H. Kanagawa

Viability of Vitrified Mouse Embryos Using Various Cryoprotectant Mixtures

Abstract Mouse morulae and blastocysts were cryopreserved by vitrification using six types of solutions. Each solution was composed of two types of cryoprotectants, glycerol (GL) + ethylene glycol (EG), GL + propylene glycol (PG), GL + dimethyl sulfoxide (DMSO), EG + PG, EG + DMSO, and PG + DMSO at an each cryoprotectant concentration of 25% v/v. Embryos were exposed to each type of vitrification solutions, which had been diluted 50% in PBS, for 5 minutes at room temperature, then for another 5 minutes at 4 °C. The embryos were loaded into straws containing vitrification solution at 4°C and plunged into liquid nitrogen within 30 seconds. After warming in water at 0°C and following one-step dilution of the cryoprotectant in 0.5 M sucrose+PBS, the embryos were cultured in vitro. The survival rates of morulae were 51, 16, 78, 44, 79 and 50%, respectively, for the six solutions. The survival rates of the morulae using GL + DMSO and EG + DMSO were significantly higher than those of the other solutions (P

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos.

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose.

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.

Development of reconstituted mouse embryos produced from bisected and electrofused pronuclear-stage embryos

The present study was designed to investigate the in vitro and in vivo development potential of reconstituted mouse embryos produced by bisection and electrofusion of pronuclear stage embryos (PN-E). Pronuclear-stage ICR and F1 (C57BL x CBA) strain mouse embryos were bisected manually with a fine glass needle under the dissecting microscope to produce karyoplasts (KP) and cytoplasts (CP). The KP of ICR PN-E and CP of F1 PN-E (KP: ICR + CP:F1) or the KP of F1 PN-E and CP of F1 PN-E (KP:F1 + CP:ICR) were attached using phytohemagglutinin-P (PHA-P) and then electrofused. High fusion rates of the KP and CP of PN-E were obtained (93.5%). The fused embryos were encapsulated in alginate gel and cultured for 72 or 96 hours. The cleavage rates of reconstituted embryos were also high (98.8%). Developmental rates to the blastocyst stage in vitro for the 96-hour culture of reconstituted embryos were 68.9% (KP:ICR + CP:F1) and 78.4% (KP:F1 + CP:ICR). Furthermore, the developmental ability of reconstituted embryos in vivo was investigated, and some live young were obtained (KP:ICR + CP:F1, 7.5% and KP:F1 + CP:ICR, 10.8%). In this study, it was confirmed that reconstituted embryos produced by bisection and electrofusion of pronuclear stage embryos were able to develop into blastocysts in vitro and into live young in vivo.

Influences of electrode geometry and field strength on the fusion and subsequent development of mouse two-cell embryos

Abstract The electrofusion and subsequent development of mouse 2-cell embryos were studied with respect to electrode geometry and field strength using a wire chamber (W-chamber) or a rectangular chamber (R-chamber) with 1-mm gaps. Field strengths of 1.0–2.5 kV cm−1 were examined. Fusion rates were high (92.6±2.4–98.9±1.1%) regardless of electrode geometry and field strength. However, when an R-chamber was used, significantly lower development rates were obtained at field strengths of 2.0 and 2.5 kV cm−1. Fusion progressed quickly using the R-chamber and applying the higher field strengths. Blastocyst formation by fused embryos in the R-chamber was faster than in the W-chamber and control embryos. In the R-chamber, the number of embryos with visible vacuoles increased, especially at 2.0–2.5 kV cm−1 (4.9-8.8%), of which 78.6% lysed at the one- or two-cell stage. Most of the fused embryos had a tetraploid chromosome constitution, but eight embryos (1.6%) were diploid. The mean cell number of tetraploid blastocysts was about half that of diploid control embryos, and was reduced with increasing field strength in the R-chamber. These results suggest that the W-chamber can be successfully applied across a relatively wide range of field strengths, providing stable conditions for fusion and subsequent development. However, when the R-chamber is used, the developmental capacity of fused embryos can be affected by an increase in field strength.

Fusion and development rates of single blastomere pairs of mouse two- and four-cell embryos using the electrofusion method

The present study was undertaken to find suitable conditions for blastomere fusion of mouse two- and four-cell embryos using the electrofusion method to simplify the nuclear transfer procedure. Single blastomeres of ICR and F1 (C57BL/6J x CBA/N) two-cell embryos or ICR four-cell embryos and F1 two-cell embryos were paired and treated with electric stimulus under different fusion conditions. Two hours after electrofusion treatment, the fused blastomere pairs were encapsulated in alginate gel and cultured for 96 hours to observe their developmental potential. When the single blastomere pairs of two-cell embryos were exposed to electric pulses of 1.0, 1.5 and 2.0 kV/cm for 30, 60 and 90 mu sec, high fusion rates were obtained (84.6 to 100%). However, when two-cell blastomere were paired with four-cell blastomere and then treated under the same conditions, the fusion rates (27.5 to 87.5%) were lower than that of single blastomere pairs of two-cell embryos regardless of the duration and strength of the d.c. pulses. The blastocyst developmental rate after in vitro culture of the fused blastomere pairs of two-cell embryos using the above electrofusion conditions was high (81.8 to 100%). Lower blastocyst developmental rates were obtained on the fused blastomere pairs of two- and four-cell embryos (46.4 to 76.2%). Based on the results of this study, a pulse duration of 60 mu sec and a pulse strength of 1.0kV/cm were the most suitable conditions for single blastomere pair fusion of two-cell or two- and four-cell embryos. The study further showed that alginate gel is a good substitute for zonae pellucidae for encapsulating zona-free embryos.