Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro‐matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM‐199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1 kV/cm for 30 μs in 0.3 M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium‐3 (PZM‐3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM‐3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine‐porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat‐porcine and porcine‐bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

Porcine fibroblasts were transferred into enucleated bovine oocytes for the interspecies nuclear transfer (NT). After NT, the embryos were cultured in three different culture systems. The media used for the experiment were CR1aa and NCSU23. The culture systems used for the experiment were: 1. Culture in CR1aa for 7 days (CR). 2. Culture in CR1aa for 2 days and subsequently in NCSU23 for 5 days (CR-NC). 3. Culture in NCSU23 for 7 days (NC). Bovine (intraspecies) NT group was used as a control. The oocytes in bovine NT group were treated the same as interspecies NT embryos except using bovine fibroblasts as nuclear donors. Regardless of their nuclear origin (interspecies vs bovine), the embryos in CR (68.4% vs 77.2%) and CR-NC (67.8% vs 70.5%) showed better developmental competence to the 2-cell stage (p<0.05) than those in NC (41.0% vs 10.0%). Bovine NT embryos in CR-NC did not develop over the 4-cell stage after the medium replacement, while interspecies NT embryos in CR-NC continued to develop and could reach over the 8-cell stage (12.2%). Blastocysts were only found in bovine NT group (17.4%), but no blastocyst was found in interspecies NT group. This study suggests that the development of interspecies NT embryos mostly depends on their recipient cytoplasm during the culture in vitro.

본 연구는 이종간 핵이식란의 생산성 향상에 기여하기 위한 기초연구로서 핵이식 수정란의 융합과 활성화 과정에 있어서 수핵난자 및 전기적 융합조건이 핵이식 수정란의 융합 및 체외 발달에 미치는 요인들을 조사하기 위하여 실시하였다. 도축되어지는 소 및 돼지의 난소에서 난포란을 채취하여 TCM-199 및 NCSU-23에 혈청 및 호르몬을 첨가하여 39℃, 5% CO₂ 배양기내에서 24 및 48시간 체외성숙을 실시하여 수핵난자를 준비하고, 공여세포의 준비는 산양의 귀세포를 채취하여 0.25% Trypsin-EDTA의 처리로 세포를 분리, 배양하여 사용하였으며, 계대배양과 함께 세포는 TCM-199 ＋ 10% FBS ＋ 10% DMSO로 동결을 실시하였다. 핵이식은 성숙된 난자의 극체 및 전핵을 laser system으로 투명대를 drilling 하여 제거하고 준비된 공여세포를 핵이 제거된 난자에 주입하여 전기적 자극으로 융합을 실시하여 융합된 난자는 전기적 자극으로 활성화를 유도하였다. 활성화가 이루어진 복제 수정란은 수핵란이 소난자의 경우 monolayer가 형성된 10% FBS가 첨가된 TCM199 배양액에서 7∼9일 동안 체외배양하였으며, 수핵란이 돼지의 경우 10% FBS가 첨가된 NCSU-3 배양액으로 6∼8일 동안 체외배양을 실시하여 배반포기로 유도하였다. 본 연구에서 얻은 결과를 요약하면 다음과 같다. 전기자극의 세기를 1.95 kv/cm와 2.10 kv/cm로 주었을 때 수핵란이 소 난자의 경우 융합율은 47.7및 44.6%였으며, 분할율도 41.9 및 54.5%로써 차이가 없었다. 수핵란이 돼지 난자인 경우는 융합율은 51.3 및 46.1%로써 차이가 없었으며, 분할율도 75.0및 84.9%로써 차이가 없었다. 전기자극 시간을 30 또는 60μsec, 횟수는 1 또는 2회 주었을 때 수핵란이 소 난자의 경우 융합율은 30 μsec 1회(50.8%) 와 2회(31.0%) 간에 차이가 없었으나, 60μsec 1회(19.3%)가 가장 낮았다(P＜0.05). 융합란의 분할율은 30μsec 1회(53.3%)와 2회(50.0%) 간에 차이가 없었으나, 60μsec 1회(18.2%)보다 유의적(P＜0.05)으로 높게 나타났다. 돼지 난자의 경우 융합율은 30μsec 1회(48.1%), 2회(45.2%)및 60μsec 1회(48.6%)간에 차이가 없었으며, 분할율은 30μsec 1회(78.4%)와 60μsec 1회 (79.4%)간에 차이가 없었으나, 30μsec 2회(53.6%)보다 유의적(P＜0.05)으로 높게 나타났다. 이종간 핵이식란의 체외발달에 있어서 수핵란이 소 난자의 경우 상실배와 배반포기로의 발달율이 22.6%로써 단위발생란 30.6%와 차이가 없었으며, 돼지 난자의 경우는 이종간 핵이식란이 5.1%로써 단위발생란 37.4%보다 유의적(P＜0.05)으로 낮게 나타났다. 이상의 실험결과로 보아 산양의 체세포를 이용한 이종간 핵이식 복제수정란의 생산을 위하여 수핵란으로 소와 돼지를 사용하여 복제수정란의 발달을 확인할 수 있었으며, 이종간 핵이식에 있어서 수핵란, 공여세포, 융합, 활성화 및 배양조건 등 아직 초보 수준에 있으며, 앞으로 보다 많은 연구를 통하여 이러한 문제들이 해결되면 멸종위기 상태에 있는 동물들의 종 보존에도 활용이 가능할 것으로 생각된다.

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 ㎍/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at 39℃, 5% CO₂ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).