Animals

Approval for animal studies was obtained from the ethics committee. Female ICR mice were obtained from SPF (Beijing) Biotechnology Co., Ltd. Standard conditions were maintained for housing, including unrestricted access to food and water, a controlled environment with appropriate temperature and humidity, and a 12-h light–dark cycle. As previously mentioned [28,29,30], the female mice aged 3 weeks were subcutaneously injected with 60 mg DHEA/kg body weight (MCE, USA) dissolved in 0.2 ml of sesame oil (MCE, USA) to induce the PCOS mouse model, while the control mice received a daily injection of 0.2 ml sesame oil. After 21 days of DHEA treatment, the injections were discontinued, and DHEA-treated mice were randomly assigned to two experimental groups. One group received NR (Jiangsu Medicience Biological Medicine Co., Ltd) supplementation at a dose of 400 mg/kg/day for a period of 14 days, while the other group maintained a standard diet. This dose was selected based on previous studies, which demonstrated that it effectively increased NAD+ levels and improved mitochondrial function [26, 31,32,33,34]. At the end of the 14-day intervention, all three groups of mice were processed simultaneously and under identical experimental conditions.

NAD+ content detection

For the assessment of ovarian NAD+ levels, we utilized the NAD+/NADH assay Kit (Abcam, UK). The procedure involved lysing ovaries in buffer, followed by centrifugation to obtain the supernatant. To this supernatant, 25 μl of NAD+ extraction buffer was added and incubated at 37 °C for 15 min. Subsequently, a mixture comprising 25 μl of NADH extraction buffer and 75 μl of NAD+/NADH reaction mix was introduced. The mixture was then incubated for 2 h at room temperature in darkness. Total NAD+ levels were quantified using the Varioskan Flash Multimode Reader (Thermo Fisher Scientific, USA) via colorimetric analysis, with excitation and emission wavelengths set at 540 nm and 590 nm, respectively.

NAD+ levels in ovarian stromal cells were quantified using the NAD+/NADH Quantification Kit (Sigma Aldrich, USA). The procedure involved harvesting and lysing the cells in NADH/NAD+ extraction buffer, followed by centrifugation to obtain the supernatant. To this supernatant, 100 μl of the master reaction mix was added and incubated at room temperature for 5 min. Subsequently, 10 μl of NADH developer was introduced, and the mixture was incubated in darkness at room temperature for 4 h. The absorbance was then determined at 450 nm using the microplate reader (Thermo Fisher Scientific, USA).

Ovarian ATP content detection

Ovarian ATP content was measured using the ATP Assay Kit (Beyotime, China). In brief, 100 µl of ATP detection lysis buffer was added to each ovary, followed by ultrasonic homogenization. The homogenates were then centrifuged at 12000 g for 5 min at 4 °C, and the supernatant was collected and stored on ice for further use. Subsequently, 100 µl of ATP detection working solution was added to each well and incubated at room temperature for 5 min. Then, 20 µl of the sample or standard was added to each well. Finally, the relative light units (RLU) or counts per minute (CPM) were measured using a luminometer or liquid scintillation counter.

Estrous cycle and histological analysis

Estrous cycle monitoring, as detailed previously [35], involved a two-week analysis of vaginal cytology. Phosphate-buffered saline (PBS) was employed for vaginal douching and harvesting exfoliated vaginal cells. These cells, suspended in PBS, were evenly spread onto slides, air-dried, and then subjected to fixation using 95% ethanol. Subsequent to hematoxylin and eosin (H&E) staining, a microscopic examination of the slides was conducted utilizing an optical microscope (Nikon, Japan). Ovaries were harvested from three distinct groups of mice during diestrus and 15 h post-human chorionic gonadotropin (hCG) treatment. The tissues were fixed overnight in 4% (w/v) paraformaldehyde at 4 °C, then embedded in paraffin. Sectioned of 5 μm thick slices were prepared and subjected to H&E staining. An optical microscope (Nikon, Japan) was used for the analysis of these stained sections.

Oocytes collection and ROS levels detection

Oocyte collection was performed via ovulation induction in mice from the three experimental groups. The protocol involved an intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin (PMSG), followed by 7.5 IU hCG 47.5 h later. Cumulus-oocyte complexes (COCs) were harvested from the oviductal ampullae 15 h post-hCG administration. To obtain denuded oocytes, the COCs were treated with 0.1% hyaluronidase (Sigma Aldrich, USA). ROS levels were quantified using ROS assay kits. Metaphase II (MII) oocytes collected from three different groups of mice were incubated with MitoSOX™ Red reagent (Thermo Fisher Scientific, USA) under conditions of 37 °C and 5% CO2 for 30 min. Following incubation, the oocytes underwent three washes in the M2 medium. Oocyte images were acquired using the Zeiss LSM 700 Confocal laser scanning microscope (Germany). ROS levels were quantified by analyzing the fluorescence intensity using Image J software.

Mitochondrial distribution and mitochondrial membrane potential (∆Ψm) assessment

For the analysis of mitochondrial distribution, MII oocytes from three groups of mice were incubated with MitoTracker™ Red (Invitrogen, USA) at 37 °C and 5% CO2 for 30 min. Following incubation, the oocytes underwent three washes in the M2 medium. The mitochondrial distribution within these oocytes was subsequently examined using the Zeiss LSM 700 Confocal laser scanning microscope (Germany). The oocytes were then categorized based on the presence of normal or aberrant mitochondrial distribution. Mitochondrial membrane potential (∆Ψm) was assessed in MII oocytes from the three experimental groups. Oocytes were incubated with JC-1 (Beyotime, China) at 37 °C and 5% CO2 for 30 min, then washed thrice in the M2 medium. Images were captured using the Zeiss LSM 700 Confocal laser scanning microscope (Germany). The fluorescence intensity was quantified using Imaging J software, with the ∆Ψm expressed as the ratio of red to green fluorescent pixels.

Spindle assembly analysis

Oocytes from three groups were initially collected, followed by fixation in 4% paraformaldehyde containing 0.5% Triton X-100 for 30 min to allow permeabilization. Subsequent to fixation, the oocytes underwent a blocking step with 1% BSA for 20 min. They were then subjected to an overnight incubation at 4 °C with an anti-α-tubulin antibody (Sigma Aldrich, USA). Following this, a series of washes with 1% BSA was performed thrice, after which the oocytes were subjected to incubation with anti-mouse 488 fluorescent secondary antibody (Invitrogen, USA) droplets at 37 °C for 1 h. Another round of three washes with 1% BSA ensued. The oocytes were then transferred to 10 μl of antifade mounting medium with DAPI (Vector, USA). Spindle morphology and chromosome alignment were visualized and analyzed using the Zeiss LSM 700 Confocal laser scanning microscope (Germany).

In vitro fertilization (IVF) and embryo culture

Male ICR mice aged 4 months were euthanized to extract spermatozoa from dissected epididymides, which were then suspended in an HTF culture medium (Nanjing Aibei Biotechnology, China). Following a 1-h capacitation period at 37 °C and 5% CO2, the spermatozoa were diluted to a concentration of 1–5 × 106/ml and introduced into IVF fertilization medium (COOK, New Zealand). The fertilization process was carried out at 37 °C and 5% CO2 for 6 h. Oocytes exhibiting two pronuclei post-fertilization were considered successfully fertilized and subsequently transferred to the KSOM culture medium (Nanjing Aibei Biotechnology, China). Over the course of the following 4 days, the rates of 2-cell embryo formation and blastocyst development were observed.

PSR staining

Picrosirius Red Staining (PSR) was performed using a Picrosirius Red Stain Kit (Polysciences, USA) according to the manufacturer's protocol. Sections were sequentially treated with solution A (phosphomolybdic acid) for 2 min at room temperature, followed by solution B (Picrosirius Red F3BA Stain) for 1 h. After washing with solution C (0.1 N hydrochloric acid), slides were dehydrated in 70% alcohol and mounted in neutral resin. Images were captured using a Nikon optical microscope (Japan). Ovarian fibrosis quantification was conducted using Image J software. Images were converted to 8-bit, and a threshold was established based on DHEA-treated mice ovarian staining. This threshold was consistently applied across all groups. The fibrosis area was determined by normalizing staining intensity to stromal areas, excluding follicles and corpora lutea.

Immunohistochemistry staining

Paraffin-embedded ovarian sections were deparaffinized by washing twice with 100% xylene, followed by rehydration through a graded series of alcohol solutions and a brief wash in distilled water. Antigen retrieval was carried out by heating the sections in sodium citrate buffer (pH 6.0) at 95 °C for 15 min. After antigen retrieval, the sections were permeabilized and blocked with 1% BSA containing 0.5% Triton X-100 for 1 h at room temperature. The primary antibody, Anti-alpha smooth muscle actin (Abcam, UK), was applied and incubated overnight at 4 °C. Following a PBS wash, horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO, China) were applied. For visualization, 3,3'-diaminobenzidine (DAB) chromogen (ZSGB-BIO, China) was used to develop the color. The sections were counterstained with hematoxylin, followed by dehydration through a graded series of alcohols, and cleared in 100% xylene. Finally, the sections were mounted with a resinous mounting medium and cover slipped. The stained sections were observed and analyzed under a Nikon optical microscope (Japan).

Isolation of stromal cells

Ovaries retrieved from each mouse were collected 15 h post-hCG injection. Utilizing insulin needles under a microscope, corpora lutea and follicles were meticulously removed, leaving behind residual ovarian tissue. These remaining tissues were then transferred to an L15 medium (Thermo Fisher Scientific, USA) supplemented with 0.1% collagenase (Sigma Aldrich, USA) and subjected to incubation at 37 °C and 5% CO2 for 30 min with agitation every 5 min. Following this, stromal cells were isolated using 40 μm filters (Biologix, China) and subsequently centrifuged at 2000 rpm for 5 min. The absence of significant expression of granulosa cell (Fshr) and oocyte (Bmp15) markers was confirmed (Fig. 4D and E).

RNA isolation and quantitative real-time PCR

Total RNA was extracted from ovarian stromal cells using the RNeasy Mini Kit (Qiagen, Germany), and cDNA synthesis was performed with HiScript III RT SuperMix for qPCR (Vazyme, China). Quantitative real-time PCR was conducted using a CFX96 Touch Deep Well Real-Time PCR Detection System (BIO-RAD, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). Primer sequences are listed in Table S2. All experiments were performed in triplicate. Gene expression levels were calculated using the ∆CT method and normalized to Gapdh.

Detection of ROS levels and mitochondrial membrane potential in stromal cells

Ovarian stromal cells were treated with MitoSOX™ Red reagent (Thermo Fisher Scientific, USA) for 30 min at 37 °C and 5% CO2. After three washes with pre-heated PBS buffer, a minimum of 10,000 cells were examined to determine ROS levels by flow cytometry (FCM). For the evaluation of mitochondrial membrane potential, ovarian stromal cells were exposed to JC-1 (Beyotime, China) for 30 min at 37 °C and 5% CO2. After three washes with pre-heated PBS buffer, at least 10,000 cells were scrutinized to assess mitochondrial membrane potential by FCM.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, USA). The experimental design included distinct groups of mice, with data collected from individual mouse ovaries or pooled cells from multiple mice, as detailed in the figure legends. Each measurement was derived from separate samples. Data are presented as mean ± SD. Comparative analyses among the Control, DHEA, and DHEA + NR supplementation groups were conducted using one-way ANOVA. The purity of stromal cell isolation was evaluated using Student's t-test. Statistical significance was defined as P < 0.05.