NAD+ Repletion Rescues Female Fertility during Reproductive Aging

Abstract

Reproductive 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.

Keywords: SIRT2; aging; embryo development; female fertility; in vitro fertilization; infertility; nicotinamide adenine dinucleotide (NAD+); nicotinamide mononucleotide (NMN); oocyte; reproductive aging.

Figure 1.. Oocyte NAD⁺ Levels Decline with Age and Are Increased with NMN Treatment

(A and B) Multispectral imaging (A) to determine NAD(P)H content in oocytes from young (4- to 5-week-old) or aged (12-month-old) mice treated with NMN (drinking water, 2 g/L, 4 weeks) (scale bar is 20 μm), quantified in (B) (one-way ANOVA 1.454 (2, 71), *p = 0.0165, 0.0287 by Dunnett’s multiple comparison test, n = 21–27 oocytes per group). (C and D) Mass spectrometry of the whole ovary (C) shows no change in NAD(H) levels between the ages of 1 and 14 months (n = 6 mice per group); however, (D) NMN increased ovarian NAD(H) in 14-month-old mice (1 h following 400 mg/kg oral gavage, *p = 0.0123, **p = 0.0072, two-tailed t test, n = 20 mice per group).

Figure 2.. NMN Treatment Restores Oocyte Quality, Follicle Dynamics, Embryo Development, and Live Birth Rates

(A) NMN treatment (drinking water, 2 g/L, 4 weeks) with NMN from 14 months restores spindle assembly in immunostained oocytes (β-tubulin in green, Hoechst for DNA in blue, kinetochores in red, p = 0.0503 by Fisher’s exact test, n = 23–25 per group). Disordered spindles with lagging chromosomes are indicated by arrows, and normal, barrel-shaped bipolar spindles with DNA aligned along the metaphase plate are indicated by arrowheads. (B and C) Oocyte yield following ovarian stimulation in (B) aged (12-month-old) C57BL/6JAusb mice (*p = 0.0211, two-tailed t-test, n = 29–30 animals per group) and (C) 14- to 16-month-old Swiss albino mice (Kruskal-Wallis 16.31, ***p = 0.0004, *p = 0.0295 by Dunnett’s mulitple comparison test, n = 4–11 per group). (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). (F) Oocyte diameter following NMN treatment (2 g/L, drinking water, 4 weeks) in aged (12-month-old) or young (4- to 6-week-old) C57BL/6 females (F(2,71) = 8.504, ***p = 0.0002, *p = 0.0332 by Dunnett’s multiple comparison test, n = 21–27 oocytes per group). (G) In a parallel cohort, oocytes were used for IVF, with blastocyst formation at day 6 of embryo development (each datapoint is cumulative data from one independent experiment). (H) 12-month-old C57BL/6 females were treated for the indicated times with NMN in drinking water (2 g/L), and MII oocytes were subjected to IVF. At day 6, inner cell mass was assessed (Kruskal-Wallis 11.93, *p = 0.0337 by Dunn’s multiple comparison test, n = 8–26 oocytes per group). Previously untreated 13-month-old C57BL/6 females (n = 15–17 per group) were treated for 4 weeks with NMN in drinking water at two doses (0.5 and 2 g/L), following which a male was introduced and breeding performance was assessed over the next 9 weeks. (I–M) Breeding performance was assessed by (I) cumulative time to pregnancy, (J) cumulative time to live birth (log-rank test, **p = 0.0059), (K) overall proportion achieving live birth (Fisher’s exact test, *p = 0.0253 ctrl versus 0.5 g/L NMN), (L) cumulative number of pups born over time (repeated-measures ANOVA, NMN F(2, 43) = 4.925, p = 0.0119, Dunnett’s multiple comparison ctrl versus NMN 0.5, ****p < 0.0001, error bars show SEM), and (M) overall number of pups per female (Kruskal-Wallis 9.220, p = 0.0100, Dunn’s multiple comparison, *p = 0.0491 ctrl versus 0.5 g/L NMN).

Figure 3.. SIRT2 Transgenic Mice Have Improved Oocyte Quality

(A and B) Oocytes from 14-month-old Sirt2^Tg/+ C57BL/6 mice were subjected to (A) immunostaining for spindle assembly (β-tubulin in green, Hoechst for DNA in blue, kinetochores in red). Disordered spindles with lagging chromosomes are indicated by arrows, and normal, barrel-shaped bipolar spindles with DNA aligned along the metaphase plate are indicated by arrowheads; this is quantified in (B) (p = 0.0131, n = 10–13 oocytes per group, Fisher’s exact test). (C) Oocyte yield from aged (14-month-old) Sirt2^Tg/+ and wild-type Sirt2^+/+ littermates (**p = 0.0030, two-tailed t-test, n = 9 animals per group). (D) Aneuploidy rates in oocytes from young (2-month-old) and aged (16-month-old) Sirt2^Tg/+ and Sirt2^+/+ littermates (n = 26 young, n = 5–7 aged). (E–G) Oocytes from Sirt2^Tg/+ mice had decreased ROS levels as determined by (E) H₂DCFDA staining, quantified in (F) (****p < 0.0001, two-tailed t-test, n = 29 oocytes per group), with (G) increased G6PD enzyme activity (**p = 0.0019, two-tailed t-test, n = 11–13 using 5 pooled oocytes per sample from 4 animals per group). (H) Mating trials from 15 months of age to determine cumulative pregnancy rates (p = 0.1319 after 5 mating rounds, n = 8 animals per group, Fisher’s exact test). (I) Spindle assembly in oocytes from Sirt2^−/− knockout animals (n = 6, 14 oocytes per group).

Figure 4.. In Vitro NMN Treatment Enhances Embryo Formation

(A) Oocytes from aged (12-month-old) mice were subjected to IVF, and embryos maintained in 1 μM NMN until day 6 of embryo development (paired two-tailed t test, day 4 *p = 0.0138, day 5 **p = 0.0086, day 6 **p = 0.0014, n = 9 independent experiments). (B) Treatment with the NAMPT inhibitor FK866 causes embryo death at day 6, which can be rescued by the NAD⁺ precursors NMN, nicotinic acid mononucleotide (NaMN), nicotinic acid riboside (NaR), and NR (Kruskal-Wallis 50.65, p < 0.0001, ****p < 0.0001, **p = 0.0048, *p = 0.0197, ***p = 0.0004, **p = 0.0014, *p = 0.0156, n = 4–21 embryos per group as shown). (C) Treatment with sirtinol or splitomicin inhibits blastocyst formation (supplemental), with decreased cell count in blastocysts (one-way ANOVA F(6,162) = 22.44, ***p = 0.0002, ****p < 0.0001 by Dunnett’s multiple comparison test, n = 5–67 per group as shown by raw datapoints). (D) Co-treatment of sirtinol-treated embryos with NMN rescues this reduction (two-way ANOVA F(1,88) sirtinol = 17.66, NMN = 12.59, *p = 0.0360, 0.0374, ****p < 0.0001 by Sidak’s multiple comparison test, n = 22–23 per group). (E) Treatment with the p53 inhibitor pifithrin rescues cell count and blastocyst formation (supplemental) in embryos treated with sirtinol (pifithrin F(2,392) = 22.37,0020sirtinol F(1,392) = 49.81, ****p < 0.0001 by Sidak’s multiple comparison test, n = 72–78 per group).