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The 14-3-3 (YWHA) is a highly conserved, ubiquitously expressed protein family regulating important cellular processes including cell cycle. This work, for the first time, explored the differential expression and roles of 14-3-3 isoforms during mouse oocyte maturation.
All seven mammalian 14-3-3 isoforms were identified in mouse eggs and ovarian follicular cells including oocytes, by Western blotting. Immunocytochemical and immunohistochemical staining confirmed the presence of all 14-3-3 isoforms in oocytes, eggs and ovarian follicles with characteristic similarities and differences in their distributions.
Mammalian oocytes are arrested at meiosis prophase I by an inhibitory phosphorylation on Cyclin-Dependent Kinase I (CDK1), released by CDC25B phosphatase which is bound and inactivated in phosphorylated form by 14-3-3 in oocyte cytoplasm. Here, in situ Proximity Ligation Assays (PLA) revealed that all 14-3-3 isoforms interact with CDC25B in oocytes, with reduced interactions in eggs. Phosphorylation of CDC25B at Ser-149 was found to be reduced in eggs compared to oocytes. Microinjection of a translation-blocking morpholino oligonucleotide against 14-3-3eta mRNA caused germinal vesicle breakdown in significantly higher percentage of oocytes compared to oocytes injected with morpholinos targeting other 14-3-3 isoforms. Thus, interaction of 14-3-3eta with CDC25B is required for maintaining prophase I arrest in oocytes.
Protein 14-3-3eta was observed to accumulate and co-localize with alpha-tubulin at both meiosis I and II spindles during mouse oocyte maturation in vivo as well as in vitro. It interacts directly with alpha-tubulin with an accumulation of the interactions at meiotic spindles, detected by in situ PLA. In a significant 76% of mouse oocytes microinjected with the morpholino against 14-3-3eta mRNA, meiotic spindles were deformed or absent with clumped chromosomes, no accumulation of 14-3-3eta and no polar body formation. All control eggs showed normal, bipolar spindles with accumulation of 14-3-3eta. Therefore, 14-3-3eta is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with alpha-tubulin, to regulate the assembly of microtubules.
These studies reveal 14-3-3 isoform-specific interactions with key proteins involved in mouse oocyte maturation, such as CDC25B and alpha-tubulin. The results help to elucidate the roles of 14-3-3 proteins in mammalian oogenesis and reproductive development.
14-3-3, YWHA, Spindle, CDC25B, Phosphorylation, Interaction
Animal Sciences | Biology | Cell and Developmental Biology | Cell Biology | Developmental Biology | Life Sciences | Physiology
De, Santanu. Protein 14-3-3 (YWHA) isoforms and their roles in regulating mouse oocyte maturation..