The NAD+ precursor nicotinamide riboside protects against postovulatory aging in vitro

Abstract

Purpose: Postovulatory aging (POA) of oocytes is clinically significant as it mirrors the degeneration observed in maternally aged oocytes, leading to substantial impairments in oocyte quality and the success rates of artificial reproductive technology (ART). The molecular alterations associated with POA, such as the degeneration of the first polar body, an increase in perivitelline space, reactive oxygen species (ROS) accumulation, energy depletion, and chromosomal and DNA damage, underscore the urgency of finding interventions to mitigate these effects. This study aims to identify whether nicotinamide riboside (NR) can prevent POA during the process of in vitro culture and raise the success rates of ART.

Method: Taking advantage of an in vitro postovulatory oocyte aging model, we examined the morphological integrity and NAD⁺ levels of ovulated mouse MII oocytes after 24 h of culturing. Following in vitro fertilization, we assessed the embryonic developmental potential of oocytes affected by POA. Using immunofluorescence and confocal microscopy, we measured the levels of ROS, mitochondrial function, and γH2AX. We also evaluated spindle assembly and chromosome alignment. Additionally, we detected the distribution of cortical granules to assess the metabolic and quality changes in POA oocytes with the supplementation of NR. To further our analysis, quantitative real-time PCR was conducted to measure the mRNA expression levels of antioxidant enzymes Sod1 and Gpx1 in the oocytes.

Results: With 200 μM NR supplementation during in vitro culture for 24 h, the oocytes from POA demonstrated reduced signs of aging-related decline in oocyte quality, including reduced ROS accumulation, improved mitochondrial function, and corrected mis-localization of cortical granules. This improvement in oocyte quality is likely due to the inhibition of oxidative stress via the NAD⁺/SIRT1 signaling pathway, which also helped to restore normal spindle assembly and chromosome alignment, as well as reduce the elevated levels of γH2AX, thereby potentially enhancing the embryonic development potential.

Conclusion: Current research provides evidence that NR is an efficient and safe natural component that prevents the process of POA and is thus a potential ideal antiaging drug for raising the success rates of ART in clinical practice.

Keywords: ART; Antioxidant; Nicotinamide riboside; Postovulatory oocyte aging.

Fig. 1

Effects of NR treatment on the NAD⁺ levels and quality of POA oocytes. A Timeline diagram of NR treatment of oocytes and hormone administration for superovulation of oocytes. B Percentages of abnormal oocytes among the fresh oocytes and among the oocytes cultured for 24 h with or without NR. n = 664 in the fresh group, n = 675 in the POA group, and n = 249, 636, and 187 in the groups treated with 100, 200, and 500 μM, respectively. C Morphology of oocytes in the fresh group and oocytes after 24 h of culture with or without NR. Bar, 100 μm. D NAD⁺ levels in fresh, POA, and NR oocytes (n = 200 for each group). E Morphology of blastocysts in the fresh, POA, and NR groups. Bar, 100 μm. F Percentages of blastocysts in the fresh, POA, and NR groups. n = 84 in the fresh group, n = 74 in the POA group, and n = 75 in the NR group. The data in (C) and (F) are shown as the means ± standard errors, n.s., P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001, by two-tailed unpaired Student t test or one-way ANOVA

Fig. 2

Effects of NR administration on ROS levels and spindle/chromosome structures in mouse oocytes in vitro. A Fluorescently stained images of ROS content in fresh, POA, and NR-treated oocytes. Bar, 200 μm. B Fluorescence intensity quantification of ROS content in fresh, POA, and NR-treated oocytes. n = 67 in the fresh group, n = 75 in the POA group, and n = 98 in the NR group. C Representative images of spindle and chromosome morphologies in oocytes from the three groups. Bar, 20 μm. D Percentages of oocytes with intact and abnormal spindles and chromosomes among fresh, POA, and NR-treated oocytes. n = 64 in the fresh group, n = 43 in the POA group, and n = 67 in the NR group. The data in (B) and (D) are shown as the means ± standard errors, n.s., P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001, by two-tailed unpaired Student t test or one-way ANOVA

Fig. 3

Effects of NR supplementation on mitochondrial function of mouse oocytes in vitro. A Fluorescently stained images of mitochondrial distribution in fresh, POA, and NR-treated oocytes. Bar, 50 μm. B Percentages of oocytes with abnormal distribution among fresh, POA, and NR-treated oocytes. n = 48 in the fresh group, n = 23 in the POA group, and n = 37 in the NR group. C Images of mitochondrial membrane potential in fresh, POA, and NR-treated oocytes fluorescently stained with the mitochondria-specific probe JC-1. JC-1 aggregate, red. JC-1 monomer, green. Bar, 200 μm. D The ratio of red to green JC-1 fluorescence intensity was used to quantify the mitochondrial membrane potential in fresh, POA, and NR-treated oocytes. n = 31 in the fresh group, n = 36 in the POA group, and n = 38 in the NR group. E Fluorescently stained images revealing the ATP levels in fresh, POA, and NR-treated oocytes. Bar, 200 μm. F Fluorescence intensity quantification of the ATP levels in fresh, POA, and NR-treated oocytes. n = 41 in the fresh group, n = 46 in the POA group, and n = 78 in the NR group. The data in (D) and (F) are shown as the means ± standard errors, n.s., P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001, by two-tailed unpaired Student t test or one-way ANOVA

Fig. 4

Effects of NR treatment on DNA damage and LCA localization in mouse oocytes in vitro. A Fluorescently stained images of DNA damage in fresh, POA, and NR-treated oocytes. DNA, blue. γH2AX, green. Bar, 50 μm. B Fluorescence intensity quantification of γH2AX signals in fresh, POA, and NR-treated oocytes. n = 49 in the fresh group, n = 37 in the POA group, and n = 47 in the NR group. C Fluorescently stained images of the CG distribution in fresh, POA, and NR-treated oocytes. CGs, red. DNA, blue. Bar, 50 μm. D Fluorescence intensity quantification of the abnormal localization of LCA in fresh, POA, and NR-treated oocytes. n = 29 in the fresh group, n = 38 in the POA group, and n = 31 in the NR group. E The expression of selected genes associated with antioxidation pathways and the NAD⁺/SIRT1 axis was examined in fresh, POA, and NR-treated oocytes by RT‒PCR. The data in (B) and (E) are shown as the means ± standard errors, n.s., P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001, by two-tailed unpaired Student t test or one-way ANOVA