germ cell protocols volume 254 || parthenogenesis and nuclear transfer in rabbit oocytes

Rabbit Parthenogenesis and Nuclear Transfer 195

195

From: Methods in Molecular Biology, vol. 254: Germ Cell Protocols, Volume 2:Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning

Edited by: H. Schatten © Humana Press Inc., Totowa, NJ

11

Parthenogenesis and Nuclear Transfer in Rabbit Oocytes

Yukio Tsunoda and Yoko Kato

1. IntroductionRabbits are widely used as experimental animals in various fields of

research. However, unlike mice and rats, rabbits with a homologous geneticbackground are difficult to obtain, as few inbred strains have been establishedbecause of the fact that inbreeding decreases reproductive ability. Moreover,an embryonic stem (ES) cell line with germline chimeras has not been estab-lished in rabbits; and thus, there are no reports on production of transgenicrabbits by gene targeting.

The most ideal, but unrealistic, method of producing genetically homolo-gous animals is parthenogenetic activation of oocytes. A variety of methodshave been used to stimulate mammalian oocytes, including those of rabbits, todevelop to blastocysts at a high rate and to fetuses if transferred to recipients(1–4). But fetuses originating from parthenogenotes die near midgestationowing deficient expression of paternally derived imprinted genes (5).

One method of producing clones is the separation or bisection of preimplan-tation embryos into several groups following transfer to recipients. Identicaloffspring in a multitude of mammals, including rabbits, have been produced(6,7), but the number of identical offspring is usually limited to two.

Another method of clone production utilizes nuclear transfer (NT) technol-ogy. Cloned rabbits have been produced by NT of blastomeres from eight-cellto morula-stage embryos into oocytes whose maternal chromosomes were pre-viously removed (8–11; Table 1). The procedures for parthenogenetic activa-tion have an important role in the developmental potential of NT oocytes.Theoretically, many clones can be produced if NT oocytes that develop to the

196 Tsunoda and Kato

Table1Successful Production of NT Rabbits

No. of offspring/no.Origin of donor cells of embryos transferred (%) Reference

Preimplantion embryo 5/164 3% (8)1/85 1% (9)

23/110 21% (10)8/243 3% (11)

Adult somatic cell 6/371 2% (21)

eight-cell to morula stage are used for serial NT, (12,13). The maximum num-ber of clones actually produced, however, is less than 10.

Since the first report, where a normal sheep was produced after NT of so-matic cells obtained from an adult and cultured in vitro (14), several clonedsheep, mice, bovine, goats, and pigs have been produced (7). Although no stud-ies have examined the maximum quantity of clones produced by somatic cellNT, there may be no limit. Somatic cell NT technology is also used for theproduction of gene targeting transgenic sheep (15) and pigs (16,17). The invitro developmental potential of NT rabbit oocytes receiving somatic cells ishigh, but live cloned offspring were only recently produced (3,18–21). Clonedrabbits were recently produced after transfer of NT oocytes receiving adultsomatic cells to asynchronous, but not to synchronous recipients (21). Yet, thesuccess rate for the production of somatic cell clones, is still low, and furthertechnical improvement for NT of rabbit somatic cells is required. In this chap-ter, parthenogenetic and NT procedures are discussed.

2. MaterialsAlthough the materials used in our laboratory are listed, equivalents can

also be used.

2.1. Superovulation

1. Mature Japanese white or New Zealand white rabbits.2. Follicular-stimulating hormone (FSH).3. Human chorionic gonadotrophin (hCG).

2.2. Recovery of Oocytes and Embryos

1. Medium for in vitro maturation and in vitro culture of oocytes (complete EBSS;18): bicarbonate-buffered Earle’s balanced salt solution (EBSS) supplementedwith nonessential and essential amino acids (NEAA and EAA), 1 mML-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS).


2. TCM-199. (Gibco-BRL, cat. no. 31100-027, Eggenstein, Germany).3. FBS: inactivate the FBS at 56°C for 30 min.4. Bovine serum albumin (BSA; Sigma Chemical Co., cat. no. A-4378, St. Louis, MO).5. Hyaluronidase, (Sigma Chemical Co., cat. no. H-3506) with a final concentration

of 300 U/mL Dulbecco’s Ca2+ and Mg2+-free phosphate buffered saline (PBS), isstored at –20°C.

2.3. Donor Cell Preparation for Preimplantation Embryos

1. Medium for embryo recovery and manipulation: PBS supplemented with 20% FBS.2. 1% Pronase with 1% polyvinylpyrrolidon (PVP; Sigma Chemical, Co.cat. no.

PVP-40) in PBS: dialyze against a large volume of PBS for 1 d.

2.4. Donor Cell Preparation for Somatic Cells

1. Dulbecco’s modified Eagle medium (DMEM).2. Disruption for subculture: 0.05% trypsin and 0.05% EDTA in PBS.3. Cell proliferation kit (Amersham, code RPN 20, Buckinghamshire, UK).4. Chromosomal analysis: 0.075 M KCl, hypotonic solution, fixative solution

(methanol 3 : acetic acid 1), 3% Giemsa solution (pH 6.4), 3 μg/mL nocodazole(1000X stock solution, stored at –20°C).

5. Gelatin-coated culture dish: 0.1% gelatin solution in PBS, four-well multidish(cat. no. 176740, Nunck, Denmark).

6. Freezing medium: Cell Culture Freezing Medium-dimethyl sulfoxide (DMSO;Gibco-BRL, cat. no. 1110-011).

2.5. Preparation of Microtools

1. Microforge, MF-9 with a 10× or 20× objective (Narishige, Tokyo, Japan).2. Pipet puller, Narishige, PN-3 or Sutter Instruments, (cat. no. P-97/IVF, Novato)3. Micromanipulator system (Narishige).4. Capillary beveling device, EG-40 (Narishige).5. Glass capillary: holding pipet (G-1, 1 × 90 mm, Narishige), enucleation/injection

pipet (Sutter Instruments, cat. no. 120-90-10), needle (10 μL microdispenser,Drummond, cat. no. 510G-310G-210G).

2.6. NT

1. Inverted microscope (Nikon TE300, Tokyo, Japan or Olympus IX70, Tokyo,Japan).

2. Electrofusion system (Electro Cell fusion Model LF101, BEX, Tokyo, Japan).3. Manipulation chamber: microdrops are made on this chamber with mineral

oil (Fig. 1).4. Fusion chamber (Fig. 2): two stainless wires 1.0 mm apart attached to polished

borosilicate glass dish (93 mm inner diameter, 13 mm depth).5. Medium for micromanipulation: EBSS supplemented with 10% FBS.6. Warm/cool plate for inverted microscope (Thermoplate MATS 555 RT, Tokai

Hit, Tokyo, Japan).


Fig. 1. Manipulation chamber with two microdrops under oil.

Fig. 2. Fusion chamber with two wires and glass needle under inverted microscope.

7. 7.5 μg/mL Cytochalasin B (CB): A 1 mg/mL stock solution in DMSO, storedat –20°C.

8. Demecolcine (colcemide): A 10 μg/mL stock solution in PBS, stored at4°C.

9. 5 μg/mL Hoechst 3342 (Calbiochem-Behring, cat. no. 382065, San Diego, CA).A 1 mg/mL stock solution in ddH2Oin an opaque vessel can be stored at 4°Cfor 1 mo.


Table 2The Composition of Zimmerman Cell Fusion Medium

Compound Molarity g/L

Sucrose 0.28 M 95.84Mg(C2H3O2)2·4H2O 0.5 mM 0.107Ca(C2H3O2)2 0.1 mM 0.016K2HPO4(anh) 1.0 mM 0.174Glutathione 0.1 mM 0.031BSA 0.01 mg/mL 0.01PH 7.0

2.7. Parthenogenetic Activation

1. Fusion medium: Zimmerman cell fusion medium (Table 2) or 0.3 M mannitol,Ca2+ and M2+-free EBSS containing 3 μg/mL BSA.

2. Activation medium: complete EBSS with 2 mM 6-dimethylaminopurine(6-DMAP; Sigma Chemical Co.).

2.8. In Vitro Culture

1. Complete EBSS.2. Light mineral oil.

2.9. Embryo Transfer

1. Anesthetic: Nembutal sodium solution (Abbott Laboratories, North Chicago, IL).

3. Methods3.1. Superovulation and Recovery of Ovulated Oocytes (See Note 1)

1. Because female rabbits sometimes ovulate during transportation, it is essential tokeep them individually housed for 15 d before the superovulation treatment.

2. Inject 0.5 IU FSH into mature females subcutaneously six times 12 h apart.3. Inject 100 IU hCG intravenously 12 h after the final dose of FSH (Fig. 3).4. Flush oviducts with EBSS containing 10% FBS 14–15 h after hCG administra-

tion (Fig. 4). Then, under a microscope, collect oocytes surrounded by cumuluscells. The cumulus cells are removed by treatment with hyaluronidase.

3.2. Superovulation and Recovery of Follicular Oocytes

1. Inject 0.5 IU FSH subcutaneously six times 12 h apart.2. Remove ovaries 12 h after the final FSH dose.3. Fully grown oocytes are collected by rupturing follicles 1–3 mm in diameter in

TCM199 supplemented with 3 mg BSA/mL.4. Fewer than 50 oocytes are cultured in 500 μL of complete EBSS in an atmo-

sphere of 5% CO2 at 39°C for 10–14 h.


Fig. 3. Intravenous injection of hCG.

Fig. 4. Flushing oviduct with medium.


5. The cumulus cells are removed by hyaluronidase treatment. Oocytes with a firstpolar body are considered to be matured.

3.3. Donor Cell Preparation for Preimplantation Embryos (See Note 2)

1. The mucin coat and zona pellucida of eight-cell to morula-stage embryos areremoved by 1% pronase treatment.

2. Zona-free embryos are separated into single blastomeres by pipetting in Ca2+ andMg2+-free PBS.

3. Separated blastomeres are maintained in EBSS supplemented with 10% FBS atroom temperature until NT.

3.4. Donor Cell Preparation for Somatic Cells (See Note 3)

1. The various tissues obtained from fetus or adult are cut into 1–3-mm fragmentsand placed in 1 mL DMEM supplemented with 10% FBS in gelatin coated dishes.

2. After 7–14 d when cell growth extends to 70–90% of the bottom of the culturedishes, cells are dispersed with PBS containing trypsin and EDTA.

3. Cells are centrifuged at 300g for 5 min at 4°C; 4 mL DMEM supplemented with10% FBS is added to the precipitation.

4. One milliliter of cell suspension is placed in a fresh gelatin-coated dish (passage 1),and cells are used for NT within seven passages, with each passage occurringevery 3–5 d.

5. To induce a quiescent state, semiconfluent cells are cultured in DMEM with 0.5%FBS for 3–14 d (serum starvation) or by culture in 10% FBS for 5–21 d (contactinhibition).

6. To determine whether cells are in the mitotic phase, the BrdU incorporation isanalyzed using a cell proliferation kit following the manufacturer’s instructions.Cells are cultured in DMEM with BrdU substituted for thymidine. At the end ofBrdU exposure, cells are washed and fixed. After immunocytochemical detec-tion with reconstituted nuclease/anti-5-bromo-2'-deoxyuridine and peroxidaseanti-mouse IgG2a, specimens are immersed in diaminobenzidine (DAB) solu-tion containing substrate/intensifier. To delineate the cell borders, 0.5% eosin for2–3 min is used as a counterstain. BrdU-positive cells in S-phase are stainedblue-black. When serum-starved or contact-inhibited cells are cultured with BrdUovernight, the incorporation rate is low (0.5–1.0%).

7. The detailed cell cycle is analyzed using flow cytometry.8. To analyze chromosome constitution, cells are treated with nocodazole for 5–6 h

and cells with a round shape are collected from the culture dish. The cells arecentrifuged at 300 g for 5 min, and 2 mL hypotonic solution is added to theprecipitate and treated for 10 min at 39°C. The cell suspension is centrifuged, 2mL fixative is added, and the cells are fixed for 10 min at room temperature.After centrifugation, 2 mL fixative is added, and the cells are mounted on a slide.


After drying, cells are stained with Giemsa for 10 min to count the chromosomenumber.

9. If necessary, 1 mL freezing medium is added to the cell precipitate in a 2-mLcryotube, directly placed into a deep freezer at –70°C and can be preserved for atleast 1 mo. For thawing, cryotubes are plunged into water at 37°C for 30–45 suntil half the ice melts and the contents are immediately transferred to a tissueculture tube with 10 mL DMEM that is cooled to 4°C and then centrifuge at 300gfor 5 min. The cells are suspended in DMEM supplemented with 10% FBS andcultured.

3.5. Preparation of Microtools

3.5.1. Holding Pipet

1. Heat the glass capillary over a flame and pull rapidly by hand so that the outerdiameter is 100–150 μm.

2. Set the capillary on a microforge vertically against the platinum–iridium wirefilament.

3. Heat the filament of the microforge so that the capillary tip is reduced in size.

3.5.2. Enucleation and Injection Pipet

1. Pull thin-walled glass capillary using a pipet puller.2. Set the capillary on a microforge horizontally against the wire filament.3. Touch the tip of the capillary with an outside diameter of 10–25 μm to the glass

bead on the tip of the wire.4. Heat the wire slightly and stop the heating so that it breaks the capillary at the

point of contact.5. Set the capillary on a microforge vertically again and round the tip slightly using

the filament.6. Bevel the capillary tip at a 40°–45° angle on a rotating grinding wheel (See Note 4).

3.5.3. Needle

1. Heat the glass capillary over a flame and pull rapidly by hand. The diameter ofthe tip is not critical.

2. Set the capillary on a microforge vertically against the wire filament.3. Pull the tip of the capillary down during low heating of the wire filament.4. Pull the tip up and down two or three times during low heating of the filament,

resulting in a fine glass needle. All microtools are slightly bent (30° angle) approx1 mm from the tip by brief exposure to the heating wire, and, depending on themicromanipulator system used, one more bend must be made by hand with asmall flame.

3.6. Nuclear Transfer

Unlike in other mammals, the zona pellucida of rabbit preimplantationembryos is necessary for normal development. Therefore, the slit on the zona


pellucida during NT should be small. EBSS supplemented with 10% FBS isused as the medium during micromanipulation.

3.6.1. Removal of the Maternal Chromosomes Using the ConventionalMethod (see Note 4)

1. Ovulated or in vitro matured oocytes are incubated with 7.5 μg/mL CB supple-mented medium at 39°C for 15 min.

2. Oocytes are moved to a drop of CB-supplemented medium in a micromanipula-tion chamber (Fig. 1).

3. Hold the oocyte with a holding pipet using a micromanipulator under an invertedmicroscope (Fig. 5A).

4. Insert the beveled enucleation pipet into the perivitelline space and aspirate asmall amount of oocyte cytoplasm close to the first polar body (Fig. 5B).

5. Stain the aspirated cytoplasm individually with 5 μg/mL Hoechst for 15 min andconfirm the presence of the metaphase II chromosomes under ultraviolet light(Fig. 5C, arrow). The remaining oocyte whose aspirated cytoplasm has the chro-mosomes can be used as a recipient oocyte.

3.6.2. Removal of the Maternal Chromosomes Usingthe Chemical-Assisted Method (see Note 5)

1. When oocytes are treated with the medium supplemented with 0.6 μg/mLdemecolcine for 30 min to 1 h at 39°C, membrane protrusion, where condensedmaternal chromosomes are located, occurs (Fig. 6A,B , arrows).

2. Oocytes with a protruding membrane are moved to the medium supplementedwith 7.5 μg/mL CB and 0.6 μg/mL demecolcine, and then the protrusion is re-moved with a beveled pipet (Fig. 7A–C).

3.6.3. Donor Nucleus Incorporation

1. Oocytes whose chromosomes have been removed are washed several times withmedium and placed in a microdrop of the medium on a manipulation chamber.

2. Place the donor blastomeres or somatic cells in the second microdrop of the me-dium on the manipulation chamber (Fig. 1).

3. Aspirate donor blastomeres or somatic cells into the injection pipet and move theinjection pipet to the droplet that contains recipient oocytes (Fig. 8A).

4. Insert the injection pipet into the perivitelline space of the recipient oocytethrough the slit in the zona pellucida and introduce a single blastomere or so-matic cell (Fig. 8B,C).

5. Press the donor cell carefully against the recipient ooplasm using the tip of theinjection pipet.

3.6.4. Fusion of Donor Cell with Recipient Oocyte

1. The manipulated oocytes are allowed to equilibrate for 3 min in a 1:1 mixture ofEBSS and Ca2+-free Zimmerman cell fusion medium (see Note 6).


Fig. 5. Removal of chromosomes from oocyte (see text).

2. Move oocytes to 100% Ca2+-free Zimmerman cell fusion medium for 3–5 min.3. Move a single or several oocytes to a fusion chamber that is overlaid with fusion

medium (Fig. 9A ).4. Move oocytes so that the agglutination plane of oocytes with donor cells is ori-

ented in parallel with the electrodes using a glass needle attached to microman-ipulator (Fig . 9B).


Fig. 6. Oocytes treated with demecolcine (see text).

5. Apply two1.5 kV/cm direct current pulses for 25 μs with a 0.1-μs interval to fusedonor cells with enucleated oocytes in Ca2+-free Zimmerman cell fusion me-dium.

6. After electrical stimulation, reconstructed oocytes are washed in Ca2+ and Mg2+-free EBSS medium containing 3 mg BSA/mL and are left for 15 min (see Note 7;Fig. 9C).

3.7. Parthenogenetic Activation

1. Before parthenogenetic activation, fused oocytes are cultured for 1–2 h in com-plete EBSS supplemented with CB to facilitate nuclear remodeling.


2. Activate fused oocytes by two 1.5-kV/cm direct current pulses for 60 μs at a20-min interval in the cell fusion medium.

3. Culture activated oocytes in complete EBSS supplemented with 2 mM 6-DMAPfor 2 h in an atmosphere of 5% CO2 in air at 39°C.

3.8. In Vitro Culture

1. Wash reconstructed activated oocytes with complete EBSS several times.2. Culture oocytes in complete EBSS for 5–6 d in an atmosphere of 5% CO2 in air

at 39°C.

Fig. 7. Removal of chromosomes from oocytes treated with demecolcine (see text).


3.9. Embryo Transfer

Because the potential of rabbit embryos to develop into fetuses decreasesdrastically with the duration of in vitro culture, NT oocytes should be trans-ferred to recipient females soon after NT.

1. Inject 30 IU of hCG intravenously into recipient females 12 h before embryotransfer.

2. Intravenously inject 1.5 mL nembutal solution into a marginal ear vein.3. Shave the flank on both sides and clean with 70% ethanol.

Fig. 8. Injection of donor cell into perivitelline space of enucleated oocytes (see text).


Fig. 9. Electric fusion of donor cell with enucleated oocyte (see text).

4. After flank incision, the oviduct is pulled to the exterior by grasping fatty tissuewith forceps.

5. Aspirate NT oocytes with a minimum volume of the medium into the pipet.6. Insert the pipet into the infundibulum and gently expel the oocytes. (Fig. 10).7. A penicillin solution is sprayed into abdomen and the abdomen is closed.8. Pregnancy diagnosis is possible after d 10 of pregnancy by palpation. If neces-

sary, the presence of fetuses can be determined after d 15 of pregnancy by middledissection.


4. Notes1. Because the timing of ovulation varies from 10.5–14.0 h following leutinizing

hormone injection of (LH or hCG) (22), it is sometimes difficult to collect alarge number of rabbit oocytes soon after ovulation. When oocytes are recovered14 h after hCG injection, gently move ovaries with oviducts to the medium, rinseovaries initially to recover oocytes sticking to the surface, and then flush ovi-ducts with the medium.

2. The cell cycle of donor nuclei and condition of recipient oocytes are importantfor the normal development of NT oocytes (23). Two different combinations canbe used for successful NT. One method is to fuse donor cells at the G1/G0 stagewith oocytes of the second metaphase. The other method is to fuse donor cells atany stage of the cell cycle with previously activated oocytes. In the studies on NTof blastomeres of preimplantation rabbit embryos, no attention has been paid tothe combination of donor cell cycle and the condition of recipient oocytes.

3. The direct exposure of donor chromosomes to nonactivated oocyte cytoplasm isessential for the reprogramming of somatic cell nuclei (24).

4. When thin-walled glass capillaries are used, a sharp point on the tip of the smoothbevel is unnecessary for penetration of the zona pellucida.

5. When oocytes are treated with demecolcine for 30 min–1 h, more than 70% ofoocytes have a membrane protrusion, and the chromosome mass migrates to acortical location (25). Removing the chromosomes with a small volume of cyto-plasm is easy, and the success rate is high.

6. If manipulated oocytes are moved directly to fusion medium, some blastomerescome out from the slit in the zona pellucida.

Fig. 10. Transfer of NT oocyte into the infundibulum (see text).


7. If reconstituted oocytes are moved to EBSS containing Ca2+ and Mg2+ after elec-trical stimulation in a Ca2+-free cell fusion medium, some oocytes might be par-thenogenetically activated.

AcknowledgmentsThe authors thank to Dr. X. J. Yin for his cooperation and T. Tani for his

help in preparing the photographs of micromanipulation. This work was sup-ported by grants from the Program for Promotion of Basic Research Activitiesfor Innovative Biosciences (PROBRAIN) and Research Grants-in-Aid fromthe Ministry of Education, Science and Culture of Japan (13308054, 14034259,15039233).

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germ cell protocols volume 254 || parthenogenesis and nuclear transfer in rabbit oocytes

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