Background: Post-ovulatory aging (POA) of oocytes is related to a decrease in the quality and quantity of oocytes caused by aging. Previous studies on the characteristics of POA have investigated injury to early embryonic developmental ability, but no information is available on its effects on mitochondrial fission and mitophagy-related responses. In this study, we aimed to elucidate the molecular mechanisms underlying mitochondrial fission and mitophagy in in vitro maturation (IVM) oocytes and a POA model based on RNA sequencing analysis. Methods: The POA model was obtained through an additional 24 h culture following the IVM of matured oocytes. NMN treatment was administered at a concentration of 25 μM during the oocyte culture process. We conducted MitoTracker staining and Western blot experiments to confirm changes in mitochondrial function between the IVM and POA groups. Additionally, comparative transcriptome analysis was performed to identify differentially expressed genes and associated changes in mitochondrial dynamics between porcine IVM and POA model oocytes. Results: In total, 32 common genes of apoptosis and 42 mitochondrial fission and function uniquely expressed genes were detected (≥ 1.5-fold change) in POA and porcine metaphase II oocytes, respectively. Functional analyses of mitochondrial fission, oxidative stress, mitophagy, autophagy, and cellular apoptosis were observed as the major changes in regulated biological processes for oocyte quality and maturation ability compared with the POA model. Additionally, we revealed that the activation of NAD+ by nicotinamide mononucleotide not only partly improved oocyte quality but also mitochondrial fission and mitophagy activation in the POA porcine model. Conclusions: In summary, our data indicate that mitochondrial fission and function play roles in controlling oxidative stress, mitophagy, and apoptosis during maturation in POA porcine oocytes. Additionally, we found that NAD+ biosynthesis is an important pathway that mediates the effects of DRP1-derived mitochondrial morphology, dynamic balance, and mitophagy in the POA model.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Chemicals and animals
    Porcine immature oocyte collection and in vitromaturation (IVM)
    In vitro aging
    MitoTracker staining and quantification of mitochondrialmorphology
    Protein extraction and western blotting analysis
    RNA sequencing (RNA-seq) analysis
    Statistical analysis
RESULTS
    Gene expression alteration in POA-derived porcineoocytes after IVM
    Expression patterns of DRP-1 mediated mitochondrialfission genes in POA porcine oocytes
    NMN influences the gene expression of mitochondrialfission, autophagy, and apoptosis in POA porcineoocytes
DISCUSSION
CONCLUSION
REFERENCES

• Ji-Hyun Shin(Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea)
• Seul-Gi Yang(DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea, Department of Companion Animal Industry, Daegu University, Gyeongsan 38453, Korea)
• Hyo-Jin Park(Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea) Corresponding author
• Deog-Bon Koo(Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea, Department of Companion Animal Industry, Daegu University, Gyeongsan 38453, Korea) Corresponding author