STUDY OF PRIMORDIAL FOLLICLE, CORPORA LUTEA AND Fmr1 mRNA LEVELS FROM OVARIUM OF Fmr1 GENE PREMUTATION MICE - Diponegoro University | Institutional Repository (UNDIP-IR)

(1)

STUDY OF PRIMORDIAL FOLLICLE, CORPORA

LUTEA AND

Fmr1

_{mRNA LEVELS FROM OVARIUM}

OF

Fmr1

_{GENE PREMUTATION MICE}

THESIS

A thesis submitted for the degree of Master of Biomedical Science majoring on Genetic Counseling

by SANTOSO G4A007053

POST GRADUATE PROGRAM

DIPONEGORO UNIVERSITY

(2)

STUDY OF PRIMORDIAL FOLLICLE, CORPORA

LUTEA AND

Fmr1

_{mRNA LEVELS FROM OVARIUM}

OF

Fmr1

_{GENE PREMUTATION MICE}

THESIS

A thesis submitted for the degree of Master of Biomedical Science majoring on Genetic Counseling

By SANTOSO G4A007053

POST GRADUATE PROGRAM

DIPONEGORO UNIVERSITY

(3)

ii THESIS

STUDY OF PRIMORDIAL FOLLICLE, CORPORA LUTEA AND Fmr1

mRNA LEVELS FROM OVARIUM OF Fmr1_{GENE PREMUTATION}

MICE

By SANTOSO G4A007053

Has been defended in front of the defence committee On July 6th, 2010

and has been approved by

The Netherland Indonesia

Principal Supervisor, Principal Supervisor,

Rob Willemsen, PhD Prof. Dr. dr. Sarjadi, Sp.PA (K)

NIP. 130352547 Supervisor,

dr. Tri Indah Winarni, Msi.Med NIP. 132163892

Recognition,

Head of Masters Degree Program in Biomedical Science

Post Graduate Program Diponegoro University

(4)

(5)

iv

DECLARATION

I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgement is made in the text.

Semarang, July 2010

(6)

v

ACKNOWLEDGEMENTS

First of all, I deeply thank God for his blessing and guidance so I could finish my master program. In completion of this thesis, Ive got a lot of support from my supervisors, colleagues, friends and family. I would like to express my deepest gratitude to my supervisors Rob Willemsen, PhD, Professor Dr. dr. Sarjadi, SpPA (K), and dr. Tri Indah Winarni, MSi.Med.

My special thanks for Rob Willemsen, PhD, my principal supervisor in The Netherlands, I gratefully acknowledge for sharing your experiences through giving ideas and valuables comments for every chapter of this thesis. To my principal supervisor in Indonesia, Professor Dr. dr. Sarjadi, SpPA (K), thank you for the time you spent for teaching and guiding me and for reading my thesis. My gratitude also to dr. Tri Indah Winarni, MSi.Med for your guidance, endless encouragement and you concern for myself.

I would like to thank to Professor dr. Sultana M.H. Faradz, PhD. Head of Master Biomedical Science Majoring on Genetic Counseling Diponegoro University Semarang, thank you for your kindest attention for me, sharing your experiences. You give me inspiring suggestion and motivation to be the best.

Special thanks for financial support from Bureau of Planning and International Cooperation, Ministry of National Education The republic

Indonesia, who fully provided Beasiswa Unggulan scholarship for my master

(7)

vi

I would like to thanks to Professor dr. Ben Oostra PhD, Chairman of Research Department of Clinical Genetics, Erasmus MC Rotterdam, The Netherlands. Thank you for the opportunity to undertake research in your laboratory, as well as your warm and stimulating interest and friendship. My gratitude also for Renate Hukema, thank you for your patience in training me to do RT and Q PCR, Western Blotting and give me tips to always remember sample id when do RT and Q PCR. Thank you for all of my colleagues at Clinical Genetic Department Erasmus MC, Rotterdam, The Netherlands, especially to Lies-Anne Severijnen, Asma Azmani, Ronald Buijsen, Linda, Christina and Jeannette Lokker.

I would like to extend my acknowledgement to Prof. Dr. dr. Susilo Wibowo, Ms.Med., Sp.And, Rector of Diponegoro University Semarang, dr. Soejoto SpKK (K), Dean of the Faculty of Medicine Diponegoro University Semarang, Dr. dr. Winarto, SpMK, SpM (K) Head of Masters Degree Program in Biomedical Science Post Graduate Program Diponegoro University Semarang, and dr. Kusmiyati D.K, M.Kes, Head of the Biochemistry Department, Faculty of Medicine, Diponegoro University Semarang.

I would like to express my sincere gratitude to my family, my parents and all of my sisters who supported me in many ways until I completed this study.

Last but not least, I would like to thank all others who have contributes to this work.

(8)

vii

ABBREVIATIONS

: Alfa

AIRE : Autoimmune Regulator Polyglandular Failure gene

AMH : Anti Mullerian Hormone

APECED : Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal

Dystrophy

ATM : Ataxia Telangiectasia Mutated gene

AT2 : Angiotensin II Subtype 2 gene

: Beta

BLM : BecQ Protein like 3-DNA Helicase gene

Bl/6 : Black 6

BMP15 : Bone Morphogenetic Protein 15 gene

BMP4 : Bone Morphogenetic Protein 4 gene

Cbl : Cas-Br-M (murine) ecotropic retroviral transforming sequence gene

CGG : Cytosine-Guanin-Guanin

Ct : Cycle time

CYP17A1 : 17- Hydroxylase/17,20-lyase enzyme

: Delta

dFMR1 : Drosophila FMR1

DIAPH2 : Diaphanous 2 gene

(9)

viii

DNA : Deoxyribonucleic acid

dNTP : Deoxyribonucleotide triphosphate

Dpc : Days post coitum

EDTA : Ethylenediaminetetraacetic Acid

EIFB2B-2 : Eukaryotic Translation Initiation Factor 2B, Subunit 2

gene

exCGG : Expanded Cytosine Guanin Guanin

FAD : Fragile X Associated Disorders FMRP : Fragile X Mental Retardation Protein

FMR1 : human Fragile X Mental Retardation 1 gene

Fmr1 : mice Fragile X Mental Retardation 1 gene

FOXL2 : Forxhead Transcription Factor gene

FSH : Follicle Stimulating Hormone

FSHRH1 : FSH Receptor gene

FXS : Fragile X Syndrome

FXPOF : Fragile X Associated Premature Ovarian Failure FXPOI : Fragile X Associated Primary Ovarian Insufficiency FXTAS : Fragile X Associated Tremor Ataxia Syndrome

(10)

ix

GALT : Galactose 1-Phosphate Uridyl Transferase gene

HCl : Hydrogen Chloride

HF : High Fidelity

Homrep : Homozygous Repeat

HSP : Stress Response Protein

INHA : Inhibin - subunit gene

ko : Knock Out

LHCGR : Luteinizing Hormone/Choriogonadotrophine Receptor

Lhx1 : Lim Homeobox 1 gene

LIF : Leukemia Inhibitory Factor gene

mRNA : Messanger Ribonucleic acid

NaCl : Natrium Chloride

PA : Polyacrilamide

PCR : Polymerase Chain Reaction

PGCs : Primordial Germ Cells

POF : Premature Ovarian Failure

POI : Primary Ovarian Insufficiency

POLG : Polymerase

P5 : Postnatal 5 (5-day-old)

P25 : Postnatal 25 (25-day-old)

Q and RT PCR : Quantitative and Reverse Transcriptase PCR

SCF : Stem Cell Factor gene

(11)

x

Sf1 : Steroidogenic factor 1 gene

SOX3 : SRY (Sex Determining Region Y) Box 3 gene

STAR : Steroidogenic Acute Regulatory Protein gene

TBE : Tris-Borate-EDTA

T1A : Antibody against human FMRP

UV : Ultraviolet

wt : Wild type

Wt1 : Wilms Tumour 1 gene

XIST : X (Inactive) Specific Transcript gene

2F5 : Antibody against human FMRP

20wks : 20 week old

(12)

xi

TABLE OF CONTENTS

COVER ... i

APPROVAL SHEET ... ii

MONITORING SHEET ... iii

DECLARATION ... iv

LIST OF APPENDICES ... xvi

ABSTRACT ... xvii

ABSTRAK ... xviii

CHAPTER I. INTRODUCTION ... 1

1.1. Background ... 1

1.2. Research questions ... 3

1.3. Research purposes ... 4

1.4. Research benefit ... 4

(13)

xii CHAPTER 2.

LITERATURE REVIEW ... 6

2.1. Definition and prevalence of the FMR1 premutation ... 6

2.2. Premutation-associated primary ovarian insufficiency ... 7

2.3. Premutation mice is an excelent model to study primary ovarian insufficiency ... 16

CHAPTER 3. THEORETICAL FRAMEWORK, CONCEPTUAL FRAMEWORK AND HYPOTHESES ... 20

3.1. Theoretical framework ... 20

3.2. Conceptual framework ... 20

3.3. Hypotheses ... 21

CHAPTER 4. RESEARCH METHODS ... 22

4.1. Research field ... 22

4.2. Setting/location and research periods ... 22

4.3. Research design ... 22

4.4. Population and samples ... 22

4.5. Research variables ... 23

4.6. Operational definitions ... 24

4.7. Research protocols ... 25

4.8. Framework of research protocol ... 28

(14)

xiii

4.10. Ethical clearance ... 30

CHAPTER 5. RESULTS AND DISCUSSION ... 31

5.1. Results ... 31

5.2. Discussion ... 37

CHAPTER 6. CONCLUSION AND RECOMMENDATION ... 42

6.1. Conclusion ... 42

6.2. Recommendation ... 42

REFERENCES ... 43

CURRICULUM VITAE ... 48

(15)

xiv

LIST OF TABLE

1. Table 1.1. Matrix of research originality.

2. Table 2.1. Causes of primary ovarian insufficiency.

3. Table 5.1. Means of primordial follicle number premutation and wt mice in several ages.

4. Table 5.2. Percentage of primordial follicle depletion from P6 to varying age in premutation and wt mice.

5. Table 5.3. Proportion of presence recent corpora lutea from ~20wk premutation mice compared to wt mice.

6. Table 5.4. Proportion of presence recent corpora lutea from ~40wk premutation mice compared to wt mice

(16)

xv

LIST OF FIGURES

1. Figure 2.1. Expression of FMR1 in normal women, premutation carriers, and full mutation carriers.

2. Figure 2.2. Relationship of primary ovarian insufficiency by CGG repeat length. Based on a data of 429 FMR1 premutation carriers and 517 non-carriers.

3. Figure 3.1. Theoretical framework 4. Figure 3.2. Conceptual framework

5. Figure 4.1. Framework of research protocol

6. Figure 5.1. Photomicrograph of ovarian histology of ~20wk premutation mice 7. Figure 5.2. Photomicrograph of ovarian histology of P6 premutation mice 8. Figure 5.3. Primordial follicle population in P6, P25, ~20wk, and ~40wk

premutation and wt mice.

9. Figure 5.4. Percentage of primordial follicle depletion from P6 to varying age in premutation and wt mice.

(17)

xvi

LIST OF APPENDICES

1. Appendix 1. Database of Primordial Follicle number, presence of recent corpora lutea, and Fmr1 mRNA from premutation mice and wild type. 2. Appendix 2. Output of SPSS analysis results.

(18)

xvii Abstract

Background : The FMR1 gene involves in fragile X-associated disorders (FAD), including the fragile X mental retardation syndrome, primary ovarian insufficiency (POI) and Fragile X associated tremor/ataxia syndrome. Primary ovarian insufficiency (POI) affect on 13-26% female premutation carrier, characterized by early depletion of ovarian follicles before the age of 40 years. The underlying mechanisms of POI are still unclear. This study focuses on number of primordial follicles, ovulation and ovarian Fmr1 mRNA levels in Fmr1

gene premutation mice as animal models.

Method : This research was a cross sectional study using a Fmr1 gene premutation mice model (exCGG mice) and wt mice. The number of primordial follicles were determined at different ages (P6, P25, ~20wk, and ~40wk). The presence of recent corpora lutea (CL) were determined at ~20wk and ~40wk.

Fmr1 mRNA levels in ovaries from P25 premutation mice were determined and compared to wild type (wt) mice.

Results : Approximately a four fold elevated level of Fmr1 mRNA was found in ovaries of premutation mice. No differences in number of primordial follicles was found in ovaries from P6 and ~20wk premutation mice compared to wt mice (p=0.052 and p=0.515, respectively). Significant higher number of primordial follicles was found at P25, and ~40wk premutation mice compared to wt mice (p=0.018, and 0.006, respectively). Furthermore reduced ovulation, determined by presence of recent corpora lutea, in 20 and 40-week-old premutation mice was found.

Conclusion : The fragile X premutation does not cause a reduction in the initial pool of primordial follicles from birth but might be involved in mechanisms influencing the folliculogenesis results in decreased ovulation..

---Keyword : Primary ovarian insufficiency (POI), FMR1 gene, FMRP, FMR1

(19)

xviii Abstrak

Latar Belakang : Gen FMR1 merupakan penyebab Fragile X-associated

disorders (FAD), yang meliputi Fragile X Syndrome (FXS), Primary ovarian

insufficiency (POI) dan tremor/ataxia syndrome. 13-26% wanita pembawa alel

premutasi mengalami POI, yang ditandai dengan berkurangnya jumlah cadangan folikel dalam ovarium sebelum berusia 40 tahun, sementara itu patogenesis dari POI pada wanita premutasi sampai sekarang masih belum diketahui. Penelitian ini bertujuan untuk mengetahui jumlah cadangan folikel primordial, ovulasi dan konsentrasi Fmr1 mRNA pada ovarium tikus premutasi gen Fmr1.

Metode : Penelitian ini merupakan penelitian cross sectional dengan menggunakan tikus premutasi dan wild type (wt). Jumlah folikel primordial dihitung dari kelompok tikus pada usia yang berbeda (6 hari, 25 hari, ~20 minggu, dan ~40 minggu). Adanya ovulasi dideteksi dengan adanya recent

corpora lutea pada kelompok tikus usia ~20 minggu dan ~40 minggu.

Konsentrasi Fmr1 mRNA diukur dari ovarium kelompok tikus premutasi usia 25 hari yang dibandingkan dengan kelompok wt.

Hasil : Terdapat peningkatan empat kali lipat konsentrasi Fmr1 mRNA pada ovarium tikus premutasi dibandingkan dengan kelompok wt. Tidak terdapat perbedaan jumlah folikel primordial pada kelompok tikus premutasi usia 6 hari (p=0.052) dan ~20 minggu (p=0.515) dan terdapat peningkatan jumlah folikel primordial pada usia 25 hari (p=0.018) dan ~40 minggu (p=0.006). Selain itu, ditemukan penurunan jumlah ovulasi yang dideteksi dengan adanya recent

corpora lutea, pada kelompok usia ~20 minggu dan ~40 minggu.

Kesimpulan : Premutasi gen Fmr1 tidak menyebabkan pengurangan jumlah folikel primordial. Ditemukan gangguan folikulogenesis yang menyebabkan pengurangan jumlah ovulasi pada tikus premutasi.

--- Kata Kunci : Primary ovarian insufficiency (POI), gen FMR1, FMRP, FMR1