Effectiveness of rabbit anti-14-3-3 eta (AHP1046, AbD Serotec) to detect the protein isoform in mouse oocytes and eggs

Results Summary

Examination of mouse oocytes matured in vitro demonstrated that 14-3-3 eta accumulates in both meiosis I and II spindles and co-localizes with alpha-tubulin, with the accumulation at the spindle region gradually increasing along the course of oocyte maturation. Oocytes were collected in HEPES-buffered MEM alpha containing dibutyryl-cAMP, and then allowed to mature in vitro by incubation in bicarbonate-buffered MEM alpha without dibutyryl-cAMP 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. We examined 15 cells at each stage of maturation and found the following pattern in all cells examined. The 14-3-3 eta protein was found to be present throughout the cytoplasm during oocyte maturation at all stages (Figure 2A-J). Accumulation of 14-3-3 eta 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 14-3-3 eta with alpha-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 14-3-3 eta was found at the broad midbody microtubules during formation of the first polar body (Figure 1G-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 14-3-3 eta was shown to accumulate in the region of the MII spindle (Figure 2I-J) as well as associated with alpha-tubulin in the first polar body (Figure 2K-L). Control cells processed simultaneously following procedures in absence of primary antibody for 14-3-3 eta detected no protein, and background fluorescence was minimal. These results indicated a potential functional role of 14-3-3 eta in regulating meiotic spindle assembly during mouse oocyte maturation.