We investigated the lethal effect of
Ay gene in embryos at the preimplantation stage
in vitro. First, the development until the blastocyst stage and the division of individual cells from 8-cell stage embryos were examined. No difference in development was detected between embryos from the experimental cross (
Ay/a ×
Ay /a) and those from the control cross (
a/a ×
a/a). Therefore, it seems that the abnormality of the
A y/
Ay embryo does not appear until blastocyst formation
in vitro. We subsequently examined the hatching from zona pellucida of the blastocysts. The hatching ratio of the embryos from the experimental cross was significantly lower than that of the control crosses (
Ay/a ×
a/a, a/a ×
a/a: p<0.05). Our observation indicates that deficiency of the
A y/
Ay embryo can be detected
in vitro at hatching. In order to elucidate the mechanism of the gene action of the
Ay, we attempted to rescue the lethal embryos from decreased hatching ratio
in vitro. When dbcAMP at the concentration of 1 mM was added to the culture medium, the hatching ratio of blastocysts from the experimental cross increased until the level of those from the control crosses. Since this result indicates that the cAMP content in
Ay homozygote seemed to be lower than those in
a/a and
Ay /a, the cAMP content in individual blastocyst was quantified. It is found that
Ay homozygosity was associated with lower level of cAMP. When adenylate cyclase was activated by forskolin and cholera toxin, the hatching,ratio was increased. These results seem to suggest that
Ay homozygote embryos posses a defect in signal transduction system mediated by adenylate cyclase during hatching.
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