Embryo production by in vitro culture (IVC) is an established assisted reproductive technology for fertility preservation (Yang et al., 2020). During in vitro maturation (IVM), metaphase Ⅱ (MⅡ) stage oocytes may exhibit the phenomenon known as the overripeness of the oocyte (Nicholas et al., 2023). Overripeness of the oocyte is described as a condition wherein oocytes kept in culture for prolonged periods experience a decline in quality and viability (Nagamatsu, 2023). This process manifests as morphological changes, diminished developmental potential, increased chromosomal abnormalities, and impaired ability to fertilize and support embryonic development (Moghadam et al., 2022). Moreover, overmature oocytes are not fertilized or activated in time; they undergo a time-dependent process of aging according to postovulatory aging (POA), both in vitro and in vivo (Wen et al., 2023).

The POA model inevitably impairs the quality of oocytes (Sun et al., 2019a). POA is associated with reduced fertilization rates, poor embryo quality, implantation failure, and abnormalities in the offspring (Di Nisio et al., 2022). As post-ovulation culture time increases during assisted reproductive technology (ART) procedures that are widely used in infertility treatment, it inevitably induces POA from the oocyte (Kim et al., 2022). Therefore, POA in oocytes before fertilization is a major cause of early pregnancy failure in mammals, including humans (Wilcox et al., 1998; Chen et al., 2022). Researchers have employed various strategies to mitigate overripeness and preserve oocyte quality during IVC (Casillas et al., 2018; Hu et al., 2023).

Not surprisingly, postovulatory aged oocytes exhibit various defects, including spindle abnormalities, loss of mitochondrial function, and DNA damage (Xing et al., 2023). To date, the mechanisms controlling porcine POA have not been well defined in the in vitro production process. However, the female aging process related to oocyte maturation is accompanied by the overproduction of reactive oxygen species (ROS) in mitochondria (Kim et al., 2022). During the progression of IVM, the balance between ROS production and antioxidant enzymes is disrupted, which in turn causes oxidative stress (Combelles et al., 2009). Acute oxidative stress promotes extensive mitochondrial fission and dysfunction, ultimately leading to elevated intracellular and mitochondrial ROS levels, loss of mitochondrial function, and mitochondria-mediated apoptotic cell death (Guo et al., 2013).

Nicotinamide adenine dinucleotide (NAD⁺) is involved in a variety of fundamental biological processes, including cellular bioenergetic metabolism, lifespan regulation, DNA repair, aging, and cell death mechanisms (Covarrubias et al., 2021). This age-related loss of oocyte quality is accompanied by declining levels of the prominent metabolic cofactor NAD⁺ (Bertoldo et al., 2020). Reproductive aging in female mammals is an irreversible process associated with declining oocyte quality, in which aged females lack oocyte-sirtuin 1 (Sirt1) due to age-related changes, such as reduced NAD⁺ synthesis (Bertoldo et al., 2020; Iljas et al., 2020).

Fusion and fission responses in mitochondria permanently counterbalance each other in mammalian cells; the inactivation of one leads to an unopposed action by the other, and the subsequent imbalance controls mitochondrial structure and functions (Liu et al., 2020). As mitochondrial regulators, mitofusins 1 and 2 (Mfn1/2) and dynamin-related protein 1 (Drp1) play important roles in mitochondrial dynamics (Chen et al., 2003; Liang et al., 2021; Chen et al., 2023; Wang et al., 2023). Mfn1/2 promotes mitochondrial fusion, whereas Drp1 promotes mitochondrial fission which is well-known (Hall et al., 2014). According to previous studies, the expression of Mfn1/2 and Drp1 tends to decrease with age in humans (Chen et al., 2023; Ye et al., 2023). Mitochondrial function, which involves various metabolic processes associated with mitochondrial dysfunction, including dynamic balance and mitophagy, has become relevant to aging (Moreira et al., 2017). Aging is also associated with mitochondrial dysfunction due to increased ROS production, which causes oxidative damage, leading to reduced mitophagy and adenosine triphosphate (ATP) generation (Srivastava, 2017). Moreover, a previous study showed for the first time a novel link between NAD⁺ metabolism and mitochondrial dynamics in aged mice (Klimova et al., 2019b; Hong et al., 2020).

However, it remains to be determined whether NAD⁺ generation is involved in the oocyte aging process via the correlation between mitochondrial dynamics and mitophagy in the POA model. Therefore, in the present study, we discovered that oocyte quality defects in the POA model were due to a disrupted mitochondrial dynamic response balance, which induces oxidative stress, mitophagy inactivation, and apoptosis in pigs. Furthermore, NAD⁺ sufficiency induced by nicotinamide mononucleotide (NMN) treatment in porcine oocytes with POA recovers the balance between mitochondrial dynamics and mitophagy.

Chemicals and animals

All the chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Porcine ovaries were obtained from prepubertal sows (6-month-old female pigs; Yorkshire/Landrace (♀) × Duroc (♂), 100 kg) at a local slaughterhouse (Gyeongsan and Daegu, Korea). No experiments were performed on live animals.

Porcine immature oocyte collection and in vitro maturation (IVM)

Prepubertal porcine ovaries were obtained at a local slaughterhouse and transported to the laboratory in 0.9% saline supplemented with 75 μg/mL potassium penicillin G at 38.5℃. Immature cumulus-oocyte complexes (COCs) were aspirated from a follicle (3-6 mm diameter) using an 18-gauge needle connected to a 10 mL syringe. COCs were washed thrice in Tyrode’s lactate-N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (TL-HEPES) and IVM medium. Approximately 50 COCs that were surrounded by at least three layers of compact cumulus cells were selected for maturation in 500 μL of IVM medium in four-well multi-dishes (Nunc, Roskilde, Denmark) at 38.5℃ under 5% CO₂ for 22 h. North Carolina State University-23 (NCSU-23) medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL human chorionic gonadotropin (hCG), 0.57 mM cysteine, 10 ng/mL β-mercaptoethanol, 10 ng/mL epidermal growth factor (EGF), and 10% follicular fluid was used for oocyte maturation. To prepare mature oocytes in vitro, a group of 50 COCs was transferred to 500 μL of maturation medium (NCSU-23) to culture at 38.5℃ in a humidified atmosphere of 5% CO₂. After culturing for 22 h, the COCs were matured in IVM medium without PMSG and hCG for an additional 22 h at 38.5℃ under 5% CO₂.

In vitro aging

For in vitro oocyte aging of pigs, matured oocytes at 44 h after IVM were cultured for an additional 24 h under 5% CO₂ in an incubator at 38.5℃. To determine whether NMN affects oocyte quality during in vitro aging periods, porcine oocytes were cultured in IVM medium at 25 μM NMN for a minimum of 24 h to a maximum of 68 h under different incubation conditions. The concentration of NMN was selected based on previous studies (Pollard et al., 2021; Miao et al., 2022; Song et al., 2022).

MitoTracker staining and quantification of mitochondrial morphology

Matured oocytes were denuded by softly pipetting in 0.1% hyaluronidase. Denuded oocytes were washed thrice in PBS containing polyvinyl alcohol (PVA) and incubated in IVM medium containing 1 μM MitoTracker Green (Invitrogen, CA, USA) for 30 min at 38.5℃. After washing three times with PVA in PBS, the oocytes were fixed in 3.7% formaldehyde at overnight 4℃. The oocytes were washed and mounted on glass slides with DAPI solution (Vector Laboratories, Burlingame, CA, USA). Finally, the stained oocytes were mounted on glass slides and observed under a laser scanning confocal fluorescence microscope (LSM 800; Zeiss, Jena, Germany). To measure the fluorescence intensity, signals from both control and treated oocytes were acquired by performing the same immunostaining procedure and setting up the same parameters as those used for confocal microscopy. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to define the region of interest (ROI), and the average fluorescence intensity per unit area within the ROI was determined. Independent measurements using identically sized ROIs were performed on the cell membrane and cytoplasm. The average values of all measurements were used to compare the final average intensities of the control and treatment groups. Quantification of mitochondrial morphology analyzed at least 35 oocytes per sample and the mitochondrial morphology was categorized into the elongation (more than 3 μM, > 3 μM) and fragments form (less than 1 μM, < 1 μM). All images were obtained at the same intensity and exposure time.

Protein extraction and western blotting analysis

Matured 50 COCs from the IVM and POA groups were collected and placed in PRO-PREP protein lysis buffer (iNtRON, Daejeon, Korea). The protein concentration in each sample was estimated using a Bradford dye-binding assay. Total protein was separated by 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Pall Life Sciences, NY, USA). Separated protein bands were transferred onto nitrocellulose membranes (Pall Life Sciences, Port Washington, NY, USA). The membranes were incubated with the primary antibodies: anti-pDRP1-Ser616 (Cell Signaling, MA, USA), anti-DRP1 (Santa Cruz, CA, USA), anti-cytochrome C (Abcam, Cambridge, England), and anti-β-actin (Santa Cruz). The membranes were then probed with horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit IgG (Thermo, Rockford, IL, USA) or an anti-goat IgG (AbFrontier, Seoul, Korea) secondary antibody on 4℃ for overnight. The blots were developed using an enhanced chemiluminescence (ECL) kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. For signal quantification, the bands were scanned using ImageJ software (NIH).

RNA sequencing (RNA-seq) analysis

The total RNA of 200 porcine oocytes in the IVM and POA groups was isolated using TRIzol reagent (Invitrogen). RNA quality was assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND-2000 Spectrophotometer (Thermo Inc., DE, USA). For each RNA sample, the construction of the library was performed using QuantSeq 3’ mRNA Seq Library Prep Kit (Lexogen Inc., GmbH, Austria) according to the manufacturer’s instructions. High-throughput single-end 75 sequencing was performed using NextSeq 500 (Illumina Inc., CA, USA). We investigated the differentially expressed genes (DEGs) that displayed a greater than 1.5-fold change after POA in the entire transcriptome. DEGs were analyzed using ExDEGA software (Excel-based differentially expressed gene analysis version 3.0; Ebiogen Inc., Seoul, Korea). Gene classification was based on searches of databases for annotation, visualization, and integrated discovery (DAVID, http://david.abcc.ncifcrf.gov/, accessed March 03, 2024), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http://www.genome.jp/kegg/mapper.goml/, accessed March 03, 2024). Heatmap-related genes in embryos were analyzed using the Multi Experiment Viewer (Mev) software.

Statistical analysis

Each experiment was repeated at least thrice. All data are presented as mean ± standard deviation (SD). Western blot content is presented as mean ± standard error of the mean (SEM). The results were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. All data were analyzed using GraphPad Prism software (version 5.0; Sand Diego, CA, USA). Histogram densitometry values were measured using the ImageJ software (NIH). The level of significance was set at p < 0.05.

Gene expression alteration in POA-derived porcine oocytes after IVM

After inducing porcine oocytes into POA compared to the IVM groups, as described in Fig. 1A, a comprehensive transcriptomic analysis revealed significant changes in RNA-seq log₂ expression. RNA-seq analysis of the POA model revealed numerous categories of enriched genes, including biological processes, cellular components, and molecular functions (Fig. 1B). The classifications of apoptosis (24 genes) and autophagy (13 genes) were remarkably altered (> 1.5-fold) in the biological process of the POA model. In the cellular components, mitochondria-associated genes (mitochondria, 29 genes; mitochondrial inner membrane, 9 genes; and mitochondrial outer membrane, 8 genes) were considerably altered (> 1.5-fold). Additionally, in the molecular function classification, oxidative stress-related genes (oxidoreductase: 25 genes, peroxidase: 7 genes, and antioxidant: 5 genes) showed significant changes in expression (> 1.5-fold). We performed a KEGG pathway enrichment analysis of the DEGs associated with oxidative stress, mitochondria, autophagy, and apoptosis in POA oocytes compared to IVM porcine oocytes (Fig. 1C-1F). We selected the top 10 categories and revealed the accuracy of enriched expression in the control and aged oocytes groups. We present detailed changes in genes selected through previous gene ontology (DAVID) and KEGG pathway enrichment analyses using heatmap cluster illustrations (Fig. 1G-1J). The RNA expression of various genes in response to oxidative stress, mitophagy and autophagy, mitochondrial function or fission, and apoptotic processes were differentially expressed (> 1.5-fold, upregulated: red; downregulated: blue) between the control and POA groups.

Figure 1.Analysis of differentially expressed genes (DEGs) in POA porcine oocytes. (A) Schematic diagram of experimental design. All the porcine oocytes were cultured for 44 of IVM (Control) or 68 h of IVM (POA or in vitro aging groups; with 24 h additional culture after 44 h of IVM). (B) DEGs or the number of genes showing differential expression between IVM and POA porcine oocytes. Functional annotation analysis of DEGs in porcine oocytes from POA group. Biological processes (red), cellular components (green), and molecular functions (blue) are indicated. (C-F) Gene ontology (GO) enrichment analysis of the DEGs associated with the expression of “Oxidative stress,” “Mitochondrial functions and fission,” “Mitophagy and autophagy,” and “Apoptotic process,”-related genes in POA-induced model. (G-J) POA compared with IVM porcine oocytes. Each heatmap cluster illustrates the expression of various genes differentially expressed (upregulated: red; downregulated: blue) between the IVM and POA-induced model. Heatmap clusters are analyzed to derive the “Oxidative stress,” “Mitochondrial functions and fission,” “Mitophagy and autophagy,” and “Apoptotic process” categories.

Expression patterns of DRP-1 mediated mitochondrial fission genes in POA porcine oocytes

To investigate changes in mitochondrial morphology and length in POA oocytes, we stained denuded oocytes at 44 h of IVM (Con) and 68 h of IVM (POA) using MitoTracker Green staining (Fig. 2A). The mitochondrial fragmentation (< 1 μm) of POA oocytes significantly decreased (p < 0.05) and an increasing opposite pattern (p < 0.05) of mitochondrial elongation (> 3 μm) was found when POA oocytes were cultured at 68 h of IVM compared to the control group (Fig. 2B). MitoTracker green fluorescence was also used to evaluate mitochondrial distribution patterns and intensity in the cytoplasm of POA oocytes. The mitochondria of the POA oocytes showed a homogeneous distribution (Fig. 2C). In addition, the POA oocytes reduced (p < 0.001) in fluorescence intensity compared to IVM porcine oocytes (Fig. 2D). To investigate the protein levels of mitochondrial fission factors (pDRP1-Ser616 and DRP1) and mitochondria-mediated apoptosis factors (Cytochrome C), we performed western blot analysis in porcine oocytes from the POA model (Fig. 2E--2H). The protein levels of pDRP1-Ser616 (p < 0.01) and total DRP1 (p < 0.001) were significantly decreased in POA oocytes, whereas cytochrome C protein expression was significantly increased (p < 0.05). These results indicate that POA oocytes show a loss of DRP1-related mitochondrial fission due to additional maturational progression.

Figure 2.Changes in DRP1-mediated mitochondrial fission on POA porcine oocytes. Changes of mitochondrial dynamics in porcine POA oocytes compared with IVM oocytes. (A, B) Stained porcine oocytes of IVM (44 h of IVM) and POA group (68 h of IVM) exhibited mitochondrial fragmentation (< 1 μm) and elongation (> 3 μm) using MitoTracker green staining. Scale bar: 50 μm. (C, D) Investigation of mitochondria fluorescence intensity from POA porcine oocytes using MitoTracker green staining. Scale bar: 50 μm. (E-H) Representative Western blot and quantitative analyses of the protein levels of mitochondrial fission regulator [pDRP1-Ser616 and DRP1] in IVM porcine oocyte and POA aged oocytes. Western blot analysis of cytochrome C protein expression as a mitochondrial-mediated apoptotic factor in POA model compared to IVM oocytes. The relative expression of all proteins is normalized to that of β-actin. Image data and Western blot data were respectively expressed as mean ± SD/SEM and were analyzed using a t-test. Differences are considered significant at *p < 0.05, **p < 0.01 and ***p < 0.001.

NMN influences the gene expression of mitochondrial fission, autophagy, and apoptosis in POA porcine oocytes

As depicted in Fig. 3A, we determined the three groups after 25 μM NMN was supplied with various treatment conditions according to the IVM period for 0-44 h (M; maturation), 44-68 h (A; aging), and 0-68 h (M + A) of IVM. We performed RNA-seq to analyze the transcriptomes of porcine oocytes after NMN treatment from different groups, including aged (POA model), M, A, and M + A. Gene ontology (GO) enrichment analysis of RNA-seq data was performed to investigate the biological process functions that were important after IVM or POA. Here, only genes that decreased or increased by a fold of 1.5 or more were used in the analysis (Fig. 3B). As expected in Fig. 1B, the enriched biological process, molecular function, and cellular component of DEGs are related to POA oocytes, such as “Oxidative stress,” “Mitochondria,” “Autophagy,” and “Apoptosis” (Fig. 3C-3F and 4). We investigated the changes in various genes using heatmap cluster illustrations (Fig. 3C-3F). Genes with significant changes (> 1.5-fold) in expression between the NMN-treated group and POA groups were identified (Fig. 4 and 5). As depicted in Fig. 4C and 4D, RNA-seq log₂ expression (p-value) of genes of “mitophagy and autophagy” and “apoptosis process” were remarkably altered in POA oocytes according to the NMN treatment. Based on these results, we identified that “oxidative stress,” “mitochondrial function and fission,” “mitophagy and autophagy,” and “apoptotic process” were the main factors affecting IVM and POA (Fig. 5). These findings suggest that the increased NAD⁺ content induced by NMN supplementation for mitochondrial fission and the mitophagy response may be associated with the recovery of the impaired quality of POA oocytes.

Figure 3.Alteration of DEGs by RNA-seq analysis in POA porcine oocytes according to 25 μM NMN exposure. (A) Schematic diagram of experimental design in NMN exposed porcine oocytes from IVM and POA model. All the porcine oocytes were cultured for 44 of IVM (Con) and 68 h of IVM (POA), and we determined the three groups of NMN treatment after 25 μM NMN was supplied with IVM period for 0–44 h (M), 44–68 h (A), and 0–68 h (M + A) of IVM. (B) Functional annotation analysis of DEGs in porcine oocytes from POA group after 25 μM NMN exposure. Biological processes (red), cellular components (green), and molecular functions (blue) are indicated. (C–F) Gene ontology (GO) enrichment analysis of the DEGs associated with the expression of “Oxidative stress,” “Mitochondrial functions and fission,” “Mitophagy and autophagy,” and “Apoptotic process”-related genes in POA-induced model under NMN supplement. (G-J) POA compared with IVM porcine oocytes. Each heatmap cluster illustrates the expression of various genes differentially expressed (upregulated: red; downregulated: blue) between the IVM and POA-induced model compared with NMN exposed groups (0–44 h: M, 44–68 h: A, and 0–68 h: M + A of IVM). Heatmap clusters are analyzed to derive the “Oxidative stress,” “Mitochondrial functions and fission,” “Mitophagy and autophagy,” and “Apoptotic process” categories.

Figure 4.The number of genes changed according to the DAVID analysis in POA oocytes after NMN treatment. (A–D) Categories are divided to derive the “Oxidative stress,” “Mitochondrial functions and fission,” “Mitophagy and autophagy,” and “Apoptotic process.” The values in the square box under each category showed the accuracy of RNA-seq log₂ expression. The graphs indicate the number of various genes (upregulated: red; downregulated: blue; Total: green) between the IVM and POA-induced model compared with NMN exposed groups (0–44 h: M, 44–68 h: A, and 0–68 h: M + A of IVM). The graphs were considered genes with more than 1.5-fold and adjusted p-value < 0.05.

Figure 5.Graphical summary. The involvement of the DRP1-derived mitochondrial fission and morphological changes in POA pig oocytes was confirmed by experiments using Western blotting and MitoTracker staining compared with IVM porcine oocytes. Taken together, we systematically identified DEGs caused by POA of pig oocytes using the RNA-seq technique.

As an increasing number of aging women pursue ART, including in vitro fertilization (IVF), and oocyte vitrification, the importance of oocyte quality and maturation capacity in aged women has been emphasized (Zhao et al., 2020; Köroğlu and Aydın, 2023). POA models that can occur either in vivo or in vitro focus on the time-dependent degradation/inactivation of proteins in oocytes and early embryos (Miao et al., 2018). These factors are closely involved in nuclear and cytoplasmic remodeling and play a key role in fertilization failure and the disruption of embryo viability (Zhao et al., 2020). Many studies have shown that porcine oocytes are suitable for studying oocyte maturation and fertilization during IVC (Agung et al., 2013; Appeltant et al., 2016). During IVC, oocytes experience delayed degradative progression, which is referred to as POA. POA is a common disorder characterized by mitochondrial dysfunction caused by ROS and various reproductive phenomena associated with aging (Kim et al., 2021). In the current study, comparative RNA-Seq data provided informative insights response to DRP1-mediated mitochondrial fission and mitophagy into mitochondrial morphology and the responses of porcine IVM- and POA-induced oocytes. From this perspective, in the present study, we demonstrated that DRP1-mediated mitochondrial fission and mitophagy activation are required for oocyte quality in the POA model compared to IVM porcine oocytes.

Although oocyte maturation has been explored for many years, little is known about the different mechanisms between IVM and POA oocytes in pigs during IVM progression. In this study, we analyzed the transcriptomes of porcine IVM and POA oocytes to explore the differences at the transcriptional level using RNA-seq. Unfortunately, there have been limited discussions regarding methodologies to suppress this problem by mitochondrial fission, changing morphology, and the mitophagy system in the POA model, even though ROS and mitochondria are directly connected to aging in female reproduction. In the present study, we demonstrated that DRP1-mediated mitochondrial fission requires IVM porcine oocytes, compared to the POA model.

The relationship between aging and ROS/oxidative stress due to mitochondrial function has been reported in oocytes and embryos (Sasaki et al., 2019). Reproductively, in vitro aged oocytes, which follow an increase in intracellular ROS levels or oxidative stress, show a decline in the fidelity of protective mechanisms in antioxidative systems against ROS (Mihalas et al., 2017; Martin et al., 2022). In addition, various mitochondria-derived metabolic pathways have become therapeutic targets for infertility caused by factors such as poor oocyte quality and aging (Jiang and Shen, 2022). Numerous studies have reported that POA has detrimental effects, such as mitochondrial distribution, dysfunction, and ROS production, leading to DNA damage and apoptosis, and further harming fertilization and embryo development (Sun et al., 2019b; Liu et al., 2022).

Mitochondria sustain normal physiological functions by maintaining a steady-state or balanced interplay between two contradictory fission and fusion events to maintain a healthy mitochondrial network. Fusion and fission events concurrently determine the overall form, size, and population of mitochondria by regulating mitochondrial dynamics (Zerihun et al., 2023). In addition, our previous study confirmed that the regulation of mitochondrial fission and fusion has the potential to improve blastocyst developmental competence via mitochondria-specific ROS reduction and improved ATP production in porcine preimplantation embryos (Yang et al., 2018). However, the correlation between POA- and DRP1-mediated mitochondrial fission in porcine oocytes remains unclear. Therefore, our study aimed to confirm this process by focusing on mitochondrial fission and morphological changes in post-ovulatory aged porcine oocytes.

As reported above, in pigs, IVM and post-ovulatory aged oocytes exhibit different categories of biological processes, molecular functions, and cellular components. However, the differences in the selectively degraded transcripts between IVM and POA porcine oocytes have not been explored in detail. Genes with more than 1.5-fold and adjusted p-value < 0.05 were considered significant DEGs. Remarkable transcriptomic patterns were observed in the number of apparent total transcripts compared with POA oocytes: oxidative stress (27 genes), mitochondrial fission and function (10 genes), mitophagy (20 genes), and apoptosis (32 genes) (Fig. 5). Thus, we examined the mitochondrial morphology and fission marker protein expression in aged porcine oocytes. We conducted a western blot analysis of p-DRP1 and DRP1 proteins in POA oocytes compared to IVM oocytes, as oxidative stress is the main mechanism that worsens the balance of mitochondrial dynamic responses, as mentioned earlier.

Further research using NMN is required to investigate mitophagy, which triggers mitochondrial fission and oxidative stress (Jang et al., 2012). A previous study reported that NMN supplementation effectively improved the quality of oocytes from naturally aged mice by recovering NAD⁺ levels (Klimova et al., 2019a). NMN supplementation not only increases ovulation in aged oocytes but also enhances their meiotic competency and fertilization ability (Miao et al., 2020). We hypothesized that NMN supplementation may affect the RNA expression levels of mitochondrial fission and mitophagy in POA oocytes. As expected, the results of the RNA-seq analysis were associated with mitochondrial fission, function, and mitophagy in porcine POA oocytes. We have presented results showing that the number of changed total transcript genes was similar among the four categories, but only changes in transcripts of the apoptosis category were higher than those in other groups.

In summary, critical factors involved in aged oocyte maturation and quality were differentially expressed between DRP1-mediated mitochondrial fission and morphology (Fig. 5). IVM and POA porcine oocytes exhibit divergent transcriptomes, indicating the differential molecular responses to oocyte maturation via mitochondrial fission and oxidative stress. Moreover, NMN-exposed porcine POA oocytes according to different treated periods exhibited a higher ratio of common DEGs of apoptosis compared to IVM or POA oocytes, which were enriched for various biological processes targeted to mitochondrial fission and functions that play important roles during aged oocyte maturation. These findings indicate significant differences in gene expression profiles via DRP1-mediated mitochondrial fission between IVM and POA porcine oocytes.

None.

Conceptualization, J-H.S., H-J.P., and D-B.K.; methodology, J-H.S. and S-G.Y.; investigation, J-H.S. and S-G.Y.; data curation, J-H.S., S-G.Y., and H-J.P.; writing - original draft, J-H.S. and S-G.Y.; writing - review & editing, S-G.Y., H-J.P., and D-B.K.; supervision, H-J.P. and D-B.K.; project administration, J-H.S., S-G.Y., and H-J.P.; funding acquisition, H-J.P. and D-B.K.

This research was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (NRF-2021R1C1C2009469 and NRF-2022R1A2C1002800), and the Basic Science Research Program through the NRF funded by the Ministry of Education (RS-2023-00246139), Republic of Korea.

Not applicable.

Not applicable.

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Not applicable.

No potential conflict of interest relevant to this article was reported.