YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation

Alaa A Eisa,Srinivasan Vijayaraghavan,Douglas Kline,Alexander C Ignatious,Ariana Detwiler,Santanu De,Eva Gilker

doi:10.1186/s12861-019-0200-1

Abstract

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.

Highlights

IntroductionProphase arrest is dependent on the production of cAMP within the oocyte
Oocytes are held in the first meiotic prophase within the mammalian ovary
Pairwise comparison of each of the Ywha isoform morpholino injection conditions with the nonsense morpholino control revealed no significant differences in percent germinal vesicle breakdown (GVBD) between the control and the experimental group (P > 0.05), with the exception of Ywhah (P < 0.0001). These results suggested that among the seven mammalian isoforms of Tyrosine 3-Monooxygenase/Tryptophan 5Monooxygenase Activation protein YWHAB (YWHA) proteins, all of which were found to bind to cell division cycle 25B (CDC25B), YWHAH may be important for maintaining the prophase I arrest of mouse oocytes through interaction with CDC25B or other proteins

Summary

Introduction

Prophase arrest is dependent on the production of cAMP within the oocyte. Meiotic arrest is maintained by cAMP-dependent activation of protein kinase A (PKA), in part, by phosphorylating and activating the oocyte-specific kinase WEE2 ( known as WEE1B) [5,6,7,8]. WEE2, in turn, is thought to phosphorylate and inactivate cyclin-dependent kinase 1 (CDK1), which together with regulatory cyclin B1 (CCNB1), forms maturation promoting factor (MPF) reviewed in [9, 10]. Immature 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).

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