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

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
SEMARANG
2010

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
SEMARANG
2010

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
Principal Supervisor,

Indonesia
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 Master’s Degree Program in Biomedical Science
Post Graduate Program Diponegoro University

Dr. dr. Winarto Sp.MK, SpM(K)
NIP. 130675157

ii

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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

Santoso

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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, I’ve 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
degree.

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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 Master’s 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.
Semarang, July 2010
Santoso

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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

DMSO

: Dimethyl Sulphoxide

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DNA

: Deoxyribonucleic acid

dNTP

: Deoxyribonucleotide triphosphate

Dpc

: Days post coitum

EDTA

: Ethylenediaminetetraacetic Acid

EIFB2B-2

: Eukaryotic Translation Initiation Factor 2B, Subunit 2
gene

EIFB2B-4

: Eukaryotic Translation Initiation Factor 2B, Subunit 4
gene

EIFB2B-5

: Eukaryotic Translation Initiation Factor 2B, Subunit 5
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
: Gamma

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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

SDS

: Sodium Dodecyl Sulfate

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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

: Wilm’s Tumour 1 gene

XIST

: X (Inactive) Specific Transcript gene

2F5

: Antibody against human FMRP

20wks

: 20 week old

40wks

: 40 week old

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TABLE OF CONTENTS
COVER .................................................................................................................... i
APPROVAL SHEET .............................................................................................. ii
MONITORING SHEET ........................................................................................ iii
DECLARATION ................................................................................................... iv
ACKNOWLEDGEMENT ...................................................................................... v
ABBREVIATIONS .............................................................................................. vii
TABLE OF CONTENTS ....................................................................................... xi
LIST OF TABLES ............................................................................................... xiv
LIST OF FIGURES .............................................................................................. xv
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
1.5. Research originality ......................................................................................... 4

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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
4.9. Data analysis ................................................................................................ 29

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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
APPENDICES ...................................................................................................... 53

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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
7. Table 5.5. Fold change of Fmr1 mRNA levels from ovaries P25 premutation
mice compared to wt mice in the same age.

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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 noncarriers.
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.
10. Figure 5.5. Photomicrograph of ovarian histology of premutation mice.

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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.
3. Appendix 3. Ethical Clearance from ErasmusMC Erasmus University
Rotterdam, The Netherland

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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
mRNA, primordial follicle, folliculogenesis

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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
mRNA, folikel primordial, folikulogenesis.

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