Intermediate phenotypes could also be found, which emphasized the need to assess the effect of demecolcine by fluorescence microscopy. For the third category of PB phenotypes, Hoechst-stained chromatin in the oocyte at the site of the budding PB was apparent at both low and high magnification, indicating that enucleation had not been successful (Fig. 2, g’ and h). In oocytes with either long flattened or two closely apposed PBs, both IE (Fig. 2, b and e) and compart-mentalization (Fig. 2, c and f) of chromatin were apparent in roughly equal proportions. In two replicate preliminary experiments, the percentages of oocytes scored as IE or compartmentalized declined significantly as the onset of a 30-min treatment with demecolcine was delayed relative to the time of activation (% IE and % compartmentalized, respectively, for combined sample [n] at treatment onset time [t]: 43% and 24%, n = 51 at t = 0; 28% and 19%, n = 51 at t = 5 min; and 18% and 15%, n = 49 at t = 10 min; P < 0.01, linear logistic regression). Thus, in subsequent experiments, demecolcine treatment was initiated immediately after activation. natural asthma treatment
The effect of changing the duration of demecolcine treatment was considered next. Oocytes were treated for 15, 30, and 45 min after activation in five replicate experiments whose results were combined (Table 2). With increasing time of exposure, no significant trend was observed for the proportion of oocytes scored as IE. However, the proportion of compartmentalized oocytes decreased as the duration of treatment increased (P < 0.001, linear logistic regression). Increasing the length of exposure to the drug also resulted in a higher incidence of DNA fragmentation (data not shown). Proportions of IE and compartmentalization in this series of experiments remained consistent between replicates, although compared with earlier trials, the average rate of IE had decreased to between 20% and 25%. This rate was observed in all subsequent experimental trials when the duration of demecolcine-treatment was standardized at 15 min.