Monozygotic twin embryos which can efficiently be produced by blastomere separation and aggregation of early cleavage stages of embryos using commercially provided well-of-the-well (WOW) culture dish. Phytohaemagglutinin (PHA) is a plant lectin that binds to and aggregates on the surface of animal cells, but also contains toxicity that causes food poisoning. The present study was conducted to evaluate the toxicity to embryos and the effect to development of isolated blastomeres on PHA-supplemented WOW culture. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and invitro culture (IVC). The tissue culture medium 199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10mgmL−1 bovine serum albumin, and CR1aa medium containing 5% CS were used for each culture step. For the evaluation of PHA toxicity, 89 embryos that developed to the 5-8-cell stage were obtained at Day 2 after insemination. Each embryo was cultured in a droplet of 5 µL/embryo IVC culture medium supplemented with or without PHA. For the evaluation of PHA to development of isolated blastomeres, 111 of 8-cell stage embryos were obtained 48-54h post-insemination. Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into single blastomeres by gentle pipetting in IVC medium. Each four blastomeres were formed in the shape of a bunch inside the thin cylinder at the tip of the Pasteur pipette by gentle pipetting. Then, each mass of blastomeres in each 60 masses was cultured individually in 5-µL droplets of IVC medium supplemented with or without PHA on the flat surface of a tissue culture dish. On the other hand, each four blastomeres were introduced into a single conical micro-well each having a diameter and depth of ~287µm and 168µm (Dai Nippon Printing). This culture of blastomeres was performed covered with a droplet of 2.5µL well−1 IVC medium supplemented with or without PHA in each 50 or 52 wells. In all of investigations, PHA was used at 50µgmL−1 (Akagi et al. 2011 J. Reprod. Dev. 57). Statistical analysis was performed using Student's t-test and analysis of variance. The blastocyst formation rate (71.1±2.3% vs. 72.7±1.7%), total cell number (120 vs. 122), and inner cell mass cell number (47 vs. 51) at Day 7 after IVF did not differ between PHA-supplemented and PHA-free group in the toxicity test, respectively. In the blastomere culture, the blastocyst formation rate was very low (10.0±5.9% vs. 5.0±2.9%) regardless of the PHA supplementation in drops on the flat surface of a tissue culture dish. On the other hand, blastocyst formation was improved using the WOW culture dish (24.0±3.6% vs. 40.4±7.6%) but there was no difference with or without PHA supplementation. Although nontoxicity of PHA and efficacy of WOW culture for isolated-aggregated blastomeres were confirmed, no improvement of PHA supplementation on development was observed in this study. Subsequently, experiments on the optimum concentration of PHA for aggregation and development of blastomeres in WOW culture are required.
Somatic Embryogenesis and Plant Regeneration from Immature Persimmon (Diospyros kaki Thunb.) Embryos
