Protein 14-3-3 eta (YWHAH) is essential for normal meiotic spindle assembly during in vitro maturation of mouse oocytes

The 14-3-3 (YWHA) proteins are known to be central mediators in various cellular signaling pathways regulating development and growth including cell cycle regulation. We previously found that all seven mammalian 14-3-3 isoforms are expressed in mouse oocytes and eggs, and that 14-3-3 eta (YWHAH) accumulates and co-localizes in the region of meiotic spindle in mouse eggs matured in vivo[De, S. et al., 2012, BMC Res Notes. 5(1), 57]. Examination of oocytes matured in vitrodemonstrated that 14-3-3 eta accumulates in both meiosis I and II spindles. To examine the role of 14-3-3 eta in meiotic spindle formation, we microinjected mouse oocytes with a ~0.1mM translation-blocking morpholino oligonucleotide against 14-3-3 eta mRNA. The morpholino-injected oocytes were held in prophase I arrest for 24 hours to allow reduction of existing 14-3-3 eta protein and then allowed to mature in vitro. Meiotic spindles in those cells were examined by immunofluorescence staining of 14-3-3 eta and alpha-tubulin along with observation of chromosomes stained with Hoechst dye. Following injection of the morpholino, the spindle was often found to be absent or abnormally formed along with no or reduced accumulation of 14-3-3 eta. In those cells, chromosomes were clumped or disorganized and first polar body formation was absent. Immunofluorescence staining of 14-3-3 eta and alpha-tubulin in control eggs matured in vitrofrom uninjected oocytes and oocytes microinjected with the ineffective, inverted form of a morpholino against 14-3-3 eta, a morpholino against 14-3-3 gamma or deionized water showed normal, bipolar meiosis II spindle assemblies. To further examine the interaction of 14-3-3 eta with alpha-tubulin in meiotic spindles, we performed an in situproximity ligation assay (Duolink In-cell Co-IP; Olink Bioscience) that can detect protein-protein interactions at the single molecule level directly in cells and allows visualization of the actual sites of the interactions. In control eggs, the proximity ligation assay revealed an accumulation of sites of interaction between 14-3-3 eta and alpha-tubulin at the normal meiotic spindles and in the cell cortices adjacent to the spindles, confirming the co-immunofluorescence finding we reported before. However, in cells matured in vitrofrom oocytes microinjected with the morpholino against 14-3-3 eta, there was a marked reduction in the interaction sites for 14-3-3 eta and alpha-tubulin in the regions of the disrupted spindles. These results suggest that 14-3-3 eta is essential for formation of the normal meiotic spindle during in vitromaturation of mouse oocytes, in part by interacting with alpha-tubulin.