GoKawiilAnimals
All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation of the RIKEN Centre for Biosystems Dynamics Research. C57BL/6 (B6), JF1/Ms (JF1), BDF1 (C57BL/6 × DBA/2) and R26R-H2B-EGFP+/− (C57BL/6 × DBA/2 background)43 mice, aged 8–10 weeks, were used to produce oocytes and sperm. Surrogate pseudopregnant females used as embryo transfer recipients (see ‘Embryo transfer’) were ICR strain mice mated with vasectomized males of the same strain. BDF1 mice were purchased from Japan SLC Inc.
Oocyte collection
Mature oocytes were collected from the oviducts of 8- to 10-week-old female mice that had been induced to superovulate with 5 IU of equine chorionic gonadotropin (ASKA Pharmaceutical) followed by 5 IU of human chorionic gonadotropin (ASKA Pharmaceutical) 48 h later. Cumulus-oocyte complexes were collected from the oviducts approximately 16 h after human chorionic gonadotropin injection. Cumulus-oocyte complexes were placed in M2 medium and treated with 0.1% (w/v) bovine testicular hyaluronidase. After several minutes, the cumulus-free oocytes were washed twice and then transferred to Chatot, Ziomek and Bavister medium (CZB). Mature MII oocytes were subjected to ICSI.
Micromanipulation
For cytoplasmic removal (Extended Data Fig. 3a,b), micromanipulation was performed as described previously44 with some modifications. Briefly, oocytes at the MII stage were transferred to M2 medium supplemented with 5 μg ml−1 cytochalasin B (Sigma-Aldrich) for 10 min. Then, the zona was cut using the LYKOS laser system (Hamilton Thorne) in a micromanipulation chamber placed on a warmed stage (37 °C) in an inverted microscope (Olympus). After cutting the zona, we held each oocyte with a holding pipette at the 9-o’clock position, and then rotated the oocyte until the hole of the zona was at the 3-o’clock position. After inserting a fire-polished injection pipette (inner diameter 30 µm) through the hole of the zona, we aspirated half the cytoplasmic volume and pinched off (Supplementary Video 3). The volume of the aspirated cytoplasm was controlled by an ocular micrometer. To obtain control oocytes, we aspirated half the total cytoplasmic volume and returned it to the oocyte.
To generate doubled oocytes (Extended Data Fig. 3a,b), we performed micromanipulation based on previously described methods45 with some modifications. Briefly, oocytes at the MII stage were transferred to M2 medium supplemented with 5 μg ml−1 cytochalasin B. A hole was made in the zona pellucida by applying several piezo pulses using an enucleation pipette. The MII spindle was aspirated into the pipette with a minimal volume of ooplasm. After enucleation, oocytes were cultured in CZB medium for at least 15 min for recovery. The enucleated oocytes and intact oocytes were treated with acid Tyrode’s solution (pH 2.1–2.5, Irvine Scientific) for 30 s to dissolve the zona. After several washes, the zona-free oocytes were cultured in CZB medium for at least 30 min, and then transferred to M2 medium containing 0.05 mg ml−1 phytohaemagglutinin (Wako Pure Chemical Industries) for 1 min. The oocytes were transferred to M2 medium on a micromanipulation chamber, in which an intact oocyte and an enucleated oocyte were attached to each other by micropipettes. The attached oocytes were cultured in CZB medium for at least 15 min, washed three times with fusion medium (0.3 M mannitol, 0.1 mM MgSO 4 and 0.1% polyvinyl alcohol) then transferred to the same solution placed between parallel electrodes and separated by 0.5 mm in a chamber. Current was applied from an electric cell fuser (LF101; BEX Co.) (15 V alternate current at 1 MHz for 1 s to line up the attached oocytes, 30 V direct current pulse for 20 µsec to induce cell fusion). The oocytes were washed and then cultured in CZB medium for 30 min for recovery and cell rounding (Supplementary Video 4).
Fertilization
IVF was performed according to the manufacturer’s instructions using CARD MEDIUM (Cosmo Bio). To limit the time of fertilization, we set the insemination time to 1 h.
ICSI with sperm heads was carried out as described previously46. Briefly, the sperm head was separated from the tail by applying several piezo pulses to the neck region, and then injected into an oocyte. After 20 min of recovery at room temperature, injected oocytes were cultured in CZB.
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