertilization rate of blastocysts among ICSI timing after the first PB extrusion, the blastocyst rate at 1-2 h was lowest among groups at 2-4 h, 4-6 h, 6-8 h, and 8-10 h. Comparing ICSI to IVF, the fertilization rates obtained in ICSI were higher than in IVF (p.0.05). No significant difference was noted in the cleaved blastocyst rate between ICSI and IVF. Following the transfer of 37 ICSI blastocysts, 4 of 20 recipients became pregnant, while with the transfer of 21 6-cell- to 8-cell-stage ICSI embryos, 3 of 8 recipients became pregnant. Four healthy offspring were produced and grew normally. These are the first marmoset offspring produced by ICSI, making it an effective fertilization method for marmosets.
Funding: A part of this study was the result of Highly Creative Animal Model Development for Brain Sciences and Construction of System for Spread of
Primate Model Animals supported by the Strategic Research Program for Brain Science, Grant-in-Aid for Scientific Research A from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT; http://www.mext.go.jp/english/), Japan, PRESTO of the Japan Science and Technology Agency to E.S., Funding
Program for World-Leading Innovative R&D on Science and Technology (FIRST) program Strategic Exploitation of Neuro-Genetics for Emergence of the Mind
from the Cabinet Office (http://www.brain.riken.jp/first-okano/en/index.html), government of Japan to E.S. and H.O., and the Global COE Program for Education
and Research Center for Metabolomic Systems Biology from the MEXT to M.S. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
The common marmoset (Callithrix jacchus) is a small New World
primate that has been used in biomedical research because of its
physiological similarity to humans, its small body size, and its
prolificacy. Marmosets show specific reproductive characteristics
including a relatively short gestation period (about 144 days),
reaching sexual maturity at 1218 months, and female marmosets
ovulate two or three oocytes in each ovarian cycle . Utilization
of this reproductive efficiency and the successful production of
transgenic marmosets with germline transmission has been
reported  and can be applied to genetically modified
nonhuman primate models in life sciences .
In vitro production techniques of preimplantation embryos
increase our understanding of the physiology of early embryonic
development and improve animal production. In marmosets,
in vitro fertilization (IVF) with fresh ejaculated sperm has achieved
over 50% fertilization rates . However, IVF requires
highquality and abundant sperm, which are difficult to obtain from
infertile males, and it often gives rise to polyspermic embryos. To
avoid these problems, intracytoplasmic sperm injection (ICSI)
could be applied as an inseminating technique in marmosets.
The ICSI procedure has improved assisted reproduction
technologies in rabbits , cattle [8,9], mice , rhesus
monkeys , and humans [12,13], providing opportunities to
investigate fundamental aspects of fertilization, such as the
mechanisms of gamete interaction and sperm-induced oocyte
Sequence of the labeled primer (5939)
Sequence of the non-labeled primer (5939)
activation. Furthermore, ICSI is a useful technique for efficient
animal production of genetically modified animals or infertile
male animals. This technology could be applied to transgenic
animals via intracytoplasmic sperm injection-mediated
transgenesis (ICSI-Tr) . ICSI-Tr can be used to insert very large DNA
fragments into the host genome. Currently, in non-human
primates, the generation of transgenic animals has been reported
using only lentiviral systems, which have limited insertional DNA
sizes. ICSI-Tr can be applied to create various transgenic animal
models, such as marmosets.
In marmosets, blastocyst stage embryos have been produced by
ICSI and IVF using in vivo-matured oocytes . However, since
the production of offspring from the ICSI embryos has not been
reported, the developmental competence to neonate of ICSI
embryos remains unknown. Furthermore, ICSI embryos from
in vitro-matured oocytes have not yet been described.
Oocyte maturation depends on nuclear meiotic progression and
is influenced by a quality and maturity of ooplasm [15,16].
Previous studies observed frequent chromatin aberrations and
decreasing developmental rates of the blastocyst stage when sperm
insemination to oocytes was performed immediately after MII
arrest [17,18]. Therefore, essential cytoplasmic changes may occur
during the MII arrest period, and successful embryo development
depends on the proper timing of oocyte maturation, as well as
This study aimed to determine the suitable timing of sperm
injection into oocytes after in vitro maturation (IVM) and to
investigate developmental competence of blastocysts of ICSI
embryos using in vitro-matured oocytes in marmosets. Finally, we
performed a quality evaluation of ICSI embryos using embryo
transfer, reporting the first birth of a normal infant from ICSI
ICSI time post-first
No. of oocytes
No. (%) *2 of embryos developed to
*1Numbers in parentheses were calculated from total oocytes.
*2Numbers in parentheses were calculated from fertilized oocytes.
Materials and Methods
A total of 66 adult marmosets, purchased from a marmoset
breeding company for experimental animals (CLEA Japan Inc.,
Tokyo, Japan), were used in this study. The body weights and ages
of the marmosets ranged from 284534 g and 28 years,
respectively. The animals were not sacrificed for the current
experiments. All animal experiments were approved by the
Institutional Animal Care and Use Committee of the Central
Institution for Experimental Animals (CIEA) (CIEA approval no:
11028A) and were performed in accordance with the CIEA
guidelines that agree with the Guidelines for Proper Conduct of
Animal Experiments by the Science Council of Japan (2006).
Animal care was conducted in accordance with the
recommendations of the Guide for the Care and Use of Laboratory Animals
(Institute of Laboratory Animal Resources, 1996). The marmosets
were housed in pairs in stainless steel living cages (39655670 cm)
with wire mesh floors maintained at 2526uC with 4555%
humidity and illumination for 12 h per day. Wood perches for
locomotion and gouging and a platform for a bed were placed in
each cage for environmental enrichment. Marmosets were kept
healthy and well-nourished with a balanced diet (CMS-1M; CLEA
Japan Inc.), including mixed L (+)-ascorbic acid (Nacalai Tesque,
Tokyo, Japan), vitamins A, D3, and E (Duphasol AE3D; Kyoritsu
Seiyaku Co., Ltd., Tokyo, Japan), and honey (Nihonhatimitsu Co.,
Ltd., Gifu, Japan). In addition, chicken liver boiled in water (DBF
Pet Co., Ltd., Niigata, Japan) was given as a supporting meal once
a week. The animals were supplied with tap water ad libitum from
Anesthesia and Postoperative Care
Animals were pre-anesthetized with an intramuscular injection
of 0.04 mg/kg of medetomidine (Domitor; Nippon Zenyaku
Kogyo, Koriyama, Japan), 0.40 mg/kg of midazolam (Dormicam
10 mg; Astellas Pharma, Tokyo, Japan), and 0.40 mg/kg of
butorphanol (Vetorphale; Meiji Seika Pharma, Tokyo, Japan).
They were also administered 15 mg/kg ampicillin (Viccillin; Meiji
Seika Pharma, Co., Ltd.) and hydrated subcutaneously with
2 mL/head of fluid (KN No.1 injection; Otsuka Pharmaceutical,
Tokyo, Japan). Thereafter, animals were anesthetized by
inhalation with 1.03.0% isoflurane (Forane; Abbott Japan, Tokyo,
Japan) via a ventilation mask. Anesthetization management was
performed by spontaneous respiration during the operation,
monitoring the heart rate and the arterial oxygen saturation.
After oocyte collection or embryo transfer, 0.20 mg/kg
atipamezole (Antisedan; Nippon Zenyaku Kogyo) was administered
intramuscularly into the animals. For postoperative analgesia
and infection control, 1.2 mg/kg ketprofen and 15 mg/kg
ampicillin were administered once daily for 3 consecutive days
following the operations.
Ovarian Stimulation and Collection Oocytes
Thirty-three donor female adult marmosets were used in the
present study. For oocyte collection from ovaries, donor female
marmoset ovarian stimulation was performed using the follicular
stimulation protocol, as described previously . Briefly, ovarian
cycles were monitored based on plasma progesterone levels using
an enzyme immunoassay (EIA) kit (TOSO Progesterone Kit;
TOSO, Tokyo, Japan). Luteolysis was induced with 0.8 mg of
cloprostenol, an analog of prostaglandin F2a (PGF2a; Estrumate;
Schering-Plough Animal Health, Union, NJ), which was
administered by intramuscular injection. After injection, onset of the
follicular phase was confirmed based on blood progesterone levels
Values within the same column with different letters (a, b) differ significantly (p,0.05), x2-test.
*1Numbers in parentheses were calculated from total oocytes.
*2Numbers in parentheses were calculated from fertilized oocytes.
No. (%) *2 of embryos developed to
No. of total blastocyst
No. (%) * of embryos developed to blastocyst
*Numbers in parentheses were calculated from total blastocysts.
Day 0: Day of fertilization.
the day after PGF2a injection. Marmoset follicles were stimulated
by intramuscular injection of recombinant human
follicle-stimulating hormone (rhFSH; 50 international units (IU); FOLYRMON
P injection; Fuji pharma Co., Ltd, Tokyo, Japan) for 9 days at
10:00, and human chorionic gonadotropin (hCG; 75IU;
Gonatropin; ASKA Pharmaceutical. Co. Ltd., Tokyo, Japan) was
administered on the day of the ninth FSH administration at 17:30.
Thirty hours after hCG injection, animals were anaesthetized as
described above and follicular aspiration was performed. Oocytes
were collected in porcine oocyte medium (POM; Research
Institute for the Functional Peptides, Yamagata, Japan) .
In Vitro Maturation (IVM)
Collected oocytes were washed three times with mPOM (POM
supplemented with 5% heat-inactivated calf serum (FBS; Gibco,
Carlsbad, CA) and 100 IU/mL FSH (Folyrmon-P; FujiPharma,
Tokyo, Japan). In vitro oocyte maturation was performed by
incubation in an 80 mL mPOM drop covered with mineral oil
(Nacalai Tesque, Tokyo, Japan) under a gas phase of 5% CO2, 5%
O2, and 90% N2 at 37.5uC. To measure the suitable ICSI timing
after the first PB extrusion, cumulus cells were degraded with
1 mg/mL hyaluronidase (Sigma) for 1 min after 19 h of IVM,
oocytes were changed to independent cultures and the first PB
extrusion observed was used in microscopy every 30 min. The
time of the first PB extrusion was recorded for each oocyte and
divided randomly into five groups 12 h, 24 h, 46 h, 68 h and
810 h followed by ICSI.
When comparing ICSI with IVF, cumulus cells were partially
removed by gentle pipetting after 26 h of IVM and the first PB
extrusion was evaluated. The oocytes from each animal were
divided into two groups, IVF and ICSI. For ICSI, the cumulus
cells were completely degraded with hyaluronidase. Twenty-seven
hours after the IVM, ICSI and IVF were performed.
Sperm Collection and Preparation
Eight male adult marmosets were used in the present study. Of
the eight male animals, three were selected based on their physical
conditions for each experiment. Marmoset semen was collected as
described previously . The animal was restrained in an upright
position, and penile vibratory stimulation was performed using a
FertiCare personal vibrator (Fertility Healthcare and Supplies,
Inc., Silverado, CA, USA) in three sexually mature donor animals.
Pre-equilibrated 700 mL TYH medium (Mitsubishi Chemical
Medience Corp., Tokyo, Japan) was immediately added to the
ejaculate and the suspension was incubated at 37.5uC for
approximately 30 min to disperse sperm from the coagulum.
The semen was washed twice with 700 mL of TYH medium by
aspiration of the supernatant after centrifugation for 5 min at
4006g, after which 200 mL of fresh TYH medium was added. The
resuspended sample was then supplied to the bottom of a conical
tube containing 500 mL fresh TYH. A total of 700 mL TYH with
sperm suspension was incubated for 30 min at 37.5uC under a gas
phase of 5% CO2, 5% O2, and 90% N2 to allow the sperm to
swim upwards. After incubation, 400 mL of supernatant was
collected and the quality of sperm samples was measured using the
Sperm Motility Analysis System (SMAS; Ditect, Tokyo, Japan)
based on viability, motility, and concentration. Samples of the
highest quality were chosen for IVF and ICSI, and the sperm
suspension was divided into two samples. For the IVF study, the
sperm suspension was adjusted to a final sperm concentration of
3.66106 sperms/mL and then into 30 mL drops. For the ICSI
study, the sperm suspension was washed with TYH and
centrifuged for 5 min at 4006g. After washing, the sperm pellet
was resuspended in M2 medium (Sigma, St. Louis, MO) and
adjusted to a final sperm concentration of 16104 sperms/mL.
Intracytoplasmic Sperm Injection (ICSI)
An oil-covered micromanipulation chamber containing a 20 mL
drop of M2 for oocytes and a 10 mL drop of M2 medium
containing 10% polyvinyl pyrrolidone (PVP; Sigma) for
spermatozoa was prepared. Approximately 1 mL of sperm suspension was
transferred into M2 medium containing 10% (w/v) PVP drops
and mixed thoroughly. One to three oocytes were placed in the
M2 medium drop. Using an inverted microscope with
micromanipulators (Narishige, Tokyo Japan), only motile sperm with
normal morphology were selected and immobilized by pressing
the tail with the injection needle tip against the dish bottom prior
to injection. A single sperm was aspirated from the sperm drop
and moved to a droplet containing oocytes. An oocyte was
captured by the holding pipette and immobilized with its PB at
either the 6 or 12 oclock position, and the zona pellucida was
drilled using piezo pulses. The pipette was inserted deeply into the
oocyte and a single piezo pulse was applied. A spermatozoon was
then injected into the cytoplasm. After ICSI, oocytes were placed
in ISM1 medium drops (Medicult; Nosan Corp., Kanagawa,
Japan) and washed three times. The day of performing ICSI was
designated as day 0.
In Vitro Fertilization (IVF)
After sperm preparation, the matured oocytes were washed
three times with 50 mL of TYH medium drops. The oocytes were
transferred into 30 mL drops of TYH medium containing 3.66106
sperms/mL and incubated for 18 h under a gas phase of 5% CO2,
5% O2, and 90% N2 at 37.5uC. After 18 h, embryos were washed
three times with 70 mL ISM1 drops. The day of performing IVF
was designated as day 0.
In Vitro Culture (IVC)
Human embryo culture medium ISM 1 and 2 (ORIGIO,
Malv Denmark) were used in this study to culture marmoset
embryos. Pronuclear formations of embryos followed by IVF or
ICSI were confirmed under microscopic observation. Embryos
were transferred in 70 mL fresh drops of ISM1 and cultured under
mineral oil cover at 37.5uC in 5% CO2, 5% O2, and 90% N2 for
3 days. On day 3, embryos were transferred to 70 mL of ISM2
drops covered with mineral oil and were placed in the incubator at
37.5uC in a humidified atmosphere of 5% CO2, 5% O2, and 90%
N2 until day 12. The media exchange and checking of embryo
development were performed every 2 days. The embryos that
developed to blastocysts were examined at days 9, 10, and 11.
Non-surgical Embryo Transfer
Twenty-eight recipient female adult marmosets were used in the
present study. The ovarian cycles of donor and recipient animals
were synchronized using PGF2a, and ovarian cycles were
monitored based on plasma progesterone levels. The embryos
produced by ICSI were transferred to surrogate mothers using
nonsurgical embryo-transfer techniques, as described previously
with modified instruments . The three different
diametersized, 7.5-cm-long glass tubes were newly developed for the
noninvasive embryo transfer technique. The thinnest glass tube
size was tapered to 4.0 mm with a 4.2 mm diameter, the middle
size glass tube was tapered to 5.0 mm with a 6.0 mm diameter,
and the widest size glass tube was tapered to 5.3 mm with a
7.0 mm diameter at one end. The Fluon ETFE 20-gauge (G) was
comprised of a cannula 108 mm in length (a blunt/tapered
cannula; 0.8-mm inner diameter and 1.10-mm outer diameter;
Oviraptor; Altair Corp., Yokohama, Japan). The 23-G had
120mm-long blunt-end stainless steel stylets and a polyethylene
160mm-long cannula (inner diameter 0.28 mm and outer diameter
0.61 mm; Oviraptor; Altair Corp.). These devices were developed
to reduce invasiveness to the animals and improve the insertion of
the cannula into the uterus. All instruments were sterilized before
use with a hydrogen peroxide gas plasma sterilizer (Sterrad 50;
Advanced Sterilization Products, Irvine, CA), and the surgeon and
surgical assistants wore sterile surgical gloves.
Vaginal dilation of the anesthetized recipients was performed
gradually by serial introduction and removal of three sizes of
75mm-long glass tubes (from thinnest to widest). The widest glass
tube was placed in the vagina to manipulate the endoscope and
cannula to prevent vaginal injury. An endoscope (1.6 mm in
diameter; TESALA AE-C1; AVS Co., Ltd., Tokyo, Japan) was
inserted into the glass tube to observe the ostium uteri externum. A
blunt/tapered Fluon ETFE 20-G outer cannula combined with a
23-G, 120-mm-long blunt-end stainless steel stylet were inserted
into the cervix via the glass tube. After inserting the Fluon ETFE
20-G outer cannula/23-G blunt-end stainless steel stylet into the
uterus, the blunt inner stainless steel stylet was removed. At this
time, the polyethylene cannula was inserted into the Fluon ETFE
20-G outer cannula as the dummy inner cannula. The uterus was
observed using linear ultrasound probe (Prosound a7: Hitachi
Aloka Medical, Ltd., Tokyo, Japan) by longitudinally placing it
onto the abdomen to confirm insertion of the dummy inner
cannula via the outer cannula into the uterus. Approximately 2 mL
of medium containing one to three embryos was loaded into a new
polyethylene cannula that attached to a 50-mL glass syringe
(Hamilton Co., Reno, NV). After removing the dummy inner
cannula, the inner catheter containing the embryos was inserted
into the outer cannula. When the inner catheter was
approximately 3 mm from the distal end of the uterus, the outer cannula
was pulled back until the cannula tip reached the proximal end of
the uterus from the cervix. The embryos were then delivered into
the uterine lumen by gentle pressure on the glass syringe. The
cannula and catheter were withdrawn after embryo transfer and
washed with medium to confirm that no embryos remained in the
cannula and catheter.
The recipients were tested for pregnancy based on plasma
progesterone measurements once a week until the time at which
pregnancy could be monitored using ultrasonography in the
The blastocyst stage embryos underwent embryo transfer
12 days after ICSI and the 6-cell- to 8-cell-stage embryos were
transferred into a surrogate mother uterus at 5 days after ICSI.
Genotyping of Neonates Using Microsatellite Markers
Parental testing based on microsatellite polymorphisms for the
delivered offspring was performed using 10 microsatellite markers
(Table 1). Genomic DNA was extracted from hair root for live
animals or frozen skin for dead animals using the DNA Micro Kit
(Qiagen KK, Tokyo, Japan). To detect microsatellite
polymorphisms, polymerase chain reaction (PCR) amplification was
performed using the extracted genomic DNA as template. The
PCR product was loaded directly on an ABI 31306l Genetic
Analyzer (Life Technologies, NY, USA) along with the GS500 LIZ
dye Size Standard (Life Technologies, NY, USA). The
electrophoresis data was processed using GeneMapper 4.0 software (Life
Technologies), and alleles were assigned according to the PCR
To evaluate differences between experimental groups, a x2 - test
was performed. Differences at p,0.05 were considered significant.
Fertilization Competence after PB Extrusion
PB extrusions were observed approximately 2024 h after IVM.
To determine the optimal timing of performing ICSI, 104 in
vitromatured oocytes derived from 12 female marmosets were divided
into five groups and subjected to ICSI at various time points: 1
2 h, 24 h, 46 h, 68 h, and 810 h after extrusion of the first PB
(Figure 1A, Table 2). Although no significant differences were
observed in the fertilization and blastocyst rate, the blastocyst rate
tended to be low among the groups when ICSI was performed at
12 h after PB extraction (5.0%, 31.6%, 16.0%, 26.7%, and
Developmental Competence of Oocytes Fertilized by ICSI
To compare embryo developmental competencies, ICSI and
IVF were performed (Table 3). Since relatively high embryo
development was shown beyond 2 h after PB extrusion, ICSI and
IVF were performed after 27 h following IVM that fit to 37 h
after PB extrusions. In total, 178 in vitro-matured oocytes derived
from 21 female marmosets were divided into two groups for ICSI
or IVF. The fertilization and developmental rates during the
blastocyst stage of ICSI and IVF embryos are shown in Figure 1B
and Table 3. The fertilization rate of the ICSI embryos (93.2%)
was significantly higher than that of the IVF embryos (82.2%, p,
0.05). No significant differences in developmental rate were
observed between ICSI and IVF embryos (35.4% and 39.2%,
respectively) in the blastocyst stage.
The length to reach the blastocyst stage of embryos using ICSI
and IVF are shown in Table 4.
Although the developmental speed to blastocyst was not
significantly different, ICSI embryos tended to develop later than
Developmental Abilities of the Embryos to Neonates
To assess the in vivo developmental potential of ICSI-derived
blastocysts (Figure 1B), 37 blastocysts after 12 days of culture were
transferred into 20 recipient surrogate mothers by nonsurgical
embryo transfer (Figure 2). Four of 20 recipients receiving
blastocysts produced by ICSI were pregnant. Although one
normal and healthy offspring (Figure 3, Table 5, Table 6) was
delivered, other recipients interrupted pregnancy by spontaneous
abortion at days 83, 104, and 105. Therefore, to investigate
whether the in vivo developmental potential of relatively early ICSI
embryos was higher, 21 embryos from the 6-cell- to 8-cell stage
after 5 days of culture were transferred into eight recipients
(Table 5). We found that three of eight recipients were pregnant
and six offspring were delivered. Although three offspring were
dead a few days after delivery by caesarean section, the other three
offspring grew to be healthy. The birth rate of the 6-cell- to
8-cellstage embryo transfer was 28.6%, which was significantly higher
than that of the blastocyst stage embryo transfer (2.7%, p,0.05).
Parentage Evaluation Tests Using Microsatellite Markers
The results of genotyping of neonates using microsatellite
markers clearly demonstrated that all offspring were derived from
donor embryos (Table 7). For offspring 635, the genotype with
The neonates ID
Body weight (g)
Status after birth
dead on day 5
dead on day 16
dead on day 1
CJ060-PET, CJ077-VIC, CJ081-VIC, CJ103-NED, CJ003-NED,
and CJ083-VIC microsatellite markers demonstrated that this
neonatal animal was derived from donors 3464 and 691. The
genotype with CJ060-PET, CJ081-VIC, and
CJ103-NEDmicrosatellite markers indicated that offspring 731, 732, 733 and 734
was not derived from the recipients, but rather from donors 3525
and 666. Similarly, the paternity testing indicated that offspring
737 and 640 were from the donor animals.
The present study is the first to report the birth of common
marmoset offspring using ICSI with oocytes matured in vitro. Based
on the results of genotyping tests using microsatellite markers, all
neonates were derived from the ICSI embryos.
Since ICSI can control the timing of fertilization, the optimal
fertilization timing through oocyte maturation was determined.
Oocyte nuclear maturation implies re-initiation and completion of
the first meiotic division from the GV stage to MII stage. Besides
these nuclear aspects of oocyte maturation, cytoplasmic aspects are
also important for fertilization and development of the oocyte .
These two processes are completely independent events .
The study of human, mouse, and bovine oocytes indicated that
cytoplasmic maturation may occur during MII arrest [17,18]. In
humans, IVM oocytes require at least 1 h incubation for
cytoplasmic maturation after the first PB extrusion to fertilize by
ICSI [26,27]. In this study, the fertilization and embryonic
developmental rates of the marmoset ICSI embryos after IVM
oocytes at various time intervals following extrusion of the first PB
showed no significant differences. Although significant differences
were not observed, the blastocyst rate in 12 h groups was lowest
among the groups, suggesting that the optimal timing of
fertilization was more than 2 h after PB extrusion in marmosets.
In this study, both ICSI and IVF were performed after 27 h
following IVM, which were adjusted to 37 h after PB extrusion,
and both embryos showed comparable developmental rates.
Furthermore, the observation of marmoset oocyte PB extrusions
approximately 2024 h after IVM (data not shown) was consistent
with previous reports . The ICSI procedure bypasses the
normal fertilization process through IVF of zona penetration and
fusion of sperm and oocyte membranes. Thus, the initial steps
involved in oocyte activation may also be bypassed. In several
species such as mice, humans, and rabbits, ICSI can activate
oocytes for further embryonic development, comparable to IVF
[10,28,29]. In bovine and porcine embryos, the developmental
competencies of embryos followed by ICSI were low. To improve
the development of embryos, ICSI has been combined with
artificial stimuli, such as exposure to ethanol, ionomycin,
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dimethylaminopurine (DMAP), and electric stimulation. [30,31].
Our results demonstrated that highly efficient marmoset
fertilization and blastocyst development occurred using ICSI with no
stimulation, indicating that sperm injection alone is sufficient to
activate oocytes, similar to that of humans, mice and rabbits.
Although the amount of total blastocysts between IVF and ICSI
was not significantly different, the time for embryos to reach the
blastocyst stage tended to be longer when using ICSI than IVF. In
our previous study, IVF embryos showed a 12% birth rate after
embryo transplantation . In the present study, the birth rate
following the transplantation of ICSI embryos was 28.6%.
Although experimental conditions in the previous study differed
from this study in two aspects, (POM medium without FBS and
embryo transfer method), the birth rate of ICSI and IVF embryos
Our results indicate that the birth rate from cleavage-stage
embryo transfers was significantly higher than that of blastocyst
transfers. Three of six offspring obtained from 6-cell- to
8-cellstage transfer were dead after birth. These three offspring were
quadruplets and derived by caesarean section. However, three of
four offspring were dead on the first day, day 5, and day 16 after
birth, respectively. The average weight of these offspring was
25.6 g lower than the average weight of normal neonates (33.3 g).
Thus, the postnatal deaths of these neonates did not result from
low embryo quality, but from premature birth of the quadruplets.
These quadruplets were obtained from a recipient female that
received three embryos. Parental testing demonstrated that all
offspring were from donor embryos. Interestingly, offspring #731,
#733 and #734 showed the same genotyping and may have been
identical triplets, indicating that one ICSI embryo split in the
recipient uterus. One reason for the low birth rate of blastocyst
embryo transfer may have been the long-term embryo culture
in vitro. In this study, human embryo culture mediums ISM 1 and
2 were used to culture marmoset embryos. We examined various
human IVC mediums using in vivo fertilized marmoset embryos
collected from female marmosets uteri and chose ISM2, which
showed the highest developmental ability to the blastocyst stage. In
human IVC, embryos reach the blastocyst stage in approximately
six days, whereas it takes 10 days in marmosets. A previous study
reported in vivo marmoset embryos reach the blastocyst stage
approximately eight days after ovulation . The prolonged
developmental period suggested this culture condition was not
optimized for marmosets. Consequently, the application of other
animal embryo culture conditions would not be adequate for
marmosets. Therefore feasible long-term culture conditions for
marmoset embryos need further investigation. Additionally, the
long incubation period of the embryos after reaching the blastocyst
stage on days 911 probably affected the birth rate of blastocysts
because all embryos were transferred on day 12 and, as described
above, the embryo culture condition was not optimized, thus
potentially affecting the embryo quality.
The marmoset is the only nonhuman primate that has been
used to generate transgenic animals with the lentiviral system and
a exogenous gene, which encoded a green fluorescent protein
(GFP) and were germline-transmitted . This lentiviral system is
the most successful transgenic method to efficiently obtain
offspring in several species . However, the major drawback
of this technique is the limited size of the transgene (up to 8 kb)
. To overcome this limitation, ICSI-Tr combined with
recombinases or transposases could be a powerful technique to
introduce very large DNA transgenes with relatively highly
efficient integration into the host genomes [37,38]. This ICSI-Tr
technique would be applicable to the marmoset and facilitate
In conclusion, in the marmoset, embryos produced by ICSI
using in vitro-matured oocytes could develop to blastocysts and
neonates. Several offspring were successfully derived from embryo
transfer after ICSI, which is a suitable fertilization method in
We thank Toshio Ito (Central Institute for Experimental Animals) for
animal management. We also thank Ryo Oiwa, Akiko Shimada, and
Takeshi Kuge (all from the Central Institute for Experimental Animals) for
their technical assistance.
Conceived and designed the experiments: TT KH HS ES. Performed the
experiments: TT CY MY ES. Analyzed the data: TT CY MY ES.
Contributed reagents/materials/analysis tools: TT TI KS AS JO HS CY
MY TE YK HO MS ES. Wrote the paper: TT TI HS HO MS ES.
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