Although somatic cell nuclear transfer (SCNT)-derived embryonic stem cells (ESCs) in pigs have great potential, their use is limited because the establishment efficiency of ESCs is extremely low. Accordingly, we tried to develop in-vitro culture system stimulating production of SCNT blastocysts with high performance in the colony formation and formation of colonies derived from SCNT blastocysts for enhancing production efficiency of porcine ESCs. For these, SCNT blastocysts produced in various types of embryo culture medium were cultured in different ESC culture medium and optimal culture medium was determined by comparing colony formation efficiency. As the results, ICM of porcine SCNT blastocysts produced through sequential culture of porcine SCNT embryos in the modified porcine zygote medium (PZM)-5 and the PZM-5F showed the best formation efficiency of colonies in α-MEM-based medium. In conclusion, appropriate combination of the embryo culture medium and ESC culture medium will greatly contribute to successful establishment of ESCs derived from SCNT embryos.

Dormant blastocysts during delayed implantation exhibit heightened autophagic activation. Activation of autophagy, the self-eating process within cells, was suggested as an adaptive response to unfavorable environment of prolonged survival in utero. During the course of this study, we observed by transmission electron microscopy that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation of implantation by estrogen. MVBs are the late endosomes which are characterized by the presence of diverse internal vesicles within a large vesicle. Autophagosomes fuse with MVBs during autophagic activation, and efficient autophagic degradation requires functional MVBs. Biogenesis of MVBs depends on a dynamic network of ESCRT complexes 0, I, II, and III. Tsg101 (a component of the ESCRT-I complex) and CD63 are often used as a marker of MVBs. Lysobisphosphatidic acid (LBPA) is an abundant lipid in MVBs and required for the formation of MVBs. In this study, we performed immunofluorescence staining for detection of MVB makers in dormant and activated embryo. In dormant blastocysts, expression of Tsg101 and LBPA exhibited a uniform pattern throughout the trophectoderm. In contrast, expression of both markers prominently increased in the mural trophectoderm of activated blastocysts. To investigate the relationship with MVB formation and autophagy activation in activated blastocyst, 3-MA, a widely used inhibitor of autophagy, was daily injected intraperitoneally to ovx mice. Interestingly, 3-MA injection to block autophagy during delayed implantation led to a reduction of the signal of MVB markers, suggesting that prolonged activation of autophagy in dormant blastocysts is associated with MVB formation upon activation of implantation. Collectively, these results show that expression of MVB makers increase in the trophectoderm of blastocysts upon activation of implantation and that the formation of MVB is associated with heightened autophagy during delayed implantation.