BisBenzimide H 33342 Trihydrochloride (Hoechst) Review

Results Summary

Given that 14-3-3η is concentrated at the metaphase II spindle of mouse eggs, we examined the co-localization of 14-3-3η with α-tubulin during the process of in vitro oocyte maturation by additional confocal indirect immunofluorescence studies. Oocytes were collected in HEPES-buffered MEM α containing dbcAMP, and then allowed to mature in vitro by incubation in bicarbonate-buffered MEM α without dbcAMP for 4.5 hours (early spindle formation), 7.5 hours (MI pro-metaphase), 9 hours (MI metaphase) and 12 hours (MI telophase to MII metaphase) before fixation [6]. We examined 15 cells at each stage of maturation and found the following pattern in all cells examined. The 14-3-3η protein was found to be present throughout the cytoplasm during oocyte maturation at all stages (Figure 2A-J). Accumulation of 14-3-3η at the meiotic spindle region was detected around prometaphase I at 7.5 hours after release from prophase I arrest (Figure 2C-D). By 9 hours of maturation, a marked co-localization of 14-3-3η with α-tubulin was observed at the metaphase I spindle with condensed chromosomes aligned at the mid-spindle region (Figure 2E-F). At telophase I, prominent accumulation of 14-3-3η was found at the broad midbody microtubules during formation of the first polar body (Figure 2G-H). At the end of 12 hours of the maturation, telophase I is followed quickly by the formation of the metaphase II spindle (Figure 2I) and again 14-3-3η was shown to accumulate in the region of the MII spindle (Figure 2I-J) as well as associated with α-tubulin in the first polar body (Figure 2K-L).