title: Evidence for the requirement of 14-3-3eta (YWHAH) in meiotic spindle assembly during mouse oocyte maturation.
abstract: 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, 14-3-3η (YWHAH) accumulates and co-localizes in the region of meiotic spindle in mouse eggs matured in vivo. Therefore, we investigated the role of 14-3-3η in spindle formation during mouse oocyte maturation.
  
  
  RESULTS
  Examination of oocytes matured in vitro demonstrated that 14-3-3η accumulates in both meiosis I and II spindles. To explore if 14-3-3η interacts directly with α-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 14-3-3η and α-tubulin at the metaphase II spindle. To demonstrate a functional role for 14-3-3η in oocyte maturation, mouse oocytes were microinjected with a translation-blocking morpholino oligonucleotide against 14-3-3η mRNA to reduce 14-3-3η protein synthesis during oocyte maturation. Meiotic spindles in those cells were examined by immunofluorescence staining of 14-3-3η and α-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 14-3-3η in the spindle region. Those cells contained clumped chromosomes, with no polar body formation. Immunofluorescence staining of 14-3-3η and α-tubulin in control eggs matured in vitro from uninjected oocytes and oocytes microinjected with the ineffective, inverted form of a morpholino against 14-3-3η, a morpholino against 14-3-3γ, or deionized water showed normal, bipolar spindles.
  
  
  CONCLUSIONS
  The results indicate that 14-3-3η is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with α-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.
authors.affiliation: Department of Biological Sciences, Kent State University, Kent, OH 44242, USA.
authors.forename: Santanu
authors.initials: S
authors.lastname: De
authors.forename: Douglas
authors.initials: D
authors.lastname: Kline
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D005455:Fluorescent Antibody Technique; D008540:Meiosis; D051379:Mice; D018613:Microscopy, Confocal; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D014404:Tubulin / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D012333:RNA, Messenger; D014404:Tubulin
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