보조생식술 과정에서 생식세포 산화-환원 항상성 조절 기전 = Mechanisms of redox homeostasis regulation in germ cells during assisted reproductive technologies

다국어 초록 (Multilingual Abstract)

Chapter 1 In vitro embryo production (IVEP) has become an essential platform in reproductive biotechnology, providing a controllable system that minimizes dependence on live animal experimentation. Despite its expanding applications in assisted reproduction, species conservation, and toxicological assessment, conventional IVEP protocols still struggle to reproduce the tightly regulated physiological microenvironment of early gametogenesis and embryogenesis. A major inhibitory factor is the supraphysiological accumulation of reactive oxygen species (ROS) under artificial in vitro conditions, which induces redox imbalance, mitochondrial dysfunction, and cellular deterioration, ultimately compromising oocyte maturation, fertilization, and preimplantation development. Similar redox-associated limitations are observed in semen cryopreservation, where oxidative stress impairs membrane stability, motility, and genomic integrity. Strategic supplementation with antioxidants has therefore emerged as a promising intervention. However, the biological efficacy of antioxidants varies substantially depending on their molecular targets and regulatory mechanisms, underscoring the need for stage-specific, organelle-targeted, and species-adapted approaches rather than empirical or non-specific ROS scavenging. This chapter synthesizes current insights into the mechanistic interplay between oxidative stress and impaired gamete and embryo development in IVEP systems, evaluates the effectiveness of antioxidant-based strategies, and proposes refined protocols aimed at enhancing cellular integrity and developmental potential. Advancing antioxidant-driven optimization of IVEP not only improves experimental precision and reproducibility but also contributes to the replacement and reduction of animal experimentation in reproductive research.

Chapter 2 evaluates the application of MnTBAP during porcine semen cryopreservation. MnTBAP was incorporated into the freezing extender at 0, 50, 100, and 150 µM, and post-thaw sperm were examined using CASA, fluorescence assays, and gene expression profiling. Supplementation with 100 µM MnTBAP produced the most favorable outcomes, significantly improving motility, viability, acrosome integrity, and membrane lipid arrangement while decreasing apoptosis and ROS levels (P < 0.05). Gene expression analysis confirmed downregulation of BAX and NOX5 and upregulation of Bcl-2, ROMO1, SMCP, SMOX, and SOD1 (P < 0.05). However, MMP, DNA fragmentation, and caspase activity did not exhibit significant changes. IVF using frozen-thawed sperm treated with 100 µM MnTBAP resulted in higher cleavage and blastocyst rates than the control. These results demonstrate that MnTBAP exerts broad ROS scavenging and protects multiple sperm structures simultaneously during cryopreservation.

Chapter 3 explores the effect of Tiron supplementation on porcine semen cryopreservation. Tiron (0, 5, 50, and 500 nM) was added to freezing extender, and post-thaw sperm characteristics were quantified. The 50 nM group displayed the highest motility, viability, acrosome integrity, and capacitation-associated plasma membrane patterns while showing the lowest apoptosis and ROS levels. Gene expression profiling comparing control vs. 50 nM Tiron revealed downregulation of BAX, ROMO1, SMOX, Keap1, and NOX5 and upregulation of Bcl-2, SMCP, catalase, Cu/ZnSOD, and Gpx1, indicating strong improvement of antioxidant defense pathways. These results demonstrate that Tiron efficiently removes mitochondrial-origin superoxide near the outer mitochondrial compartment during the post-thaw recovery phase and contributes to improved functional resilience of frozen-thawed sperm.

Chapter 4 investigates how the mitochondrial-targeted antioxidant SkQ1 regulates mitochondrial metabolism and developmental competence during porcine IVM. Cumulus-oocyte complexes were treated with 0, 0.02, 0.2, and 2 µM SkQ1, and 0.2 µM was the most effective concentration for the MII rate, cumulus-oocyte complexes maturation-related gene expression (GDF9, BMP15, PTGS2, Has2), cleavage and blastocyst formation rates, total blastocyst cell numbers, distribution of cortical granules, glutathione levels, mitochondrial activity (MMP, MitoSOX Green/Red), and mtDNA stability while reducing apoptosis and ROS. SkQ1-mediated transcriptional responses varied distinctly across developmental cell types during IVEP. In cumulus cells, SkQ1 simultaneously upregulated catalase, Gpx4, CPT1A, Nrf2, cytochrome B, Mfn2, POLG, TFAM, PINK1, and PARKIN, demonstrating the broad activation of mitochondrial biogenesis, dynamics, and mitophagy as part of an extensive mitochondrial quality-control network. In oocytes, many of the antioxidant and mitochondrial-supportive genes elevated in cumulus cells were similarly increased, whereas caspase-3, PINK1, and PARKIN were significantly downregulated, indicating that the primary regulatory shift at this stage favors mitochondrial functional stabilization and suppression of apoptotic signaling rather than expansion of mitochondrial turnover. In blastocysts, antioxidant and anti-apoptotic gene expression remained elevated together with persistent enhancement of mitochondrial dynamics, while POLG and TFAM did not differ significantly from the control, suggesting that late-stage embryos rely predominantly on sustaining mitochondrial functionality rather than further inducing biogenesis. These data show that SkQ1 enhances oocyte quality by reinforcing mitochondria-dependent redox homeostasis and promoting coordinated nuclear and cytoplasmic maturation, driving a highly efficient transition from oocyte to embryo.

Taken together, this thesis demonstrates that antioxidant efficacy in reproductive systems depends on aligning the mode of action with the biological source and developmental context of oxidative stress rather than indiscriminate ROS elimination. By defining mechanistic criteria for antioxidant selection across IVEP and sperm cryopreservation that range from broad-spectrum redox stabilization to mitochondrial-centered mtROS regulation, this work enhances developmental reproducibility while supporting ethical strategies to reduce animal use in reproductive biotechnology.

목차 (Table of Contents)

• Contents ⅰ
• List of Tables iii
• List of Figures iv
• Abstract vi
• Chapter 1. Antioxidant-Based in vitro embryo production systems and sperm cryopreservation 1
• 1. Introduction 2
• 2. IVEP refinement as an alternative to animal models 4
• 3. Redox dysregulation in reproductive cells 8
• 4. Antioxidant application and mechanisms in IVEP 14
• 5. Emerging antioxidant delivery technologies in IVEP 17
• 6. Translational applications of antioxidant-enhanced IVEP 19
• 7. Conclusion 21
• 8. References 23
• Chapter 2. Effects of MnTBAP on porcine semen cryopreservation and capacitation 31
• Abstract 32
• 1. Introduction 34
• 2. Materials and Methods 37
• 3. Results 48
• 4. Discussion 53
• 5. Conclusion 58
• 6. References 59
• Chapter 3. Effects of Tiron on Porcine Semen Cryopreservation 77
• Abstract 78
• 1. Introduction 80
• 2. Materials and Methods 81
• 3. Results 88
• 4. Discussion 92
• 5. Conclusion 94
• 6. References 95
• Chapter 4. Mitochondria-targeted antioxidants SkQ1 promote redox homeostasis and embryonic development in porcine 105
• Abstract 106
• 1. Introduction 108
• 2. Materials and Methods 110
• 3. Results 120
• 4. Discussion 128
• 5. Conclusion 132
• 6. References 134
• Abstract in Korean 155
• Acknowledgment 157