YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation
YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation
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DOI：
10.1186/s12861-019-0200-1
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发表时间：
2019-10-22
影响因子：
--
通讯作者：
Kline, Douglas
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作者：
Eisa, Alaa A.;De, Santanu;Kline, Douglas
未成熟的哺乳动物卵母细胞被细胞周期蛋白依赖性激酶1 （CDK1）的抑制性磷酸化阻滞在减数分裂的前期I。这种减数分裂停滞和生发囊泡破裂的释放依赖于CDK1蛋白的去磷酸化，细胞周期分裂25B （CDC25B）。有证据表明，磷酸化的CDC25B与未成熟卵母细胞细胞质中的YWHA（14-3-3）蛋白结合，从而保持非活性形式。YWHA在减数分裂中的重要性需要进一步的研究。结果在小鼠卵母细胞和卵子中检测到YWHA蛋白家族多个亚型的信使RNA。先前报道的在小鼠卵母细胞中表达的所有7种哺乳动物YWHA亚型都被发现与CDC25B相互作用，原位近距离结扎试验证明了这一点。通过福斯特共振能量转移（FRET）显微镜观察YWHAH与CDC25B的相互作用。卵母细胞胞浆内显微注射R18（一种已知的合成的、非同型特异性的ywha阻断肽）可促进生发囊泡破裂。这表明抑制YWHA蛋白与其结合伙伴之间的相互作用可使卵母细胞免于减数分裂停滞。微量注射同种异构体特异性、翻译阻断的morpholino寡核苷酸来敲低或下调卵母细胞中YWHA蛋白的合成，这表明一种特定的YWHA异构体在维持减数分裂停滞中起作用。然而，更明确的是，与敲除实验相反，卵母细胞特异性和整体缺失YWHA两种亚型，YWHAH （14-3-3 eta）或YWHAE （14-3-3 epsilon）表明，完全缺失其中一种或两种亚型并不会改变卵母细胞的发育和从减数分裂前期I阻滞中释放出来。结论小鼠卵母细胞和卵子中存在多种YWHA蛋白同工型表达，并与细胞周期蛋白CDC25B相互作用，但YWHAH和YWHAE同工型对正常小鼠卵母细胞成熟、受精和早期胚胎发育不是必需的。
Background 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). 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. Results Messenger 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 Forster 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. Conclusions Multiple 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.