Chromosome transfer in mature oocytes

Article (PDF Available)inFertility and sterility 97(5):e16 · May 2012with26 Reads
DOI: 10.1016/j.fertnstert.2012.03.048 · Source: PubMed
Abstract
To demonstrate step-by-step micromanipulation procedures required for transfer of spindle-chromosomal complexes between mature oocytes. Video presentation of reproductive biology study. In vitro fertilization and embryo manipulation laboratory. Rhesus (Macaca mulatta) primates. Transplantation of the genetic material between mammalian oocytes offers many opportunities to study various aspects of nuclear-cytoplasmic interactions during oogenesis, fertilization and embryo development. We demonstrate the feasibility of isolation and transfer of chromosomes between mature metaphase II (MII) primate oocyte. After fertilization, manipulated oocytes were capable of producing healthy offspring or embryonic stem cells. In this video, we show micromanipulation procedures required for isolation and transfer of spindle-chromosomal complexes between rhesus MII oocytes. In brief, the spindle is visualized using a polarized microscope and extracted into a membrane enclosed karyoplast. Karyoplasts are then reintroduced into an enucleated recipient oocyte (cytoplast, derived from an another female) by karyoplast-cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA from another. This video demonstrates the protocol developed for primate oocytes that successfully allowed of isolation and transfer of chromosomes between mature metaphase II (MII) oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Video is available at http://fertstertforum.com/2012974tachibana/.

Figures

p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
1138
|
VOL.5 NO.6
|
2010
|
NATURE PROTOCOLS
INTRODUCTION
Transplantation of genetic material in mammalian oocytes
and embryos
During oogenesis, mammalian oocytes undergo two subsequent
meiotic divisions that result in a single, haploid egg. The first mei-
otic division begins in the fetal ovary, but oocytes arrest at prophase
I of the first meiotic cell cycle. Primary oocytes at this stage have a
distinct large nucleus known as a germinal vesicle (GV). At puberty,
oocytes resume meiosis and undergo germinal vesicle breakdown,
followed by condensation of chromosomes and segregation of the
first polar body. Mature oocytes arrest again at the metaphase II
(MII) stage. Completion of meiosis and separation of chromo-
somes into the second polar body are incited by sperm entry at
fertilization. Transplantation of genetic material between mam-
malian oocytes offers many opportunities to study various aspects
of nuclear-cytoplasmic interactions during oogenesis, fertilization
and embryo development
1,2
. Such technologies may also have far-
reaching clinical applications for overcoming cytoplasmic defects
in human oocytes. Particularly, new assisted reproductive options
have been sought that would prevent the transmission of mito-
chondrial diseases, caused by mutations in mitochondrial DNA
(mtDNA), from affected women to their children
3
. Furthermore,
whereas the mechanisms responsible for reproductive aging in older
women are unclear, assisted reproductive technology (ART) results
show that women even in their sixties can have healthy children as
long as they use oocytes donated by younger women
4
. If the factors
responsible for oocyte aging are confined to the cytoplasm and not
to the nucleus itself, the nuclear transfer strategy may well prove
valuable for overcoming this form of reproductive aging and allow
older women to have their own biological children.
In model animals, successful nuclear transfer has been accom-
plished between GV oocytes (GVT)
5
. The choice of this particular
stage of oocytes has been mainly dictated by the visibility of the
nucleus, and by the possibility of isolating and transplanting intact
nuclear material surrounded by a nuclear membrane. Similarly,
nuclear transfer techniques have also been expanded to pronuclear-
stage zygotes
6
. Until recently, transfer of genetic material in mature
oocytes was thought to be unattainable because of the unique
biological characteristics of MII-arrested oocytes. However, trans-
plantation of MII chromosomes has several clear advantages over
GV oocytes or pronuclear-stage zygotes. (i) In contrast to GV
oocytes, mature eggs do not require in vitro maturation before
fertilization. In humans, in vitro maturation of GV-intact oocytes
is inefficient and associated with poor developmental competence
following fertilization. Mature MII eggs, however, are routinely
retrieved and used in clinical in vitro fertilization (IVF) programs.
(ii) Pronuclear transfer in fertilized human zygotes is associated
with serious ethical and moral issues involving the destruction of
human embryos. (iii) Nuclear transplantation in GV oocytes and
pronuclear-stage zygotes inevitably results in significant mtDNA
carryover because of an uneven concentration of mitochondria in
the perinuclear space
6–9
. Thus, transmission of mtDNA from nuclear
donor oocytes generates a notable heteroplasmy in embryos and
offspring, rendering these approaches inappropriate for patients
with mtDNA mutations. Our recent findings indicate that mito-
chondria are evenly distributed in MII oocytes, and that chromo-
some transfer does not cause any detectable mtDNA heteroplasmy
in resulting embryos and offspring
10
.
Despite these advantages, one of the main difficulties in nuclear
transplantation in mature oocytes is related to the detection of
nuclear material in mature eggs, using conventional micro-
scopes. This is because of the fact that a nuclear membrane in MII
oocytes is absent and chromatin is condensed into chromosomes.
Furthermore, metaphase chromosomes and the spindle apparatus
in MII oocytes are prone to damage or premature resumption of
meiosis and abnormal segregation of chromosomes during manip-
ulations. Early attempts to transfer MII chromosomes in human
MII oocytes resulted in limited success because of low fertilization
rates, pronuclear anomalies and poor embryo development
11,12
.
The developmental potential of such embryos was only monitored
in vitro by blastocyst formation rates.
Chromosome transfer in metaphase II oocytes
We recently implemented several methodological advances during
manipulation of rhesus macaque MII oocytes to surmount these
Chromosome transfer in mature oocytes
Masahito Tachibana
1,5
, Michelle Sparman
1,5
& Shoukhrat Mitalipov
1–4
1
Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon, USA.
2
Oregon Stem Cell Center, Oregon Health and Science
University, Beaverton, Oregon, USA.
3
Department of Obstetrics and Gynecology, Oregon Health and Science University, Beaverton, Oregon, USA.
4
Department of
Molecular and Medical Genetics, Oregon Health and Science University, Beaverton, Oregon, USA.
5
These authors contributed equally to this work. Correspondence
should be addressed to S.M. (mitalipo@ohsu.edu).
Published online 27 May 2010; doi:10.1038/nprot.2010.75
In this article, we describe detailed protocols for the isolation and transfer of spindle–chromosomal complexes between mature,
metaphase II-arrested oocytes. In brief, the spindle–chromosomal complex is visualized using a polarized microscope and extracted
into a membrane-enclosed karyoplast. Chromosomes are then reintroduced into an enucleated recipient egg (cytoplast), derived
from another female, by karyoplast–cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material
from one female and cytoplasmic components, including mitochondria and mitochondrial DNA (mtDNA), from another female.
This approach yields developmentally competent oocytes suitable for fertilization and producing embryonic stem cells or healthy
offspring. The protocol was initially developed for monkey oocytes but can also be used in other species, including mouse and
human oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA
mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Chromosome transfer between the cohorts of
oocytes isolated from two females can be completed within 2 h.
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
NATURE PROTOCOLS
|
VOL.5 NO.6
|
2010
|
1139
biological barriers
10
. We incorporated and adopted the spindle
imaging system (Oosight from CRi) for detection and isolation of
MII spindle–chromosomal complexes. This innovative approach
allowed efficient, noninvasive visualization and removal of intact
MII spindles into small karyoplasts with nearly 100% efficiency.
Furthermore, this technique also enables isolation of karyoplasts
containing very small amounts of cytoplasm, thus minimizing the
amount of cotransferred mtDNA. Another modification was imple-
mented to avoid spontaneous activation and premature segrega-
tion of MII chromosomes during introduction of karyoplasts into
recipient cytoplasts by electrofusion. We developed the karyoplast–
cytoplast fusion method that uses the natural cell membrane fusion
property of the viral envelope isolated from the Hemagglutinating
virus of Japan (HVJ, also referred to as inactivated SeV).
Using these modifications, we recently showed that reconstructed
MII oocytes are capable of supporting normal fertilization, embryo
development and production of healthy offspring or embryonic
stem cells
10
. Moreover, genetic analysis confirmed that mtDNA in
all analyzed offspring and stem cells originated exclusively from
cytoplasts with no contribution of spindle donor mtDNA. In this
study, we describe detailed methods for chromosome transfer in
MII-arrested oocytes in a clinically relevant nonhuman primate
model. Protocols include specific reagents, supplies and equipment
required to conduct these procedures. We also illustrate step-by-
step micromanipulation techniques that involve isolation of chro-
mosomes into karyoplasts, followed by transfer to and fusion with
recipient cytoplasts. Descriptions of these procedures are supported
by illustrations in figures and a video. Because of close similari-
ties in oocyte biology and embryo development between rhesus
macaques and humans, described protocols are directly appli-
cable to human oocytes in standard clinical IVF settings. Most
supporting ART techniques required for chromosome transfer
are routine in clinical IVF laboratories. These include collection
of MII oocytes, fertilization by intracytoplasmic sperm injection
(ICSI) and in vitro embryo culture. Therefore, we focused here on
the detailed description of manipulation procedures specifically
required for chromosome transfer. In addition to standard IVF
equipment, our procedures require a spindle imaging system and
a laser objective.
We also believe that the described manipulation procedures are
universal, meaning that critical equipment, reagents and micro-
manipulation steps described here are optimal to successfully carry
out chromosome transfer in other species. Therefore, our proto-
cols can be applied without further adaptations to other species,
including mice and farm animals. During somatic cell nuclear
transfer procedures, MII spindles in selected mouse strains can be
located and removed without a special imaging system. However,
unlike cloning, in which nuclear material is normally discarded,
spindle-chromosomal integrity during chromosome transfer
must be maintained. Therefore, we find that spindle imaging is
extremely helpful to maintain intact mouse MII spindles during
the enucleation step.
In addition to the specific chromosome transfer steps, we also
included a detailed description of monkey oocyte and embryo cul-
ture media preparations and fertilization by ISCI. These media and
protocols are specific to the rhesus monkey (particularly in our
laboratory) and should be replaced with appropriate protocols for
other species.
MATERIALS
REAGENTS
Rhesus macaque MII-stage oocytes and semen were used (detailed protocols
for controlled ovarian stimulations, oocyte and sperm collections are avail-
able in ref. 13). All our animal procedures were approved by the institutional
animal care and use committee at the Oregon National Primate Research
Center. ! CAUTION Experimenters must comply with national regulations
about animals and their use.
Talp HEPES medium (TH; see REAGENT SETUP and Table 1)
Talp HEPES medium + 0.3% BSA (TH3; see REAGENT SETUP)
Hamster embryo culture medium (HECM-9; see REAGENT SETUP and
Table 1)
Cytochalasin B (Sigma, cat. no. C6762)
Dimethylsulfoxide (DMSO; Sigma, cat. no. D2650)
HVJ Envelope Cell Fusion Kit (Ishihara Sangyo Kaisha Ltd, cat. no.
GenomONE-CF EX)
Mineral oil (SAGE Assisted Reproduction Products, cat. no. ART-4008)
High-viscosity silicon oil (Fluka, cat. no. 85416)
EQUIPMENT
Inverted microscope with Hoffman or Relief Contrast optics (Olympus
IX70 or IX71)
Dissecting stereomicroscope (Olympus SZX-7)
Heated microscope stages (Tokai Hit, MATS-U55R30 and
MATS-U4020WF)
Micromanipulators (Narishige, cat. no. MMO-202ND)
Micropipette holders (Narishige, cat. no. IM-7)
Microinjection system and microsyringe (Narishige, cat. no. IM-5B;
Hamilton, cat. no. 81101, 1725LT 250 µl SYR)
Teflon tubing (Narishige, cat. no. CT-2)
Holding pipettes, inner diameter (i.d.) 25–30 µm, outer diameter (o.d.)
120–140 µm, 20° angle (Humagen Fertility Diagnostics,
cat. no. MPH-LG-20)
Enucleation pipettes (Biomedical Instruments, ES blastocyst injection
pipette, with spike, i.d. = 13.4–14.1
µm (size 38 or 39), L = 5.5 cm,
BA = 20°, bending orientation C, BL 1 mm)
CRITICAL The size of
holding and enucleation pipettes may vary between species. For spindle–
chromosomal complex isolations in the mouse, we recommend to use
enucleation pipette i.d. = 12–13
µm. It is larger than the enucleation pipette
routinely used during mouse somatic cell nuclear transfer. The holding
pipette size for mouse oocytes is o.d. = 90–100
µm and i.d. = 15–20 µm.
Intracytoplasmic sperm injection (ICSI) pipette (Humagen Fertility
Diagnostics, cat. no. MIC-NSP-0)
Oosight spindle imaging system (CRi)
XYClone RED-i laser objective (Hamilton Thorne)
CO
2
/O
2
incubator to maintain 6% CO
2
, 5% O
2
and 89% N
2
embryo culture
conditions
Alternatively, premixed gas can be used to fill up the modular incubator
chamber (Billups-Rothenberg, cat. no. MIC-101)
FluoroDish Cell Culture Dish, Oosight-compatible glass-bottom
micromanipulation dishes (World Precision Instruments, cat. no.
FD5040-100).
Nunclon four-well dishes (Thermo Sci Nunc, cat. no. 176740)
50 × 9-mm Petri dishes (Becton-Dickinson Falcon, cat. no. 35-1006)
REAGENT SETUP
Preparation of base Talp HEPES medium To prepare 1,000 ml of TH medium,
you should add exact amounts of chemicals listed in Table 1 to 1,000 ml of Milli-
Q water. Adjust the pH of the TH medium to 7.4 and osmolarity to 275–290.
Filter the TH medium through a 0.22-µm filter and store up to 1 month at 4 °C.
TH is used as a base medium for TH3 or for diluting reagents. CRITICAL TH
and HECM manipulation and embryo culture media are specific to the rhesus
monkey. These media should be replaced with appropriate 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES)-buffered manipulation and embryo
culture media for different species. For example, we routinely use M2 medium
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
1140
|
VOL.5 NO.6
|
2010
|
NATURE PROTOCOLS
for mouse embryo manipulations, and potassium simplex optimization medium
for mouse embryo culture. CRITICAL It is important to use highly purified
embryo-tested water during all medium and solution preparations.
Preparation of TH3 manipulation medium Add 3 mg ml
1
BSA (Sigma,
cat. no. A3311) to the TH medium, filter through a 0.22-µm filter and store
up to 1 week at 4 °C. However, once warmed, use within 24 h. We usu-
ally prepare and warm up this medium the evening before an experiment.
This medium is used for oocyte and sperm handling during collection and
manipulations. CRITICAL Except TH and HECM media, it is crucial that
a recommended supplier, manufacturer, reagent or catalog number is used
for all reagents rather than than alternative. These reagents were meticu-
lously tested in our laboratory over the years and alternative sources may not
produce desirable results.
Preparation of hamster embryo culture medium To prepare 1,000 ml
HECM-9 base medium, you should add exact amounts of chemicals listed
in Table 1 to 1,000 ml Milli-Q water. Adjust the pH to 7.4 and osmolarity to
277 ± 5. Sterilize by filtering through a 0.22-µm filter and store up to 1 week
at 4 °C.
Preparation of 100× amino acid stock solution To prepare 100 ml of stock
solution, add amounts of amino acids listed in Table 1 to 100 ml of Milli-Q
water. Filter through a 0.22-µm filter and aliquot into 500 µl. This stock
solution can be stored for up to 3 months at 20 °C.
Preparation of HECM-9 + AA medium To prepare 10 ml HECM-9 +
amino acid (AA) medium, you should add 0.1 ml AA 100× stock solution to
9.9 ml base HECM-9 medium. Preequilibrate in an incubator at 37 °C in 5%
CO
2
for a minimum of 4 h before use. This medium is used for culture of
monkey oocytes and embryos up to the eight-cell stage.
Preparation of HECM-9 + AA + 5% FBS Add 0.1 ml of AA 100× stock and
0.5 ml fetal bovine serum (FBS; Hyclone, cat. no. SH30070.03) to 9.4 ml of
base HECM-9 medium. Preequilibrate in an incubator at 37 °C in 5% CO
2
for a minimum of 4 h before use. This medium is used for culture of monkey
embryos from the eight-cell stage to blastocysts.
Preparation of TH + Polyvinylpyrrolidone Reconstitute a vial of lyophi-
lized polyvinylpyrrolidone (PVP; Irvine Scientific, cat. no. 99219) with 1 ml
of base TH medium and divide into 20-µl aliquots. TH + PVP can be stored
for up to 3 months at 4 °C. This medium is used for sperm immobilization
during ICSI.
Preparation of cytochalasin B To prepare 1,000× stock solution of cyto-
chalasin B (CB), it is recommended to reconstitute a vial containing 1 mg CB
(Sigma) with 200 µl DMSO (Sigma). Divide this master stock of CB
(5 mg ml
1
) into 5-µl aliquots and store for up to 6 months at 20 °C. For
preparation of micromanipulation medium, it is recommended to add 1 µl
CB master stock solution to 1 ml TH3 medium (final concentration
5 µg ml
1
). All described chromosome transfer manipulations are carried out
in this medium. CRITICAL Prepare this micromanipulation medium fresh
just before use. CRITICAL Avoid multiple freezing and thawing of CB stock
solution to maintain its activity. The CB working concentration may vary
from 2.5 to 7.5 µg ml
1
depending on species and requirement of different
manipulation procedures.
Preparation of HVJ-E solution To prepare HVJ-E solution, you should
reconstitute a vial of freeze-dried inactivated Sendai virus envelope (Ishihara
Sangyo Kaisha Ltd) with 260 µl of suspension buffer (comes with the kit).
Keep the solution on ice during preparation. Aliquot into 5-µl vials and store
at 80 °C for up to 3 months. For fusion of karyoplast/cytoplast couples,
thaw a vial of HVJ-E solution just before manipulations and use undiluted.
CRITICAL Avoid multiple freezing and thawing of HVJ-E stock solution to
maintain its activity.
EQUIPMENT SETUP
Setting up micromanipulators and micropipettes The micromanipulation
station consisting of an Olympus inverted microscope, Narishige microma-
nipulators, a microinjector, a spindle imaging system and a laser objective
is depicted in Figure 1a. Attach a metal micropipette holder (Narishige) to
Teflon tubing connected to a 20-ml plastic syringe mounted on a stand
(Fig. 1b). Insert and tighten a holding glass micropipette into a metal micro-
pipette holder. Fill in approximately half of the holding micropipette with
TH3 + CB manipulation medium. Air-fill the rest of the holding line.
Attach a second metal micropipette holder to a Teflon tubing connected to
a 200–250-µl volume microsyringe controlled by a microinjector (Narishige;
Fig. 1c). Fill the entire system with water. Load the enucleation micropipette
completely with high-viscosity silicon oil to improve control over aspirations
and injections. Insert and tighten an enucleation micropipette into a metal
micropipette holder. CRITICAL The entire line including microsyringe,
tubing and enucleation micropipette must be completely free of
air bubbles.
Setting up the micromanipulation chamber Place two 20-µl micromanipu-
lation drops of TH3 medium containing CB (5 µg ml
1
) and one 5-µl drop
of HVJ-E solution in the center of glass-bottom dish as shown in Figure 2a.
Cover the dish with approximately 3.2 ml SAGE oil. CRITICAL Separate
micromanipulation drops are necessary to keep apart oocytes derived
from different females. Inactivated SeV extract (HVJ-E) must be thawed
immediately before use as its activity quickly declines, resulting in low
fusion rates. ! CAUTION The micromanipulation equipment, settings
and techniques may vary in each laboratory. We recommend the described
settings because these techniques have been tested in our laboratory
over the years on several species and for many types of micromanipulation
needs.
PROCEDURE
Karyoplast isolation TIMING 0–1 h
1| Place three to five oocytes from each of the two females into separate manipulation drops and mount the chamber on the
microscope stage. Incubate oocytes in CB containing manipulation medium for at least 5 min before manipulations. Set up
the Oosight and laser system (next step) while waiting. We suggest an experimental plan for serial chromosome transfer between
several oocytes from two different females as shown in Figure 2b. Step-by-step manipulations of an entire chromosome
transfer procedure can also be found in Supplementary Video.
CRITICAL STEP Oocytes and embryos are temperature sensitive and must be maintained at 37 °C at all times during
retrieval, scoring, manipulation and culture. All oocyte manipulation procedures should be conducted in a dimly lit and warm
room (30 °C).
2| Switch the condenser turret to a slot with a circular polarizer and interference filter and insert a crystal universal com-
pensator into the analyzer slot of the microscope. Open the side camera port in the microscope and start Oosight control
software. Run a background acquisition using a 20× laser objective and initiate the Oosight mode according to the manual.
Start XYClone laser control program to operate the laser objective. Adjust the laser power output to 100 and adjust pulse to
a range of 100–300 µs.
CRITICAL STEP Do not use full XYClone software to operate the laser during enucleation; instead use the laser control
panel mode that is integrated with Oosight software.
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
NATURE PROTOCOLS
|
VOL.5 NO.6
|
2010
|
1141
3| Lower both the holding and enucle-
ation pipettes into the first manipula-
tion drop and immobilize an individual
oocyte on the holding pipette, with
the sharpest spindle image situated
in the equatorial plane close to the 3
o’clock position. Rotating an oocyte is
essential to find the spindle (Fig. 3a;
spindle positioning; see also
Supplementary Video).
? TROUBLESHOOTING
4| Navigate the holding pipette with
an attached oocyte to the visible REDi
laser target and position the target
to the zona pellucida adjacent to the
spindle.
5| Slightly lower the holding pipette
with oocyte attached and gently press
an oocyte to the bottom of the plate
to stabilize it during chromosome
isolation.
6| Focus the objective on the spindle
and bring the tip of the enucleation
pipette to the same focal plane.
CRITICAL STEP Ensure that the
enucleation pipette and spindle are
in the same equatorial focal plane
by gently poking an oocyte with the
pipette without piercing the zona
pellucida, as a result of which the
spindle should move away
(see Supplementary Video).
7| Drill a hole in the zona next to the
spindle with the laser, starting from
the outer layer of the zona, using
the lowest laser settings (Fig. 3a;
laser-assisted zona drilling).
CRITICAL STEP Avoid making
a large gap in the zona and make
sure to leave a thin intact inner
layer in the zona pellucida. This
will prevent leakage of the cytoplast
from the zona.
? TROUBLESHOOTING
8| Slowly insert the pipette through the slit in the zona pellucida without piercing the plasma membrane and navigate its
opening close to the spindle.
9| Slowly aspirate the spindle with a minimum amount of the underlying cytoplasm into the enucleation pipette. Confirm
the presence of the spindle in the pipette, which can be easily observed within the pipette (see Supplementary Video;
karyoplast isolation). There is no need for any further confirmation of enucleation by DNA staining.
? TROUBLESHOOTING
TABLE 1
|
Reagents and quantities for media preparations.
Reagents
TH medium
(1,000 ml)
HECM-9
(1,000 ml)
AA 100× stock
(100 ml)
NaCl (Sigma, cat. no. S5886) 6.660 g 6.639 g
KCl (Sigma, cat. no. P5405) 0.239 g 0.2240 g
CaCl
2
-2H
2
O (Sigma, cat. no. C7902) 0.294 g 0.2790 g
MgCl
2
-6H
2
O (Sigma, cat. no. M0250) 0.102 g 0.1020 g
Na
2
HPO
4
(Sigma, cat. no. S5136) 0.048 g
Glucose (Sigma, cat. no. G6152) 0.900 g
Na lactate (Sigma, cat. no. L7900) 1.870 ml 0.6320 ml
Phenol Red (Sigma, cat. no. P3532) 0.010 g
NaHCO
3
(Sigma, cat. no. S5761) 0.168 g 2.1000 g
Gentamycin Sulfate (Sigma, cat. no. G1264) 0.050 g 0.0100 g
HEPES (Sigma, cat. no. H6147) 2.603 g
Na pyruvate (Sigma, cat. no. P4562) 0.060 g
PVA (Sigma, cat. no. P8136) 0.1000 g
HCl, 1 M (Sigma, cat. no. H7020) 1.4000 ml
Taurine (Sigma, cat. no. T8691) 0.626 g
Asparagine (Sigma, cat. no. A4159) 0.013 g
Cytseine (Sigma, cat. no. C6852) 0.018 g
Histidine (Sigma, cat. no. H5659) 0.021 g
Lysine (Sigma, cat. no. L8662) 0.018 g
Proline (Sigma, cat. no. P5607) 0.012 g
Serine (Sigma, cat. no. S4311) 0.011 g
Aspartic acid (Sigma, cat. no. A4534) 0.013 g
Glycine (Sigma, cat. no. G8790) 0.008 g
Glutamic acid (Sigma, cat. no. G5889) 0.017 g
Glutamine (Sigma, cat. no. G8540) 0.292 g
Pantothenic acid (Sigma, cat. no. P5155) 0.007 g
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
1142
|
VOL.5 NO.6
|
2010
|
NATURE PROTOCOLS
10| Slowly withdraw the pipette from
the slit in the zona pellucida. As the
pipette is pulled away from the egg,
the plasma membrane will stretch and
form a thin bridge between the egg
and the karyoplast. A rapid movement
of the enucleation pipette toward
the 6 o’clock position will break this
membrane bridge (Fig. 3a; karyoplast
isolation).
? TROUBLESHOOTING
11| Expel the karyoplast into the
manipulation drop next to the enu-
cleated egg (cytoplast). Move both
pipettes to the second manipulation
drop containing eggs from the second
female. Repeat the enucleation steps
described above for the first oocyte in
this group. Keep the karyoplast inside
the pipette.
CRITICAL STEP To avoid accidental mixing, store oocytes, cytoplasts and karyoplasts from each female in separate drops.
CRITICAL STEP Avoid contact between the karyoplast and oil in the pipette as this may cause lysis.
CRITICAL STEP We highly recommend that our experimental plan be followed (see Fig. 2b). To minimize manipulation
steps (expelling karyoplasts from the pipette during an intermediate step) and avoid karyoplast lysis, we recommend the
immediate transfer of a karyoplast from one female into an enucleated recipient oocyte from another female. Leaving
isolated karyoplasts in the manipulation drop for an extended time may cause sticking of the membrane to the bottom of
the dish and eventual lysis. However, the first karyoplast from the first female must be expelled from the pipette and placed
into the drop until the last oocyte from the second female is enucleated.
? TROUBLESHOOTING
Karyoplast transfer and oocyte assessment TIMING 2–3 h
12| Release an enucleated egg from the second female and lift up the holding pipette from the manipulation chamber. Move
the enucleation pipette containing the karyoplast into the middle HVJ-E drop.
13| Gently expel the karyoplast from the pipette into the drop to soak in the HVJ-E for approximately 10 s (Fig. 3b).
Aspirate the karyoplast again into the pipette and return the pipette to the first manipulation drop containing oocytes from
the first female.
14| Lower the holding pipette into the drop and immobilize previously prepared cytoplasts with the first polar body
positioned at 9 o’clock, but avoid holding over the hole in the zona made previously during spindle isolation.
PC
Laser
a
b c
CRi camera
20 ml syringe
Pipette holder
Pipette holder
Stand
Teflon tube
(air-filled)
Teflon tube
(water-filled)
Microinjector
(IM-5B)
Figure 1
|
Equipment and pipette setting for
chromosome transfer. (a) Micromanipulation
station consisting of an inverted microscope,
two micromanipulators, microinjectors, spindle
imaging and laser systems controlled by a PC.
(b) Setting up the holding pipette. The entire line
consists of a metal Narishige pipette holder, Teflon
tubing and a 20-ml plastic syringe filled with air.
Syringe is mounted on a stand. (c) Setting up the
enucleation pipette. The entire line consists of
a metal Narishige pipette holder, Teflon tubing
and a 250-µl Hamilton glass syringe filled with
water. The syringe is mounted on the Narishige
microinjection system.
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
NATURE PROTOCOLS
|
VOL.5 NO.6
|
2010
|
1143
15| Drill the hole in the zona in the 3 o’clock position, using the laser as described above (Fig. 3b; laser-assisted zona drilling).
16| Insert the transfer pipette through the zona slit and gently eject the karyoplast into the perivitelline space (Fig. 3b;
karyoplast transfer).
CRITICAL STEP It is important to place a karyoplast as close as possible to the cytoplast, ensuring good contact between
the membranes. Avoid injecting an excessive amount of medium that would create a large pocket in the perivitelline space
during karyoplast transfer (see Supplementary Video; karyoplast transfer).
? TROUBLESHOOTING
17| Isolate the spindle from the second oocyte of the first female. Soak the karyoplast in HVJ-E and transfer into the first cytoplast
from the second female.
18| Repeat swapping of chromosomes between the two female oocytes. During the last step, transfer the karyoplast isolated
from the first oocyte into the last cytoplast from the second female.
19| Rinse reconstructed oocytes at least three times in TH3 to remove the CB residue and place into embryo culture medium
(HECM-9) at 37 °C in 5% CO
2
for 20–30 min.
CRITICAL STEP Manipulated oocytes must be thoroughly rinsed to remove CB residues as it may cause difficulties during
the ICSI procedure.
20| Evaluate fusion in 20–30 min after
karyoplast transfer by noting the pres-
ence or absence of karyoplasts in the
perivitelline space. Separate fused and
unfused couples into different wells.
? TROUBLESHOOTING
21| Return unfused oocytes into
culture for an additional 30 min and
check fusion again.
Karyoplast isolation
Karyoplast isolation
Karyoplst transfer
Cytoplast donor oocyte
(spindle free)
Cytoplast donor oocyte
(spindle free)
Laser target
Laser target
Zona
Slit
Intact layer
Laser-assisted zona drilling
Karyoplast transfer
Karyoplast exposure of HVJ-E
Zona slit made for karyoplast isolation
Spindle positioning
Laser-assisted zona drilling
Same focal plane
Chromosome donor oocyte
Chromosome donor oocyte
Chromosome donor oocyte
a
b
Figure 3
|
Schematic diagram of the chromosome
transfer procedure. Chromosome transfer in
MII oocytes consists of two chief procedural
steps: (a) isolation of the spindle–chromosomal
complex into a karyoplast and (b) introduction
of the karyoplast into a recipient cytoplast. Step-
by-step manipulations of rhesus monkey oocytes
can also be found in Supplementary Video.
Covered with mineral oil
a b
Female 1 eggs
Female 2 eggs
CB-TH3
CB-TH3
1
7
3
2
4
6
5
HVJ-E
CB-TH3
20 µl
CB-TH3
20 µl
HVJ-E
5 µl
Glass-bottom dish
A
a
b
c
B
C
Figure 2
|
Experimental design for serial
spindle–chromosomal complex transfer between
two cohorts of oocytes. (a) Suggested layout of
micromanipulation drops on the manipulation
chamber. (b) Recommended experimental plan for
serial chromosome transfer between oocytes from
two females. Step 1: Isolate a karyoplast from the
first (A) oocyte of female 1 and place it next to
the cytoplast until the last transfer. Step 2: Isolate
a karyoplast from the first (a) oocyte of female 2,
briefly soak in HVJ-E and transfer into a perivitelline
space of cytoplast A from female 1. Step 3: Isolate
and transfer a karyoplast from the second (B) oocyte
of female 1 and transfer it into the cytoplast (a) of
female 2. Proceed in a similar manner for Steps 4,
5 and 6. During the last step (Step 7), pick up the
karyoplast isolated from the oocyte (A) of female 1
and transfer into the last cytoplast (c) of female 2.
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
1144
|
VOL.5 NO.6
|
2010
|
NATURE PROTOCOLS
CRITICAL STEP Fusion rates in our laboratory are nearly 100%. However, if fusion does not occur within 60 min, unfused
oocytes can be directly exposed to HVJ-E solution for a brief time (5–10 s). Oocytes should be placed back into culture and
evaluated for fusion after 20–30 min.
Intracytoplasmic sperm injection and ICSI preparation TIMING 4.5–5.5 h
22| Wash collected spermatozoa twice by resuspension in TH3 medium, followed by centrifugation of the liquid portion of the
ejaculate for 7 min, at 200g.
23| Take an aliquot and determine motility and concentration and adjust sperm concentration to 1 × 10
6
motile spermatozoa
per ml in TH3 medium, then store for approximately 3 h at room temperature before ICSI.
24| Set up an inverted Olympus IX70 or IX71 microscope equipped with Hoffman or Relief Contrast optics, heating stage
(set at 37 °C) and micromanipulators ready for the ICSI procedure.
25| Set up micromanipulators and micropipettes in the same manner as for chromosome transfer, except use a smaller-diameter
ICSI pipette instead of an enucleation pipette.
CRITICAL STEP Use CB-free TH3 medium for manipulations during ISCI to avoid difficulties in penetrating the oocyte
membrane.
26| Fill approximately half the holding micropipette with TH3 medium before the micromanipulation procedure.
27| Dilute a small aliquot of sperm with 10% PVP (1:4) and place a 5-µl drop in the center of the glass-bottom dish.
It is not critical to use a glass-bottom manipulation dish for ISCI; you can use the plastic lid of 50 × 9-mm Falcon dish. Next,
place a 30-µl drop of TH3 adjacent to the sperm drop. Slowly cover the manipulation dish with 3.2 ml of prewarmed
(37 °C) SAGE oil.
28| Transfer manipulated oocytes into the 30-µl drop of TH3 manipulation medium and place the dish on the heated stage
(37 °C) of the microscope.
29| Lower the ICSI pipette into the sperm drop. Select a motile sperm and quickly immobilize it by striking the midpiece
with the tip of the pipette. Slowly aspirate the immobilized sperm into the ISCI pipette tail first.
30| Move the ISCI pipette to the manipulation drop containing oocytes and lower the holding pipette.
31| Immobilize an individual oocyte on the holding pipette with the polar body positioned at either the 12 o’clock or 6 o’clock
position, but avoid holding over the hole in the zona made previously during chromosome transfer.
32| Slightly press down the holding pipette with oocyte attached until it touches the bottom of the plate to stabilize the
egg during injection.
33| Focus the objective on the oocyte membrane and then bring the tip of the ICSI pipette into sharp focus next to the
3 o’clock position on the oocyte.
34| Slowly push the sperm to the pipette tip using the microsyringe and insert the ICSI pipette through the zona pellucida and
‘into’ the oocyte across approximately one-third of the egg’s diameter. Piercing the plasma membrane during the ICSI procedure is
critical but often difficult to achieve, as the needle can grossly invaginate the membrane without breaking through.
? TROUBLESHOOTING
35| Pierce the plasma membrane by slowly aspirating egg cytoplasm into the ICSI pipette (as far back as the needle junc-
tion with the zona pellucida) until the plasma membrane breaks. A ‘pop’ or sudden movement of cytoplasm into the pipette
will indicate the release of membrane tension. Once the membrane is penetrated, expel the sperm into the cytoplasm with a
minimal amount of medium.
? TROUBLESHOOTING
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
NATURE PROTOCOLS
|
VOL.5 NO.6
|
2010
|
1145
Embryo culture to the blastocyst stage TIMING 7–9 d
36| After ICSI, transfer embryos into HECM-9 + AA culture medium (containing amino acids) covered with SAGE oil and
culture at 37 °C, in 6% CO
2
, 5% O
2
and 89% N
2
.
37| Check for fertilization by noting the presence of pronuclei the next morning. This should be carried out no later than
14–16 h after ICSI. The presence of two pronuclei indicates normal fertilization. You will find the second polar body on the
opposite side of the first polar body.
? TROUBLESHOOTING
38| Monitor embryos daily for cleavage and embryonic development. Eight-cell–stage embryos should be transferred into
fresh HECM-9 medium containing amino acids and 5% FBS and cultured for a maximum of 9 d with medium changes every
other day. On reaching the blastocyst stage, embryos can be transferred into surrogate females or used for the derivation of
embryonic stem cells.
? TROUBLESHOOTING
TIMING
Steps 1–11, karyoplast isolation: 2–4 min per oocyte
Steps 12–16, karyoplast transfer: 1–2 min per oocyte
Steps 17 and 18, repeat serial chromosome transfer for remaining oocytes: 3–6 min × number of oocytes,
30–60 min (for a total of 10 oocytes)
Step 19, rinse oocytes: 3 min
Step 20, evaluation of fusion: 30 min after reconstruction
Step 21, preculture before ICSI: further 30 min
Steps 22 and 23, preparation of sperm for ICSI: 3–4 h before ICSI
Steps 24–35, fertilization by ICSI: 2–3 min per oocyte
Step 36, transfer embryos back into culture media: immediately after ICSI
Step 37, fertilization check: no later than 14–16 h after ICSI
Step 38, embryo culture: up to 9 d
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 2.
TABLE 2
|
Troubleshooting table.
Step Problem Possible reason Solution
3 Complete absence of
or poor spindle image
Incorrect setting of the imaging
system
Improper position of an oocyte
Immature oocytes or
premature activation
Suboptimal temperature
Make sure that all Oosight parts are properly installed (polarizer, CRi
lens, camera port, etc.) and the camera port is open. Run background
acquisition and make sure that all Oosight modes are working properly.
Refer to the manual
The spindle of primate oocytes is much smaller than in rodents
and can only be seen at a certain position within an oocyte. Slow
rotation of oocytes in different directions is always required to visualize
spindles
If a meiotic mid-body is present adjacent to the first polar body, the
oocyte is still completing meiosis I. Place oocytes back into
culture medium for 30–60 min and attempt imaging again. Wash
oocytes before culture to remove cytochalasin B residue. If a second
PB is observed, meiosis has been completed, possibly due to premature
activation
The microtubules of the spindle apparatus are temperature
sensitive. Always maintain and operate oocytes at 37 °C
(continued)
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
1146
|
VOL.5 NO.6
|
2010
|
NATURE PROTOCOLS
ANTICIPATED RESULTS
All techniques described above will require certain micromanipulation skills, necessitating diligent practice before attempt-
ing this procedure. Chromosome transfer in MII oocytes itself does not have any adverse effect on fertilization or embryo
development. If a highly skilled person performs manipulations, spindle visualization and karyoplast isolation can be suc-
cessfully achieved at a rate of 90% or higher. If one is having difficulties with spindle/karyoplast isolation, it is possible
that the positioning of an oocyte on the holding pipette is not appropriate. Spend more time rotating an oocyte with your
holding pipette so that you can position the metaphase spindle exactly where it should be; this will save you a lot of time when
proceeding to remove the spindle with the enucleation pipette. Importantly, this will allow you to isolate chromosomes into
TABLE 2
|
Troubleshooting table (continued).
Step Problem Possible reason Solution
7 Difficulties in the
laser-assisted zona
pellucida drilling
Incorrect setting(s) on the laser
system
Laser power level set
too low
Check all connections of the laser system. Contact Hamilton Thorne
representatives for troubleshooting
Increase laser output gradually
9 Difficulties in
isolation of spindles
Enucleation pipette is out of
focus
The pipette is too small
Make sure to bring the spindle into an equatorial plane close to the
3 o’clock position and move the pipette tip to the same focal plane. Proper
alignment can be confirmed by gently poking the spindles through the
zona with the pipette tip. You should be able to push the spindle away
The optimal size of the pipette is important. The spindle may not fit
easily if the pipette is too small. However, too-large pipettes may
remove excess volume of cytoplasm during karyoplast isolation
10 Lysis of cytoplasts
during spindle
isolation
Incorrect concentration of CB
Sharp edges of the
enucleation pipette
Check the CB concentrations and make sure to use fresh CB stock
The tip of enucleation pipettes must be polished to avoid cutting the
membrane. This is normally done by the pipette manufacturer
11 Karyoplast lysis Incorrect concentration of CB
Sticking of the karyoplast
membrane to the bottom of the
dish, pipette and pipette oil
Check the CB concentrations and make sure to use fresh CB stock
Avoid keeping isolated karyoplasts too long within the pipette or in the
manipulation drop. Transfer karyoplasts immediately into recipient cyto-
plasts. Avoid contact between a karyoplast and oil in the pipette
16 ‘Leaking’ of the
cytoplasm out of the
opposite hole in the
zona pellucida during
karyoplast transfer
Excessive pressure on the zona
pellucida during insertion of the
pipette
Too large a hole in the zona
Avoid squishing the zona during karyoplast transfer. See
Figure 4d
Minimize the size of the zona slit and always leave a thin intact layer
in the zona wall. Review Step 7 and see Figure 4a and b
20 Poor fusion rates Insufficient contact
between karyoplasts and
cytoplasts
Inactive HVJ-E
Make sure to remove extra fluid surrounding karyoplasts from the
perivitelline space to ensure sufficient membrane contact. Review
Step 16 and see Figure 4e and f
Thaw HVJ-E just before use
34 ‘Leaking’ of cytoplasm
out of the hole in the
zona pellucida during
ICSI
Excessive pressure on the zona
pellucida during insertion of the
pipette
Too large a hole in the zona
Poking with injection pipette away from the center of the oocyte may
cause cytoplasmic leakage during ICSI. To prevent this, you may insert
the pipette through the gap made earlier during chromosome transfer
Once again, minimize the size of the zona slit and always leave a thin
intact layer in the zona wall as we suggest during Steps 7 and 15. See
Figure 4a and b
35 Difficulties of break-
ing oocyte membrane
during ICSI
Excessive softening of oocytes
due to CB residue
ICSI should be performed in CB-free media and oocytes must be thor-
oughly rinsed after chromosome transfer manipulations
37, 38 Poor fertilization and
embryo development
Premature activation
Inadequate culture condition
Check your reconstructed oocytes carefully before ICSI. If you observe
the second PB at the time of ICSI, oocytes are prematurely
activated and have completed meiosis
Check the quality of all reagents, media and culture conditions
p
uo
r
G
gn
i
h
s
i
lb
uP eru
t
a
N
010
2
©
natureprotocols
/
m
o
c
.
e
r
u
t
a
n
.
w
w
w
/
/
:
pt
t
h
PROTOCOL
NATURE PROTOCOLS
|
VOL.5 NO.6
|
2010
|
1147
a karyoplast with a minimum amount
of cytoplasm. To avoid mtDNA carryover
during chromosome transfer, we recom-
mend that the size of the karyoplast
should be minimized. Moreover, large
karyoplasts (more that 1% of oocyte
size) are difficult to transfer, often
resulting in lysis during manipulation. It is often difficult to see spindles in freshly matured MII oocytes (within 30 min of
polar body extrusion). We recommend waiting for an additional 1–2 h before attempting chromosome transfer with these
oocytes. Transferring spindles back into enucleated oocytes should be relatively smooth and quick once you have prac-
ticed for a while. Experimental examples of good and poor outcomes during chromosome transfer are shown in Figure 4. To
increase fusion rates, it is suggested to literally squeeze out all extra media that were transferred to the perivitelline space
with the karyoplast. If the karyoplast and plasma membrane are in tight contact, fusion usually occurs within 20–30 min at a
90–100% rate. We do not recommend electrofusion because of accidental activation of MII spindle–chromosomal complexes
induced by electric pulses
10
. We consider fertilization and cleavage rates of 80–90% acceptable, although we always strive
for 100%. As a control for chromosome transfer procedures, we recommend monitoring the development of nonmanipulated
ICSI embryos from the same cohort of oocytes. Blastocyst development may vary between experiments because of variation
in oocyte quality following controlled ovarian stimulation protocols in nonhuman primates, but you should consistently be
producing blastocysts from each experiment.
4. Paulson, R.J. et al. Pregnancy in the sixth decade of life: obstetric
outcomes in women of advanced reproductive age. JAMA 288, 2320–2323
(2002).
5. Takeuchi, T., Ergun, B., Huang, T.H., Rosenwaks, Z. & Palermo, G.D. A
reliable technique of nuclear transplantation for immature mammalian
oocytes. Hum. Reprod. 14, 1312–1317 (1999).
6. Sato, A. et al. Gene therapy for progeny of mito-mice carrying pathogenic
mtDNA by nuclear transplantation. Proc. Natl Acad. Sci. USA 102,
16765–16770 (2005).
7. Meirelles, F.V. & Smith, L.C. Mitochondrial genotype segregation in a
mouse heteroplasmic lineage produced by embryonic karyoplast
transplantation. Genetics 145, 445–451 (1997).
8. Meirelles, F.V. & Smith, L.C. Mitochondrial genotype segregation during
preimplantation development in mouse heteroplasmic embryos. Genetics
148, 877–883 (1998).
9. Wilding, M. et al. Mitochondrial aggregation patterns and activity in human
oocytes and preimplantation embryos. Hum. Reprod. 16, 909–917 (2001).
10. Tachibana, M. et al. Mitochondrial gene replacement in primate offspring
and embryonic stem cells. Nature 461, 367–372 (2009).
11. Cohen, J. et al. Ooplasmic transfer in mature human oocytes. Mol. Hum.
Reprod. 4, 269–280 (1998).
12. Tanaka, A. et al. Metaphase II karyoplast transfer from human in-vitro
matured oocytes to enucleated mature oocytes. Reprod. Biomed. Online
19, 514–520 (2009).
13. Mitalipov, S.M. & Wolf, D.P. Nuclear transfer in nonhuman primates.
Methods Mol. Biol. 348, 151–168 (2006).
a
b
c
d
e
f
Karyoplast
Karyoplast
Figure 4
|
Experimental examples of good
and poor outcomes during chromosome transfer.
(a) Optimal (partial) zonal drilling with laser,
leaving a thin intact layer. This will prevent
cytoplasm leakage during further manipulations.
(b) An example of too large a gap in the zona
pellucida. (c) Examples of small (desirable,
depicted by arrow) and oversized karyoplasts.
(d) Cytoplasmic leakage during karyoplast transfer
(or ICSI) as a result of a large zona gap. (e) Good
contact between karyoplast and cytoplast that
is critical for efficient fusion. (f) An example of
poor karyoplast–cytoplast contact due to a large
pocket in the perivitelline space.
Note: Supplementary information is available via the HTML version of this article.
ACKNOWLEDGMENTS
This work was supported by start-up funds from the Oregon
National Primate Research Center, the Oregon Stem Cell Center and grants from
the National Institutes of Health HD057121, HD059946, HD063276, HD047721,
HD047675, RR0000163 and U54 HD18185.
AUTHOR CONTRIBUTIONS S.M., M.S. and M.T. developed and wrote this protocol.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Published online at http://www.natureprotocols.com/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
1. Cui, L.B., Huang, X.Y. & Sun, F.Z. Transfer of germinal vesicle to ooplasm
of young mice could not rescue ageing-associated chromosome
misalignment in meiosis of oocytes from aged mice. Hum. Reprod. 20,
1624–1631 (2005).
2. Liu, H., Wang, C.W., Grifo, J.A., Krey, L.C. & Zhang, J. Reconstruction of
mouse oocytes by germinal vesicle transfer: maturity of host oocyte
cytoplasm determines meiosis. Hum. Reprod. 14, 2357–2361 (1999).
3. Roberts, R.M. Prevention of human mitochondrial (mtDNA) disease by
nucleus transplantation into an enucleated donor oocyte. Am. J. Med.
Genet. 87, 265–266 (1999).
    • "The UK approved a resolution aiming to legalize the clinical use of mitochondrial replacement therapy (MRT), which could soon enable women with mutated mtDNA to have children with normal mitochondria (Gorman et al., 2015a). Two related procedures are currently being pursued – maternal spindle transfer (Tachibana et al., 2010), and pronuclear transfer (Craven et al., 2010). Both techniques combine the nuclear genetic material of the two parents with the mitochondrial genome of a donor woman with healthy mitochondria, representing a breakthrough in the prevention of genetic diseases. "
    [Show abstract] [Hide abstract] ABSTRACT: Once considered exclusively the cell's powerhouse, mitochondria are now recognized to perform multiple essential cellular functions beyond energy production, impacting most areas of cell biology and medicine. Since the emergence of molecular biology and the discovery of pathogenic mitochondrial DNA defects in the 1980's, research advances have revealed a number of common human diseases which share an underlying pathogenesis involving mitochondrial dysfunction. Mitochondria undergo function-defining dynamic shape changes, communicate with each other, regulate gene expression within the nucleus, modulate synaptic transmission within the brain, release molecules that contribute to oncogenic transformation and trigger inflammatory responses systemically, and influence the regulation of complex physiological systems. Novel “mitopathogenic” mechanisms are thus being uncovered across a number of medical disciplines including genetics, oncology, neurology, immunology, and critical care medicine. Increasing knowledge of the bioenergetic aspects of human disease has provided new opportunities for diagnosis, therapy, prevention, and in connecting various domains of medicine. In this article, we overview specific aspects of mitochondrial biology that have contributed to – and likely will continue to enhance the progress of modern medicine.
    Full-text · Article · Jul 2016
    • "Early attempts using this technology were largely unsuccessful, with poor rates of fertilization and poor embryo development. However, recent methodological advances have allowed proof of principle experiments to be conducted in primates (Tachibana et al., 2009Tachibana et al., , 2010). In this novel study by Tachibana et al. (2009), reconstructed MII oocytes were capable of normal fertilization, normal embryo development and four live and healthy offspring were produced. "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondrial DNA (mtDNA) mutations are a relatively common cause of progressive disorders that can be severe or even life-threatening. There is currently no cure for these disorders; therefore recent research has been focused on attempting to prevent the transmission of these maternally inherited mutations. Here we highlight the challenges of understanding the transmission of mtDNA diseases, discuss current genetic management options and explore the use of germ-line reconstruction technologies to prevent mtDNA diseases. In particular we discuss their potential, indications, limitations and possible safety concerns. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
    Full-text · Article · Nov 2014
    • "Reconstructed oocytes were next fertilized by ICSI, placed into 4-well dishes (Nalge Nunc) containing embryo culture medium and cultured at 37°C in 6% CO 2 , 5% O 2 , and 89% N 2 (Tachibana et al., 2010). Complete mitochondrial gene replacement by the ST procedure and embryo transfers were carried out as we previously described (Tachibana et al., 2010; Tachibana et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.
    Full-text · Article · May 2012
Show more

  • undefined · undefined
  • undefined · undefined
  • undefined · undefined