Karyotype analysis is a major work in the process of triploid abalone production for the purpose of productivity and quality improvement. However, the metaphase spreads for karyotype analysis have been prepared just from the larvae at trochophore stage, which has restricted the spectrum of sample correction inhibiting more efficient analysis. Here, we investigated the feasibility of preparing metaphase spreads from the larvae at veliger stage that is the next developmental stage of trochophore. For this, diploid and triploid larvae at trochophore and veliger stages from Pacific abalone (Haliotis discus hannai ) were subjected to metaphase spread preparation and its efficiencies were measured and compared each other. As the results, although the efficiencies of metaphase spread preparation were significantly lower in the larvae at veliger stage compared to the ones at trochophore stage regardless of ploidy status, we found that the preparation of metaphase spreads, which showed the clear chromosomal images containing the normal number of chromosomes, was possible from the veliger stage larvae. On the other hands, all larvae used in this study regardless of developmental stage and ploidy did not show colchicine sensitivity. Moreover, no significant difference was observed in cell cycle distribution of the cells comprising larvae between two developmental stages regardless of ploidy status. These suggested that the details of protocol to prepare metaphase spreads from abalone larvae should be optimized depending on its developmental stages. Taken together, we demonstrated the feasibility of preparing metaphase spreads from H. discus hannai veliger stage larvae for karyotype analysis.

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM. This is approximately 500～1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (~50 M) and butyrolactone (～100 M). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone H1 kinase and MAP kinase in mouse oocytes.

The present study investigated the effect of treatment with nocodazole on the efficiency of cell cycle synchronization and development of mouse 4-cefl embryos. In addition, developmental ability of reconstituted embryos that received nuclei from 4-cell embryos synchronized with nocodazole at M phase was examined in vitro and in vivo. (1) When 4-cell blastorneres exposed to culture medium containing lg /ml nocodazole for 2, 4, 6 and 8 hrs, 40% (40/10l), 75% (l19/159), 85% (87/102) and 97% (155/160) of nuclei were synchronized at M phase, respectively. (2) Treated with nocodazole for 4 hrs, the proportion of 4-cell embryos developed to blastocysts (98%, 60/61) was not significanUy different from that of the control embryos (98%, 196/201). However, the developmental ability of 4-cell embryos treated for 8 (87%, P<0.05)and 12 hrs (76%, P

미성숙의 Germinal Vesicle(GV 단계에서 성숙한 Metaphase II(MII) 단계가 되는 난자성숙 과정은 핵과 세포질의 성숙을 통해 이루어지며, 이를 통해 수정과 배 발달을 할 수 있는 능력을 갖게 된다. GV 난자는 prophase I 단계에 arrest 되어 있다가 meiosis 과정을 거쳐 성숙한 MII로 되는데 이를 조절하는 기작에 대해서는 거의 알려져 있지 않다. 따라서 본 연구는 미성숙 난자와 성숙 난자간의 유전자 발현의 차이

소맥, 대두, 면화 및 대맥의 근단에 대하여 저온(0℃ ~2℃ 에 24 또는 12시간) 8-Hydroxyquinolin (0.03과 0.1%에서 2시간 또는 10시간) 및 Colchicine(0.2%와 1.0%에서 2 또는 10시간) 처리를 하고 mitotic cell의 수와 이에 대한 meitaphase의 출현빈도를 검토하였다. 1 저온처리는 모든 작물에 대하여 mitotic cell의 수와 metaphase의 출현빈도를 현저히 증가시켰다. 저온의 정도와 처리시간은 작위에 따라 달라 본시험에서는 소맥을 제외한 타작물에서는 최적온도와 시간은 시험된 시간보다도 짧은 범위 이내에 있는 것으로 생각된다. 2. 8-Hydroxyquinolin의 효과도 작물에 따라 다르나 0.03% 2시간 정도의 처리에서는 대체로 metaphase의 빈도를 증가시키나 mitotic cell의 수를 감소시키는 경향이 있다. 3. Colchicine은 mitotic cell의 증가 및 metaphase 빈도 증가에 항상 유효하였으며 이용에 있어서는 타처리에 비하여 같거나 우수하였다. 4. 본 시험에서의 각종 처리는 염색체를 수축시켰는데 그 정도가 가장 심한 것은 Colchicine이었고 그 다음이 8-Hydroxyquinolin 및 저온처리의 순이었다. 5. 재료에 따라 적당한 온도와 처리시간이 검토된다면 저온처리가 유효하게 적용될 수 있는 가능성이 논의되었다.