Osmolarity of culture media is one of the most important factors affecting in vitro development. This study was conducted to investigate the DNA methylation status of Pre-1 and satellite sequence in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaCl or 0.05M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. Previous our studies have reported that pNT embryos cultured in both hypertonic media showed significantly higher blastocyst formation rate than that of control. The DNA methylation status of the satellite sequences in blastocyst was characterized using bisulfite-sequencing technology. The satellite region had a similar methylation pattern of in vivo blastocyst among two culture groups excepting the control group. Each level of methylation is that the satellite DNA moderately methylated (43.10% of PZM-3; 56.12% of NaCl; 55.06% of sucrose; 60.00% of in vivo embryos). As a result of the sequence of PRE-1, CpG methylation pattern was similar to three groups, including in vivo group. In case of the satellite DNA region, the osmolarity conditions were affected CpG DNA methylation status while PRE-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. These results indicate that the modification of osmolarity in a culture media may influence to spatially change of DNA methylation of repetitive sequence for pNT embryo development.

This study was conducted to investigate the development and gene expression in miniature pig nuclear transfer (mNT) embryos produced under different osmolarity culture conditions. Control group of mNT embryos was cultured in PZM-3 for 6 days. Treatment group of mNT embryos was cultured in modified PZM-3 with NaCl (mPZM-3, 320 mOsmol) for 2 days, and then cultured in PZM-3 (270 mOsmol) for 4 days. Blastocyst formation rate of the treatment group was significantly higher than the control and the apoptosis rate was significantly lower in treatment group. Bax- and caspase-3 mRNA expression were significantly higher in the control than the treatment group. Also, the majority of imprinting genes were expressed aberrantly in in vitro produced mNT blastocysts compared to in vivo derived blastocyst H19 and Xist mRNA expression were significantly lower in the control than the treatment group or in vivo. IGF2 mRNA expression was significantly higher in the control than the treatment group or in vivo. IGF2r mRNA expression was significantly lower in the control. Methylation profiles of individual DNA strands in H19 upstream T-DMR sequences showed a similar methylation status between treatment group and in vivo. These results indicate that the modification of osmolarity in culture medium at early culture stage could provide more beneficial culture environments for mNT embryos.

In vitro development of porcine embryo is affected by culture condition. One possible factor is osmolarity of culture medium. This study examined whether high osmolarity of culture medium at the early culture stage improves development of preimplantation porcine in vitro fertilization (IVF) and nuclear transfer (NT) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (250～270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sorbitol or 0.05 M sucrose (300～320 mOsmol, sorbitol or sucrose group) for the first 2 days, and then cultured in PZM-3 for further culture. NT embryos cultured in sucrose group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the sorbitol (p<0.05). For IVF, sucrose group showed a significantly increased the blastocyst formation rate with a decreased apoptosis rate compared to the control (p<0.05). This study represents that the high osmolarity in the early embryo culture stage can enhance the in vitro development of porcine NT and IVF embryos to the blastocyst stage with reduced apoptosis of cells.

본 연구는 배양액의 종류에 따른 돼지 난자의 성숙 및 단위발생란의 배반포 형성율을 검토하였으며, 배양액의 삼투압과 발달 시기에 따른 배양액의 삼투압 변화가 돼지 단위발생란의 발달에 미치는 영향을 검토하였다. 실험 1에서 난포란을 NCSU-23 mWM 및 mKRB에 각각 성숙배양한 결과 성숙률은 62.1~71.3%로 배양액에 따른 차이가 없었다. 실험 2에서는 각각의 배양액으로 성숙된 난자를 활성화 처리 후 동일한 배양액으로 6일간 배양하여 발달율을 검토한 결과, 배반포 발육율은 NCSU-23에 배양 시 22.9%로 타 그룹(0~0.6%)보다 유의적으로 높게 나타났다(P<0.05). 실험 3에서는 단위발생란을 NaCl 안에 의해 256, 280 및 300 mOsmol(mOsm)로 조정한 NCSU-23에 6일간 배양한 결과, 배반포 발육율은 11.0~14.4%로 실험군 간에 유의적인 차이는 없었으나 삼투압이 낮을수록 난자의 fragment 비율이 높게 나타났다(P<0.05). 실험 4에서는 단위발생란을 삼투압이 조정된 세 종류의 NCSU-23에 48시간 배양한 후 삼투압이 높거나 낮은 NCSU-23으로 옮겨 4일간 추가 배양한 결과 배반포 형성율은 배양 48시간 후에 배양액의 삼투압을 낮춰 주었을 때(21.0%)가 높여 주었을 때 (11.8%) 보다 유의적으로 높게 나타났다(P<0.05).본 실험의 결과는 돼지 단위발생란의 발육이 배양액의 종류 및 삼투압에 의해 영향을 받으며, 배양액의 삼투압은 돼지 단위발생란의 발육 단계별로 영향을 주어, 초기에는 높은 삼투압의 배양액에서 배양하고 일정 시간 후 낮은 삼투압의 배양액으로 배양함으로써 발육이 증진될 수 있음을 시사한다.