YWHA (14-3-3) Protein Isoforms and Their Interactions with CDC25B Phosphatase in Mouse Oogenesis and Oocyte Maturation

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 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. 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

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

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

Noncanonical necrosis in 2 different cell types in a <i>Caenorhabditis elegans</i> NAD⁺ salvage pathway mutant

AbstractNecrosis was once described as a chaotic unregulated response to cellular insult. We now know that necrosis is controlled by multiple pathways in response to many different cellular conditions. In our pnc-1+Caenorhabditis eleganspnc-1C. elegansmec-4dced-4pnc-

Comparative metabolomic profiling reveals that dysregulated glycolysis stemming from lack of salvage NAD⁺ biosynthesis impairs reproductive development in Caenorhabditis elegans

Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD(+) consumers to resynthesize NAD(+), resulting in a reduction in global NAD(+) bioavailability. We manipulate NAD(+) levels to demonstrate that a minor deficit in NAD(+) availability is incompatible with a normal pace of gonad development. The NAD(+) deficit compromises NAD(+) consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD(+) biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD(+) salvage biosynthesis for the purposes of inhibiting tumor growth