체외 환경에서 생산되는 배아 (Embryo)는 활성산소종 (Reaction oxygen species, ROS) 수준이 일정 수준을 초과함에 따라 산화적인 손상을 받게 된다. 선행연구에 따르면, 항산화제는 ROS를 감소시켜주는 효과를 가지기 때문에 ROS로부터 오는 배아의 단백질, DNA의 손상, 세포 자멸사를 방지하여 배아의 발달률을 향상시킨다. 이전연구에 따르면 항산화제로써 엘라그산 (Ellagic acid, EA)은 ROS를 효과적으로 제거하고, 난자의 산화스트레스를 방지하는 효과를 가지고 있다고 보고되었다. 그리하여, 본 연구를 통해 우리는 소의 수정란 배양체계 중 in vitro culture (IVC) 단계에서 EA의 농도 (0, 5, 10 μM) 별 첨가가 소의 수정란 발달률과, 질적 수준에 미치는 영향을 조사하고자 실험을 진행하였다. 결과적으로, 배반포의 단계별 발달 수준에서 cleavage 형성률은 EA첨가군과 대조군 간의 차이를 발견할 수 없었으나 배반포 형성률에서는 모든 EA 첨가군들이 대조군보다 높았고 EA 첨가군 중에 5 μM 첨가군이 가장 높았다 (p < 0.05). 생산된 배반포의 총 세포 수는 5 μM EA 첨가군이 대조군과 10 μM EA 첨가군 보다 유의적으로 높았으며, 대조군과 10 μM EA 첨가군 사이의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 137 ± 7.90 vs. 163.2 ± 7.42 vs. 138.8 ± 6.67, p < 0.05). 세포 자멸사 세포 수는 모든 EA 첨가군들이 대조군보다 유의적으로 낮았다 (Control vs. 5 μM vs. 10 μM; 22.65 ± 4.08, 9.61 ± 1.55, 6.14 ± 0.90, p < 0.05). ROS 수준에서 모든 EA 첨가군들과 대조군 간의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 6.81 ± 1.31, 3.86 ± 0.23, 4.11 ± 0.18, p < 0.05). qRT-PCR 실험 결과에서 Nrf2 gene expression은 대조군과, 5 μM 첨가군에서 유의적 차이가 없었으나, 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다. Keap1 gene expression은 5 μM 첨가군에서 유의적으로 하향 조절된 것을 관찰하였다. 하지만 EA의 농도가 10 μM으로 높아짐에 따라 발현 수준이 증가한 것을 관찰할 수 있었다. CAT gene expression은 5 μM 첨가군에서 유의적으로 상향조절 되었으나 10 μM 첨가군에서는 유의적인 차이를 보이지 않았다. SOD1 gene expression은 대조군과 5 μM 첨가군은 유의적인 차이를 보이지 않았으나 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다.

Matrix Metalloproteinases (both MMP2 and -9) play a pivotal role of the embryos hatching and implantation. Therefore, the objective of this study was carried out to investigate the influence of MMP2 and MMP9 on embryo development potential and subsequent effect at molecular level. There was no significant difference of cleavage rate among the groups. The development competence of blastocyst was significantly higher (P<0.05) in MMP9 treatment (39.81±16.61) than that to the combined treatment of MMP2 and –9 (23.68±0.27), but there was no significant difference among the control vs. MMP2 vs. MMP9 (35.05±2.74 vs. 32.71±6.18 vs. 39.81±16.61, respectively). On the other hand, the hatching rate of blastocysts was significantly lower (P<0.05) in combined group of MMP2 and –9 (12.55±0.09) (Table1). The expression level of MMP2 and MMP9 was significantly lower (P<0.05) in the entire treatment groups than that in the control group. But the expression of MMP9 was significantly higher (P<0.05) when compared in the entire treatment groups. The relative expression embryonic developmental gene, IFNt expression level significantly lower (P < 0.05) in the MMP9 embryos. The placenta establishment genes, PLAC8 and SSLP1, expression were significantly higher (P < 0.05) in the MMP2 embryos compared to other groups. Transcription regulation gene, HNRNPA2B1, was higher (P < 0.05) in the combined group of MMP2+MMP9 than that in the other groups. In conclusion, our results suggest that MMPs to culture medium improves the blastocyst development rate and further impact on target gene expression analysis.

Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups (41.46 ± 10.66 vs. 37.73 ± 8.92 vs. 45.11 ± 11.41% vs. 41.59 ± 11.88, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups (79.84 ± 12.63 vs. 83.3 ± 17.46 vs. 78.55 ± 14.48% vs. 72.02 ± 14.09). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.