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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.
During oogenesis, mammalian oocytes undergo two subsequent meiotic divisions that result in a single, haploid egg. The first meiotic 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 chromosomes into the second polar body are incited by sperm entry at fertilization. Transplantation of genetic material between mammalian oocytes offers many opportunities to study various aspects of nuclear-cytoplasmic interactions during oogenesis, fertilization and embryo development1,2. 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 mitochondrial diseases, caused by mutations in mitochondrial DNA (mtDNA), from affected women to their children3. 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 women4. 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 accomplished 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 zygotes6. 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, transplantation 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 space6–9. 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 mitochondria are evenly distributed in MII oocytes, and that chromosome transfer does not cause any detectable mtDNA heteroplasmy in resulting embryos and offspring10.
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 microscopes. 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 manipulations. Early attempts to transfer MII chromosomes in human MII oocytes resulted in limited success because of low fertilization rates, pronuclear anomalies and poor embryo development11,12. The developmental potential of such embryos was only monitored in vitro by blastocyst formation rates.
We recently implemented several methodological advances during manipulation of rhesus macaque MII oocytes to surmount these biological barriers10. 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 implemented to avoid spontaneous activation and premature segregation 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 cells10. 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 chromosomes 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 similarities in oocyte biology and embryo development between rhesus macaques and humans, described protocols are directly applicable 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 protocols 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 culture 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.
Rhesus macaque MII-stage oocytes and semen were used (detailed protocols for controlled ovarian stimulations, oocyte and sperm collections are available 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.
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 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.
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 usually 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 meticulously tested in our laboratory over the years and alternative sources may not produce desirable results.
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.
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.
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% for a minimum of 4 h before use. This medium is used for culture of CO2 monkey oocytes and embryos up to the eight-cell stage.
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% CO2 for a minimum of 4 h before use. This medium is used for culture of monkey embryos from the eight-cell stage to blastocysts.
Reconstitute a vial of lyophilized 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.
To prepare 1,000× stock solution of cytochalasin 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.
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.
The micromanipulation station consisting of an Olympus inverted microscope, Narishige micromanipulators, 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 micropipette 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.
Place two 20-μl micromanipulation 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.
All techniques described above will require certain micromanipulation skills, necessitating diligent practice before attempting 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 successfully 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 a karyoplast with a minimum amount of cytoplasm. To avoid mtDNA carryover during chromosome transfer, we recommend 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 practiced 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 pulses10. 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.
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.
Note: Supplementary information is available via the HTML version of this article.
AUTHOR CONTRIBUTIONS S.M., M.S. and M.T. developed and wrote this protocol.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
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