Abstract
Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and the establishment of metaphase II arrest.
Highlights
Mammalian oocytes undergo a protracted and discontinuous developmental programme that begins during fetal life and is not completed until postnatal adulthood [1]
Entry into meiosis I is marked by germinal vesicle (GV) breakdown (GVBD), a readily identifiable morphological change that can be robustly inhibited using phosphodiesterase inhibitors such as 3-isobuyl 1-methylxanthine (IBMX)[2, 13,14,15]
By 2 h post-release, control GVBD rates increased to ~80% and by this stage GVBD had increased markedly in NAM-treated oocytes to 67%, this remained significantly lower than controls (Fig 1A)
Summary
Mammalian oocytes undergo a protracted and discontinuous developmental programme that begins during fetal life and is not completed until postnatal adulthood [1]. The majority of this time is spent in a prophase I-arrested state with an intact nucleus, termed the germinal vesicle (GV) in oocytes, equivalent to a late G2-phase arrest [2, 3]. Unlike other HDACs, sirtuin-mediated deacetylation involves a unique enzymatic reaction requiring NAD+ cleavage into nicotinamide (NAM) and an ADP-ribose peptide-imidate intermediate, the resolution of which culminates in release of the deacetylated substrate [6]. Sirtuin activity can be positively modulated by increasing NAD+ availability and negatively regulated through increasing NAM
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