Administration of nicotinamide mononucleotide improves oocyte quality of obese mice

Abstract Objectives Obesity has become a common health concern around the world. Maternal obesity could cause poor reproductive outcomes due to chronic ovarian inflammation and decreased oocyte quality. However, the strategies to improve the poor reproductive outcomes of obese females have not been fully studied. In this study, we aimed to explore the effects and underlying mechanisms of nicotinamide mononucleotide (NMN) on oocyte quality and reproductive performance of obese mice. Materials and Methods The obese mouse model was established by feeding high‐fat diet which was confirmed by body weight record, fasting blood glucose test and oral glucose tolerance test. The expression of ovary development related genes and inflammation related genes, including Lhx8, Bmp4, Adgre1, Ccl2, TNF‐α, Gal‐3, Clec10a and IL‐10 in ovaries and the expression of Bax and Sod1 in oocytes were detected using quantitative reverse transcription PCR (RT‐qPCR). The adipose size of abdominal fat tissue was determined with haematoxylin and eosin (H&E) staining. Immunofluorescence staining was performed to measure the ROS level, spindle/chromosome structure, mitochondrial function, actin dynamics and DNA damage of oocytes. Results The administration of NMN restored ovarian weight and reduced the adipose size of abdominal fat tissue and ovarian inflammation in high fat diet (HFD) mice. Furthermore, NMN treatment improved the oocytes quality partially by restoring the mitochondrial function and actin dynamics, reducing meiotic defects, DNA damage and ROS level and lipid droplet distribution of oocytes in HFD mice. On the long‐term effect, NMN restored offspring body weight of HFD mice. Conclusion NMN could improve the oocyte quality of HFD‐induced obese mice.

mitochondrial dysfunction, meiotic abnormalities and oxidative stress caused by obesity. 5 The oocyte mitochondrial dysfunction of obese mice can be transmitted to offspring, increasing the risk of insulin resistance in offspring. 6 The adverse effects of maternal obesity on oocytes and preimplantation embryos may lead to long-term metabolic diseases in offspring. 7,8 Thus, low-quality of oocytes is the main reason for the poor reproductive outcomes of obese women. 5 However, the therapeutic strategies to improve oocyte quality of obese women have not been well explored.
Nicotinamide adenine dinucleotide (NAD + ) is an important cofactor which participates in various biological processes, including energy metabolism, DNA repair, genomic stability and cell survival. 9,10 The NAD + level decreased with age and metabolic abnormalities, such as obesity and diabetes. 11 Study has shown that the loss of NAD + decreased oocyte quality and compromised early embryonic development in obese mice. 12 Nicotinamide mononucleotide (NMN), a key precursor of NAD + , can enhance NAD + biosynthesis and reverse defects caused by insufficient NAD + . Therefore, administration of NMN can alleviate physiological abnormality in ageing mice. 13 NMN can also benefit the patients of type 2 diabetes induced by high-fat diet and enhance liver insulin sensitivity. 11 In 2020, Miao et al. and Bertoldo et al. separately reported that NMN supplementation recovered NAD + level and improved the oocyte quality in ageing mice. 14,15 Despite progress has been made in the application of NMN towards ageing and obesity, the effect of NMN on oocyte quality and female fertility in obese mice has not been fully explored.
In our study, we established obesity model by high-fat diet feeding, and further investigated the effect of NMN on oocyte quality and reproductive performance of obese mice. Our results showed that the in vivo administration of NMN reduces ovarian inflammation, improves oocyte quality and restores offspring weight of obese female mice. Furthermore, the NMN supplementation improved oocyte quality of obese mice by recovering mitochondrial function, reducing the accumulated ROS and suppressing apoptosis.

| Fasting blood glucose and oral glucose tolerance test
For fasting blood glucose, the blood glucose level of mice was measured after they were fasted for 12 h. For oral glucose tolerance test (OGTT), mice were fasted for 4 h followed by a glucose administration (2 g/kg) by gavage, then the blood glucose level was measured at 0, 30, 60 and 120 min after the glucose load. The blood glucose was detected with one Touch Ultra glucose meter (ACCU-CHEK Performa, Roche Diabetes Care GmbH).

| RNA isolation and quantitative real-time polymerase chain reaction
Total RNA was exacted from 50 oocytes and reversed to cDNA using SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen).
The total RNA of ovary was extracted using TRIzol (Takara, Japan) and reversed to cDNA using RevertAid Master Mix (https://www.

| Histological analysis of fat
Mouse abdominal fat used for histological analysis was collected and fixed in 10% neutral formalin buffered solution for at least 24 h, dehydrated, and embedded in paraffin. Paraffin-embedded fat was then sectioned at a thickness of 5 μm for further haematoxylin and eosin (H&E) staining.

| Immunofluorescence staining
Indirect immunofluorescence was performed as previously described with some modifications. 16 The zona pellucida of oocytes was removed using acidic tyrode solution (Sigma) and oocytes were then fixed with

| Statistical analysis
All values from at least three independent replicates were expressed as mean ± SEM. Data were analysed by t-test or chi-squared test using GraphPad Prism 7 statistical software. P < 0.05 was considered as significant.

| Establishment of obese mouse model
To establish the obese mouse model, 4-week-old mice were fed with normal diet (ND) or high fat diet (HFD) for 12 weeks and the body weight of mice was measured every 2 weeks. The body weight of mice fed with high fat diet (HFD mice) is significantly higher than that of mice fed with normal diet (ND mice) ( Figure 1A,B). Consistently, we found the FGB value of HFD group is higher than that of ND group ( Figure 1C). Furthermore, the OGTT results indicated that the HFD mice are glucose intolerant ( Figure 1D,E). These results indicated that the obese mice model is established successfully.

| NMN protected against abnormal metabolism in HFD mice
After 12 weeks of dietary intervention, the ND mice and HFD mice were intraperitoneally injected with PBS or NMN for 10 consecutive days ( Figure 2A). The body weights of HFD mice treated with PBS were gradually increased, but there was no dramatic change on the body weights of HFD mice treated with NMN ( Figure 2B). The adipose size of abdominal fat tissue was significantly larger in HFD mice than in ND mice ( Figure 2C), however, NMN administration reduced the adipose size of HFD mice ( Figure 2D). These results showed that the administration of NMN protected against abnormal metabolism in HFD mice.

| NMN improved the ovary quality of HFD mice
Next, we sought to investigate the effect of NMN on the ovary physiology of obese female mice. There was no obvious morphological difference T A B L E 1 Primer sequences of genes for quantitative realtime PCR

Gene
Primer sequence on the ovaries among the ND, HFD and HFD + NMN groups ( Figure 3A). Given that actin dynamics is the key index of the oocyte quality, we then performed immunofluorescence experiment on the MII oocytes to examine whether HFD exerts detrimental effect on actin dynamics to further decrease oocyte quality. The results showed that the actin fluorescence intensity at the plasma membrane of HFD oocytes was lower than that of the ND group, and the supplement of NMN could significantly increase the actin fluorescence intensity at the plasma membrane of the HFD oocytes ( Figure 5H,I). These results indicated that NMN could improve oocytes quality partially by maintaining the integrity of cytoskeleton.

| NMN restored lipid droplet distribution of HFD mice
High fat diet could induce abnormal lipid droplet distribution in oocyte, which may be one of the reasons for the decline of oocyte quality. Therefore, we detected the lipid droplets in the indicated oocytes. Our results showed that the lipid droplet fluorescence intensity in HFD oocytes is slightly higher than that in ND oocytes, and the administration of NMN could reduce the fluorescence intensity of lipid droplets ( Figure 6A,B).

| NMN restored offspring weight of HFD mice
We next sought to investigate the longer effect of NMN treatment on embryo development. The offspring numbers and weights in three groups were then assessed. There was no significant difference in the pregnancy probabilities of mice in three groups ( Figure 7A). The female mice showed a similar litter size between three groups ( Figure 7B). However, the birth weight of pups in the HFD group was significantly lower than that in the ND group, and the NMN supplement could rescue the reduced birth weight of pups caused by HFD ( Figure 7C).

| DISCUSSION
The obesity induced by high fat diet does harm to female fertility due to the possible damage on oocyte quality. 5 Multiple drugs, such as melatonin, phycocyanin and resveratrol, have been developed to improve obese mice fertility. [19][20][21] Recent studies reported that the administration of nicotinic acid (NA) and nicotinamide riboside (NR) could alleviate subfertility of obese mice by elevating NAD + level. 12,22 NMN is a key NAD + intermediate which has been shown effective in the improvement of oocyte quality in ageing mice. 14,15 However, the exact function of NMN on the obese mice fertility are not fully studied. In our study, we investigated the effect of NMN on ovaries and oocytes of HFD-induced obese mice. Previous studies have shown that obesity induced by high fat diet could cause ovarian dysfunction and affect ovarian genes involved in folliculogenesis, thus reducing oocyte quality. 23 We found that the ovary weight and ovarian organ index were lower in HFD group than that in ND group. We wondered if high fat diet and NMN administration would change the expressions of those genes such as Bmp4 and Lhx8, which were related to ovarian follicular development. 24,25 We found that the mRNA level of Bmp4 and Lhx8 were reduced in HFD group and NMN administration recovered the expression of these two genes. Above results revealed that the NMN administration reversed the abnormal ovary weight and the abnormal expression of folliculogenesis-related genes including Lhx8 and Bmp4.
It has been reported that high fat diet leads to inflammation which plays important role in oocyte maturation and ovulation. 26,27 NMN has been shown to relieve LPS-induced inflammation by suppressing pro-inflammatory cytokine production. 28 Our results showed that the expression of pro-inflammatory genes including Adgre1, Ccl2, TNF-α and Gal-3 was upregulated but the expression of antiinflammatory genes including Clec10a and IL-10 was downregulated in HFD group. Importantly, the NMN administration reversed their abnormal expression profile.
Normal spindle-chromosome structure is essential for oocyte maturation and fertilization. 29 The defective spindle-chromosome complex (SCC) during meiosis can lead to aneuploidy, pregnancy loss and genetic diseases. 30,31 Studies have shown that the structure of the SCC in HFD mouse oocytes was disrupted. 5,19 Consistently, in our The fluorescence intensity of actin filaments on the membrane was quantified in ND, HFD and HFD + NMN mice oocytes. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 damage and apoptosis. 5 The distribution pattern of mitochondrion has been considered as one of the important indicators for the oocyte cytoplasmic maturation because of its vital role for cell development. 33 Previous studies on the effect of NMN on maternally aged oocytes have shown that NMN could eliminate the ROS level, recover mitochondrial function and reduce DNA damage. 14 In order to assess the function of NMN on the quality of HFD oocytes, we then measured the ROS level, mitochondrial function and DNA damage of oocytes in ND group, HFD group and HFD + NMN group. We found that HFD induced a higher ROS level, damaged mitochondrial function and more DNA damage, and NMN treatment could recovered these indexes. These data indicated that NMN inhibited the ROS accumulation in oocytes of HFD mice which further improved oocyte quality of HFD mice. It was reported that the actin dynamics is involved in oocyte quality control and NMN could recover the abnormal actin dynamics in porcine oocytes. 34 In line with this, we found that high fat diet disturbed the integrity of actin in oocytes and NMN could restore the actin dynamics in the HFD mice oocytes.
In our study, there was no significant difference on the pregnancy rate among the three groups. Moreover, previous study has shown that HFD mice ovulated smaller number of MII oocytes and produced less litter size. 20 However, in our study, HFD mice could ovulated similar number of MII oocytes and produced a similar litter size compared with ND mice. Although there was no significant difference in litter size between the ND and HFD group, HFD mice showed a declined tendency in the litter size. The possible reason is that the genetic background of the mouse strains used in our study differed from that used in previous study. 20 Furthermore, the mice age might also reduce the difference of the ovulation rate and litter size as these mice reached an older stage (more than 17 weeks) after the HFD treatment. Moreover, the little number of mice used in the experiment might affect the results. However, our results showed that the birth weight of pups in HFD group was lower than that in the control group, and NMN administration could help to recover their birth weight.
Altogether, we provided in vivo evidence to show that NMN has a capability to improve the quality of oocytes in HFD-induced obese mice and revealed a potential mechanism that NMN could restore mitochondrial function, reduce ROS accumulation, DNA damage and lipid droplet distribution of HFD oocytes. Therefore, our work will provide a theoretical reference for improving the reproductive outcome of female obese people with the administration of NMN.