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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.


Spindle assembly and chromosome alignment during meiosis I.Immunofluorescence staining of oocytes for chromosomes (DNA), kinetochores (labelled using anti-centromere antibody; ACA) and spindle microtubules (β-tubulin) at 2 h, 4 h and 8 h post-GVBD, representing ball, intermediate and bipolar stages, respectively, in controls (A), and NAM-treated (B) oocytes. 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.g003: Spindle assembly and chromosome alignment during meiosis I.Immunofluorescence staining of oocytes for chromosomes (DNA), kinetochores (labelled using anti-centromere antibody; ACA) and spindle microtubules (β-tubulin) at 2 h, 4 h and 8 h post-GVBD, representing ball, intermediate and bipolar stages, respectively, in controls (A), and NAM-treated (B) oocytes. 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: Next we examined the effect of NAM on spindle assembly following GVBD by immunostaining oocytes at defined stages during meiosis I. In mouse oocytes, bipolar spindle assembly occurs in the absence of centrosomes and is a protracted process lasting 6–8 h [4, 22–24]. By 1–2 h post-GVBD, clumped chromosomes localize to the surface of a microtubule “ball”, the earliest spindle morphology during which kinetochore pairs of recombined homologous chromosomes lie adjacent to one another (Fig 3Ai). By 8 h post-GVBD, a barrel-shaped bipolar spindle has formed with chromosomes aligned at the spindle equator (Fig 3Aiii). At this stage, chromosomes have become stretched with kinetochore pairs directed towards opposite spindle poles. By 4 h post-GVBD, spindle assembly is intermediate between the above two extremes with chromosomes scattered throughout the spindle, some of which have become stretched (Fig 3Aii).


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)

Spindle assembly and chromosome alignment during meiosis I.Immunofluorescence staining of oocytes for chromosomes (DNA), kinetochores (labelled using anti-centromere antibody; ACA) and spindle microtubules (β-tubulin) at 2 h, 4 h and 8 h post-GVBD, representing ball, intermediate and bipolar stages, respectively, in controls (A), and NAM-treated (B) oocytes. 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4418673&req=5

pone.0126194.g003: Spindle assembly and chromosome alignment during meiosis I.Immunofluorescence staining of oocytes for chromosomes (DNA), kinetochores (labelled using anti-centromere antibody; ACA) and spindle microtubules (β-tubulin) at 2 h, 4 h and 8 h post-GVBD, representing ball, intermediate and bipolar stages, respectively, in controls (A), and NAM-treated (B) oocytes. 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: Next we examined the effect of NAM on spindle assembly following GVBD by immunostaining oocytes at defined stages during meiosis I. In mouse oocytes, bipolar spindle assembly occurs in the absence of centrosomes and is a protracted process lasting 6–8 h [4, 22–24]. By 1–2 h post-GVBD, clumped chromosomes localize to the surface of a microtubule “ball”, the earliest spindle morphology during which kinetochore pairs of recombined homologous chromosomes lie adjacent to one another (Fig 3Ai). By 8 h post-GVBD, a barrel-shaped bipolar spindle has formed with chromosomes aligned at the spindle equator (Fig 3Aiii). At this stage, chromosomes have become stretched with kinetochore pairs directed towards opposite spindle poles. By 4 h post-GVBD, spindle assembly is intermediate between the above two extremes with chromosomes scattered throughout the spindle, some of which have become stretched (Fig 3Aii).

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.