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Nicotinamide impairs entry into and exit from meiosis I in mouse oocytes.

Riepsamen A, Wu L, Lau L, Listijono D, Ledger W, Sinclair D, Homer H - PLoS ONE (2015)

Bottom Line: We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1.NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE).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.

View Article: PubMed Central - PubMed

Affiliation: School of Women's & Children's Health, University of New South Wales, Sydney, New South Wales, Australia.

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.

No MeSH data available.


Effect of NAM treatment on securin and Cdc20 levels and kinetochore Mad2 localisation during meiosis I.(A) Western blot of securin and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD and plotted as a graph. (B) Western blot of Cdc20 and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD. (C, D) Changes in kinetochore Mad2 levels during meiosis I. Immunofluorescence staining for chromosomes (DNA), kinetochores (ACA), Mad2 and spindle microtubules (β-tubulin) at 2 h (upper panels) and 8 h (lower panels) post-GVBD in controls (C), and NAM-treated (D) oocytes. Note that at 2 h post-GVBD when spindles are at the ball stage, kinetochores recruit high levels of microtubules and that by 8 h post-GVBD when a bipolar spindle is present, Mad2 is undetectable at kinetochores in both groups. Panels to the extreme left are whole-oocyte images whilst panels to the right are magnified images of the regions enclosed by the dashed white squares. Scale bars = 10 μm.
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pone.0126194.g005: Effect of NAM treatment on securin and Cdc20 levels and kinetochore Mad2 localisation during meiosis I.(A) Western blot of securin and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD and plotted as a graph. (B) Western blot of Cdc20 and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD. (C, D) Changes in kinetochore Mad2 levels during meiosis I. Immunofluorescence staining for chromosomes (DNA), kinetochores (ACA), Mad2 and spindle microtubules (β-tubulin) at 2 h (upper panels) and 8 h (lower panels) post-GVBD in controls (C), and NAM-treated (D) oocytes. Note that at 2 h post-GVBD when spindles are at the ball stage, kinetochores recruit high levels of microtubules and that by 8 h post-GVBD when a bipolar spindle is present, Mad2 is undetectable at kinetochores in both groups. Panels to the extreme left are whole-oocyte images whilst panels to the right are magnified images of the regions enclosed by the dashed white squares. Scale bars = 10 μm.

Mentions: This prompted us to examine APC-Cdc20 activation during late meiosis I in NAM-treated oocytes. In mouse oocytes, destruction of two APC-Cdc20 substrates, securin and cyclin B1, are required for exit from meiosis I [25, 26]. The pattern of securin destruction has therefore been used as a surrogate marker of APC-Cdc20 activity in mouse oocytes with the onset of securin destruction correlating with initial APC-Cdc20 activation [16, 27]. Using immunoblotting we found that in control oocytes, securin declined precipitously between 6 h and 9 h post-GVBD, with a further decline by 14 h post-GVBD (Fig 5A), indicating that APC-Cdc20-mediated destruction commences between 6 h and 9 h post-GVBD. Notably, securin levels also declined markedly from 6 h to 9 h post-GVBD, with an additional decline from 9 h to 14 h post-GVBD, in NAM-treated oocytes (Fig 5A). Moreover, the pattern of decline in NAM-treated oocytes was similar to that in controls (Fig 5A).


Nicotinamide impairs entry into and exit from meiosis I in mouse oocytes.

Riepsamen A, Wu L, Lau L, Listijono D, Ledger W, Sinclair D, Homer H - PLoS ONE (2015)

Effect of NAM treatment on securin and Cdc20 levels and kinetochore Mad2 localisation during meiosis I.(A) Western blot of securin and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD and plotted as a graph. (B) Western blot of Cdc20 and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD. (C, D) Changes in kinetochore Mad2 levels during meiosis I. Immunofluorescence staining for chromosomes (DNA), kinetochores (ACA), Mad2 and spindle microtubules (β-tubulin) at 2 h (upper panels) and 8 h (lower panels) post-GVBD in controls (C), and NAM-treated (D) oocytes. Note that at 2 h post-GVBD when spindles are at the ball stage, kinetochores recruit high levels of microtubules and that by 8 h post-GVBD when a bipolar spindle is present, Mad2 is undetectable at kinetochores in both groups. Panels to the extreme left are whole-oocyte images whilst panels to the right are magnified images of the regions enclosed by the dashed white squares. Scale bars = 10 μm.
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Related In: Results  -  Collection

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pone.0126194.g005: Effect of NAM treatment on securin and Cdc20 levels and kinetochore Mad2 localisation during meiosis I.(A) Western blot of securin and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD and plotted as a graph. (B) Western blot of Cdc20 and actin in control and NAM-treated oocytes at 6 h, 9 h and 14 h post-GVBD (30 oocytes per sample; shown is a representative blot of 3 replicates). Band intensities from Westerns were quantified and normalised against the maximal intensity at 6 h post-GVBD. (C, D) Changes in kinetochore Mad2 levels during meiosis I. Immunofluorescence staining for chromosomes (DNA), kinetochores (ACA), Mad2 and spindle microtubules (β-tubulin) at 2 h (upper panels) and 8 h (lower panels) post-GVBD in controls (C), and NAM-treated (D) oocytes. Note that at 2 h post-GVBD when spindles are at the ball stage, kinetochores recruit high levels of microtubules and that by 8 h post-GVBD when a bipolar spindle is present, Mad2 is undetectable at kinetochores in both groups. Panels to the extreme left are whole-oocyte images whilst panels to the right are magnified images of the regions enclosed by the dashed white squares. Scale bars = 10 μm.
Mentions: This prompted us to examine APC-Cdc20 activation during late meiosis I in NAM-treated oocytes. In mouse oocytes, destruction of two APC-Cdc20 substrates, securin and cyclin B1, are required for exit from meiosis I [25, 26]. The pattern of securin destruction has therefore been used as a surrogate marker of APC-Cdc20 activity in mouse oocytes with the onset of securin destruction correlating with initial APC-Cdc20 activation [16, 27]. Using immunoblotting we found that in control oocytes, securin declined precipitously between 6 h and 9 h post-GVBD, with a further decline by 14 h post-GVBD (Fig 5A), indicating that APC-Cdc20-mediated destruction commences between 6 h and 9 h post-GVBD. Notably, securin levels also declined markedly from 6 h to 9 h post-GVBD, with an additional decline from 9 h to 14 h post-GVBD, in NAM-treated oocytes (Fig 5A). Moreover, the pattern of decline in NAM-treated oocytes was similar to that in controls (Fig 5A).

Bottom Line: We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1.NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE).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.

View Article: PubMed Central - PubMed

Affiliation: School of Women's & Children's Health, University of New South Wales, Sydney, New South Wales, Australia.

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.

No MeSH data available.