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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 at metaphase II and of flavopiridol treatment at 9 h post-GVBD and at metaphase II.(A) GV-stage oocytes were matured in vitro to metaphase II stage and then incubated in 10 mM NAM for 20 h before being fixed and immunostained. (B, C) GV-stage oocytes were cultured in vitro in standard medium either till 9 h post-GVBD (B) or till metaphase II (C) and were then transferred into medium containing 5 μM flavopiridol for 20 h before being fixed and immunostained. Shown are representative confocal images of oocytes from the different treatment groups immunostained for chromosomes (DNA), kinetochores (ACA), and spindle microtubules (β-tubulin). Note that in the NAM-treated oocytes, a well-formed bipolar spindle bearing condensed chromosomes is readily discernible in the oocyte (A). In contrast, following flavopiridol treatment, microtubules form a diffuse network in the oocyte, a persistent central spindle connects oocyte and PB (white arrows) and decondensed chromatin is observed within a nuclear structure (white arrowheads) (B, C). Significantly, in the 9 h flavopiridol group, there is a single PB (B) whereas in the metaphase II flavopiridol group, three PBs are visible (numbered 1–3). The initial PB that was present before flavopiridol was added divided to form PB1 and PB3 following treatment since neither one is connected to the oocyte by a central spindle; interestingly, they remain connected to one another by a persisting central spindle. Following flavopiridol treatment, PB2 was extruded from the oocyte and remains connected via a second central spindle. Only the phenotype in (B) replicates that observed in oocytes treated with NAM from the GV-stage (see Fig 4B). Scale bars = 10 μm.
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pone.0126194.g006: Effect of NAM treatment at metaphase II and of flavopiridol treatment at 9 h post-GVBD and at metaphase II.(A) GV-stage oocytes were matured in vitro to metaphase II stage and then incubated in 10 mM NAM for 20 h before being fixed and immunostained. (B, C) GV-stage oocytes were cultured in vitro in standard medium either till 9 h post-GVBD (B) or till metaphase II (C) and were then transferred into medium containing 5 μM flavopiridol for 20 h before being fixed and immunostained. Shown are representative confocal images of oocytes from the different treatment groups immunostained for chromosomes (DNA), kinetochores (ACA), and spindle microtubules (β-tubulin). Note that in the NAM-treated oocytes, a well-formed bipolar spindle bearing condensed chromosomes is readily discernible in the oocyte (A). In contrast, following flavopiridol treatment, microtubules form a diffuse network in the oocyte, a persistent central spindle connects oocyte and PB (white arrows) and decondensed chromatin is observed within a nuclear structure (white arrowheads) (B, C). Significantly, in the 9 h flavopiridol group, there is a single PB (B) whereas in the metaphase II flavopiridol group, three PBs are visible (numbered 1–3). The initial PB that was present before flavopiridol was added divided to form PB1 and PB3 following treatment since neither one is connected to the oocyte by a central spindle; interestingly, they remain connected to one another by a persisting central spindle. Following flavopiridol treatment, PB2 was extruded from the oocyte and remains connected via a second central spindle. Only the phenotype in (B) replicates that observed in oocytes treated with NAM from the GV-stage (see Fig 4B). Scale bars = 10 μm.

Mentions: To differentiate between these two possibilities, we first treated in vitro matured metaphase II-arrested oocytes with NAM. We found that 36 of 37 oocytes cultured in 10 mM NAM for >20 h maintained a well formed bipolar spindle with condensed chromosomes (Fig 6A), consistent with previous reports of ovulated mouse eggs treated with 20 mM NAM [9]. Thus, whereas 18–24 h of NAM treatment commencing at the GV-stage induced an interphase-like state (Fig 4B), treatment for similar periods after PBE had occurred did not, suggesting that unlike loss of c-Mos [33, 34], NAM does not compromise maintenance of metaphase II arrest to induce parthenogenesis.


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 at metaphase II and of flavopiridol treatment at 9 h post-GVBD and at metaphase II.(A) GV-stage oocytes were matured in vitro to metaphase II stage and then incubated in 10 mM NAM for 20 h before being fixed and immunostained. (B, C) GV-stage oocytes were cultured in vitro in standard medium either till 9 h post-GVBD (B) or till metaphase II (C) and were then transferred into medium containing 5 μM flavopiridol for 20 h before being fixed and immunostained. Shown are representative confocal images of oocytes from the different treatment groups immunostained for chromosomes (DNA), kinetochores (ACA), and spindle microtubules (β-tubulin). Note that in the NAM-treated oocytes, a well-formed bipolar spindle bearing condensed chromosomes is readily discernible in the oocyte (A). In contrast, following flavopiridol treatment, microtubules form a diffuse network in the oocyte, a persistent central spindle connects oocyte and PB (white arrows) and decondensed chromatin is observed within a nuclear structure (white arrowheads) (B, C). Significantly, in the 9 h flavopiridol group, there is a single PB (B) whereas in the metaphase II flavopiridol group, three PBs are visible (numbered 1–3). The initial PB that was present before flavopiridol was added divided to form PB1 and PB3 following treatment since neither one is connected to the oocyte by a central spindle; interestingly, they remain connected to one another by a persisting central spindle. Following flavopiridol treatment, PB2 was extruded from the oocyte and remains connected via a second central spindle. Only the phenotype in (B) replicates that observed in oocytes treated with NAM from the GV-stage (see Fig 4B). Scale bars = 10 μm.
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Related In: Results  -  Collection

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pone.0126194.g006: Effect of NAM treatment at metaphase II and of flavopiridol treatment at 9 h post-GVBD and at metaphase II.(A) GV-stage oocytes were matured in vitro to metaphase II stage and then incubated in 10 mM NAM for 20 h before being fixed and immunostained. (B, C) GV-stage oocytes were cultured in vitro in standard medium either till 9 h post-GVBD (B) or till metaphase II (C) and were then transferred into medium containing 5 μM flavopiridol for 20 h before being fixed and immunostained. Shown are representative confocal images of oocytes from the different treatment groups immunostained for chromosomes (DNA), kinetochores (ACA), and spindle microtubules (β-tubulin). Note that in the NAM-treated oocytes, a well-formed bipolar spindle bearing condensed chromosomes is readily discernible in the oocyte (A). In contrast, following flavopiridol treatment, microtubules form a diffuse network in the oocyte, a persistent central spindle connects oocyte and PB (white arrows) and decondensed chromatin is observed within a nuclear structure (white arrowheads) (B, C). Significantly, in the 9 h flavopiridol group, there is a single PB (B) whereas in the metaphase II flavopiridol group, three PBs are visible (numbered 1–3). The initial PB that was present before flavopiridol was added divided to form PB1 and PB3 following treatment since neither one is connected to the oocyte by a central spindle; interestingly, they remain connected to one another by a persisting central spindle. Following flavopiridol treatment, PB2 was extruded from the oocyte and remains connected via a second central spindle. Only the phenotype in (B) replicates that observed in oocytes treated with NAM from the GV-stage (see Fig 4B). Scale bars = 10 μm.
Mentions: To differentiate between these two possibilities, we first treated in vitro matured metaphase II-arrested oocytes with NAM. We found that 36 of 37 oocytes cultured in 10 mM NAM for >20 h maintained a well formed bipolar spindle with condensed chromosomes (Fig 6A), consistent with previous reports of ovulated mouse eggs treated with 20 mM NAM [9]. Thus, whereas 18–24 h of NAM treatment commencing at the GV-stage induced an interphase-like state (Fig 4B), treatment for similar periods after PBE had occurred did not, suggesting that unlike loss of c-Mos [33, 34], NAM does not compromise maintenance of metaphase II arrest to induce parthenogenesis.

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.