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

Abstract

BackgroundThe 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.ResultsExamination 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.ConclusionsThe 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.

14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes

“…Evidence suggests that this role of the Aurora B–14-3-3 pathway may be conserved also in mammalian oocytes and that Ncd may not be the only target. Depletion of the mouse 14-3-3η in oocytes results in spindle disorganization ( De and Kline, 2013 ). In mitotic anaphase/telophase mammalian cells, the kinesin-6 MKlp1 is regulated by a similar mechanism ( Douglas et al, 2010 ), which is potentially used also in oocytes.…”

A NudE/14-3-3 Pathway Coordinates Dynein and the Kinesin Khc73 to Position the Mitotic Spindle

“…Par-1 is essential for several cellular processes including the maintenance of the dynamic state of microtubules (reviewed in (Hayashi et al, 2012), and among its diverse set of substrates are several microtubule-associated proteins including the microtubule stabilizing protein Tau (reviewed in (Drewes et al, 1997; Matenia and Mandelkow, 2009)). Also essential for several cellular functions, 14-3-3 proteins are ubiquitous adapter proteins that are involved in cell cycle control (reviewed in (Gardino and Yaffe, 2011)), microtubule maintenance (Zhou et al, 2010), and spindle assembly and regulation (De and Kline, 2013). Because of their various roles in spindle-related processes, disruption of the 14-3-3 proteins and Par-1 may have compounding effects on Pins-mediated spindle positioning, producing the random phenotype we observe.…”

microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells

“…For example, MIB1 and PAX6 were implicated in regulating neural stem cell differentiation. 31 , 32 YWHAH has recently been implicated in regulating cell division during meiosis, 33 while ZWILCH has been shown to be essential for kinetochore functions during cell division. 34 However, our data revealed an additional role of these proteins in bone marrow adipogenesis.…”