Evidence for the requirement of 14-3-3eta (YWHAH) in meiotic spindle assembly during mouse oocyte maturation

Background: The 14-3-3 (YWHA) proteins are central mediators in various cellular signaling pathways regulating development and growth, including cell cycle regulation. We previously reported that all seven mammalian 14-3-3 isoforms are expressed in mouse oocytes and eggs and that, 14-3-3. (YWHAH) accumulates and co-localizes in the region of meiotic spindle in mouse eggs matured in vivo. Therefore, we investigated the role of 14-3-3. in spindle formation during mouse oocyte maturation. Results: Examination of oocytes matured in vitro demonstrated that 14-3-3. accumulates in both meiosis I and II spindles. To explore if 14-3-3. interacts directly with a-tubulin in meiotic spindles, we performed an in situ proximity ligation assay that can detect intracellular protein-protein interactions at the single molecule level and which allows visualization of the actual interaction sites. This assay revealed a marked interaction between 14-3-3. and a-tubulin at the metaphase II spindle. To demonstrate a functional role for 14-3-3. in oocyte maturation, mouse oocytes were microinjected with a translation-blocking morpholino oligonucleotide against 14-3-3. mRNA to reduce 14-3-3. protein synthesis during oocyte maturation. Meiotic spindles in those cells were examined by immunofluorescence staining of 14-3-3. and a-tubulin along with observation of DNA. In 76% of cells injected with the morpholino, meiotic spindles were found to be deformed or absent and there was reduced or no accumulation of 14-3-3. in the spindle region. Those cells contained clumped chromosomes, with no polar body formation. Immunofluorescence staining of 14-3-3. and a-tubulin in control eggs matured in vitro from uninjected oocytes and oocytes microinjected with the ineffective, inverted form of a morpholino against 14-3-3., a morpholino against 14-3-3., or deionized water showed normal, bipolar spindles. Conclusions: The results indicate that 14-3-3. is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with a-tubulin, to regulate the assembly of microtubules. These data add to our understanding of the roles of 14-3-3 proteins in mouse oocyte maturation and mammalian reproduction.