Long-term culture induces Bax-dependent apoptosis in rat preimplantation embryos.

Although rat preimplantation embryos are necessary for producing genetically modified rats, their in vitro culture remains a challenge. Rat zygotes can develop from the one-cell stage to the blastocyst stage in vitro; however, long-term culture reduces their developmental competence via an unknown mechanism. In this study, we examined how in vitro conditions affect rat preimplantation embryos, which may explain this reduced competence. Comprehensive gene expression analysis showed that genes related to apoptosis and energy metabolism were differentially expressed in rat embryos cultured long-term in vitro compared with those developed in vivo. Furthermore, we found that the expression of Bak1 and Bax, which are responsible for mitochondrial outer membrane permeabilization, were more upregulated in embryos cultured in vitro than those developed in vivo. Similarly, apoptosis-dependent DNA fragmentation was also exacerbated in in vitro culture conditions. Finally, gene disruption using CRISPR/Cas9 showed that Bax, but not Bak1, was responsible for these effects. These findings suggest that long-term in vitro culture induces Bax-dependent apoptosis through the mitochondrial pathway and may provide clues to improve the long-term culture of rat preimplantation embryos for genetic engineering research.