Parthenogenetic Activation Of Mouse Oocytes By Ethanol And 6-DMAP

To decrease the toxicity of ethanol and 6-DMAP to oocytes and establish a good method of oocytes activation,in the promotion of oocyte activation by combination of ethanol with 6-DMAP,the temporal window for the action of 6-DMAP and the influence of 6-DMAP on the development of the parthenogenetic embryos were studied in the present paper. Several experiments were conducted. Oocytes were cultured with CZB containing 6-DMAP for various periods after treatment with ethanol,and were cultured in 6-DMAP-free CZB before and after treatment with 6-DMAP. The total time for treatment and culture was 6 hr. The results obtained are as follows:1. The rates of oocyte activation after treatment with 10% ethanol for 5 min and 10 min were 57.9% and 85.6%,respectively,and the rates were 41.3% and 63.7%,respectively,after treated with 5% ethanol for 5 min and 10 min.2. After the oocytes collected 18 hr post hCG injection were treated with 6-DMAP for 2,4 and 6 hr,the rates of activation were 12%,25% and 40%,respectively.3. When oocytes recovered 18 hr post hCG were treated with 6-DMAP for 6 hr after treatment with 10% ethanol for 5 min,the activation rates reached 100%,which was significantly higher than that obtained when oocytes were treated with 10% ethanol for 5 min or 6-DMAP alone for 6 hr.4. When the oocytes 18 hr post hCG were treated with 6-DMAP for 0,1,2,4 and 6 hr after treatment with 10% ethanol for 5 min,the activation rates were 53%,61%,88%,93% and 89%,respectively. When the oocytes were treated with 6-DMAP for 1 hr at 6 different sequential points after treatment with 10% ethanol for 5 min,the activation rates were 53%,87%,92%,54%,60% and 59% respectively. When the oocytes were treated with 6-DMAP for 0.5 hr at 4 sequential points starting from 3th to 6th 0.5 hr after treatment with ethanol,the activation rates were 95%,91%,90% and 97%,respectively. Thus,the temporal window for 6-DMAP action in the oocytes 18 hr post hCG was at the 2nd and 3rd 1 hr after treatment with ethanol.When the oocytes collected 15 hr post hCG were treated with 6-DMAP for 0,1,2,4 and 6 hr after treatment with 10% ethanol for 10 min,the activation rates were 47%,49%,82%,78% and 80%,respectively. When the oocytes were treated with 6-DMAP for 1 hr at 6 sequential points after treatment with 10% ethanol for 10 min,the activation rates were 52%,62%,57%,88%,87% and 41%,respectively. Thus,the temporal window for action of 6-DMAP in the oocytes 15 hr post hCG was at the 4th and 5th 1 hr after treatment with ethanol.When the oocytes 13 hr post hCG were treated with 6-DMAP for 0,1,2,4 and 6 hr after treatment with 10% ethanol for 10 min,the activation rates were 0 %,15%,35%,72% and 81%,respectively. When the oocytes were treated with 6-DMAP for 1 hr at 6 sequential points after treatment with 10% ethanol for 10 min,the activation rates were 27%,31%,24%,28%,25% and 23%,respectively. This might indicate that the temporal window of 6-DMAP action widened with the increase of the egg age.From the above results,we concluded that the temporal window for action of 6-DMAP on the oocytes depended on the egg age of oocytes but independent of the starting time of the treatment with ethanol.5. With the prolonging of treatment with 6-DMAP,the developmental ability of parthenogenetic embryos decreased. When the oocytes treated with 6-DMAP for 1 hr,the percentage of morula and blastocysts (72%) were significantly higher than that of oocytes treated with 6-DMAP for 6 hr (28%).6. With the prolonging of treatment with 6-DMAP after treatment with ethanolthe ratio of 2-cell activated oocytes decreased and reduced to 0% after treatment with 6-DMAP for 6 hr.