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.

Introduction

In the context of female reproduction, the quality of the oocyte is a pivotal factor influencing the potential for successful embryonic development [1–4]. Oocyte aging is associated with many deleterious effects, such as decreased fertilization potential, decreased in vitro fertilization (IVF) success rates, impaired developmental competence, and an increased incidence of embryonic resorption, while the underlying mechanism of oocyte aging remains unknown [5]. Generally, there are two kinds of oocyte aging that have been represented and studied, namely, reproductive or maternal aging and POA. If fertilization is not completed within 12 h for rodents and 24 h for monkeys and humans after ovulation, the unfertilized oocytes arrested at metaphase II (MII) stage will remain in the oviduct in vivo or remain arrested under culture systems in vitro and experience time-dependent deterioration of quality via a process named postovulatory oocyte aging [6–8]. POA is clinically inevitable. To be more specific, during the process of ART in humans, oocytes that are collected may undergo an extended culture phase before fertilization, which can diminish the potential for the embryos to thrive. Both natural conception and procedures involving POA have been linked to decreased rates of successful fertilization, suboptimal embryo development, unsuccessful implantation, and the occurrence of congenital issues in the offspring [8]. Similar to maternally aged oocytes, POA oocytes show morphological and molecular changes, such as first polar body (PBI) degeneration, an increased perivitelline space, ROS accumulation, energy depletion, chromosome separation disruption, and DNA fragmentation [7–9]. Accumulating evidence has demonstrated that the POA process severely deteriorates oocyte quality and subsequent fertilization and early embryo developmental competence, which remains one of the most intractable problems associated with ART failure [10]. Although some drugs such as caffeine, melatonin, and resveratrol have been shown to extend the window for fertilization, the current strategies of preventing postovulatory oocyte aging are not widely applied due to the insufficient efficacy and unknown side effects of these drugs [7, 11–13].

The metabolite nicotinamide adenine dinucleotide (NAD⁺) is a prominent redox cofactor that is indispensable for DNA repair, energy metabolism, autophagy, genomic stability, and epigenetic homeostasis [14, 15]. Over the last several decades, an increasing number of studies have reported that NAD⁺ levels decline with age across multiple tissues, and loss of NAD⁺ is implicated in various diseases associated with aging, including metabolic diseases, neurodegenerative diseases, and cancer [15, 16]. Accordingly, supplementation with NAD⁺ precursors has been shown to reverse the declines in NAD⁺ content in aged organs and display beneficial effects against aging to maintain late-life health [16, 17]. NR is one of the NAD⁺ precursors. Once it enters the cell, NR is converted to NAD⁺ through a two-step reaction. Initially, it is catalyzed by nicotinamide riboside kinases (NRKs) and metabolized into nicotinamide mononucleotide (NMN); this step is followed by the production of NAD⁺ by nicotinamide mononucleotide adenylyltransferase (Nmnat) [18]. In addition, NR can be found in some foods, such as milk [19, 20], which constitutes a dietary source for NAD⁺ supplementation. Studies have demonstrated that NR treatment ameliorates metabolic and age-related disorders characterized by defective mitochondrial function [21, 22]. Recently, many studies have reported that supplementation with NR or NMN, which increases ovarian NAD⁺ levels, can mitigate the age-related decline in female fertility in mice, reverse egg aging, and improve the fertility rates of older female mice [23–25]. This offers hope to women struggling with conception. Notably, a recent study suggests that NR can ameliorate POA [26]. Although significant progress has been made in using NR to combat aging and delay POA, further in-depth investigation is required to understand the specific effects of NR supplementation on postovulatory oocyte aging and the underlying mechanisms, ensuring the clinical application of NR [26, 27]. In this study, we took advantage of an in vitro postovulatory oocyte aging model to explore the influences of NR supplementation on postovulatory aged oocytes in mice. We demonstrated that NR supplementation in vitro attenuated features of the aging-related deterioration of oocyte quality, such as mitochondrial dysfunction, mis-localization of cortical granules, and the subsequent decline in embryonic development potential, likely by inhibiting oxidative stress through NAD⁺/SIRT1 signaling. Our study provides evidence that NR is an efficient natural component that prevents the process of POA and is thus an ideal antiaging drug for ART in clinical practice.

Materials and methods

The present study was approved by the Institutional Review Board of Peking University Third Hospital.

Animals and reagents

The mice used here were treated in line with the guidelines for the care and use of laboratory animals. The animals used in this study were 8- to 10-week-old ICR mice (CD-1, Animal Center of Medical College of Peking University). The mice were housed and bred in the Animal Center of the Medical College of Peking University, and they were allowed to acclimate for 7 days before the experiments began. M2 medium was purchased from Sigma‒Aldrich (Saint Louis, MO, USA). Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were obtained from the Ningbo Second Hormone Factory (China).

NR supplementation

NR was purchased from BioChemPartner (China, catalog number 1341–23-7), dissolved in DMSO and diluted with maturation medium (M2 medium) to a final concentration of 100, 200, or 500 μM. We selected the concentrations of NR after a comprehensive literature review and preliminary experiments, ensuring they were within a range explored in other studies and biologically relevant for observing dose-dependent effects in our specific cell models and assays.

Oocyte collection and culture

Female ICR mice were superovulated by intraperitoneal injection with 10 IU of PMSG, which was followed by injection with 10 IU of hCG 48 h later. Cumulus–oocyte complexes were collected from oviductal ampullae approximately 14 h after hCG injection. Mice were sacrificed via cervical dislocation, and the cumulus cells were removed by brief incubation with 0.2% hyaluronidase. For the in vitro aging process, denuded oocytes were observed under stereomicroscopy, and mature oocytes displaying a first polar body were collected and further cultured in M2 medium with or without NR under liquid paraffin oil at 37 °C in a humidified atmosphere in an incubator gassed with 5% CO₂ in air, while the groups without NR were cultured with DMSO as a control. We cultured 20–30 oocytes and embryos together in a single culture drop, with a volume of 50 µL of culture medium per drop. This volume was selected to ensure adequate nutrient availability while maintaining appropriate gas exchange for the developing embryos. Furthermore, we implemented a quality control procedure where dead oocytes and embryos were carefully monitored and subsequently removed from the culture drops to prevent any potential negative impact on the viability and development of the remaining oocytes and embryos. After 24 h of culturing, oocytes were obtained for subsequent analysis. Fresh MII oocytes without an in vitro aging process were used for the fresh group.

Immunofluorescence and confocal microscopy

For reactive oxygen species (ROS) detection and mitochondrial membrane potential (MMP) staining, oocytes were incubated in PBS containing 10 μM DCFH-DA (Beyotime Biotechnology Company, Shanghai, China) or JC-1 buffer solution supplemented with 2 μM JC-1 probes (Beyotime Biotechnology Company, Shanghai, China) at 37 °C under 5% CO₂ for 30 min. Oocytes incubated in PBS were regarded as the negative controls. After washing three times with PBS supplemented with BSA, the oocytes were observed by laser scanning confocal microscopy, and the fluorescence intensity of the oocytes was calculated with ImageJ (ImageJ software, Bethesda, MD, USA).

For staining of α-tubulin, oocytes were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. After fixation, the oocytes were washed three times in PBS containing 0.1% BSA for 5 min and then permeabilized in 0.5% Triton X-100 for 15 min at room temperature. The oocytes were then washed and blocked in PBS containing 1% BSA for at least 30 min and stained with an anti-α-tubulin-FITC antibody (1:200) for 20 min at room temperature. Hoechst 33,342 (1:100 dilution) was used to stain DNA for 5–10 min. After washing three times with washing buffer, the oocytes were mounted in 1% BSA covered with a thin layer of mineral oil, and then, the oocytes were imaged with a confocal microscope (LSM710, Carl Zeiss, Oberkochen, Germany).

To observe the amount of DNA damage, a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, Beverly, USA, 1:200 dilution) was used for immunofluorescence staining to analyze DNA double-strand breaks. Then, the oocytes were analyzed using a confocal microscope. To determine the expression of γH2AX in oocyte samples, the stained oocytes were scanned along the Z axis, and continuous photographs were captured under a confocal laser scanning microscope. The positive γH2AX signal was calculated using an auto-counting program in Zeiss 2011 software. This program is specifically designed to enhance the accuracy and efficiency of fluorescence signal analysis. The auto-counting feature allowed us to objectively quantify the fluorescence intensity within the DNA nuclei, ensuring that the measurements were specific and not confounded by non-specific fluorescence signals that might occur outside the nuclei.

For ATP staining, we referred to articles that used this ATP detection kit, which researchers also measured the ATP content in fixed cells [28]. After fixation and permeabilization, oocytes were washed three times and incubated in PBS containing 500 nM BODIPY FL ATP (Molecular Probes, Eugene, OR) for 1 h at room temperature in the dark. Then, the oocytes were washed three times in PBS supplemented with BSA and mounted on a confocal-compatible culture dish. Images of each oocyte were captured by confocal microscopy.

To assess the distribution and dynamics of cortical granules (CGs), the specific marker LCA-FITC was used. Similarly, after fixation and permeabilization, oocytes were collected and blocked in PBS supplemented with 1% BSA for 30 min and then stained with an LCA-FITC antibody (1:100) at 4 °C overnight. Then, the oocytes were counterstained with Hoechst 33,342 (1:100) for 10 min. After washing three times with washing buffer, the oocytes were collected on glass slides and observed under a confocal laser scanning microscope (Carl Zeiss 710).

All images were acquired using a Zeiss LSM 710 confocal microscope (Carl Zeiss Microscopy GmbH, Germany). The magnification used was × 100, and the imaging conditions were consistent across all samples. The laser power and other imaging settings were optimized to minimize photobleaching and ensure the accuracy of the fluorescence measurements. The image analysis was performed using ImageJ (ImageJ software, Bethesda, MD, USA) and ZEN (Carl Zeiss Microscopy GmbH, Germany) software. The fluorescence intensity was quantified by selecting regions of interest (ROIs) within the images and measuring the mean intensity values. Care was taken to ensure that the ROIs were placed in equivalent positions across all samples. Background fluorescence was subtracted from the ROI measurements by using the rolling ball algorithm in ImageJ, which helps to account for any uneven illumination or background signal.

In vitro fertilization

Adult male ICR mice were used for sperm collection. Conventional IVF was conducted using human tubal fluid (HTF) medium at 5% CO₂, 37 °C. The cauda epididymis was harvested, and sperm were collected and suspended in HTF medium before capacitation at 37 °C under 5% CO₂ for 1 h. After capacitation, sperm were carefully added to freshly ovulated oocytes to a final concentration of 4 × 10⁵/mL, and the gametes were cocultured for 6 h. The existence of two pronuclei was regarded as an indicator of successful fertilization. After they were washed three times, the zygotes were transferred to and incubated in KSOM medium and were assessed for their blastocyst efficiency 4–5 days later. The blastocyst rate was assessed 4 days after fertilization for the fresh group, while for the groups treated with POA and NR, the assessment was made 5 days later. As for the blastocyst rates, we counted them from the initial number of oocytes.

Quantitative real-time PCR

Total RNA from 100 oocytes from the three groups was extracted using a RNeasy Mini Kit (Qiagen, MD, USA) according to the manufacturer’s instructions and then reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). Samples of each group were stored at − 20 °C until use. Each reaction used to determine the expression level of each mRNA was composed of 4 μL of cDNA template, 10 μL of SYBR Green Master Mix, 2 μL of gene-specific primers, and 4 μL of water. The units of fluorescence were converted into cycle threshold (CT) values. The expression level of RNA was calculated by the formula 2^−ΔΔCt.

Measurement of NAD+ content

NAD⁺ content was detected by the UPLC‒MS/MS (ultra-performance liquid chromatography–tandem mass spectrometry) method with some modifications as previously reported [29]. Briefly, 300 oocytes from three groups were collected. Total metabolites were extracted by using prechilled 80% methanol and then incubated at − 80 °C for 2 h. After centrifugation, all the lysate mixtures were dried to pellets using a SpeedVac. Lamivudine was diluted to a final concentration of 10 ng/mL by using prechilled water and added to each sample as an internal standard. Then, the samples were vortexed and centrifuged at 14,000 × g for 20 min at 4 °C. The supernatants were transferred to new tubes and stored in a − 80 °C freezer.

The UPLC‒MS/MS experiments were performed using a Waters Xevo TQ-S system. A 2 μL aliquot was injected into a 2.1 × 100 mm, 1.8 μm column (Waters ACQUITY UPLC HSS T3). The mobile phases comprised 5 mM ammonium acetate (pH 7.5) in water (A) and acetonitrile (B). The extracts were gradient-eluted using the following program: (1) 0% B for 1.5 min, (2) increase to 25% B at 5 min, (3) increase to 98% B at 5.5 min and hold for 2.5 min, and (5) revert to 0% B at 8.05 min and hold for 10 min. The flow rate was 0.3 mL/min. The mass spectrometer was operated in positive mode with a capillary voltage of 3.2 kV. The MS desolvation temperature and source temperature were held at 400 °C and 150 °C, respectively, with a cone gas flow rate of 150 L/hr. The UPLC‒MS/MS data were processed by software automatically. The relative intensity of NAD⁺ in each sample was the peak area of NAD⁺ divided by the peak area of the internal standard, which is also regarded as the relative response. The injection volume of each sample was 3 injections, and the average value of the 3 reactions was considered the final relative content value.

Statistical analysis

Fluorescence intensities were all measured using ImageJ software. The results were compared using SPSS 17.0 software (Chicago, IL, USA). Data derived from at least three independent replicates are shown as the mean ± SEM, and the experiment data were all analyzed by Student T-test or one-way ANOVA and considered statistically significant at values of P < 0.05. Statistics and graphs were made by the Prism6 (GraphPad Software, La Jolla) and Adobe Illustrator 2014.

Results

NR restores NAD+ levels and improves the quality of postovulatory aging oocytes

To investigate the potential involvement of NR in maintaining the quality of postovulatory aging oocytes and to confirm the appropriate concentration of NR supplementation, we first examined the morphological changes and integrity of ovulated mouse MII oocytes aged for 24 h in vitro with or without NR supplementation (Fig. 1A). Fresh MII oocytes obtained from superovulated mice were considered the fresh group. Fresh oocytes were incubated in vitro with 0 (POA group), 100, 200, or 500 μM NR. As shown in Fig. 1B, C, the aging oocytes displayed typical morphological abnormalities, including fragmentation, degeneration, and parthenogenetic activation, after 24 h of culturing. The quantitative results showed that the ratio of abnormal oocytes in the aging group was significantly higher than that of fresh oocytes (fresh 6.3 ± 0.1% vs. POA 41.3 ± 2.4%, P < 0.001; Fig. 1B). In addition, NR supplementation exerted protective effects against morphological defects of oocytes, and these protective effects were concentration dependent. In particular, 200 μM NR remarkably reduced the abnormal oocyte rate from 41.3% in the aging group to 21.1% in the NR group (POA 41.3 ± 2.4% vs. NR 21.1 ± 2.7%, P < 0.001; Fig. 1C). However, when mature oocytes were exposed to 500 μM NR, the morphological abnormalities were not improved compared with those under 200 μM NR supplementation (200 μM NR 21.1 ± 2.7% vs. 500 μM NR 22.2 ± 4.1%, P < 0.001; Fig. 1C). Therefore, we chose 200 μM NR for the subsequent study. Given that NR treatment increases NAD⁺ levels in different tissues, we then analyzed the NAD⁺ levels in aging oocytes. As expected, we detected significant declines in NAD⁺ levels in aging oocytes; however, NAD⁺ accumulation was present in aging oocytes after 200 μM NR treatment, indicating that NR supplementation might be a feasible strategy to enhance the quality of aging oocytes (fresh 1403.0 ± 34.3 vs. POA 531.9 ± 143.7, P < 0.01; NR 1107.0 ± 116.8 vs. POA 531.9 ± 143.7, P < 0.05; Fig. 1D).

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

NR rescues early embryo development in aging oocytes

Having determined the potential role of NR in the improvement of the quality of POA oocytes, we next investigated whether NR indeed elevates the embryonic development potential of POA oocytes after fertilization. We performed in vitro fertilization of oocytes in three groups and recorded the rates of early embryo development. The results showed that the blastocyst rate of POA oocytes was significantly lower than that of fresh oocytes (POA 7.9 ± 1.8% vs. fresh 64.3 ± 3.5%), while NR significantly increased the blastocyst rate (NR 19.9 ± 3.0%) (Fig. 1E, F). Collectively, these results indicate that NR supplementation is able to improve early embryonic development.

NR prevents ROS accumulation during postovulatory oocyte aging

Given that oxidative stress is induced by breaking of the balance between the generation and clearance of ROS and that ROS accumulation is considered the key primary factor underlying cellular aging in aging oocytes, we further explored whether NR alleviates oxidative stress in aging oocytes. To achieve this, we used the DCFH-DA probe to evaluate intracellular ROS levels in oocytes. As shown in Fig. 2A, B, the green fluorescence signals of DCFH-DA were hardly detected in the fresh oocytes, but they were dramatically enhanced in aging oocytes (fresh 6.2 ± 0.9 vs. POA 20.0 ± 4.0, P < 0.001), suggesting that the postovulatory aging process induced oxidative stress in oocytes. Accordingly, the ROS signals in NR oocytes were significantly weaker than those observed in aging oocytes (NR 10.1 ± 2.7 vs. POA 20.0 ± 4.0, P < 0.05) (Fig. 2A, B). Collectively, these findings reveal that NR prevents ROS accumulation during postovulatory oocyte aging in vitro.

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

NR restores normal spindle assembly and chromosome alignment during postovulatory oocyte aging

Because accumulated ROS leads to cytoskeletal abnormalities in aging oocytes and because normal spindle assembly with aligned chromosomes has been accepted as one of the most important indicators of development potential, we further explored spindle morphology and chromosome alignment in postovulatory aging oocytes. To this end, immunofluorescence staining was performed in oocytes with an anti-α-tubulin-FITC antibody to visualize spindle morphologies and Hoechst 33,342 to evaluate chromosomal arrangement. As shown in Fig. 2C, D, a large majority of fresh oocytes displayed normal barrel-shaped spindle morphologies with well-aligned chromosomes on the equatorial plate. In contrast, more than 70% of oocytes from the POA group exhibited strikingly disordered spindles and poorly aligned chromosomes (Fig. 2C, D). Specifically, the microtubules gradually drifted away from spindles, which leads to depolymerization of the spindle structure. Chromosomes in the control group formed irregular clusters, while 49.3% of NR-treated oocytes displayed normal spindles and aligned chromosomes (Fig. 2C, D). As expected, NR supplementation significantly prevented abnormalities in the spindle apparatus during postovulatory oocyte aging in vitro (fresh 10.9% vs. POA 74.4%, P < 0.001; NR 49.3% vs. POA 74.4%, P < 0.05). Collectively, these observations suggest that NR supplementation can partially restore normal spindle assembly and chromosome alignment in postovulatory aging oocytes.

NR promotes mitochondrial function during postovulatory oocyte aging

Mitochondrial oxidative phosphorylation generates cellular energy in the form of ATP as well as superoxide free radicals, making good mitochondrial dynamics a compelling indicator of competent oocytes. Mitochondrial dysfunction is closely related to the decline in the quality of aging oocytes, which is reflected in atypical localization and aggregation of mitochondria, a decrease in mitochondrial membrane potential (MMP), and a decrease in ATP production. Therefore, we next confirmed postovulatory mitochondrial function in vitro.

First, mitochondrial distribution was assessed with MitoTracker Red CMXRos and analyzed by confocal microscopy. In fresh oocytes, mitochondria generally showed a homogeneous distribution in the oocyte cytoplasm, which represented a typical pattern of mitochondrial distribution in mature oocytes (Fig. 3A). After culture in vitro for 24 h, more than 65% of aging oocytes displayed an abnormal clustering distribution of mitochondria in the cytoplasm (fresh 16.7% vs. POA 65.2%, P < 0.001; Fig. 3A, B). Compared to the aging-oocyte group, the NR-supplemented-oocyte group exhibited a significantly lower abnormal mitochondrial dynamics ratio of 24.3% (POA 65.2% vs. NR 24.3%, P < 0.01; Fig. 3A, B). Given the above observations, it is possible that the impaired mitochondrial distribution, which is associated with postovulatory aging in vitro, may be partially alleviated by treatment with NR. We next investigated whether NR exposure improved mitochondrial membrane potential. To verify this, the inner membrane potential dye JC-1 was used to evaluate the mitochondrial membrane potential. When the MMP is low, JC-1 displays green fluorescence; otherwise, it emits red fluorescence (Fig. 3C). MMP levels were calculated according to the ratio of red/green fluorescence. Statistically, the red/green ratio in aging oocytes was significantly lower than that in fresh oocytes, whereas it was totally restored in NR-treated aging oocytes (fresh 1.7 ± 0.2 vs. POA 0.3 ± 0.1, P < 0.001; NR 1.9 ± 0.2 vs. POA 0.3 ± 0.1, P < 0.001; Fig. 3C, D).

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

As stated previously, mitochondria are the main source of ATP production through the oxidative phosphorylation process, making them the key sources of the energy consumed in mature MII oocytes. In addition, ATP plays essential roles in several major events during the process of fertilization of oocytes, including Ca²⁺ oscillation, chromosome segregation, related enzyme activation, etc., making mitochondria the main energy sources in oocytes. Given that the decline in ATP content is a convincing indicator of mitochondrial dysfunction in oocytes, we further traced ATP production in oocytes after NR treatment in vitro. The quantitative results revealed that the ATP content significantly declined with aging but was improved upon NR supplementation (fresh 33.0 ± 0.7 vs. POA 15.3 ± 1.3, P < 0.001; NR 20.1 ± 0.4 vs. POA 15.3 ± 1.3, P < 0.001; Fig. 3E, F). In conclusion, these findings reveal that NR treatment can promote mitochondrial function in oocytes during postovulatory aging in vitro.

NR reduces DNA damage in aging oocytes

Since excess ROS are associated with DNA damage, we next explored the potential effect of NR on DNA damage during postovulatory oocyte aging. As γH2AX reflects DNA damage, the severity of DNA damage in oocytes was examined by investigating the fluorescence intensity of γH2AX staining. In Fig. 4A, the irregular chromosome clusters in NR oocytes are significantly fewer than in aging oocytes. For γH2AX staining, the statistical results showed that γH2AX levels were significantly elevated in oocytes following the aging process in comparison with fresh oocytes (POA 26034.0 ± 1979.0 vs. fresh 6383.0 ± 687.6, P < 0.001; Fig. 4A, B) but were significantly reduced in NR-supplemented oocytes (POA 26034.0 ± 1979.0 vs. NR 7668.0 ± 610.1, P < 0.001; Fig. 4A, B), suggesting that DNA damage during postovulatory aging might be attenuated by supplementation with NR.

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

NR restores mis-localized cortical granules in aging oocytes

Mammalian cortical granules (CGs) are membrane-bound vesicles that localize in the cortices of unfertilized oocytes. After fertilization, CGs undergo exocytosis and release their contents into the perivitelline space (PVS), and the released cortical granule proteins function to block polyspermy. CGs have long been thought to be significant indicators of oocyte cytoplasmic maturation. We next considered whether the dynamics and function were interrupted by the postovulatory aging process. Staining with LCA markers revealed that CGs were located under the subcortical region and absent from the cortical granule-free domains (CGFDs), which are associated with the meiotic spindle in fresh oocytes (Fig. 4C, D). In contrast, oocytes after the aging process displayed an abnormal distribution pattern of CGs, showing discontinuous or totally absent fluorescence signals (Fig. 4C). In addition, CGs could be detected in the CGFDs in some other aging oocytes (Fig. 4C). Furthermore, the abnormal distribution rate of CGs in aging oocytes was partially alleviated after NR supplementation (POA 92.1% vs. NR 64.5%, P < 0.01; Fig. 4C, D). Altogether, these results indicate that NR supplementation might restore the mis-localized CGs in aging oocytes.

NR improves the quality of aging oocytes via the NAD+/SIRT1 axis

We have verified the effectiveness and safety of NR in preventing the aging of oocytes from various perspectives, and as a result, the embryonic development potential of these aging oocytes is enhanced. To determine the underlying mechanisms of NR’s protective effects, we then measured whether the expression levels of antioxidant enzymes were changed. The results showed that compared with those in the oocytes of the fresh group, the mRNA expression levels of Sod1 and Gpx1 in the oocytes of the POA group were significantly decreased, while the expression levels were partially restored by NR treatment, indicating that NR can alleviate the damage to antioxidant capacity in POA oocytes (Fig. 4E). In addition, NAD⁺ levels decrease with age, which reduces the molecular function of SIRT1. The molecular mechanism that links NAD⁺ decline and aging is still unclear. The NAD⁺/SIRT1 signaling axis and its downstream molecule PGC1α may play an important role in the aging process. We tested the expression levels of Sirt1 and PGC1α in the fresh group, the POA group, and the POA group with NR. The results showed that the mRNA expression levels of Sirt1 and PGC1α were decreased in the POA group, but NR effectively reversed this change (Fig. 4E). In summary, NR can improve the quality of oocytes through antioxidation and antiaging mechanisms, thereby improving the developmental potential of embryos.

Discussion

POA is the rapid degradation of oocytes that occurs beyond the optimal fertilization window. In clinical ART, POA significantly affects the quality of retrieved oocytes, potentially leading to reduced fertilization rates, poorer embryo quality, and an increased risk of aneuploidies, thereby impacting ART outcomes. Efforts to improve ART have increasingly focused on addressing POA of oocytes, which is a critical factor influencing the success of these reproductive procedures [7–9, 11].

Numerous researches investigated various compounds that exhibit potential in delaying POA of oocytes, a process leading to a decline in oocyte quality and fertility. Antioxidants such as N-acetyl-L-cysteine (NAC), melatonin, resveratrol, coenzyme Q10 (CoQ10), and astaxanthin are highlighted for their roles in reducing ROS and oxidative stress, which are key factors in POA [7]. These antioxidants protect the oocytes by maintaining mitochondrial function, spindle organization, and chromosomal integrity. For instance, NAC is shown to improve spindle organization and decrease ROS, while melatonin enhances the distribution of cortical granules and reduces cytoplasm fragmentation [30, 31]. Resveratrol and CoQ10 similarly support spindle and chromosome alignment, crucial for oocyte health [13, 32]. Additionally, compounds like putrescine and imperatorin are noted for their ability to stabilize cellular calcium levels and modulate the activity of cell cycle regulatory factors, thereby preventing apoptosis and supporting oocyte viability [33, 34]. These antiaging chemicals collectively target the molecular mechanisms of POA, aiming to preserve the quality of oocytes and potentially increase the success rate of assisted reproductive technologies. Current methods to alleviate POA in oocytes, such as the use of antioxidants and other “antiaging” chemicals, have limitations. They may only partially reverse the effects of POA, and their mechanisms still require further in-depth investigation [7].

With advancing age, NAD⁺ levels in the ovaries decline, a change associated with the reduced efficiency of mitochondrial energy production and increased oxidative stress, which are key contributors to oocyte aging [23, 24, 27]. The NAD⁺ precursors, such as NMN and NR, have shown potential in modulating this aging process [14, 16, 35]. They can boost NAD⁺ synthesis, thereby enhancing mitochondrial function, reducing oxidative stress, and potentially improving oocyte quality [35, 36]. The restoration of NAD⁺ levels through these precursors might also upregulate Sirtuin activity, a family of proteins dependent on NAD⁺ for deacetylase action, further contributing to the delay of ovarian senescence [19, 23]. In essence, the modulation of NAD⁺ metabolism presents a promising therapeutic strategy against age-related ovarian and oocyte decline.

The most recent study showed that NR, a potent NAD⁺ precursor, plays a crucial role in combating POA in oocytes. As oocytes age, NAD⁺ levels plummet, triggering a cascade of detrimental effects, including the exacerbation of oxidative stress and apoptosis, which are hallmarks of declining oocyte quality. The pivotal study underscores that NR supplementation can effectively reverse these aging indicators by replenishing NAD⁺. This restoration not only mitigates oxidative stress and bolsters mitochondrial functionality but also attenuates DNA damage and apoptosis. NR enhances the quality of oocytes and their developmental capacity into viable embryos, thereby potentially escalating the efficacy of ART [26].

Our study also demonstrates NR can enhance oocyte quality during in vitro postovulatory aging by reducing cellular fragmentation and apoptosis. It mitigates meiotic spindle disruptions, DNA damage, and oxidative stress, indicated by decreased ROS levels, crucial for oocyte health. NR also preserves mitochondrial function, improving ATP production and MMP, suggesting a potential restoration of compromised mitochondrial function due to aging. Our research, building upon the work of others, further confirms the role of NR in mitigating POA and enhancing both oocyte quality and the embryonic developmental potential [21, 26, 35].

Compared to previous studies published in Reproduction [26], our research not only confirms the effectiveness of NR in rescuing POA but also provides new insights into the underlying mechanism by which NR achieves this rescue. Firstly, this study explores a broader range of NR concentrations (100, 200, 500 μM) compared to the lower concentrations used in Reproduction (0, 0.1, 0.5, 2.5 μM). This may reveal the different effects of NR at various dosages on the aging process, providing important information for finding the most effective therapeutic window. Despite the fact that the concentrations used in this study are significantly higher than those employed in previous work published in Reproduction, the rescuing effects observed are similar. This discrepancy in concentration yet congruence in efficacy could very well be attributed to the distinct genetic backgrounds of the mice utilized in the studies. This study used CD-1 mice as the experimental model, different from the KM mice in the Reproduction study, indicating that this study tested the effects of NR across different genetic backgrounds, thereby increasing the broad applicability of the study’s results. It is important to consider that the genetic makeup of the mouse strains might influence how they respond to the treatment, thereby potentially accounting for the observed similarities in outcomes despite the variation in concentration. Therefore, it is noteworthy that the differences in concentration may also be correlated with the different genetic backgrounds of the mice, suggesting that further investigation into strain-specific responses could be warranted. Another innovative aspect of this study is the examination of embryos 24 h after POA, focusing on long-term effects, as opposed to the 6-h examination in the Reproduction study, providing more flexible options for the future clinical application of NR.

In addition to that, this study also found an increase in ATP levels after NR treatment, which was not covered in the Reproduction study, indicating that NR may directly affect the energy metabolism of oocytes, providing new evidence for the potential role of NR in improving oocyte quality and embryonic development potential. Moreover, the improvement in cortical granule expression observed in this study offers new insights into the potential role of NR in enhancing oocyte quality. Cortical granules are pivotal to oocyte quality, with their distribution and functionality significantly impacted during POA. CGs are crucial for establishing the membrane block to polyspermy, indicating their role in fertilization success [37, 38]. Morphological changes in CGs, such as vacuolization and internalization, reflect cytoplasmic maturity and are associated with apoptosis, which reduces oocyte viability. Environmental chemicals, particularly endocrine-disrupting chemicals (EDCs), may exacerbate aging by impairing CG function, further affecting fertilization and embryo development. In essence, the state of CGs serves as a biomarker for oocyte aging and is integral to the success of fertilization and subsequent embryonic development [7, 39]. Our study found that NR protects oocytes from aging effects and supports cytoplasmic maturation, as shown by preserved CG dynamics. This leads to improved early embryonic development from aging oocytes.

Our study builds upon previous research that has shown NMN to be effective in restoring NAD⁺ levels in aged mice, thereby enhancing oocyte quality through improved ovulation, meiotic competence, and fertilization potential [24]. Previous studies have observed that ovastacin, a crucial component of CGs, is responsible for the postfertilization cleavage of ZP2, leading to zona pellucida hardening that prevents polyspermy. The supplementation with NMN has been noted to restore ovastacin levels in aged oocytes, which aligns with the hypothesis that NAD⁺ precursors like NMN play a role in maintaining oocyte cytoplasmic maturation and fertilization potential.

In light of these findings, we hypothesize that NR, similar to NMN, may also contribute to the regulation of CG distribution by elevating NAD⁺ levels. This regulation is a key factor in the hardening of the zona pellucida, which is consistent with the effects observed with NMN supplementation. However, the precise impact and mechanisms of NR on zona pellucida hardening necessitate further investigation to substantiate these preliminary observations.

In previous studies, they examined the function of the mitochondrial oxidative respiratory chain, a crucial step in energy production, and detected the mRNA expression of core proteins in the four complexes of the mitochondrial oxidative respiratory chain, including Ndufv1, Sdhb, Uqcrc2, and Atp5a1 [26]. NR can significantly enhance the expression levels of these proteins. Our study reveals that NR upregulates the expression of the antioxidant genes Sod1 and Gpx1 [40, 41], suggesting its potential to inhibit oxidative stress and reduce the production of ROS. This modulation enhances cellular antioxidant defenses, contributing to a reduction in oxidative stress and ROS that may slow the aging process and mitigate the risk of age-related diseases. The beneficial effects of NR are further linked to the NAD⁺/SIRT1 pathway, where NR’s ability to elevate NAD⁺ levels activate SIRT1, a crucial regulator of mitochondrial function and metabolic flexibility [21]. Our study provides evidence that the activation of SIRT1 and its downstream target PGC1α by NR indicates a mechanism that fortifies mitochondrial health and metabolic adaptability in oocytes. Collectively, these findings underscore the therapeutic potential of NR in combating conditions influenced by oxidative stress and highlight its promise in improving mitochondrial function and overall cellular resilience.

Existing literature compellingly demonstrates that POA induces various anomalies in the oocytes and their corresponding embryos at the morphological, molecular, genomic, and epigenetic levels. The underlying mechanisms behind these effects are progressively gaining recognition. It is evident that POA significantly contributes to a deteriorating environment for oocytes and embryos by elevating ROS levels, which in turn increases oxidative stress. This stress has the potential to impair the developmental capabilities of oocytes and embryos, which is a critical aspect of IVF and Intracytoplasmic Sperm Injection (ICSI) cycles.

Our research indicates that there may be a viable approach to mitigating the premature aging of oocytes in vitro, potentially leading to enhanced fertilization success and improved embryonic development within ART. However, it is imperative to undertake additional studies to ascertain the extent to which NR treatment can bolster the health and quality of oocytes and embryos in clinical scenarios. Furthermore, the possibility that POA could bequeath genetic and epigenetic changes to offspring underscores the necessity to delve into its underlying mechanisms, given that such alterations might precipitate subsequent health complications and pathologies. In summary, our preclinical findings underscore the potential of “antiaging” compounds like NR in the in vitro culture process to mitigate POA, which may contribute to the success rate of IVF treatments.

Author contribution

This project was conceived and coordinated by J.Q. and D.J. The experiments were carried out by T.J.L. and Y.B.W. The sample collection was coordinated by T.J.L., W.D.P., and L.F. Y.Y. joined the data analysis and discussion. The paper was written by T.J.L. and Y.B.W. and revised by Y.Y.

Funding

This project was funded by the National Science Fund for Distinguished Young Scholars (82225019), the National Key Research and Development Program of China (2021YFC2700303), the National Natural Science Foundation of China (82192873, 81971381, 82101714, and 81771580), and the Innovation and Transformation Fund of Peking University Third Hospital.

Declarations

Competing interests

The authors declare no competing interests.