BackgroundThe 14-3-3 (YWHA) proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ). These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms.ResultsWe have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ) are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development.ConclusionsWe found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the differential expression of these 14-3-3 isoforms in female germ cells and ovarian follicles provides the foundation for further investigating 14-3-3 isoform-specific interactions with key proteins involved in ovarian development, meiosis and oocyte maturation. This will lead to a better understanding of the individual functional roles of the 14-3-3 protein isoforms in mammalian oogenesis and female reproductive development.

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.

BackgroundImmature mammalian oocytes are held arrested at prophase I of meiosis by an inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1). Release from this meiotic arrest and germinal vesicle breakdown is dependent on dephosphorylation of CDK1 by the protein, cell cycle division 25B (CDC25B). Evidence suggests that phosphorylated CDC25B is bound to YWHA (14-3-3) proteins in the cytoplasm of immature oocytes and is thus maintained in an inactive form. The importance of YWHA in meiosis demands additional studies.ResultsMessenger RNA for multiple isoforms of the YWHA protein family was detected in mouse oocytes and eggs. All seven mammalian YWHA isoforms previously reported to be expressed in mouse oocytes, were found to interact with CDC25B as evidenced by in situ proximity ligation assays. Interaction of YWHAH with CDC25B was indicated by Förster Resonance Energy Transfer (FRET) microscopy. Intracytoplasmic microinjection of oocytes with R18, a known, synthetic, non-isoform-specific, YWHA-blocking peptide promoted germinal vesicle breakdown. This suggests that inhibiting the interactions between YWHA proteins and their binding partners releases the oocyte from meiotic arrest. Microinjection of isoform-specific, translation-blocking morpholino oligonucleotides to knockdown or downregulate YWHA protein synthesis in oocytes suggested a role for a specific YWHA isoform in maintaining the meiotic arrest. More definitively however, and in contrast to the knockdown experiments, oocyte-specific and global deletion of two isoforms of YWHA, YWHAH (14-3-3 eta) or YWHAE (14-3-3 epsilon) indicated that the complete absence of either or both isoforms does not alter oocyte development and release from the meiotic prophase I arrest.ConclusionsMultiple isoforms of the YWHA protein are expressed in mouse oocytes and eggs and interact with the cell cycle protein CDC25B, but YWHAH and YWHAE isoforms are not essential for normal mouse oocyte maturation, fertilization and early embryonic development.

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