Table of contents
ACKNOWLEDGMENTS... IV
TABLE OF CONTENTS ... VIII
LIST OF ABBREVIATIONS ... XII
SUMMARY ... XVI
RÉSUMÉ ... XVIII
LIST OF PUBLICATIONS AND CONFERENCE CONTRIBUTIONS ... XXI
CHAPTER 1: GENERAL INTRODUCTION ... 1
1.1 Chemotherapy and female fertility... 3
1.1.1 Mechanisms of chemotherapy induced ovarian damage: focus on alkylating agents ... 3
1.1.2 DNA Damage Response in the Ovary ... 6
1.1.3. Apoptosis within the Ovary ... 7
1.1.3.1 Mitochondrial Pathway (Internal) ... 8
1.1.3.2 Death Receptor Pathway (External) ... 9
1.1.3.3 Sphingomyelin-Mediated Apoptosis ... 9
1.1.3.4 Tap53-induced Apoptosis ... 10
1.1.4 Female Fertility Preservation ... 11
1.1.4.1 Cryopreservation approaches ... 11 1.1.4.2 Ovarian pharmacoprotection ... 11 1.2 MicroRNAs synthesis ... 14 1.2.1. Canonical Pathway... 16 1.2.1.2 Drosha-Independent Pathway ... 16 1.2.1.3 Dicer-Independent Pathway ... 17
1.2.2 MicroRNA Target Recognition ... 18
1.2.3 MicroRNA Functional Roles... 19
1.2.4 MicroRNAs in Female Reproduction ... 20
1.2.4.1 MicroRNAs’ role in Sex Determination and differentiation ... 20
1.2.4.3 MicroRNAs in Oogenesis ... 22
1.2.4.4 MicroRNAs in Oocyte-Granulosa Cells Communication ... 23
1.2.4.5 MicroRNAs in primordial follicle activation ... 24
1.2.5 MicroRNAs in DNA Damage Response ... 26
1.2.6 MicroRNAs in Ovarian Cells Apoptosis Regulation ... 27
1.2.7 MicroRNAs Expression Modulation ... 27
1.2.7.1 MicroRNAs Dysregulation in Cancer ... 28
1.2.7.2 MicroRNAs Modulation by Cytotoxic Agents ... 29
1.2.8 MicroRNAs Modulation in Reproductive Disorders ... 29
1.3. MicroRNAs in Disease Management ... 30
1.3.1 MicroRNAs as Potential Therapeutic Targets ... 31
1.3.2 Route of Administration and MicroRNA Delivery Systems ... 32
1.3.2.1 Non-Carrier Approaches ... 32
1.3.2.2 Viral MicroRNA Delivery ... 33
1.3.2.3 Non-Viral MicroRNA Delivery ... 34
1.3.2.4 Inorganic MicroRNA Nanocarriers ... 34
1.3.3 MicroRNA Therapeutics from Bench to the Bedside ... 35
1.3.4 MicroRNA Therapeutic Approaches in Onco-fertility Strategies ... 37
CHAPTER 2: OBJECTIVES OF THE STUDY ... 40
CHAPTER 3: EXPERIMENTAL STRATEGY ... 44
CHAPTER 4: STUDY RESULTS ... 50
PART 1: MIRNAS SELECTION AND IN VITRO PROTECTIVE EFFECT ... 52
4.1 Identification and efficiency assessment of potential miRNAs targets for developing new pharmacological drugs against chemotherapy-induced ovarian damage using mice model ... 54
4.1.1 Introduction ... 54
4.1.2 Results ... 54
4.1.3 Discussion ... 60
4.1.4 Conclusion ... 61
4.2.1 Introduction ... 62
4.2.2 Results ... 62
4.2.3 Discussion ... 66
4.2.4 Conclusion ... 67
PART 2: IN VIVO FOLLICULAR DEVELOPMENT AFTER EXPOSURE TO SELECTED MIRNA
... 69
4.3. Beneficial effects of let-7a mimic transfection application on oocyte quality after in vitro chemotherapy exposure and heterotopic mouse ovarian transplantation. ... 71
4.3.1 Abstract ... 71
4.3.2 Materials and Methods ... 72
4.3.3 Results ... 74
4.3.4 Discussion ... 80
4.3.5 Conclusion ... 82
CHAPTER 5. REVIEW ARTICLE ... 84
‘’MICRORNAS: FROM THE FIELD OF ONCOLOGY TO FERTILITY PRESERVATION STRATEGIES AND
TO FUTURE CLINICAL APPLICATIONS’’ ... 86
CHAPTER 6: GENERAL DISCUSSION ... 88
6.1 Animal model ... 90
6.2 Chemotherapy induced ovarian damage ... 91
6.3 Identification of miRNAs with ovarian protective properties ... 91
6.4 Let-7; tumor suppressor or oncogene? ... 93
6.5 MicroRNA delivery in vitro ... 94
6.6 In vitro miRNA functional analysis-Apoptosis evaluation ... 96
6.7 In vitro miRNA functional analysis-Follicle activation evaluation... 97
6.8 In vivo studies-Long term effects of miRNA replacement approaches ... 97
6.9 In vivo model limitations ... 98
6.10 Future in vitro and in vivo experiments ... 98
CHAPTER 7: FUTURE PERSPECTIVES... 100
7.1 Which nano-vector can serve as an ideal miRNA-delivery system? ... 102
7.2 How can the ovary be specifically targeted?... 103
7.3 How can the ovarian protective therapeutics be administered?... 104
7.4 Are the gold nanoparticles safe? ... 105
7.5 Which are the limitations and how they can be overcome? ... 106
