The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

염색체의 구조적 이상으로 인한 습관성 유산과 기형아의 출산을 예방하기 위해 착상전 배아에서 할구를 분석하여 정상적인 핵형을 가진 배아만을 이식하는 착상전 유전자 진단 (preimplantation genetic diagnosis, PGD)의 성공적인 임상 적용이 보고되고 있으며, 그 적용 범위가 확대되고 있다. 그러나 일반적인 간기의 핵상을 이용한 PGD에서는 형광직접보합법 probe의 제약으로 보인자와 정상적인 핵형을 구분할 수 없는 단점이 있다. 따라서 본 연구에서는 보다 정확한 PGD를 위해 생쥐 배아를 이용하여 분리한 할구에서 중기 염색체상을 획득하기 위해 미세소관 (microtubule) 형성 저해제를 처리하였으며, 이를 통해 확립된 방법을 인간의 PGD에 적용하고자 하였다. 과배란이 유도된 ICR 생쥐에서 4- 또는 8-세포기 배아를 수획하여 colcemid, nocodazole, vinblastine을 각각 0.1, 0.5, 1.0, 5.0M을 처리하고, hoechst 33342로 염색하여 핵상을 관찰하여 최적의 농도를 결정하였다. 또한 각 미세소관 형성 저해제를 혼합 처리하여 가장 높은 중기 염색체상을 획득할 수 있는 혼합 처리를 결정하였다. 이렇게 결정된 혼합 처리 방법을 인간의 체외 수정 및 배아 이식술에서 획득된 3PN 배아에 처리하여 중기 염색체를 획득하였다. Colcemid, nocodazole, vinblastine 모두 1 M이 최적 농도임을 확인할 수 있었다 (각각 96.3%, 92.0%, 98,4%). 미세소관 형성저해제를 혼합 처리하였을 경우 nocodazole과 vinblastine (각각 1M)을 혼합 처리했을 때 중기 염색체 획득률(97.3%)이 가장 높았다. 인간의 3PN 배아에 1M의 nocodazole과 vinblastine을 혼합 처리한 후, 113개의 할구를 분석하여 44개(38.9%)의 할구에서 중기 염색체를 확인할 수 있었다. 본 실험 결과를 통해 중기 염색체를 획득하기 위하여 미세소관 형성 저해제를 처리하는 방법은 생쥐의 배아에서는 효과적이지만, 인간의 배아에서는 그 효율이 다소 낮음을 알 수 있었다. 그러나 이 방법을 개선하여 인간의 할구에서 중기 염색체의 획득률을 높이고, 이를 염색체의 구조적 이상에 대한 착상전 유전자 진단에 적용한다면, 보인자와 정상의 핵상을 구분하여 정상의 핵상만을 갖는 배아의 이식을 통하여 더욱 정확한 착상전 유전자 진단을 시행할 수 있으리라 사료된다.

This study was carried out to investigate the aggregation rate of isolated mouse 2-, 4- and 8-cell stage blastomeres in phytohemagglutinin(PHA) solution. The morphologically normal embryos were collected from the oviduct of superovulated female mouse by flushing with M2 and the zona pellucida of embryos were removed with 0.5% pronase. The blastomeres were isolated by pipetting after plunging into Ca++-Mg++free PBS for 20 min. The result showed that aggregation rate in 0.5% (84.9~93.1%) was higher than that in 1.0% PHA(76.0~82.1%). Optimal aggregation time was 60min (83.9~100.0%) when compared with 30min (78.8~87.5%). Developmental to blastocyst in recombinated blastomeres was higher under conditions of 0.5% PHA solution and 60-min aggregation than that under other conditions.

To investigate the effect of EDTA on the in vitro development of blastomeres isolated from 2, 4, and 8-cell embryos(termed 1 /2, 1 /4 and 1 /8 blastomeres, respectively) of ICR strain mice, those were cultured in vitro in 35 mm culture dishes containing NaHCO-BMOC-3 medium supplemented with 10, 50, 100, or 500 M of EDTA at 37 for 72hrs. under the atmosphere of 5% and 95% air. EDTA supplementation of 10, 50, or 100 M to medium significantly(P<0.01) increased blastocyst formation rate compared with controls in 1 /2(58.3, 63.7, and 61.3% vs 21.6%), 1 /4(54.7, 57.5 and 62.2% vs. 2L3%), and 1 /8 blastomeres(46.2, 48.7, and 57.7% vs. 19.1%). Whereas, it was significantly(P<0.01) decreased to 4.5, 2.3, and 2.0% for 1 /2, 1 /4 and 1 /8 blastomeres, respectively by the EDTA supplementation of 500 M Both the nuclear number(P<0.05) and diameter of blastocysts(P<0.01) developed from balstomeres were significantly affected by the origin of blastomeres. The nuclear number of blastocysrs developed from 1/2, 1/4, and 1/8 blastomeres ranged 28.3i1.3, 24.18i1.2, and 19.840.9, respectively. And the diameter of those blastocysts was 87.21.1, 56.40.9, and 39.20.8 M, respectively.