T. Kono

Ovicidal effects of vitrification solution and the vitrification-warming cycle and establishment of the proportion of toxic effects on nuclei and cytoplasm of mouse zygotes

The present study was conducted to compare the resistance of the nuclei with that of the cytoplasm of mouse zygotes to damage during the vitrification-warming cycle using the technique of pronuclear transplantation. Zygotes were collected from the oviduct of superovulated F1 female mice mated with males of the same strain. They were cryopreserved by the vitrification method. After being diluted with glycerol-sucrose PB1 solution, 86% of the recovered zygotes were morphologically normal and 80% of them developed to the two-cell stage in vitro, but the proportion of zygotes which developed to blastocysts was only 27%. When zygotes were exposed to VS1 solution in the same manner as above without cooling, 61% of them developed to blastocysts. In order to examine the source of injury during vitrification, the pronuclei of vitrified zygotes were transferred into enucleated fresh zygotes and vice versa. The developmental rate of blastocysts from vitrified zygotes that were enucleated and fused with pronuclei from fresh zygotes was significantly higher than that of zygotes reconstituted reversely. These findings suggest that nuclei are apparently damaged more than cytoplasm by the vitrification-warming cycle and the toxicity of VS1 solution.

Parthenogenetic activation by electric stimulus of bovine oocytes matured in vitro

The purpose of this study was to determine the optimal conditions for parthenogenetic activation of in vitro-matured bovine oocytes by electric stimulus in vitro. Oocytes were assigned to a factorial treatment structure with direct current ranging from 0.5 to 1 KW/cm for 25 to 100musec and single or double pulses. The optimal conditions for activation were found to be direct current pulses of 1 KV for 25 musec x 2, under which 84% of stimulated oocytes formed one (70%), two (13%) or three (2%) pronuclei. When the stimulated oocytes were incubated in a culture medium containing cytochalasin B, 80% of the oocytes formed two pronuclei. A proportion of the parthenogenetic oocytes developed to the two-cell stage or higher (27%, 83 312 ) in vitro; however, this was significantly (P<0.001) lower than that of the oocytes fertilized in vitro (46%, 736 1608 ).

Culture and Freezing of the Mouse and Rabbit Embryos using Commercial Tissue Culture Medium GIT

市販の細胞培養用汎用培地GITが, マウスおよびウサギ胚の培養液あるいは凍結用保存液として使用できるかどうか検討した.その結果, GITはマウス初期胚の培養液としては適さないが, ウサギ胚では20%同種非働化血清を添加した培養液の成績と同等な結果が得られたことから培養液として利用でき, またマウス胚の凍結用保存液としても利用できる可能性のあることが示唆された.