MTNR1A 유전자의 표적 후성유전학적 조절을 통한 돼지 난자의 발달 능력 향상을 위한 새로운 전략 = A Novel Strategy to Enhanc Developmental Competence of Porcine Oocytes via Targeted Epigenetic Regulation of the MTNR1A Gene

국문 초록 (Abstract)

제1장에서는 돼지 보조생식기술(ART)의 농업적 및 의학적 중요성에 대해 기술한다. 돼지 ART의 성공을 가로막는 주된 장애물은 체외 성숙(IVM) 유래 난자의 낮은 발달 능력(developmental competence)이며, 이는 불완전한 세포질 성숙과 높은 수준의 산화적 스트레스(OS)에 기인한다. 산화적 스트레스는 난자의 품질을 저하시키는 핵심 요인이다. 난구세포에서 발현되는 멜라토닌 수용체 1A(MTNR1A)는 보호 신호전달에 관여하지만, 인위적인 IVM 환경에서는 DNA 메틸화에 의해 후성유전학적으로 침묵(silencing)될 수 있다. 본 학위논문은 표적 탈메틸화를 위한 CRISPR/dCas9-Tet1 후성유전학적 편집도구를 사용하여 MTNR1A 유전자의 발현을 복구함으로써, 돼지 난자의 발달 능력을 향상시키는 새로운 전략을 제시한다.

제2장에서는 돼지 체세포(APEFs) 모델을 사용하여 MTNR1A의 표적 탈메틸화를 위한 CRISPR/dCas9-Tet1 시스템을 확립하고 검증하였다. 세가지 후보 sgRNA 중 -291 bp 프로모터 영역을 표적으로 하는 sgRNA가 MTNR1A mRNA 및 단백질 발현을 증가시키는 데 가장 효과적이었다. 바이설파이트 시퀀싱(Bisulfite sequencing)을 통해 이러한 활성화가 프로모터 영역의 탈메틸화에 기인함을 확인하였다. 또한, MTNR1A 발현의 복구는 하위 신호전달 경로인 pAKT 및 pERK1/2의 인산화를 유의적으로 증가시켰다. 비표적(Off-target) 분석 결과, 상동 유전자인 MTNR1B 유전자에는 영향을 미치지 않아 시스템의 높은 특이성이 확인되었다.

제3장에서는 검증된 dCas9-Tet1 시스템을 IVM 과정 동안 돼지 난구-난자 복합체(COCs)에 직접 적용한 내용을 상세히 기술한다. 이 시스템은 난구세포에서 MTNR1A의 탈메틸화와 발현을 성공적으로 유도하였으며, 난구세포 팽창과 난자 핵 성숙을 향상시켰다. 특히, 분자적 분석을 통해 MTNR1A의 복구가 NRF2 인산화를 촉발하고, 이는 HO-1 및 NQO1의 전사적 상향 조절로 이어짐을 밝혔다. 이러한 활성화는 세포 내 글루타치온(GSH) 수치를 유의적으로 높여 항산화 방어 시스템을 강화하였다. 체외 수정(IVF) 결과, 이러한 산화적 균형의 개선은 난할율 및 포배 형성률의 유의적인 증가로 이어졌다. 생성된 포배는 총 세포 수의 증가와 함께 미토콘드리아 기능 및 만능성(pluripotency) 관련 유전자의 발현이 향상되는 등 우수한 품질을 나타냈다.

제4장에서는 과산화수소(H2O2)를 이용해 확립한 산화적 스트레스 모델을 사용하여 dCas9-Tet1 시스템의 치료적 잠재성을 조사하였다. H2O2 처리는 세포 내 활성산소종(ROS) 수치를 현저히 증가시켰으며, 난구세포 팽창, 난자 핵 성숙 및 이후의 배아 발달을 심각하게 저해하였다. H2O2와 dCas9-Tet1 시스템을 병용 처리한 "구제(rescue) 그룹"에서는 H2O2 단독 처리군에 비해 ROS 수치가 유의적으로 감소하였으며, 난구세포 팽창, 성숙률 및 포배 형성률이부분적으로 회복되었다. 이러한 결과는 표적 MTNR1A 탈메틸화가 정상적인 체외 성숙 조건에서는 기능 향상제(enhancer)로서 작용할 뿐만 아니라, ROS에 의해 손상된 난자의 발달 능력을 구제할 수 있는 치료적 전략으로도 기능할 수 있음을 입증한다.

다국어 초록 (Multilingual Abstract)

Chapter 1 describes the agricultural and biomedical importance of porcine Assisted Reproductive Technologies (ARTs). The primary obstacle to the success of porcine ART is the low developmental competence of oocytes derived from in vitro maturation (IVM), which is attributed to incomplete cytoplasmic maturation and high levels of oxidative stress (OS). OS is a key factor in impairing oocyte quality. Melatonin receptor 1A (MTNR1A), expressed in cumulus cells, is involved in protective signaling; however, its expression may be epigenetically silenced (via DNA methylation) under artificial IVM conditions. This dissertation presents a novel strategy to improve the developmental competence of porcine oocytes by restoring MTNR1A gene expression using the CRISPR/dCas9-Tet1 epigenetic editing tool for targeted demethylation.

In Chapter 2, a CRISPR/dCas9-Tet1 system for the targeted demethylation of MTNR1A was established and validated using a porcine somatic cell (APEFs) model. Among the three candidate sgRNAs, the one targeting the -291 bp promoter region was the most effective at increasing MTNR1A mRNA and protein expression. Bisulfite sequencing confirmed that this activation was due to demethylation of the promoter region. Furthermore, restoration of MTNR1A expression significantly increased the phosphorylation of the downstream signaling pathways, pAKT and pERK1/2. Off-target analysis confirmed the high specificity of the system, with no effect on the homologous MTNR1B.

Chapter 3 details the direct application of the validated dCas9-Tet1 system to porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM). The system successfully induced demethylation and expression of MTNR1A in cumulus cells, enhancing cumulus expansion and oocyte nuclear maturation. Crucially, molecular analysis revealed that MTNR1A restoration triggered NRF2 phosphorylation, leading to the transcriptional upregulation of HO-1 and NQO1. This activation significantly elevated intracellular glutathione (GSH) levels, thereby reinforcing the antioxidant defense. Following in vitro fertilization (IVF), this improved oxidative balance translated into significantly higher cleavage and blastocyst formation rates. The resulting blastocysts exhibited superior quality, characterized by increased total cell numbers and enhanced expression of genes related to mitochondrial function and pluripotency.

In Chapter 4, the therapeutic potential of the dCas9-Tet1 system was investigated using an oxidative stress model established with hydrogen peroxide (H2O2). H2O2 treatment markedly increased intracellular reactive oxygen species (ROS) levels and severely impaired cumulus expansion, oocyte nuclear maturation, and subsequent embryonic development. In the “rescue group,” in which H2O2 and the dCas9-Tet1 system were co-administered, ROS levels were significantly reduced, and cumulus expansion, maturation rates, and blastocyst formation were partially restored compared to the H2O2-only group. These findings demonstrate that targeted MTNR1A demethylation can function as an enhancer under normal in vitro maturation conditions and as a therapeutic strategy capable of rescuing the developmental competence of ROS-damaged oocytes.

목차 (Table of Contents)

• Chapter 1. General Introduction 1
• 1.1. Importance of Porcine Reproductive Biotechnology 2
• 1.2. Current Status and Challenges of Porcine ART 3
• 1.3. Defining Oocyte Developmental Competence 5
• 1.4. Oxidative Stress as a Major Factor in Oocyte Quality-Decline 7
• 1.5. Melatonin and the MTNR1A Receptor in Oocyte Maturation 9
• 1.6. Epigenetic Regulation as a Target for Intervention 11
• 1.7. CRISPR-Based Epigenetic Editing: The dCas9-Tet1 System 13
• 1.8. Hypothesis and Specific Objectives 15
• 1.9. Significance and Innovation of This Dissertation 16
• References 18
• Chapter 2. Establishment and Validation of a CRISPR/Cas9-based Epigenetic Editing System for Targeted MTNR1A Demethylation in Porcine Cells 26
• 2.1. Abstract 27
• 2.2. Introduction 28
• 2.3. Materials and Methods 30
• 2.3.1. sgRNA Design and Vector Construction 31
• 2.3.2. Cell culture 31
• 2.3.3. Virus Production and Transduction 31
• 2.3.4. Quantitative Real-Time PCR (qRT-PCR) 31
• 2.3.5. Western Blot Analysis 32
• 2.3.6. Bisulfite Sequencing 32
• 2.3.7. Off-target Analysis 33
• 2.3.8. Statistical Analysis 34
• 2.4. Results 34
• 2.4.1. Validation of sgRNAs for MTNR1A-Targeted Demethylation 34
• 2.4.2. dCas9-Tet1-291sgRNA Induces Targeted DNA Demethylation 35
• 2.4.3. Specificity and Robustness of the Editing System 35
• 2.4.4. MTNR1A Activation Induces Downstream AKT and ERK Signaling 36
• 2.5. Discussion 37
• References 40
• Chapter 3. Enhancement of In Vitro Maturation and Subsequent Embryonic Development of Porcine Oocytes by Targeted MTNR1A Demethylation 49
• 3.1. Abstract 50
• 3.2. Introduction 51
• 3.3. Materials and Methods 53
• 3.3.1. Collection and IVM of COCs 53
• 3.3.2. Lentiviral Transduction of COCs 54
• 3.3.3. Validation in Cumulus Cells 54
• 3.3.4. Assessment of Cumulus Expansion and Oocyte Maturation 55
• 3.3.5. Gene Expression Analysis in Oocytes and Cumulus Cells 55
• 3.3.6. In Vitro Fertilization (IVF) and Embryo Culture 56
• 3.3.7. Assessment of Embryo Development 56
• 3.3.8. Oxidative State and Gene Expression in Blastocysts 57
• 3.3.9. Statistical Analysis 57
• 3.4. Results 57
• 3.4.1. dCas9-Tet1 System Successfully Activates MTNR1A in Cumulus Cells 57
• 3.4.2. Targeted MTNR1A Demethylation Promotes Oocyte Maturation 58
• 3.4.3. MTNR1A Activation Restores Gap Junctional Communication via Upregulation of Connexin 43. 59
• 3.4.4. MTNR1A Activation Upregulates HO-1 and NQO1 Transcription via NRF2 Phosphorylation, Leading to Increased Intracellular GSH Levels 60
• 3.4.5. MTNR1A Activation Enhances Subsequent Embryonic Development 61
• 3.4.6. Blastocysts Derived from Treated Oocytes Show Improved Molecular Profiles 61
• 3.5. Discussion 62
• References 67
• Chapter 4. Therapeutic Potential of Targeted MTNR1A Demethylation for Rescuing Developmentally Compromised Oocytes from Oxidative Stress 78
• 4.1. Abstract 79
• 4.2. Introduction 80
• 4.3. Materials and Methods 81
• 4.3.1. Establishment of Oxidative Stress (OS) Model 81
• 4.3.2. Experimental Groups 82
• 4.3.3. Assessment of ROS, Maturation, and Embryo Development 82
• 4.3.4. Statistical Analysis 82
• 4.4. Results 83
• 4.4.1. H₂O₂ Treatment Induces Acute Oxidative Stress in COCs 83
• 4.4.2. MTNR1A Activation Mitigates Oxidative Damage 83
• 4.4.3. dCas9-Tet1 System Rescues Oocyte Maturation Phenotype 84
• 4.4.4. MTNR1A Activation Rescues Embryonic Developmental Competence 84
• 4.5. Discussion 85
• References 89
• Chapter 5. General Discussion 93
• 5.1. Summary and Synthesis of Findings 94
• 5.2. A Novel Strategy: Endogenous Restoration vs. Exogenous Supplementation 95
• 5.3. Implications and Context with Existing Research 97
• 5.4. Limitations of the Study 99
• 5.5. Future Directions and Concluding Remarks 101
• 5.6. Ethical and Translational Considerations 103
• References 105
• Abstract (Korean) 109
• Acknowledgements 112