Biology Faculty Proceedings, Presentations, Speeches, Lectures

Title

Interactions of YWHA (14-3-3) protein isoforms with CDC25B phosphatase in regulating mouse oocyte maturation

Event Name/Location

48th Annual Meeting of the Society for the Study of Reproduction, "Evolution of Sex" / San Juan, Puerto Rico, USA

Document Type

Conference Presentation

Publication Date

6-2015

Abstract

Immature mammalian oocytes are held in prophase I arrest by an inhibitory phosphorylation of the cyclin -dependent kinase I (CDK1) that, with the regulatory cyclin B1 (CCNB1), makes up the maturation promoting factor (MPF). Dephosphorylation of CDK1, and thus, resumption of meiosis resulting in germinal vesicle breakdown (GVBD), is performed by cell cycle division 25B (CDC25B). Evidence suggests that YWHA (14-3-3) proteins sequester phosphorylated CDC25B in the cytoplasm of immature oocytes, consequently preventing it from activating the MPF. There are seven mammalian isoforms of YWHA encoded by separate genes. To understand how release from meiotic arrest may be regulated by YWHA proteins, it is necessary to examine the expression of all YWHA protein isoforms and the interactions of each isoform with CDC25B. Using isoform-specific antibodies, we previously found that all seven mammalian isoforms of YWHA are expressed in immature oocytes and mature eggs. Here, we present results to confirm this observation and examine which of the seven isoforms may be associated with meiotic arrest. PCR results confirmed by sequence analysis show the expression of all seven YWHA isoform mRNAs in immature mouse oocytes and mature eggs. Each YWHA isoform was found to interact with CDC25B by co-immunoprecipitation experiments. To determine if the YWHA proteins are important in maintaining meiotic arrest, oocytes were microinjected with R18, a non-isoform-specific, YWHA-blocking peptide. Microinjection of R18 caused a significant increase in GVBD compared to the control oocytes. To determine which isoform(s) may be responsible for maintaining prophase I arrest, 0.1mM isoformspecific translation-blocking morpholino oligonucleotides were microinjected into the oocytes. The injected oocytes were held in prophase I arrest for 24 hours, and then incubated with media containing a threshold concentration of dbcAMP, which would normally maintain meiotic arrest. A 70% increase in GVBD in the oocytes injected with the YWHAH (14-3-3η) morpholino was seen, despite the presence of dbcAMP, compared to control eggs including those injected with morpholinos against the other isoforms. The reduction of interactions with CDC25B or perhaps other target proteins by YWHAH resulted in the release of the oocyte from meiotic arrest. Although all YWHA isoforms were found to interact with CDC25B, these results suggest that YWHAH may be key in maintaining prophase I arrest.

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