Cloned Mice: RESULTS(1)

Meiotic Resumption Following Ethanol Activation of B6D2F1 Oocytes The timing of meiotic progression after ethanol activation was characterized in oocytes recovered from B6D2F1 mice (Table 1 and Fig. 1). At 15 min pa, the majority of oocytes sampled (85%, n = 41) had reached anaphase (A) II. At 30 min pa, oocytes were roughly equal in terms of those still in AII and those that had progressed to telophase (T) II with the spindle tending to lie parallel to the plasma membrane (AII, 42%; TII; 52%; n = 40). By 60 min pa, most oocytes were in various stages of TII, with the modal peak (71%, n = 41) having undergone spindle rotation and initiation of a PB that remained open. Polar body formation was completed by virtually all oocytes (91%, n = 44) when they were next sampled at 120 min pa. However, pronuclei were only observed in 32% of oocytes (n = 40) at the next time of sampling (180 min pa). At this time, an increase was observed in the proportion of degenerate oocytes to 17% (n = 40), with degeneracy manifested by vacuolized or fragmented cytoplasm. At earlier time points, the proportion of degenerate oocytes ranged from 2% to 7%. canadian neighbor pharmacy online

Effect of Administrating Demecolcine to Ethanol-Activated B6D2F1 Oocytes

In the next series of experiments, we hypothesized that enucleation might be induced by a transient, reversible interference with spindle function during the anaphase-telo-phase (A-T) II transition. Thus, the transient administration of demecolcine to ethanol-activated B6D2F1 oocytes was assessed. Because the A-TII transition was observed to occur in these oocytes between 15 and 30 min pa, a 30-min treatment initiated either immediately or 5 or 10 min after activation (t = 0, 5, or 10 pa) was assessed. Activated oocytes were assessed 90 min pa, by which time first PBs emitted during meiotic maturation were invariably either degenerate or no longer detected. As a result, demecolcine-treated oocytes that lacked a PB or possessed a small, rounded PB corresponded to those that had either not yet released a second PB or had done so normally, respectively. This was confirmed by fluorescence microscopy (data not shown). Demecolcine-treatment induced three broad categories of PB phenotypes, irrespective of the treatment’s time of initiation of duration. These included the presence of a single elongated PB, two closely apposed PBs, or a PB accompanied by a second, budding PB that still remained connected (Fig. 2, a, d, and g).

Table 1.
Table1Cloned Mice Derived from

Fig1Cloned Mice Derived from
FIG. 1. Meiotic resumption of mouse oocytes following ethanol activation. Mouse B6D2F1 oocytes arrested at MII (a) and activated by culture in 7% ethanol for 7 min (b-f) were fixed and immunostained for microtubules (green) and microfilaments (red), with DNA stained blue by Hoechst 33342. Oocytes fixed at 0 (a) and 15 (b) min pa could be found at MII and AII, respectively. Oocytes fixed at 30 (c), 60 (d), and 90 (e) min pa could be found in various stages of TII, with the spindle located parallel to the surface or perpendicular to the surface before and after PB closure, respectively. At 180 min pa (f), pronuclei and a cytoplasmic network of microtubules could be observed. Magnification X1000.

Fig2Cloned Mice Derived from
FIG. 2. Induced enucleation and com-partmentalization of oocyte chromatin. Mouse oocytes were activated with ethanol and then transiently treated with 0.4 |xg/ml of demecolcine before being fixed and immunostained for microtubules (green) and microfilaments (red), with DNA stained by Hoechst 33342 (blue). Three PB phenotypes were associated with demecolcine treatment irrespective of the time of initiation or duration of treatment or the strain of mouse oocyte. These phenotypes included the presence of a single elongated PB (a-c), two closely apposed PBs (d-f), or the former accompanied by a second, budding PB that still remained connected (g and h). Oocytes were assessed at low magnification on an inverted microscope using visible light alone (a, d, and g) or with ultraviolet light (a’, d’, and g’). The latter clearly revealed chromatin in the oocyte cytoplasm only in association with budding PBs (g) and not with the other PB morphologies (a and d). Visualization of cortical microfilaments and microtubule spindles in oocytes at higher magnification (b, c, e, f, and h) enabled the assessment of cytoplasmic continuity between the oocyte and their PBs. Oocytes with either a long flattened (b and c) or two closely apposed PBs (e and f) were scored as compartmentalized or IE if continuity could be observed (c and f) or not (b and e), respectively. Oocyte cytoplasm-PB continuity was invariably detected when a budding PB was observed (h). Following overnight recovery, oocytes that could not be scored as IE frequently cleaved to the 2-cell stage (i). All of the oocytes shown in all panels of the figure were from the B6CBAF1 strain, although the same results were observed in B6D2F1 strain. Magnification X360 (a, d, and g), X1800 (b, c, e, f, and h), and X1000 (i).