Graphical Abstract Highlights d Declining NAD(P)H is associated with oocyte dysfunction during reproductive aging d Oocyte quality and fertility can be restored by NMN treatment in aged mice d Supplementation of embryo media with NMN improves developmental milestones d SIRT2 overexpression mimics benefits of NMN but is unlikely to mediate its effects SUMMARYReproductive aging in female mammals is an irreversible process associated with declining oocyte quality, which is the rate-limiting factor to fertility.Here, we show that this loss of oocyte quality with age accompanies declining levels of the prominent metabolic cofactor nicotinamide adenine dinucleotide (NAD + ). Treatment with the NAD + metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality in aged animals, leading to restoration in fertility, and this can be recapitulated by transgenic overexpression of the NAD + -dependent deacylase SIRT2, though deletion of this enzyme does not impair oocyte quality. These benefits of NMN extend to the developing embryo, where supplementation reverses the adverse effect of maternal age on developmental milestones. These findings suggest that late-life restoration of NAD + levels represents an opportunity to rescue female reproductive function in mammals.

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.

2Female infertility is a common and devastating condition with life-long health, emotional and 3 social consequences. There is currently no pharmacological therapy for preserving oocyte 4 quality during aging, which is the strongest risk factor for infertility. This leads to an age 5 dependent decline in natural conception and IVF success rates (1). Here, we show that this is 6 due in part to declining levels of the metabolic cofactor nicotinamide adenine dinucleotide 7 (NAD + ), and that restoring NAD + levels with its metabolic precursor nicotinamide 8 mononucleotide (NMN) rejuvenates oocyte quality and quantity in aged animals, leading to 9 improved fertility. These benefits extend to the developing embryo, where NMN 10 supplementation in embryo culture media following IVF enhances blastocyst formation in 11 older mice. The NAD + dependent deacylase SIRT2 is sufficient, but not essential, to 12 recapitulate the benefits of in vivo NMN treatment, and transgenic overexpression of SIRT2 13 maintains oocyte spindle assembly, accurate chromosome segregation, decreased oxidative 14 stress and overall fertility with ageing. Pharmacological elevation of NAD + may be an 15 effective, non-invasive strategy for restoring and maintaining female fertility during ageing, 16 and for improving the success of IVF.17 18 19 risks (4), is expensive and has a limited success rate. Repeated IVF failures are a substantial 1 source of emotional distress, and failure to conceive offspring is a substantial source of 2 relationship breakdown (5). 4The rate-limiting factors for successful pregnancies in IVF are oocyte quantity and quality, 5 both of which start to decline from the middle of the third decade of life in humans (1, 3). 6 Despite the enormous need, there are no clinically viable strategies to either preserve or 7 rejuvenate oocyte quantity or quality during ageing. There is a major need in reproductive 8 medicine for a non-invasive, pharmacological treatment to maintain or restore oocyte quantity 9 and/or quality during ageing. The effect of such a therapy would be to alleviate a rate-limiting 10 barrier to IVF success, or increase the chances of unaided conception, without having to resort 11 to IVF. 12 13The molecular basis for the decline in oocyte quality with advancing age is not clear but is 14 certainly multifactorial. The key factors thought to be involved include genome instability, 15 reduced mitochondrial bioenergetics, increased reactive oxygen species (ROS), and impaired 16 fidelity during meiotic chromosome segregation due to disrupted spindle assembly and 17 compromised function of the spindle assembly checkpoint (SAC) surveillance system (6). This 18 latter hypothesis is evidenced by an increased rate of aneuploidy in embryos with increased 19

No abstract