NAD+ repletion attenuates obesity‐induced oocyte mitochondrial dysfunction and offspring metabolic abnormalities via a SIRT3‐dependent pathway

Dear Editor, Over-nutrition in females causes subfertility and impairs offspring health, but the detail mechanisms and therapeutic strategies has not been well investigated.1–3 Nicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates mitochondrial functions.4–6 In this study, we revealed that obesity induced NAD decline in oocyte, while supplementation of NAD precursor, nicotinamide riboside (NR), in HFD-fed mice alleviated subfertility and reduced metabolic dysfunction in offspring through a NAD-SIRT3-dependent pathway. An obese mouse model was established by feeding female mice a high-fat diet (HFD) supplemented with or without NR (400 mg/kg/day) for 3 months beginning at the age of 4 weeks (Figure S1). The NAD levels in ovaries and MII oocytes from HFD mice decreased dramatically in comparison with controls. RT-PCR results showed that the key rate-limited NAD biosynthesis gene Nampt was marked decreased in the obese oocytes (Figure S4A). However, the NAD decrease was largely reduced by supplementation of NR (Figures 1A and 1B and S4A). Super-ovulated oocyte number was decreased in HFD mice compared with control mice (Figure 1C). Conversely, NR supplementation led to significantly more oocytes in HFD mice (Figure 1C) in comparison with HFD mice. Furthermore, more morphological defects as shown by fragments were observed in HFD oocytes compared with control oocytes, whereas such defects were attenuated due to supplementation of NR (Figure 1D and E). In addition, HFD mice gave birth to less offspring compared with controls. However, the subfertility of HFD mice were attenuated by administration of NR increased (Figure 1F). ROS content in MII oocytes was measured by using MitoSOX. The results showed that the ROS content was higher in HFD oocytes in comparison with controls, whereas NR supplementation significantly reduced ROS content in HFD oocytes (Figure 1G and H). MII oocytes

from young mice displayed a typical barrel-shaped spindle apparatus together with well-organised chromosomes. Conversely, oocytes from HFD mice had a lower proportion of normal spindle morphology with well-organised chromosomes, which was inhibited by NR supplementation ( Figure 1I and J). Embryonic development was monitored after in vitro fertilisation, lower fertilisation rate and 4-cell embryo formation rate in the HFD group were observed compared with the control group, whereas the decrease of embryonic development competence was alleviated after NR supplementation ( Figure 1K and L).
To elucidate the effects of NR supplementation in the improvement of HFD quality, single oocyte transcriptome sequencing was performed and analyzed from control and HFD as well as HFD with NR mice. RNA-seq data was confirmed by using RT-PCR analysis of the randomly selected genes ( Figure S2; Table S1 showed the primer sequences). Different gene expression trend was observed in HFD oocytes when comparing with controls, whereas NR treatment largely changed the obese oocyte transcriptome (Figure 2A and B). KEGG and GO analysis revealed that the differentially expressed genes were enriched in mitochondrial functions ( Figure 2C-F). Consistent with the sequencing data, mitochondrial membrane potential was impaired in HFD oocytes as shown by a lower ratio of red to green fluorescence intensity ( Figure 2G and H). Lower transcription of mitochondrial fusion and fission related genes was found in HFD oocytes ( Figure 2I). In addition, ATP level together with OXPHOS related genes were all decreased in HFD oocytes in comparison with controls ( Figure 2J and K). However, NR supplementation largely alleviated the changes in HFD oocytes. These observations further verified that NR supplementation improved mitochondrial functions in HFD oocytes.
Accumulated evidence has validated that SIRT3, whose activity is activated by NAD + , is involved in the regulation of oxidative stress homeostasis in HFD oocytes. [7][8][9] F I G U R E 1 Supplementation of NR attenuates the obesity-associated decline in oocyte quality and improves early embryonic competence by increasing the NAD + level. (A) NAD + levels in ovaries from Control, HFD and HFD supplemented with NR mice (n = 6 for Control, n = 4 for HFD and n = 6 for HFD + NR). (B) NAD + levels in single oocytes from ND, HFD and HFD supplemented with NR mice (n = 6 for Control, n = 4 for HFD and n = 6 for HFD + NR). We found that HFD induced decreased transcription of Sirt3 in oocytes but elevated by NR supplementation (Figure 3A), indicating that Sirt3 may participate the protective effects of NR on HFD oocytes. To test this possibility, Sirt3 knockout mice (genotyping was showed in Figure S3) were employed and treated as described above. We found that NR supplementation increased superovulated oocyte numbers, but the effects was vanished, the abnormal oocyte morphology rate and ROS contents were still in higher levels in HFD treated Sirt3 knockout mice ( Figure 3B-F). The proportion of meiotic defects showed by abnormal spindle assembly/chromosome mis-segregation was higher in HFD treated Sirt3 knockout mice supplied with NR than wild type mice (Figire 3G and H). As expected, decreased mitochondrial membrane potential was observed in oocytes from HFD Sirt3 knockout mice supplied with NR as compared with wild type mice ( Figure 3I, E and J). These data together indicated F I G U R E 4 Sirt3 mediated protective effects of NR supplementation on metabolism and mitochondrial function in offspring. GTT of 7to 8-week-old female (A) and male (B) mice born to HFD-fed dams, HFD+NR-fed dams, or HFD + NR fed dams (Sirt3 −/− ), the area under curve (AUC) were also calculated based on the glucose levels of each time point. * Comparison between HFD and HFD+NR;ˆComparison of Control and HFD. (C) Transcription of genes related to OXPHOS in offspring's muscle from each group. (D) Immunoblotting for proteins related to electron transport chain complex content (CI, NDUFB8; CII, SDHB; CIII, UQCRC2; CIV, MTCO 1; CV, ATP5A). Relative expression of each protein was calculated as a ratio to GAPDH levels. (E) Diagram illustrating the beneficial effects of NR supplementation to ameroliate obesity-induced oocyte dysfunction and improve offspring metabolism. Obesity decreases NAD + levels in oocytes, which leads to mitochondrial dysfunction accompanied by ROS accumulation and meiotic defects, resulting in reduced oocyte quality and earlier embryonic developmental competence. NR supplementation restores mitochondrial functions in oocytes by restoring the NAD + level and thus decreases ROS accumulation and meiotic defects via a Sirt3-dependent pathway. Importantly, administration of NR attenuates the obesity-induced decreases in early embryonic competence and the live birth rate as well as normalises the metabolism in offspring. * p < .05, ** p < .01, *** p < .001;ˆˆp < .01,ˆˆˆp < .001 that the effects of NR supplementation on improvement of mitochondrial function and HFD oocyte quality was largely dependent on Sirt3.
Because mitochondria are inherited maternally, we next investigated whether these beneficial effects of NR supplementation were transferred to offspring born to HFD mice. The results showed that glucose level was higher in offspring born to mice fed a HFD in comparison with those born to mice fed a control diet after injection of glucose. However, all of these changes were attenuated in the offspring born to mice fed a high-fat diet supplemented with NR ( Figure 4A and B). Mechanismly, NR supplementation increased expression of genes related to OXPHOS and NAD + levels ( Figure S4B) in offspring muscle born to HFD-fed mice ( Figure 4C and D), indicating that NR supplementation could alleviate obesity-induced metabolic disturbances in offspring by improving mitochondrial functions.
In summary, as shown in Figure 4E, HFD induced NAD + deficiency in oocytes, which caused mitochondrial dysfunction, accumulation of ROS and abnormal spindle assembly, thereby impairing early embryonic development, and decreasing the live birth rate. However, increasing the NAD + level by supplementation of NR to HFD mice improved mitochondrial functions in oocyte by via a Sirt3-dependent pathway. Our data suggest that increasing NAD + level is a potential therapeutic approach for treatment of obesity-related ovarian infertility and metabolic disturbance in offspring.