Nicotinamide Impairs Entry into and Exit from Meiosis I in Mouse Oocytes

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.

NAD+ Repletion Rescues Female Fertility during Reproductive Aging

“…Consequently, in oocytes from Sirt2 Tg/+ -overexpressing animals, increases in kinetochore-microtubule stability may have contributed to augmented chromosome alignment and improved fertility. These observations are in agreement with separate in vitro studies in which chemical or morpholino-mediated Sirt2 knockdown in oocytes resulted in severe spindle defects and chromosome disorganization (Qiu et al, 2018;Riepsamen et al, 2015). In contrast to those studies, we observed that oocytes from constitutive Sirt2 knockout mice maintained normal spindle assembly and chromosome organization, suggesting that SIRT2 may be dispensable for oocytes.…”

“…Oocytes from whole-body Sirt2 À/À knockout mice at the age of 5-6 months displayed normal spindle assembly and maturation ( Figure 3I), indicating that at a younger age at which NAD + is replete, SIRT2 is not essential for accurate spindle assembly or that redundancy exists in the role of SIRT2 with other yet-to-be-identified factors. These in vivo results from Sirt2 knockout animals are in contrast to in vitro studies (Qiu et al, 2018;Riepsamen et al, 2015;Zhang et al, 2014). Altogether, (D and E) Aged (12-to 14-month-old) transgenic mice overexpressing NMNAT1 have increased oocyte yield (*p = 0.0416, two-tailed t-test, n = 8-10 per group) (D) in comparison to transgenics overexpressing NMNAT3 (n = 7-11) (E).…”

NAD+repletion rescues female fertility during reproductive ageing

“…Consequently, in oocytes from Sirt2 Tg/+ overexpressing animals, increases in kinetochore – microtubule stability likely contributed to augmented chromosome alignment and improved fertility. These observations are corroborated by separate in vitro studies where morpholino-mediated Sirt2 knockdown or chemical inhibition of SIRT2 in oocytes resulted in severe spindle defects and chromosome disorganization (29, 38). These results are supported by the apparent lower rates of aneuploidy in oocytes from aged Sirt2 Tg/+ animals in the present study.…”

“…4i), indicating that at a younger age where NAD is replete, SIRT2 is not essential for accurate spindle assembly, or that there is redundancy in the role of SIRT2 with other yet to be identified factors. These in vivo results from Sirt2 knockout animals are in contrast to studies of in vitro morpholino knockdown of Sirt2 (28) or chemical inhibition of SIRT2 in oocytes (38), where depletion or inhibition results in spindle assembly defects and aneuploidy (28, 29). The discrepancy between these results may be due to the compensatory, upregulation of other factors in constitutive knockout animals, versus the acute depletion of SIRT2 in oocytes during in vitro maturation.…”

SIRT1, 2, 3 protect mouse oocytes from postovulatory aging

“…NAM acts a non-competitive pan-sirtuin inhibitor by reacting with the ADP-ribose peptideimidate intermediate to reform NAD + protein [ 31 ]. A recent study examined the effects of NAM on oocyte meiosis progression [ 32 ]. Additionally, NAM causes developmental defects and increases the level of mitochondrial ROS in preimplantation embryos [ 33 ].…”