REGULATION OF APOPTOSIS

IN THE FEMALE
REPRODUCTIVE SYSTEM

TOM MI
VA S KIVUO
Department of Obstetrics and
Gynaecology,
University of Oulu

OULU 2002

TOMMI VASKIVUO

REGULATION OF APOPTOSIS IN
THE FEMALE REPRODUCTIVE
SYSTEM

Academic Dissertation to be presented with the assent of

the Faculty of Medicine, University of Oulu, for public
discussion in the Auditorium 4 of the University Hospital
of Oulu, on May 8th, 2002, at 12 noon.

O U L U N Y L I O P I S TO, O U L U 2 0 0 2

Copyright 2002
University of Oulu, 2002

Reviewed by
Professor John E. Eriksson
Docent Jorma Toppari

ISBN 951-42-6667-6

(URL: http://herkules.oulu.fi/isbn9514266676/)

ALSO AVAILABLE IN PRINTED FORMAT

Acta Univ. Oul. D 676, 2002
ISBN 951-42-6666-8

ISSN 0355-3221

(URL: http://herkules.oulu.fi/issn03553221/)

OULU UNIVERSITY PRESS

OULU 2002

Vaskivuo, Tommi, Regulation of apoptosis in the female reproductive system

Department of Obstetrics and Gynaecology, University of Oulu, P.O.Box 5000, FIN-90014
University of Oulu, Finland
Oulu, Finland
2002

Abstract
Apoptosis is a genetically programmed mechanism for a multicellular organism to remove cells that
are unnecessary, or potentially harmful. The female reproductive system is characterised by a high
rate of cellular proliferation. At the same time, apoptosis is also abundant during the normal
physiological function of the ovary and endometrium. More than half of the 7 million oocytes that are
produced during human ovarian development are deleted before birth and only about 400 oocytes
reach the stage of ovulation during the female fertile lifespan. The fate of the non-ovulatory follicles
is atresia, occurring through the mechanism of apoptosis. The endometrium goes through radical
renewal processes during each menstrual cycle. Apoptosis has been suggested to participate in the
regulation of endometrial cellular homeostasis. Errors in this mechanism can result in endometrial
diseases such as hyperplasia and cancer. In this work, apoptosis and its regulation were studied in the
human fetal and adult ovary, normal endometrium and endometrial pathologies.
In fetal ovaries, apoptosis was already abundantly present in oocytes at 13 weeks of gestation. The
maximum rate of apoptosis was seen between the 14th and 20th weeks, after which apoptosis
decreased towards term. Ovarian Bcl-2 expression was detected in early fetal life during weeks 13
and 14. Bax expression was observed throughout the studied period, from week 13 to 40. The
expression of transcription factor GATA-4, which is linked to follicular survival, was localised to the
granulosa cells and was high in early fetal life and decreased somewhat towards term. In adult life
apoptosis was located in the granulosa cells of the growing follicles. In ovarian biopsies from women
homozygous for the inactivating C566T mutation of the FSH receptor, apoptosis or GATA-4
expression was not detected. During corpus luteum regression a peak in apoptosis was detected 10 12 days after the LH surge, and was preceded by an increase in 17HSD type 1 and TNF- expression.
During normal menstrual cycles, the highest rate of apoptosis was observed in the menstrual
endometrium. This increase in apoptosis was preceded by a decreased Bcl-2/Bax ratio. In endometrial
hyperplasia, the rate of apoptosis was similar to that seen during normal proliferation of the
endometrium, but an apparent increase was observed in grade II endometrial carcinoma. In grade III
carcinoma, the rate of apoptosis was lower than in grade II carcinoma but higher than in hyperplasia.
These results indicate that apoptosis is the mechanism behind the substantial oocyte demise during
ovarian development. During adult life, apoptosis was mainly localised to the granulosa cells of the
growing follicles which do not reach the stage of a dominant follicle. In ovaries where FSH action is
abolished, folliculogenesis was impaired and ovarian apoptosis was negligible. Apoptosis is also the
underlying mechanism of corpus luteum regression. In the endometrium, apoptosis has a role in
rejuvenating the endometrium for growth during the next endometrial cycle and in regulating cellular
homeostasis.

Keywords: ovary, apoptosis, oocyte, corpus luteum, endometrium, endometrial hyperplasia, endometrial carcinoma

Acknowledgements
This work was carried out at the Department of Obstetrics and Gynaecology during the
years 1994-2002.
I owe my deepest gratitude to my supervisor Professor Juha Tapanainen, M.D., Ph.D.,
for guiding me in the world of science. His enthusiasm and optimistic attitude towards
research and life in general have inspired me during these years. He has given me an
exceptional opportunity to carry out the present work. It has been a privilegedge to work
with you.
I also wish to thank my second supervisor, Professor Markku Heikinheimo, M.D.,
Ph.D., for sharing his vision and comments. His interest in my work has been of great
importance.
I wish to express my sincere gratitude to Professor Pentti Jouppila, M.D., Ph.D., the
Head of the Department of Obstetrics and Gynaecology, who provided excellent
conditions and an encouraging atmosphere for scientific work. I also want to thank
Professor Veli-Pekka Lehto, M.D., Ph.D., the Head of the Department of Pathology and
for providing outsanding research facilites at my disposal. I wish to thank Professor Frej
Stenbck for rewarding collaboration and his interest in my work.
I am gratefull to Docent Paavo Pkk, M.D., Ph.D., for his help during this study.
Docent Leo Dunkel, M.D., Ph.D, has provided fundamental technical assistance in the
early stages of this work, for which I am grateful. I also appreciate his genuine interest
towards my work.
I want to thank my co-authors and collaborators: Kristiina Aittomki, M.D., Ph.D.,
Mikko Anttonen, Hkan Billig, M.D., Ph.D., Marinus Dorland, M.D., Ph.D., Docent
Riitta Herva, M.D., Ph.D., Professor Ilpo Huhtaniemi, M.D., Ph.D., Veli Isomaa, Ph.D.,
Pepe Karhumaa, M.D. Ph.D., Ilkka Ketola, M.D., Olayi Oduwole, M.Sc., Professor Jan
Olafsson, M.D., Ph.D., Yoshio Osawa, M.D., Ulrika Ottander, M.D. Ph.D., Professor Juha
Risteli M.D., Ph.D., Professor Egbert te Velde, M.D., Ph.D., and Professor Pirkko Vihko,
M.D., Ph.D.
I specially want to thank Mirja Ahvensalmi for all the help in the laboratory; you were
irreplaceable in this work.
I wish to thank my friends and co-workers in the laboratory and at the clinic; Ilkka
Jrvel, M.D., Ph.D., Riitta Koivunen, M.D., Ph.D., Tuula Lujala, Laure Morin-Papunen

M.D., Ph.D., Kaarin Mkikallio, M.D., Antti Perheentupa, M.D., Ph.D., Terhi Piltonen,
docent Juha Rsnen, M.D. Ph.D., Marja Tolppanen, Erja Tomperi, Manu Tuovinen,
Mirja Vahera, Riitta Vuollet and Pivi Koukkula.
I would like to thank Liisa Krki and Seija Leskel in the photography lab for their
skilled expertise in and friendly co-operation.
Professor John Eriksson Ph.D., and Docent Jorma Toppari M.D., Ph.D., are gratefully
acknowledged for the careful review of the present manuscript.
I thank Nick Bolton, Ph.D., for the revision of the language of this thesis.
My friends Matti Hiltunen, M.D., Tapani Hr, M.D., Marko Kervinen, M.D., TimoJussi Linna, M.D., Jani Mntyjrvi, M.Sc., Jarkko Piuhola, M.D., docent Juha Rantala,
Ph.D., Jaakko Rnty, M.D., Marko Talala and Jarmo Vesala, MBA have provided superb
company occasionaly also outside the world of science.
I also thank my dear friends Otto and Kirsi Palva, Tuomas and Nina Kerola.
Marja and Pekka Laatio have supported my work in countless ways. I cant thank you
enough.
I want to thank my brother Teemu for being there.
I thank my parents Timo and Tuula for making this all possible.
Finally, my warmest thanks belong to my beloved Liisa. Thank you for everything.
This work was supported financially by Oulu University Hospital, Finnish Medical
Foundation Duodecim, the Academy of Finland, Juslius Foundation, Emil Aaltonen
Foundation, Ostrobothnian Cancer Foundation, Finnish Endocrine society and Oulu
Medical Research Foundation. All these are gratefully acknowledged.

Oulu, March, 2002

Tommi Vaskivuo

Abbreviations
AIF
Apaf-1
BAD
Bak
Bax
Bcl-2
Bcl-XL
Bcl-XS
BH
BIR
BOD
Bok
bp
CAD
C. Elegans
CARD
cAMP
CL
DAB
DD
DED
DISC
DNA
DNase
E2
ERK
FADD
FasL
FasR
FLICE
FLIP

Apoptosis-inducing factor
Apoptosis protease-activating factor 1
Bcl- XL /Bcl-2-associated death promoter
Bcl-2 homologous antagonist/killer
Bcl-2 -associated x protein
B cell leukaemia-2
B cell leukaemia-x long
B cell leukaemia-x short
Bcl-2 homology
Baculoviral inhibitory repeat
Bcl-2 -related ovarian death gene
Bcl-2 -related ovarian killer
Base pair
Caspase-activated DNase
Caenorhabditis elegans
Caspase activation and recruitment domain
Cyclic adenosine 3,5-monophosphate
Corpus luteum
Diaminobenzidine
Death domain
Death effector domain
Death-inducing signalling complex
Deoxyribonucleic acid
DNA ladder nuclease
Estradiol
Extracellular signal regulated kinase
Fas-associated death domain
Fas ligand
Fas receptor
Fas ligand-interacting cell effector
FLICE-inhibitory protein

FSH
FSHR
GDF
GnRH
hCG
17HSD
IAP
ICAD
IB
IKK
IVF
IB
kb
kDa
KL
LH
Mcl-1
MAPK
MKK
mRNA
NF- B
NIK
PBS
PI-3K
PKC
PRL
P450arom
RIP
RNA
ROS
SDS
Smac
SSC
T
TGFTNFTNFR
TRAIL
VDAC
XIAP
Z-VAD-fmk
m

Follicle-stimulating hormone
Follicle-stimulating hormone receptor
Growth differentiation factor
Gonadotrophin releasing hormone
Human chorionic gonadotrophin
17-Hydroxysteroid dehydrogenase
Inhibitor of apoptosis
Inhibitor of CAD
Inhibitor of NF- B
Inhibitor of NF- B kinase
In vitro fertilization
Inhibitor of NF- B
Kilobase
Kilodalton
Kit ligand
Luteinising hormone
Myeloid cell leukaemia-1
Mitogen-activated protein kinase
Mitogen-activated protein kinase kinase
Messenger ribonucleic acid
Nuclear factor B
NF- B inducing kinase
Phosphate-buffered saline
Phophoinositide 3-kinase
Protein kinase C
Prolactin
Cytochrome P450 aromatase
Receptor-interacting protein
Ribonucleic acid
Reactive oxygen species
Sodium dodecyl sulphate
Second mitochondria-derived activator of caspases
Saline sodium citrate
Testosterone
Transforming growth factor-beta
Tumour necrosis factor
Tumour necrosis factor receptor
TNF-related apoptosis inducing ligand
Voltage-dependent anion channel
X-linked inhibitor of apoptosis
Benzyloxycarbonyl-VAD-fluoromethylketone
Mitochondrial transmembrane potential

List of original publications

This thesis is based on the following articles, which are referred to in the text by their
Roman numerals:
I.

Vaskivuo T, Anttonen M, Herva R, Billig H, Dorland M, te Velde E, Stenbck F,

Heikinheimo M & Tapanainen J (2001) Survival of human ovarian follicles from
fetal to adult life: apoptosis, apoptosis-related proteins and transcription factor
GATA-4. J Clin Endocrinol Metab. 86:3421 - 3429.

II.

Vaskivuo T, Aittomki K, Anttonen M, Ruokonen A, Herva R, Osawa Y,

Heikinheimo M, Huhtaniemi I & Tapanainen J (2001) Effects of FSH and hCG
in Subjects with an Inactivating Mutation of the FSH Receptor. Fertil Steril. In
press.

III.

Vaskivuo T, Ottander U, Oduwole O, Isomaa V, Vihko P, Olofsson J &

Tapanainen J (2001) Role of apoptosis, apoptosis related factors and 17
hydroxysteroid dehydrogenases in human corpus luteum regression. Mol Cell
Endocrinol. In Press.

IV.

Vaskivuo T, Stenbck F, Karhumaa P, Risteli J, Dunkel L & Tapanainen J (2000)

Apoptosis and apoptosis-related proteins in human endometrium. Mol Cell
Endocrinol. 165:75 - 83.

V.

Vaskivuo T, Stenbck F & Tapanainen J (2001) Apoptosis and apoptosis related

factors Bcl-2, Bax, TNF- and NF- B in human endometrial hyperplasia and
carcinoma. Submitted.

Contents
Acknowledgements ............................................................................................................ 4
Abbreviations ..................................................................................................................... 6
List of original publications ............................................................................................... 8
Contents ............................................................................................................................. 9
1 Introduction................................................................................................................... 12
2 Review of the literature ................................................................................................. 14
2.1 Apoptosis................................................................................................................ 14
2.1.1 History............................................................................................................. 15
2.1.2 Apoptosis vs. necrosis ..................................................................................... 16
2.1.3 Common mechanisms of apoptosis................................................................. 17
2.1.4 Regulation of apoptosis................................................................................... 18
2.1.4.1 Death receptors......................................................................................... 19
2.1.4.2 NF- B ....................................................................................................... 21
2.1.4.3 Tumour suppressor p53 ............................................................................ 21
2.1.4.4 GATA transcription factors....................................................................... 22
2.1.4.5 The Bcl-2 family ...................................................................................... 23
2.1.4.6 The mitochondrial forum.......................................................................... 23
2.1.4.7 The Apaf-1 complex or apoptosome ........................................................ 25
2.1.5 Execution of apoptosis .................................................................................... 26
2.1.5.1 Caspases ................................................................................................... 26
2.1.5.2 Inhibitors of apoptosis proteins ................................................................ 28
2.1.6 Role of apoptosis in development ................................................................... 28
2.1.7 Apoptosis and tissue homeostasis ................................................................... 29
2.1.8 Apoptosis in disease ........................................................................................ 30
2.1.9 Studying apoptosis .......................................................................................... 30
2.1.9.1 DNA analysis............................................................................................ 31
2.2 Ovarian function..................................................................................................... 31
2.2.1 Ovarian development....................................................................................... 31
2.2.1.1 Postnatal trends ........................................................................................ 33
2.2.2 The adult ovary................................................................................................ 34
2.2.2.1 Hormonal regulation of follicular growth ................................................ 35

2.2.2.2 Regulation of ovarian steroidogenesis...................................................... 36

2.2.2.3 The role of FSH in the female reproduction............................................. 36
2.2.2.4 The corpus luteum.................................................................................... 37
2.2.3 Regulation of ovarian apoptosis ...................................................................... 37
2.3 Endometrial function.............................................................................................. 39
2.3.1 Uterine development ....................................................................................... 39
2.3.2 Regulation of endometrial function during the menstrual cycle...................... 39
2.3.3 Endometrial cancer and hyperplasia................................................................ 40
3 Aims of the study .......................................................................................................... 42
4 Materials and methods .................................................................................................. 43
4.1 Tissue samples........................................................................................................ 43
4.1.1 Fetal and neonatal ovaries ............................................................................... 43
4.1.2 Adult ovaries ................................................................................................... 43
4.1.3 Patients with inactivating FSHR mutation ...................................................... 44
4.1.4 The corpus luteum........................................................................................... 45
4.1.5 Cycling endometrium...................................................................................... 45
4.1.6 Endometrial hyperplasias and carcinomas ...................................................... 45
4.2 Cell culture............................................................................................................. 46
4.3 In situ 3 end labelling of apoptotic cells ............................................................... 46
4.4 Gel electrophoretic DNA fragmentation analysis................................................... 47
4.4.1 Radioactive DNA fragmentation analysis ....................................................... 47
4.4.2 Non-radioactive DNA fragmentation analysis................................................. 47
4.5 In situ hybridisation analysis.................................................................................. 47
4.5.1 Bcl-2 and Bax.................................................................................................. 47
4.5.2 GATA-4 ........................................................................................................... 48
4.5.3 17HSD type 1 and 2 ........................................................................................ 48
4.6 Northern blotting.................................................................................................... 49
4.7 Immunohistochemistry........................................................................................... 49
4.8 Western blotting ..................................................................................................... 49
4.9 FSH and hCG stimulation ...................................................................................... 50
4.10 Hormone measurements....................................................................................... 50
4.11 Histopathological analysis.................................................................................... 51
4.12 Statistics ............................................................................................................... 51
5 Results........................................................................................................................... 52
5.1 Apoptosis in the fetal ovary.................................................................................... 52
5.1.1 Regulation of apoptosis in fetal ovary............................................................. 53
5.2 Apoptosis in adult ovary......................................................................................... 54
5.2.1 Apoptosis-regulating factors in adult ovary..................................................... 55
5.3 Role of FSH in ovarian apoptosis........................................................................... 55
5.4 Apoptosis in the corpus luteum .............................................................................. 56
5.4.1 Regulation of apoptosis in the corpus luteum ................................................. 57
5.4.2 17HSD type 1 and 2 expression in the corpus luteum..................................... 57
5.5 Apoptosis in the endometrium during the menstrual cycle .................................... 57
5.5.1 Regulation of apoptosis in cycling endometrium ............................................ 58
5.5.2 Proliferation during normal endometrial cycles .............................................. 59
5.6 Apoptosis in endometrial hyperplasias and carcinomas ......................................... 59

5.6.1 Apoptosis in endometrial hyperplasia ............................................................. 59

5.6.2 Apoptosis in endometrial adenocarcinoma...................................................... 60
5.7 Functional study of C566T FSHR mutation........................................................... 61
6 Discussion ..................................................................................................................... 62
6.1 Germ cell attrition .................................................................................................. 62
6.2 Follicular atresia..................................................................................................... 64
6.3 Two-cell-two-gonadotrophin theory....................................................................... 65
6.4 Corpus luteum regression....................................................................................... 66
6.5 Endometrial apoptosis during the normal menstrual cycle .................................... 66
6.6 Regulation of endometrial homeostasis ................................................................. 67
6.7 Future perspectives................................................................................................. 68
7 Summary and conclusions............................................................................................. 70
8 References..................................................................................................................... 71
9 Original publications..................................................................................................... 83

1 Introduction
Apoptosis, or programmed cell death, describes the carefully coordinated collapse of a
cell, protein degradation and DNA fragmentation followed by rapid engulfment of the
corpses by neighbouring cells. It is an essential part of life for every multicellular
organism from worms to humans. Apoptosis has been a target of intensive research
throughout recent years. This is reflected by over 50,000 publications to date, with the
great majority of them published during the last decade (source: ISI- Web of Science).
Apoptosis plays a major role from embryonic development to senescence. During
development, apoptosis facilitates sculpturing of the fingers and toes, maturation of the
nervous and immune systems and formation of male and female reproductive organs. In
postnatal life, apoptosis regulates tissue homeostasis, immune system function and helps
to purge the body of cells that have been invaded by pathogens. In male and female
reproductive organs apoptosis is an essential component of their function.
During early ovarian development, mitotic divisions of germ cells give birth to
approximately 7 million oocytes (Baker 1971). It is well known that large numbers of
germ cells are culled from the ovary for as yet unknown reasons, resulting in less than a
third of the total number of potential oocytes being endowed in ovarian primordial
follicles at the time of birth. The process of germ cell attrition continues after birth and it
has been estimated that at the onset of puberty only 200,000 400,000 oocytes remain
(Faddy et al. 1992). In a fertile, hormonally active ovary, a few resting primordial
follicles will start growing during every menstrual cycle. Ultimately, only one of these
follicles will develop into a dominant follicle and ovulate. The fate of the non-ovulatory
follicles is atresia. After ovulation, the dominant follicle collapses and creates the corpus
luteum. If pregnancy does not take place, corpus luteum regression ensues to allow the
growth of follicles during the next menstrual cycle.
While these events have been well known for decades only the recent advances in
biochemical techniques have implicated apoptosis as the mechanism for germ cell
depletion, follicular atresia and corpus luteum regression (reviewed in Morita & Tilly
1999).
The endometrium also undergoes cyclic changes during every menstrual cycle.
Apoptosis participates in the regulation of tissue homeostasis in the endometrium. It is
well known that development of cancer can often arise from defects in mechanisms that

13
control apoptotic cell death (Wyllie 1997a). It is possible that improper regulation of
apoptosis and subsequent cellular homeostasis in the endometrium leads to development
of endometrial hyperplasia or carcinoma.
The universality and frequency of apoptotic cell death signifies the importance of its
regulation. Too little or too much apoptosis may lead to pathology, including
developmental defects, neurodegeneration, autoimmune diseases, infertility or cancer
(Wyllie et al. 1999, Nicholson 2000).
Regulation of apoptosis in reproductive organs has been studied extensively using
transgenic and knockout animal models. The pathways of cell death are also well known
in in vitro-cultured human granulosa-luteal cells (reviewed in Maruo et al. 1999).
However, these cells differ from their original precursors so much, that far reaching
conclusions about the in vivo regulation of apoptosis in follicular granulosa cells or the
corpus luteum are hard to derive. For obvious reasons, functional in vivo experiments are
extremely difficult to conduct. The aim of this work was to study the role of apoptosis
and its regulatory mechanism in the human female reproductive system, i.e. ovary and
endometrium.

2 Review of the literature

2.1 Apoptosis
Multicellular animals often need to discard cells that are superfluous or potentially
harmful. Apoptosis is an active dedicated programme that leads to regulated destruction
of a cell. It counter-balances cell division and cell migration in the upkeep of homeostasis
and ensures proper function in a variety of tissues, such as reproductive organs and the
immune system.
The process starts with a death signal. This signal can be a physiological messenger
molecule, e.g. a cytokine or hormone, but also radiation and some chemical and
pharmacological agents can trigger the molecular cascade leading to apoptosis. After the
signal is recognised by the cell, its strength is weighed against factors that promote
cellular survival (reviewed in Hengartner 2000). Mitochondria represent not only the
powerhouse of the cell, but also a central command centre that participates in the decision
as in whether the cell should commit suicide through apoptosis, or continue its business
(reviewed in Kroemer & Reed 2000). Mitochondria sequester a number of proteins that
can initiate an automatic sequence leading to cell death when they are released to the
cytoplasm. The Bcl-2 family is directly associated with actions on the mitochondrial
membrane that can lead to an increase in mitochondrial permeability and outflow of proapoptotic proteins (Gross et al. 1999). The Bcl-2 family includes pro and anti-apoptotic
members and execution of the apoptotic message is dependent on their relative
proportions (Chao & Korsmeyer 1998).
In the actual execution stage of apoptosis, proteolytic enzymes, caspases are activated.
They are responsible for the most visible stages of apoptotic cell death (Zimmermann &
Green 2001). Caspases selectively cleave target proteins, which results in both
deactivation and activation of the proteins (Dales et al. 2001). These substrates include
structural proteins, other caspases and DNase (reviewed in Grutter 2000). The subsequent
activation of other caspases functions as an enhancer of the process, creating a growing
cascade of apoptotic proteinases (Grutter 2000). The eventual activation of caspaseactivated DNase (CAD) leads to DNA laddering that is typical of apoptosis (Wyllie et al.

15
1984). In the end apoptosis reduces cells to small apoptotic bodies that are swiftly cleared
up from the scene by phagocytosis (Platt et al. 1998).

2.1.1 History
Apoptosis has been discovered and rediscovered several times. With the development of
the microscope and formulation of the cellular paradigm, the understanding of cells as the
basic biological building blocks rapidly grew. Early studies depicted proliferation of cells
taking place throughout development and in 1842 Vogt described for the first time that
cell death was present in toad development (reviewed in Clarke & Clarke 1996). In 1885,
nearly half a century later, Flemming discovered cell death in rabbit Graafian follicles
(Flemming 1885). This was the first recognition of cell death as a part of physiological
function. Later on Flemming further developed the theory and proposed that cell death
involved chemical changes within the cells. Two years later he also observed that
degeneration of testicular germ cell populations occurred through a similar mechanism
(Flemming 1887). Flemming used the term chromatolysis to describe his new
observations, and by the end of the 19th century, chromatolysis was widely accepted to
depict a distinct form of cell death that is called apoptosis today. These studies were
conducted over a century ago, and Flemming magnificently documented the
morphological features of apoptosis almost nine decades before the concept was
introduced. The importance of Flemmings findings was not understood at the time and
he himself strayed away from the study of cell death (reviewed in Clarke & Clarke 1996).
The concept of physiological cell death was kept alive by a small number of scientists,
mainly in the field of developmental biology. In 1914 the German anatomist Ludvig
Grper proposed a theorem that to compensate for mitosis there would also have to be
mechanisms to keep the continuing proliferation in check, referring to Flemmings
chromatolysis as a possible candidate (reviewed in Majno & Joris 1995). While the logic
behind Grpers idea seems obvious today, his paper on the issue was mainly ignored at
the time.
Almost sixty years later, apoptosis was rediscovered one more time by the Australian
pathologist, John Kerr. During his Ph.D. studies he observed a type of cell death in
hepatocytes that was distinguishable from necrosis and he named it shrinkage necrosis. In
1972, Kerr, Wyllie and Currie proposed the term apoptosis to describe a relatively
conserved set of morphological features observed in a wide variety of cell types during
physiological episodes of cell death (Kerr et al. 1972). The term apoptosis was suggested
by James Cormack, professor of classical Greek at the University of Aberdeen. It means
to fall away from (apo = from, ptosis = a fall), previously used to describe the falling of
leaves in the autumn. This paper, now considered as a landmark in the field of cell death,
described how developmental and homeostatic cell deaths controlled by the body could
be separated from classical, accidental cell death, necrosis. After these findings, another
research group published their observations where they found that radiation caused the
DNA in lymphocytes to break down into multiples of approximately 180 200 bp
(Yamada et al. 1981). Wyllie incorporated these findings and realised that the DNA ladder

16
formation was indeed a hallmark of apoptosis (Wyllie et al. 1984). The development of a
molecular mechanism to study apoptosis led to a rapid expansion of apoptosis research.
Since then a plethora of oncogenes and tumour suppressor genes have been found to have
a role in regulation of apoptosis, emphasizing the role of apoptosis in control of
homeostasis (reviewed in Cheng 1999). Furthermore, different transgenic and knockout
animal models have enabled detailed studies of apoptosis in a variety of physiological
functions.

2.1.2 Apoptosis vs. necrosis

Necrosis is a violent from of cell death that is caused by a range of noxious chemicals,
biological agents, or physical damage (Wyllie 1997b). It is associated with rupturing of
cellular membranes, swelling of the cells and random destruction of the cellular
structures. The cytosolic proteins of the dying cell are released into the intercellular
space, causing an inflammatory reaction (Wyllie 1997b). Necrosis typically involves large
groups of cells that have become victims of the same pathological assault (Arends &
Wyllie 1991) (Fig. 1).

Fig. 1. Two cell death pathways, necrosis and apoptosis. Necrosis involves breakdown of the
cellular membrane, which leads to leakage of intracellular proteins to the extracellular space
and subsequently, inflammation. Necrosis usually affects large groups of cells while apoptosis
typically involves single cells that undergo organised destruction of the cellular cytoskeleton
and formation of apoptotic bodies, which are phagocytosed without an inflammatory
reaction.

17

Apoptosis occurs in single cells that separate themselves from neighbouring cells
and/or the intracellular matrix (Kerr et al. 1972). Rather than swelling, an apoptotic cell
loses volume and shrinks. At the same time the cellular matrix is actively dismantled by
caspases. This is an energy-requiring, coordinated process opposite to necrosis, which
does not require energy after the initial pathological attack that sets the process in motion
(Wyllie 1997b). In the end, neighbouring cells or macrophages will take care of the
apoptotic bodies by phagocytosis, averting an inflammatory response (reviewed in Savill
& Fadok 2000) (Fig. 1).
However, as with most things in life, death also evades classification into two exact
categories. Apoptosis and necrosis are often viewed as endpoints on a sliding scale. Some
necrotic cell deaths display apoptotic properties such as chromosomal condensation or
DNA fragmentation, and sometimes a process that would be defined as apoptosis
possesses some qualities of necrosis (Majno & Joris 1995).

2.2 Common mechanisms of apoptosis

Apoptosis-regulating genes have been found in every metazoan organism. These genes
have proven to been exceptionally well conserved, throughout evolution (reviewed in Liu
& Hengartner 1999). While it has been thought that apoptosis is a mechanism that would
be confined to multicellular animals, surprisingly even some bacteria show hints of a
similar procedure to control homeostasis (Lewis 2000) (Fig. 2).

Fig. 2. Conserved cell death programme from worms to humans. Analogues of Ced genes that
control apoptosis in C. elegans have been found in humans. Death signals initiate the pathway
by directly inhibiting the actions of anti-apoptotic proteins (Ced-9 in C. elegans and Bcl-2 in
humans) or by activating factors that are capable of suppressing the actions of these proteins
(such as Egl-1 and BAD). Inhibition of Ced-9 or Bcl-2 leads to triggering of the next step in
the suicide programme. Subsequent activation of Ced-4 or Apaf-1 factors sets off the final
executors of apoptosis, Ced-3 or Caspases.

18
While a large number of important observations have been made in this field, perhaps
the greatest impact on the study of apoptosis has been the identification of the different
genes that encode the proteins which are responsible for initiation, processing and
execution of cell death. Much of what we know about the genetic basis of apoptosis in
mammalian cells has been derived from studies using the nematode Caenorhabditis
elegans, which has proven to be a Rosetta stone in deciphering the cell death programme.
In the C. elegans hermaphrodite, 131 of the 1090 somatic cells that are generated during
development undergo apoptosis. These deaths are dependent on the actions of at least
three key genes: ced-3, ced-4 and ced-9 (reviewed in Hengartner 1996). The cloning of
these three genes indicated the existence of homologues in other species, including
humans (Fig. 2).

2.2.1 Regulation of apoptosis

A wide variety of stimuli are capable of inducing apoptosis. Some are universal and can
produce apoptosis in almost any cell, while most apoptosis-inducing factors show some
selection of their targets (Rich et al. 2000). As a result of the profusion of apoptosisinducing mechanisms and the fact that virtually all eukaryotic cells can be induced to
undergo apoptosis, such a massacre has to be under tight lock and key. Apoptosisinducing signals are carefully processed and evaluated against anti-apoptotic factors in
the target cells. If the pro-apoptotic elements beat their counterparts, a dedicated death
program is then activated and the cell will undergo apoptosis (Fig. 3).

Fig. 3. Four stages leading to apoptosis according to Morita and Tilly (Morita & Tilly 1999.)
The first stage comprises of different potentially harmful stimuli that interact with a cell. In
the second stage, an early signalling molecule is activated. This signal is processed by a
regulatory mechanism, which evaluates the strength of the apoptosis inducing signal against
anti-apoptotic signals in the third stage. If the death inducers prevail the cell commits to
apoptosis and enters the fourth and final stage where specific executor proteins are
responsible for the organized destruction of the cell.

19
As apoptosis plays a part in the regulation of tissue homeostasis and a variety of other
physiological functions, it is controlled by physiological mechanisms. Death by neglect is
a classical example of this. Just as cells require extracellular growth factors and mitogens
to grow and divide, they also need survival factors to escape cell death. Failure to supply
adequate levels of survival factors leads to activation of apoptosis (reviewed in Raff 1992,
Conlon & Raff 1999). Furthermore, cells can also be driven into apoptosis by conflicting
signals that scramble the normal status of a cell (Wyllie 1997b.)
In normal physiological cell turnover the apoptotic stimuli can be represented by
cytokines, or death factors, such as Fas ligand (FasL) (reviewed in Nagata 1994) or TNFwhich is present in the ovary and endometrium (Tabibzadeh et al. 1999). Both of these
proteins belong to the same transmembrane protein subfamily that can be generated into
soluble forms by metalloproteinase-mediated cleavage (reviewed in Krammer 1999,
Schmitz et al. 2000).
In addition to physiological control mechanisms of apoptosis, a variety of pathological
insults can trigger apoptosis. Factors that are capable of causing DNA damage, such as
radiation, cytostatic drugs or genotoxic compounds, can also induce apoptosis (reviewed
in Wahl & Carr 2001, Bratton & Cohen 2001). DNA breaks are detected by transcription
factor p53, which is subsequently activated. Depending on the damage and cell type, p53
will either cause an arrest in the cell cycle or activate the apoptotic self-destruction
sequence (reviewed in Balint & Vousden 2001). The antimicroboid drug staurosporin
functions as a general kinase inhibitor and activates apoptosis via an unknown pathway
(Ojeda et al. 1995). Some chemical agents, such as hydrogen peroxide, can also trigger
the apoptotic pathway in several cell types (Madesh & Hajnoczky 2001, Gorman et al.
1997).

2.2.1.1 Death receptors

Death factors induce apoptosis through activation of specific death receptors that belong
to the growing superfamily of TNF/NGF receptors (reviewed in Schmitz et al. 2000).
These death receptors are characterized by a unique intracellular death domain (DD),
which is crucial for death ligand-induced apoptosis (Huang et al. 1996). The binding of
death ligand to its receptor leads to trimerization of the receptors. In concordance with
this, functional soluble forms of FasL and TNF- exist as trimers (Nagata 1994).
The trimerization of death receptors and subsequent association of three death
domains lead to the formation of a death-inducing signalling complex (DISC) which
leads into activation of pro-caspase-8 (Fig. 4). Activation of a death receptor can also lead
to generation of additional death signals such as ceramide (reviewed in Kronke 1999).
The message to induce apoptosis can be modulated directly at the death receptor level.
For example, glycosylation of Fas receptor has been shown to regulate the FasL-induced
apoptosis pathway (Peter et al. 1997, Keppler et al. 1999). In addition, death receptor
signalling can obviously be regulated on a transcriptional level. It has been observed that
activation of tumour suppressor p53 upregulates Fas expression (Muller et al. 1998).
Furthermore, a class of proteins named FLIPs (Thome et al. 1997) can block apoptosis by
directly interacting with the death receptor pathway (Hu et al. 1997, Bertin et al. 1997).

20

Fig. 4. Regulation of death receptor signalling. Activated Fas trimer recruits an adaptor
protein called FADD, through interaction with their respective death domains (DDs). FADD
functions as a bridge between Fas and downstream signal transduction, which is mediated by
the N-terminal region of the protein, termed death effector domain (DED). The TNFR1
mediated pathway utilizes TRADD protein in recruitment of FADD. The binding of procaspase-8 to the FAS/FADD or alternatively the TNFR1/TRADD/FADD complex activates
autoprocessing of the pro-enzyme to its active form. A mutated form of caspase-8, c-FLIP,
can also bind to FADD, thus inhibiting the binding and activation of caspase-8. Activated
caspase-8 is able to induce apoptosis through a mitochondrial pathway by cleaving BID, or
directly by activating downstream effector caspases. Mitogens and growth factors can
directly inhibit the activation of caspase-8 through an unknown pathway. Additionally,
activation of MAPK/ERK pathway can lead to phosphorylation, i.e. inactivation of BAD.
TNFR1 also utilizes an anti-apoptotic signalling pathway. TRAF2 can bind to
TNFR1/TRADD complexes and activate a pathway that leads to phosphorylation of I B and
consequently activation of transcription factor NF- B. The TNFR1/TRADD/TRAF2 complex
can also recruit a fourth protein, termed RIP, which possesses a serine-threonine kinase
domain with unknown function.

Mitogens and growth factors can also inhibit death receptor induced apoptosis.
Mitogen-activated protein kinase (MAPK) pathways can be activated by mitogens,
growth factors and environmental stress (Seger & Krebs 1995, Robinson & Cob 1997,
Lewis et al. 1998). The MAPK family consists of at least three different signalling
cascades: the ERK1/2, JNK and p38 kinase pathways. ERK1/2 has been shown to
directly inhibit death receptor-induced apoptosis by preventing caspase activation via
unknown mechanism (Holmstrm et al. 1998, 1999, Tran et al. 2001). Furthermore,
activation of the MAPK cascade can inactivate the pro-apoptotic Bcl-2 family member
BAD by phosphorylating BAD (Bonni et al. 1999, Scheid et al. 1999). Another

21
proliferation stimulating signalling cascade, PI-3K pathway, has also been shown to
inhibit apoptosis by phosphorylating BAD (Craddock et al. 1999, Wolf et al. 2001).

2.2.1.2 NF- B
NF- B is a transcription factor with a wide variety of functions ranging from
inflammatory reaction and development to cellular survival and oncogenesis (Chen et al.
2001). In their inactive forms NF- B proteins are bound to their inhibitor I B and
sequestered into the cytoplasm (Magnani et al. 2000). A response to different
extracellular signals leads to phosphorylation of I B, breakdown of I B/NF- B complexes
and subsequent translocation of NF- B dimer into the nucleus where it activates
transcription (Fig. 4).
There is growing evidence that the NF- B pathway is involved in regulation of
apoptosis. NF- B targets a variety of apoptosis regulating genes including those for TP53,
RB1, TNF- , TRAF-1 and TRAF-2 (Webster & Perkins 1999, reviewed in Pahl 1999, Foo
& Nolan 1999). Furthermore, NF- B may compete with p53 transcriptional activity,
providing a second potential mechanism for the NF- B to regulate the cell death program
(Ravi et al. 1998, Wadgaonkar et al. 1999). Thirdly, since NF- B can be activated by
TNFR pathway, it has been suspected that NF- B might be particularly involved in
interfering with TNF- -induced apoptosis.

2.2.1.3 Tumour suppressor p53

One of the main envisaged missions of apoptosis is deletion of potentially harmful cells
after DNA damage. Tumour suppressor p53 has a key role in DNA damage recognition,
DNA repair, cell cycle regulation and particularly in triggering apoptosis after genetic
injury (Robles & Harris 2001). The observation that p53 is at the crossroads of multiple
pathways of fundamental importance in the development of cancer partially explain why
its gene is the most commonly mutated one in human malignancies (reviewed in Sigal &
Rotter 2000). Furthermore, the gene encoding p53, TP53, is highly vulnerable to even a
single base change in the coding sequence and loss of a single allele of TP53 can result in
a reduction of p53 function (Sigal & Rotter 2000).
Although transcription of TP53 is regulated by a number of genes including NF- B
(Webster& Perkins 1999), the main mechanisms that govern p53 action are exerted at the
protein level (reviewed in Woods & Vousden 2001, Pluquet & Hainaut 2001). Mdm2 is a
protein that has a central role in suppressing p53 action by binding and targeting p53 for
degradation (Kussie et al. 1996, Kubbutat et al. 1997). The action of p53 induces Mdm2
expression, creating a negative feedback loop (Wu et al. 1993). Hyperproliferative signals
and activation of oncogenes induce stablization of p53 through p14ARF, which prevents
Mdm2-mediated degradation of p53 (Zhang et al. 1998, Stott et al. 1998, Honda &
Yasuda 1999). Other signals that can lead to stabilization and activation of p53 via a

22
variety of pathways include cytokines (Eizenberg et al. 1995), cytokine deprivation
(Canman et al. 1995), hypoxia and heat shock (Graeber et al. 1994), and most
importantly, DNA damage (Robles & Harris 2001). Activation of p53 induces expression
of p21WAF-1, which inhibits the function of cyclin-dependent kinases (Cdk) and
consequently induces cell-cycle arrest, providing more time for the cell to repair the
possible genetic damage before mitosis (Pluquet & Hainaut 2001). P53 also induces
transcription of a number of pro-apoptotic genes, including bax and fas (Miyashita &
Reed 1995, Owen-Schaub et al. 1995).
While the control of p53 stability is critical in the regulation of its function, its
transcriptional activity is regulated by post-translational modifications of the p53 protein
(reviewed in Woods & Vousden 2001, Pluquet & Hainaut 2001). The different patterns of
these modifications may determine p53 target genes, and explain how p53 selects
between different cellular responses, such as cell-cycle arrest and apoptosis.

2.2.1.4 GATA transcription factors

GATA transcription factors form a family of zinc finger proteins that participate in the
regulation of a large number of genes involved in differentiation and proliferation in a
variety of tissues (Molkentin 2000). GATA-binding proteins act according to their name
by binding to a consensus GATA motif, (A/T)GATA(A/G), in the promoter and enhancer
regions of target genes (reviewed in Orkin 1992, Yang & Evans 1995). GATA-1, the
founding member of the family, is a fundamental regulator of gene expression in
haematopoietic cell lineages (reviewed in Orkin 1992), where it has been reported to
protect these cells from apoptosis (Blobel & Orkin 1996, De Maria et al. 1999).
GATA-4 and GATA-6 transcription factors are expressed in mouse (Heikinheimo et al.
1997, Viger et al. 1998, Ketola et al. 1999) and human ovary and testis (Laitinen et al.
2000, Ketola et al. 2000). In addition, GATA-2 has also been recently associated with
ovarian development (Siggers et al. 2002). In mouse ovary, GATA-4 and GATA-6 have
overlapping expression patterns. GATA-4 is associated with follicular development and
its downregulation precedes ovulation and follicular atresia, i.e. granulosa cell apoptosis.
In contrast, GATA-6 expression is unaffected by ovulation or atresia and the corpus
luteum (CL) has abundant GATA-6 expression (Heikinheimo et al. 1997). GATA-4
expression is also seen in sex-cord-derived ovarian tumours, indicating a possible role in
suppressing apoptosis (Laitinen et al. 2000). Furthermore, pituitary gonadotrophins,
which are known to suppress ovarian apoptosis, induce GATA-4 expression in gonadal
tumour cell lines (Heikinheimo et al. 1997, Ketola et al. 2000). These observations
indicate a possible role for GATA-4 in regulation of granulosa cell apoptosis.

23

2.2.1.5 The Bcl-2 family

The mitochondrion is not just the cells powerhouse; it is also intimately involved in the
delicate network of apoptosis-regulating pathways. In this web, the mitochondrion is
positioned in the middle, where enhancing or silencing of apoptotic signals take place.
The key regulators that operate this cellular rheostat are members of Bcl-2 family.
Bcl-2 was discovered in B-cell neoplasms where chromosomal translocation
juxtaposed the location of the bcl-2 gene locus at chromosome segment 18q21 with the Ig
heavy chain locus at 14q32, resulting in overexpression of Bcl-2 (Tsujimoto et al. 1985,
Bakhshi et al. 1985, Cleary & Sklar 1985). It was soon realized that bcl-2 is a homologue
of the ced-9 gene, which has a key role in regulation of apoptosis in the nematode C.
elegans (Fig. 2). While C. elegans has only one ced-9-type gene, in humans the situation
proved to be different. Reports of the first two bcl-2 homologues bax and bcl-X were
published at the same time. Although Bax was found to be highly homologous to Bcl-2, it
proved to have an apoptosis-promoting action (Oltvai et al. 1993). Bcl-X was found to be
present in two splice variants that repress (Bcl-XL) or augment (Bcl-XS) cell death (Boise
et al. 1993). These observations made it evident that in humans a whole family of antiand pro-apoptotic proteins contributes to regulation of apoptosis. Today the Bcl-2 family
consists of at least 24 members (reviewed in Adams & Cory 1998, Chao & Korsmeyer
1998, Hsu & Hsueh 2000) (Fig. 5).

2.2.1.6 The mitochondrial forum

The mitochondrion seems to be the focus of the actions of the Bcl-2 family. It is also
the source of reactive oxygen species (ROS) that were initially suspected to be linked to
Bcl-2 and apoptotic cell death (Kane et al. 1993). Although ROS are hazardous for the
cell, the subsequent reports of apoptosis in the absence of oxygen suggested that they are
not essential for apoptosis (Shimizu et al. 1995, Jacobson & Raff 1995). The fog
surrounding mitochondria and apoptosis started clearing when the human Ced-4
homologue, apoptotic protease-activating factor-1 (Apaf-1), was identified (Zou et al.
1997). Surprisingly a factor initially termed Apaf-2 was recognized as cytochrome c,
which is also intimately involved in the mitochondrial respiratory chain (Liu et al. 1996).
The participation of cytochrome c in apoptosis was supported by the results of
experiments where injected cytochrome c induced caspase activation and apoptosis in
various cell types (Zhivotovsky et al. 1998). Although it has been observed that during
apoptosis many different mitochondrial proteins that might be potentially involved with
apoptosis are released into the cytoplasm, cytochrome c remains as the most probable key
inducer of apoptosis.

24

Fig. 5. The Bcl-2 family. The bcl-2 gene encodes a 2526 kDa protein that bears no obvious
structural clues to suggest the mechanism by which it controls apoptosis. It has a
hydrophobic transmembrane domain (TM) of 21 amino acids in its C-terminus that enables
the insertion of the protein into membranes. Bcl-2 also contains 4 conserved homology
regions, termed Bcl-2 homology domains 1, 2, 3 and 4 (BH1, BH2, BH3 and BH4). Most of the
Bcl-2 family members possess the TM region and variable amounts of BH regions. Through
interactions of these BH domains, members of the Bcl-2 family can form homo- and
heterodimers with each other and apparently titrate one anothers functions.

Three plausible models for the mechanism of how Bcl-2 family members regulate
cytochrome c release have been presented. First, Bcl-2 proteins have been shown to have
pore-forming capabilities (Muchmore et al. 1996) (Fig. 6). Following a conformational
change, they could form channels or even holes in the outer mitochondrial membrane
(Hengartner 2000). However, it is still unclear whether these channels would ever be big
enough for proteins to pass through. The second model suggests that Bcl-2 family
members may interact with other proteins on the mitochondrial membrane to form large
pore channels. A particular candidate for a such function is the voltage-dependent anion
channel (VDAC), as several Bcl-2 proteins can bind to it and regulate its activity
(Shimizu et al. 1999). However, the pore size of VDAC is not large enough for
cytochrome c to pass through (Shimizu et al. 1999) and this model must assume that
VDAC undergoes significant conformational change upon binding to Bcl-2 proteins. The
third model proposes that Bcl-2 members induce rupturing of the mitochondrial
membrane, which subsequently triggers caspase activation and apoptosis (Hengartner
2000).

25
Although it has been observed that deleting the TM domain renders both Bcl-2 and
Bax unable to complete their anti- or pro-apoptotic function (Tanaka et al. 1993, Zha et
al. 1996), some members of the Bcl-2 family, e.g. Bcl-XL/Bcl-2-associated death
promoter (BAD) and BID, do not possess a TM and their function does not seem to be
dependent on localization at the lipid membranes. This suggests that they exert their
function solely by dimerisation with other active members of the Bcl-2 family.

Fig. 6. Representation of possible interactions between anti-apoptotic (white) and proapoptotic (black) members of the Bcl-2 family at the outer mitochondrial membrane.
Apoptotic signals relocate Bax from the cytoplasm to the mitochondrion (1). Anti-apoptotic
members of the Bcl-2 family, such as Bcl-2 itself and Bcl-XL can block the pro-apoptotic
effects of Bax by binding it and forming heterodimers (2). However, other pro-apoptotic Bcl-2
proteins, e.g. BAD and BID, can interact with Bcl-2 and Bcl- XL and prevent their antiapoptotic function (3). Eventually, the relationship between pro-apoptotic and anti-apoptotic
factors determines the susceptibility to apoptosis. If there are more pro-apoptotic factors, the
mitochondrion subsequently loses its membrane potential and a number of apoptosispromoting molecules, such as cytochrome c and apoptosis-inducing factor (AIF) are released
into the cytoplasm (4).

2.2.1.7 The Apaf-1 complex or apoptosome

If the apoptotic-inducing fraction of Bcl-2 family outbalances their anti-apoptotic
relatives, an array of molecules is released from the mitochondrial compartment. The
principal actor in the event that follows is cytochrome c, which associates with apoptosis
protease-inducing factor-1 (Apaf-1) and procaspase-9 (Zou et al. 1997). Together these

26
three factors create a holoenzyme termed apoptosome, which is a key connection between
mitochondria and caspase activation (Fig. 7). The apoptosome proceeds to activate
caspase-3 and other death effector caspases that are required for the final stages of
apoptotic cell death (reviewed in Adrain & Martin 2001).
In addition to cytochrome c, the mitochondrion seems to contain redundant
mechanisms for induction of apoptosis. Apoptosis-inducing factor (AIF) is encoded by a
single gene located on the X chromosome (Susin et al. 1999). AIF protein is normally
contained in the mitochondria, but once dislocated from there to the cytoplasm, it induces
apoptosis via an as yet unknown pathway (reviewed by Daugas et al. 2000). However,
AIF-induced apoptosis appears to be independent of caspase activation and it cannot be
inhibited by Bcl-2 overexpression (Susin et al. 1999, Zamzami & Kroemer 1999).

2.2.2 Execution of apoptosis

Proteolytic enzymes, caspases, are responsible for the actual physical labour that is
involved in apoptosis. However, there are still fail-safe mechanisms that are designed to
keep caspases in check, and of course a counterforce to regulate this action.

2.2.2.1 Caspases
Most of the morphological changes in apoptotic cells that were observed by Kerr et al.
(1972) are the result of cleavage of cytoskeletal proteins and nuclear laminins by a family
of cysteine proteases, caspases, which are activated in cell death (Kothakota et al. 1997,
Rao et al. 1996, Buendia et al. 1999). Caspases have been conserved through evolution
and their homologues can be found in insects and nematodes (Miura et al. 1993). In
mammals these proteases form a large family that consists of at least fourteen members
(reviewed in Budihardjo et al. 1999). They all show a high degree of specificity, which is
important in apoptotic cell death as the process involves cleavage of a particular group of
proteins in a coordinated manner, rather than random proteolysis (reviewed in Grutter
2000). In most cases, caspase-mediated protein surgery results in inactivation of the target
proteins. However, caspases can also activate proteins by cleaving off an inhibitory
domain or inactivating a subunit that regulates enzyme activation.
Caspases are initially present as inactive zymogens, procaspases (reviewed in
Earnshaw et al. 1999). It is notable that procaspases themselves can be activated by
proteolysis. Consequently, once caspase activation is triggered, the effect can be
exponentially multiplied by processing of other procaspases to their active state. Thus the
most obvious way to activate a procaspase is to expose it to an activated caspase
molecule. This type of activation has been termed the caspase cascade, and it is used
extensively for activation of downstream effector caspases-3, -6 and -7. These three short
prodomain caspases are considered to be the workhorses of apoptotic cell death and they
are usually more abundant than their long prodomain cousins (Budihardjo et al. 1999).

27

Fig. 7. Activation of apoptosis through mitochondrial pathway. Extracellular signals can have
an effect on the relationship of Bcl-2 family members at the surface of mitochondria. Proapoptotic Bcl-2 proteins can release a variety of molecules from the mitochondrial
compartment. Cytochrome c is considered to be the primary mitochondrial factor in caspasemediated apoptosis. Together with Apaf-1 and procaspase-9, cytochrome c forms the
apoptosome, which is a potent activator of caspase-3. Smac/Diablo is a mitochondrial factor
that can inhibit the action of IAP proteins, which themselves can prevent caspase-3 activation
and action. AIF is also released from mitochondria and it can activate apoptosis via unknown,
caspase-independent pathway.

One of the end points in apoptosis is fragmentation of DNA into multiples of

approximately 180 bp (Wyllie et al. 1984). Recently, the enzyme responsible for this
action has been found, and it is now termed caspase-activated DNase (CAD) (Enari et al.
1998). CAD is found as an inactive complex, which is bound to an inhibitory subunit,
inhibitor of CAD (ICAD) (Sakahira et al. 1998). The finding that activation of CAD is
dependent on caspase-3-mediated cleavage of ICAD provides the final link between the
programmed cell death pathway and internucleosomal DNA cleavage. The active caspase3 molecule cleaves the inhibitory subunit, which then results in the release of the catalytic
enzyme. Subsequently, activated CAD proceeds with its intended mission to cleave the
genomic DNA.

28

2.2.2.2 Inhibitors of apoptosis proteins

When activation of one caspase can easily escalate into recruitment of the caspase
cascade and cell death, it is easy to imagine that there must exist pre-emptive measures to
prevent accidental cell death due to adventitious caspase activation (reviewed in Deveraux
& Reed 1999). A family of proteins named inhibitors-of-apoptosis (IAP), have the ability
to bind procaspases and activated caspases, blocking their processing and activity. IAPs
were first found in baculoviruses as proteins that suppress apoptosis, so allowing the virus
to replicate in infected cells (Crook et al. 1993, Birnbaum et al. 1994). They are
characterised by a novel domain of 70 amino acids, termed the baculoviral IAP repeat
(BIR) domain, which is essential for suppression of apoptosis (reviewed in Deveraux &
Reed 1999).
Interestingly, IAPs also have a controlling factor termed second mitochondria-derived
activator of caspases (Smac) or Diablo that can prevent IAPs from binding caspases,
allowing them to be activated and perform their part in apoptotic program (Verhagen et
al. 2000). Smac/Diablo is normally confined to the mitochondria, but once released, it
binds to IAPs and removes this block in the cell death pathway. Furthermore,
Smac/Diablo possesses an amino-terminal sequence that is capable of procaspase-3
activation (Chai et al. 2000).

2.2.3 Role of apoptosis in development

During the ontogeny of many organs, cells are over-produced only to be carved away.
Classical examples of apoptosis during development are found in the development of the
nervous and immune systems. Both rely on a three-phased design, which incorporates
proliferation, differentiation and cell death. The central nervous system assembles itself
according to a genetic programme, which does not contain a cellular map for placement
and connection for every neuron. Once the pieces (neurons) are in place, the set-up is
tested. Any orphan cells without adequate connections to neighbouring cells are removed
by the mechanism of apoptosis, because their survival depends on the availability of
neurotrophic factors secreted by the target cells they innervate (reviewed in Meier et al.
2000). It has been estimated that through this process more than 80% of the ganglion
cells in the cat retina and optic nerve die shortly after they are born (reviewed in Barres &
Raff 1999, Meier et al. 2000). In the immune system the wastage of cells is equally
profound (Debatin 2001).
A similar course of action takes place in many other places in the developing embryo.
Apoptosis seems to be an essential component for fusion of epithelial sheets that merge to
form the neural tube. If explanted chick embryos are treated with apoptosis inhibitors the
epithelial sheets still meet but fail to fuse to form the neural tube (Weil et al. 1997). In the
reproductive system apoptosis is responsible for removing the Wolffian duct in females
and the Mllerian duct in males (reviewed in Capel 2000). During gonadal development a
large number of germ cells is culled from the developing male and female gonads. Germ
cells migrate to the human ovary during the fifth and sixth weeks of development and

29
begin to multiply through mitotic divisions. In the testis, this process also continues in
postnatal life, renewing the germ cell reserve continuously, decreasing the effect of
apoptotic cell death in the male gonads. However, in the ovary mitotic divisions of germ
cells cease before birth and apoptotic death of the remaining oocytes has a profound
effect on the reproductive lifespan (reviewed in Morita & Tilly 1999).
Based on previous findings, it has been postulated that cell death is the default state
during the development of a metazoan organism, and that cells need to compete for
adequate survival factors (Raff 1992, Raff et al. 1993). This suggests that cells are as a
rule trapped in their specific microenvironments with sufficient levels of survival factors.
The observation that cells generally commit suicide when they are separated from their
neighbours or basal stroma support this notion (Zhang et al. 1995).

2.2.4 Apoptosis and tissue homeostasis

The maintenance of tissue homeostasis is finely tuned between cell proliferation and cell
death i.e. apoptosis. The maintenance of this balance is crucial to any multicellular
organism. Too much proliferation leads to hyperplasia and to anatomical and
physiological problems that are associated with it. The worst-case scenario is a total loss
of homeostatic control and development of cancer (reviewed in Lyons & Clarke 1997). If
apoptosis supersedes proliferation, the result is a reduction of the tissue mass. If the
process runs rampant, it eventually reaches a point where physiological function is no
longer possible (Thompson 1995). Apoptosis has been shown to function as a limiting
factor of tumour growth in early stages, when the angiogenesis is limiting the tumour
progression (Naik et al. 1996, O'Reilly et al. 1996). Furthermore, a tumours resistance to
chemotherapeutic agents has often been suggested to be dependent on expression of antiapoptotic genes, such as members of the Bcl-2 family, or loss of apoptosis-inducing
genes, such as TP53 (Minn et al. 1996, Lowe et al. 1993).
Tissues that have constant cellular proliferation, such as haematopoietic cells,
epithelium lining the intestinal crypts and male germ cells, also have a high rate of
apoptosis (Wyllie 1987, Billig et al. 1995). Similarly, tissues that have a minimal rate of
cell proliferation, such as the nervous system, heart, liver and kidney, exhibit only a very
little apoptosis (Benedetti et al. 1988). The ovary is an exception to this rule. While a
high level of granulosa cell proliferation is matched by a high rate of apoptosis, the
oocytes increase their number through mitosis only during the early fetal life, whereafter
the oocyte population is only reduced through the mechanism of apoptosis. Eventually,
the pool of resting follicles, i.e. oocytes, is depleted and menopause ensues (reviewed in
Morita & Tilly 1999).

30

2.2.5 Apoptosis in disease

A deep look into the molecular pathogenesis of human diseases has revealed an apoptotic
component that either contributes to disease progression or is responsible for the process
in a growing number of pathological conditions. While apoptosis is an essential building
block of physiological events, it seems to have an equally important function in
pathological events. Autoimmune disorders are often associated with erroneously
activated apoptosis in the target tissue (Greer et al. 2001). Ischaemic injury can trigger
apoptosis in vascular tissue or in the surrounding nerve cells in the central nervous system
(reviewed in Johnston et al. 2001). In graft versus host disease apoptosis plays a role in
the disease process (reviewed in French & Tschopp 2000). In many neurodegenerative
diseases, such as Alzheimers disease, Parkinsons disease, Huntingtons disease,
Charcot-Marie-Tooth type 1A demyelinating neuropathy and spinal muscular atrophy, an
apoptotic component that significantly contributes to the pathogenesis of the disease has
been found (Mazarakis et al. 1997).
Many pathogens have discovered a way to circumvent their hosts defences by
manipulating apoptotic inducing factors. Many viruses encode genes that prevent
apoptotic destruction of the host cell (reviewed in White 2001). On the other hand, HIVinfected lymphocytes can activate apoptosis by expressing Fas ligand (reviewed in
Kaplan and Sieg 1998). During Ebola virus infection, massive apoptosis is observed in
the cells of the vascular endothelium (Baize et al. 2000). Some parasites, such as
Trypanoma cruzi can induce cell death in T cells, which consequently inhibits the
macrophage-directed killing of parasites (Freire-de-Lima et al. 2000).
The universality of the genetic programme controlling apoptosis has helped to rapidly
analyse the regulation of apoptosis in different tissues and diseases. While there are high
expectations about possible therapeutics that might be targeted against apoptosisregulating factors, the ubiquitous nature of the event also sets limitations to the possible
measures.

2.2.6 Studying apoptosis

Apoptosis has many distinctive features that give away its presence to a trained eye.
However, the apoptotic bodies are ultimately swallowed by neighbouring cells or
macrophages (reviewed in Savill & Fadok 2000). Although the exact speed of the process
depends on the cell type, in most case the whole episode is over within a few hours
(reviewed by Wyllie 1997b, Cummings et al. 1997). Morphological changes of the
apoptotic cells, including condensation of chromatin and cytoplasm, fragmentation of the
cell and apoptotic body formation, can be detected by using light microscopy (Kerr et al.
1972). While it has been argued that the method can be as sensitive as biochemical
methods, it is highly dependent on the observer. Furthermore, careful inspection of
histological samples to detect these changes is very time consuming.
Electron microscopy can also be used in detecting apoptosis. In some respects it has
been considered as the most convincing method for accurate identification of apoptosis

31
(Kerr et al. 1994). However, for obvious reasons electron microscopy is the least feasible
method for analysis of clinical samples.

2.2.6.1 DNA analysis

Breakdown of genomic DNA into multiples of approximately 180 bp is considered to be a
hallmark of apoptosis (Wyllie et al. 1984). This cleavage of chromosomes produces a
large number of DNA breaks, and subsequently a simultaneous amount of new 3-OH
DNA ends. In normal living cells, only an insignificant number of 3-ends are present,
making this a promising target for detection of apoptosis. The enzyme terminal
deoxynucleotidyl transferase (TdT) has the capability to incorporate individual
deoxyribonucleotide triphosphates to the 3-end of double- or single-stranded DNA. This
quality can be used to detect 3-ends with nucleotides that have been labelled with
radioactive, fluorescent or digoxigenin labels. Apoptosis can then be measured
quantitatively by using gel electrophoresis, where apoptotic DNA is organized into a
typical ladder pattern of multiples of 180 bp. In situ labelling of 3-ends can be used to
qualitatively recognise apoptotic cells in immuhistochemical tissue sections.

2.3 Ovarian function

2.3.1 Ovarian development

It is thought that in the human embryo, primordial germ cells originate from embryonic
ectoderm (Buehr 1997). They migrate from their original location and during the fourth
week of development, germ cells are visible in the wall of the yolk sac. Shortly thereafter,
germ cells migrate one final time to the developing gonad where they arrive during the
fifth or sixth week after fertilization (Sadler 1990). The germ cells express a tyrosine
kinase receptor, c-kit, on their surface (Manova & Bachvarova 1991). Its ligand, stem cell
factor or kit ligand, is expressed by the cells along the germ cell migratory pathway
(Matsui et al. 1990). The interaction between c-kit and its ligand seems to be fundamental
for germ cell migration. Today it is known that in mice c-kit and kit ligand are encoded
by white spotting and Steel loci respectively. White spotting or Steel mutant mice have
ovaries with very few, if any, germ cells (Coulombre & Russel 1954, Bennet 1956, Mintz
& Russel 1957). In addition, other genetic factors are known to be essential for gonadal
development (Table 1). Wilms tumour gene, WT-1, is essential for ovarian development.
Without WT-1 action, germ cells migrate normally, but the urogenital ridge fails to
develop, leading to gonadal and kidney agenesis (Kreidberg et al. 1993). It is well known
that the male differentiation pathway is triggered by Y chromosome-encoded testisdetermining factor (SRY) (Gubbay et al. 1990, Koopman et al. 1991). Recent
observations demonstrate that the female developmental pathway requires active

32
participation of the Wnt-4 signalling pathway. In wnt-4 deficient mice the Mllerian ducts
are absent and the females are masculinized (Vainio et al. 1999).
The human ovary begins to differentiate morphologically during the 1112th weeks of
development. Before this time the germ cells have already started increasing their number
through mitotic cell divisions (reviewed by Peters 1970, Hirshfield 1991). Already at this
age, large numbers of germ cells are culled from the developing ovary. This process is
termed germ cell attrition and it is probable that this process continues in the ovary until
menopause (reviewed by Kaipia & Hsueh 1997, Morita & Tilly 1999). Evidence from
rodent ovaries suggests that this degeneration in the fetal gonad occurs via apoptosis
(Coucouvanis et al. 1993). This phenomenon seems to be universal, since all vertebrate
species that have been examined to date are born with much fever oocytes than their
maximum number during early development (Beaumont & Mandl 1961, Borum 1961,
Baker 1963, Forabosco et al. 1991).
Table 1. Mouse models of ovarian failure.
Transgenic/mutant mouse

Ovarian phenotype

Reference

c-kit deficiency

Loss of germ cells due to defect in migration

(Coulombre & Russel 1954,

and proliferation
Kit ligand deficiency

Loss of germ cells due to defect in migration

Mintz & Russel 1957)

(Bennet 1956)

and proliferation
Zfx knockout

Loss of germ cells due to defect in

(Luoh et al. 1997)

proliferation
Atm knockout

Loss of germ cells

(Barlow et al. 1996)

Dazla knockout

Loss of germ cells

(Ruggiu et al. 1997)

WT-1 knockout

Failure of gonadal development

(Kreidberg et al. 1993)

GDF-9 knockout

Arrest of folliculogenesis at primary stage

(Dong et al. 1996)

IGF-I knockout

Arrest of folliculogenesis before antral

(Baker et al. 1996)

follicle stage
FSH- subunit knockout

Arrest of folliculogenesis at preantral stage

(Kumar et al. 1997)

ER knockout

Infertility due to failure to ovulate

(Lubahn et al. 1993)

Wnt-4 knockout

Lowered germ cell number, masuclinization

(Vainio et al. 1999)

After the 10th week, epithelial precursors of human granulosa cells begin to form the
very first follicular structures. Initially, the pre-granulosa cells arrange themselves in
structures that encircle several oogonia. This loose net starts to tighten and granulosa cells
envelop most oocytes by the 24th week and all at birth. The number of oogonia increases
steadily, until by means of an unknown signal they begin to enter meiosis (Sadler 1990).
The initiation of meiosis denotes the transformation of the oogonia into an oocyte. This
process starts at around week 15 and the number of oocytes reaches its maximum of 7
million around the 20th week (Baker 1971). The meiosis of oocytes is arrested at
prophase of the first meiotic division. Meiotic division is completed just prior to
ovulation, and in humans this arrest can be 50 years long (Mira 1998). When entering
meiosis oocytes lose their ability to increase their numbers through mitosis. This inclines
the balance in favour of germ cell attrition and from this moment onwards, the number of
oocytes begins to inevitably decline (Baker 1963). Unlike spermatogenesis in males,

33
where the spermatogonia act as stem cells and constantly divide to produce gametes, the
ovary has a finite supply of oocytes that is determined at birth (reviewed in Morita &
Tilly 1999).
At birth, all oocytes are surrounded by a single layer of flat granulosa cells, which
together with the oocyte constitute the primordial follicle. The number of primordial
follicles has been estimated to be anywhere from 266,000 to 2,000,000 at the time of
birth (Block 1953, Baker 1971, Forabosco et al. 1991, Gougeon et al. 1994). These
follicles comprise the pool of resting follicles that is the basic factor in determining
postnatal ovarian life span (Gougeon 1996).

Fig. 8. Germ cell attrition and follicular atresia according to Kaipia & Hsueh 1997. Germ
cells migrate to the ovary during early embryonic development. Their number increases
through mitotic divisions but most of the oocytes formed during development do not survive
to the time of birth.

2.3.1.1 Postnatal trends

After birth the attrition of oocytes continues, although the process is attenuated.
Throughout childhood the ovary remains endocrinologically inactive, but little by little
the number of primordial follicles declines. It has been estimated that at the onset of
puberty there are at maximum 200,000 resting follicles left in the human ovary (Baker
1963, Faddy et al. 1992). In the fertile ovary, a resting follicle has two future options, it
can either undergo atresia or it can be recruited into the pool of growing follicles. While
atresia represents certain death, the fate of growing follicles is only slightly better.
Ultimately, during every menstrual cycle only one follicle survives the process, reaches
full maturation and ovulates, and only approximately 400 oocytes from the original 7

34
million will ovulate during the fertile life span (Gougeon 1996). In the end, when all the
follicles have been depleted, fertile senescence, i.e. menopause, ensues (Fig. 8).

2.3.2 The adult ovary

Throughout the entire female reproductive lifespan, the ovary relies on the reserve of
resting follicles. A small number of follicles continuously leave the resting follicle pool
and start growing. The early stages of folliculogenesis proceed very slowly. While the
exact time for the follicles to attain the preantral stage is unknown, it has been estimated
that in humans the process can take over 300 days. One of the reasons for this is the long
doubling time of granulosa cells (~250 hr) (Erickson 2000). After recruitment the follicle
begins to increase in size, both by proliferation of granulosa cells and by growth of the
oocyte. This process is at least partially guided by a gene for a factor termed growth
differentiation factor 9 (GDF-9). GDF-9 is a member of the transforming growth factor
(TGF ) superfamily that includes TGFs, bone morphogenetic proteins (BMPs), MIS,
activins and inhibin. GDF-9-deficient mice show a block in follicular development at the
one layer follicle stage (Dong et al. 1996). The observation that GDF-9 is also expressed
in human oocytes during early folliculogenesis suggests that the gene also plays a major
role in the human ovary (Aaltonen et al. 1999). It has also been proposed that proper
interaction between c-kit and kit ligand is required for this process.

Fig. 9. Folliculogenesis and classification of growing follicles in the human ovary according to
Gougeon (1996). Growing follicles enter class 2 usually in the late luteal phase, class 3
between late luteal and early follicular phases, class 4 during late follicular phase and become
recruitable class 5 follicles during late luteal phase.

35
When the follicles reach the size where they contain three to six layers of granulosa
cells, the surrounding connective tissue stratifies and differentiates into two parts. The
outer part, the theca externa, is basically similar to the stromal tissue surrounding it. In
the inner part, the theca interna, stromal precursor cells differentiate into epithelioid cells.
At this stage, the follicle is defined as a preantral follicle. Already at this stage, a
considerable proportion of growing follicles fail to survive (Fig. 9), and they degenerate
through a process termed follicular atresia. Observations in humans and in animals
suggest that apoptosis is the mechanism of follicular atresia (Kaipia & Hsueh 1997).
After the primary follicle stage, gonadotrophins especially FSH, are increasingly
important in sustaining follicular growth (reviewed by Hirshfield 1991, Zeleznik 2001,
Adashi 1994).

2.3.2.1 Hormonal regulation of follicular growth

After the initial stages, follicular growth and development are brought about by the
combined action of FSH and LH on the follicular cells. FSH and LH bind to their specific
receptors on the surface of granulosa and theca interstitial cells, respectively. The
activation of FSHR and LHR stimulates mitosis and differentiation responses in
granulosa and theca interna cells (Gougeon 1996). In addition, gonadotrophins have two
major endocrine effects. The first is that FSH and LH action stimulate the production of
estradiol specifically in the dominant follicle (Erickson 2000). According to the two-celltwo-gonadotrophin theory, both gonadotrophins and both granulosa and theca interna
cells have a specific task in this process (Fig. 10). Another endocrine response is the
marked increase in production of inhibin by FSH (Groome et al. 1994).

Fig. 10. Two-cell-two-gonadotrophin theory. LH induces androstenedione synthesis in theca

cells. Driven by the FSH stimulus, granulosa cells process androstenedione into estrone which
is further converted into estradiol by type 1 17HSD enzyme.

36
After the preantral stage, follicular fluid begins to accumulate in the follicle,
expanding it relatively rapidly (Fig. 9). The dominant follicle is selected from a cohort of
stage 5 follicles (Fig. 9) and eventually, the whole hormone-dependent stage of follicular
growth can take approximately 4050 days to complete (Gougeon 1996). Only one
follicle is selected at a time, and the fate of all remaining growing follicles is atresia. The
first evidence that apoptosis is responsible for this process was gathered from work on
rodents (Tilly et al. 1991, Tilly et al. 1992). Similarly, it was discovered that the process
is hormonally controlled. Estrogens and gonadotrophins inhibit and androgens induce
ovarian cell death (Chun et al. 1994, Billig et al. 1993, Yuan & Giudice 1997).
It has been estimated that up to 30 years of age, the loss of follicles (and oocytes) in
the human ovary is mainly due to atresia of resting follicles. After reaching a critical
threshold in the number of follicles (estimated at 25,000), the recruitment of growing
follicles is increased twofold and thereafter the loss of follicles is mainly due to atresia of
growing follicles (Gougeon 1996, Erickson 2000).

2.3.2.2 Regulation of ovarian steroidogenesis

According to the two-cell-two-gonadotrophin theory, granulosa and theca interna cells as
well as both gonadotrophins are needed for proper ovarian steroidogenesis (Fig. 10).
Driven by LH stimulation theca cells produce androgens de novo. Aromatisation of
androgens to estrogens takes place in neighbouring granulosa cells mainly under the
influence of FSH (Gougeon 1996).
During the luteal phase the CL produces progesterone and estrogens. The
physiological activities of estrogens are regulated by 17 -hydroxysteroid dehydrogenases
(17HSDs). Type 1 17HSD is responsible for the conversion of the low activity sex steroid,
estrone (E1) to the more potent form, estradiol (E2), while the type 2 enzyme catalyses the
opposite reaction (Peltoketo et al. 1999). In human ovary 17HSD type 1 is expressed in
granulosa cells (Ghersevich et al. 1994) and in the CL (Sawetawan et al. 1994), while the
presence of the type 2 enzyme has not been reported. However, the role of these enzymes
in the CL throughout the luteal phase is uncertain.

2.3.2.3 The role of FSH in the female reproduction

Recently discovered FSH and FSHR mutations in humans have markedly expanded
our knowledge about the role of FSH in reproduction (Aittomki et al. 1995, reviewed in
Themmen & Huhtaniemi 2000). FSH is an absolute requirement for proper ovarian
function in women. Women homozygous for an inactivating mutation of the FSHR gene
show hypergonadotrophic ovarian dysgenesis, amenorrhoea and infertility (Aittomki et
al. 1996). Furthermore, ovarian biopsy samples from homozygous women show a high
number of primordial and primary follicles but only occasional signs of further follicular

37
development (Aittomki et al. 1996). However, male homozygotes for the FSHR
mutation are only subfertile, with a lowered sperm count and testicular size, suggesting
that FSH is not an absolute requirement for initiation of spermatogenesis in puberty
(Tapanainen et al. 1997). Similar observations have been made in mice lacking the FSHgene (Kumar et al. 1997).

2.3.2.4 The corpus luteum

After ovulation, the dominant follicle collapses and formation of the corpus luteum (CL)
proceeds. The principal function of the CL is to produce progesterone to prepare the
endometrium for implantation. During normal menstrual cycles this process is driven by
pituitary LH. In the case of pregnancy, the trophoblasts of the early embryo start
producing hCG for maintenance of the CL until the luteo-placental shift is completed
(Niswender et al. 2000). If fertilisation does not occur, CL regression has to take place to
allow follicular development and ovulation in the next ovarian cycle. This gives a life
span of 1216 days for the CL during normal menstrual cycles.

2.3.3 Regulation of ovarian apoptosis

Apoptosis has been found to occur in three different ovarian cell types: oocytes,
granulosa cells and luteal cells. Animal studies have indicated that oocyte apoptosis is
most profound during embryonal development (reviewed in Morita & Tilly 1999). The
peak number of oocytes in mouse ovaries is reached on day 13.5 (E13.5) of gestation,
when some of the oocytes have already abandoned the mitotic cell cycle and entered the
first phases of meiotic divisions (Byskov 1986). Apoptosis has been confirmed to be the
mechanism of germ cell atresia in animals (Pesce & De Felici 1994, Ratts et al. 1995,
Morita et al. 1999). However, no reports confirming these results in humans have been
published. Furthermore, while oocyte apoptosis has clearly been established to affect
neonatal ovarian development in several species, it is not clear what physiological role
oocyte apoptosis plays in adult life. In vitro and in vivo experiments have shown that
DNA damaging agents, such as cyclic polyaromatic hydrocarbons (PAHs), and cytostatics
induce oocyte apoptosis in adult mice (Perez et al. 1997a, Matikainen et al. 2001a).
Based on these results, it is clear that oocytes of the adult ovary have retained their ability
to undergo apoptosis under a pathological insult.
Transgenic and knockout animal models have shed some light on the issue of how
oocyte cell death is regulated (Table 2). Bcl-2 knockout mice have been found to have
significantly fewer oocytes than similar age-matched wild type animals (Ratts et al.
1995). Bcl-2 can also be detected in E12.5 and E15.5 wild type mouse oocytes (Felici et
al. 1999). Another anti-apoptotic Bcl-2 family member, Bcl-XL, also seems to play a role
in determining germ cell fate. Although Bcl-XL knockout mice die at a very early stage of
fetal development, there is a clear loss of germ cells at E11.5 compared with wild type

38
animals, and this loss can be prevented in vitro by transfection with the Bcl-XL gene
(Watanabe et al. 1997). Its role in oocyte cell death is supported by the finding that the
gene is expressed in murine oocytes (Jurisicova et al. 1998). Mice deficient in the proapoptotic Bcl-2 family member Bax showed a normal number of oocytes at birth, but at
the onset of puberty, the follicle pool was threefold in size when compared with agematched wild type mice (Perez et al. 1999).
During folliculogenesis, apoptosis has been mainly observed in the granulosa cells of
the growing ovaries (Yuan & Giudice 1997). This process seems to be principally
controlled by hormones. Testosterone induces and estradiol inhibits granulosa cell
apoptosis in rodent ovaries (Chun et al. 1994). Gonadotrophins have also been shown to
be survival factors for human granulosa cells in vitro (Chun et al. 1994, Jablonka-Shariff
et al. 1996). Furthermore, the high androgen/estrogen ratio in the follicular fluid seems to
predispose human granulosa cells to cell death (Yuan & Giudice 1997). Granulosa cell
apoptosis is mediated by the Bcl-2 family of cell death-regulating factors (Tilly et al.
1995a, reviewed in Hsu & Hsueh 2000). Apaf-1 is also expressed in granulosa cells and it
is activated by Bcl-2 family-mediated release of cytochrome c (Robles et al. 1999). In
addition, the tumour suppressor genes TP53 and WT1 might have a role in regulating
granulosa cell apoptosis in rodents (Tilly et al. 1995b) and in humans (Makrigiannakis et
al. 2000).
There is accumulating evidence that apoptosis plays a part in CL regression in animals
(Bruce et al. 2001, Zheng et al. 1994) and in humans (Shikone et al. 1996, Sugino et al.
2000). Recent observations also reflect the expression of apoptosis-regulating members
of the Bcl-2 family, Bcl-2, Bcl-X and Bax, in the human CL (Rodger et al. 1995, Rodger
et al. 1998, Sugino et al. 2000). Furthermore, experiments with cultured granulosa luteal
cells suggest that caspases 3 and 9 are activated in staurosporin-induced apoptosis in
luteinized human granulosa cells (Khan et al. 2000).

Table 2. Transgenic and knockout animal models for abnormal ovarian cell death.
Transgenic/mutant mouse

Ovarian phenotype

Reference

Bcl-2 knockout

Loss of oocytes

(Ratts et al. 1995)

Bcl-2 transgene

Loss of ovarian apoptosis, enhanced

(Hsu et al. 1996)

folliculogenesis, teratomas
Bcl-XL knockout

Loss of germ cells

(Watanabe et al. 1997)

Bax knockout

Normal at birth, increased number of oocytes

(Perez et al. 1999)

Caspase-2 knockout

Increased number of primary follicles

(Bergeron et al. 1998)

Caspase-3 knockout

Normal number of primary follicles

(Matikainen et al. 2001b)

at puberty and during fertile life

39

2.4 Endometrial function

2.4.1 Uterine development

The Mllerian duct, the antecedent of the uterus, arises from coelomic mesothelium in
close association with the Wolffian duct. The uterus develops as a muscular expansion of
the Mllerian duct together with the fallopian tubules and the upper portion of the vagina,
which are also Mllerian derivatives (Healy & Hodgen 1983). As stated earlier, the
formation of the Mllerian duct is dependent on Wnt-4 action, and in Wnt-4 knockout
mice the Mllerian duct is absent (Vainio et al. 1999).
The uterus can be divided into three major functional elements. The first one is the
endometrium, a glandular membrane that lines the uterine cavity. Its main purpose is the
implantation and nourishment of the early embryo. The second component, the
myometrium is the smooth muscle body of the uterus located under the endometrium.
The third component, the cervix, opens to the vagina and contains glands that supply
mucus to the vagina. The endometrium itself consists of three independent structural
components the glands, stroma and blood vessels. In adult life, all three layers are under
cyclical regulation of ovarian steroids.

2.4.2 Regulation of endometrial function during the menstrual cycle

The endometrium undergoes cyclic phases of proliferation. To assure optimal conditions
for implantation the endometrium is swiftly discarded and regenerated if fertilization
does not occur (Lockwood et al. 1997) (Fig. 11).
Regeneration of the endometrium ensues immediately after menstruation. Significant
stromal proliferation is observed throughout the follicular phase, which persists until
ovulation. Follicular phase proliferation is characterised by upregulation of estrogen and
progesterone receptors in glandular epithelium and in the stroma. Apoptosis has been
morphologically (Hopwood & Levison 1976) and biochemically (Tao et al. 1997)
detected in human endometrium. Expression of the regulating factors Bcl-2 and Bax, has
also been observed (Otsuki et al. 1994, Koh et al. 1995, Tao et al. 1997, Tao et al. 1998).
Animal studies indicate the participation of ovarian steroids in the control of endometrial
apoptosis, since removal of steroid action leads to increased endometrial cell death (Pecci
et al. 1997).

40

Fig. 11. Changes in the endometrial tissue and hormone values during the menstrual cycle.

2.4.3 Endometrial cancer and hyperplasia

Endometrial carcinoma is one of the most frequently found carcinomas of the female
reproductive system (Purdie & Green 2001). It has been proposed that the path to
development of endometrial carcinoma goes through pre-malignant stages of endometrial
hyperplasia (Czernobilsky & Lifschitz-Mercer 1997). Endometrial hyperplasia is thought
to arise from an unopposed estrogen influence that drives endometrial cell proliferation,
while an alternative pathway, utilising improper function of the tumour suppressor p53,
has also been suggested (Kounelis et al. 2000). Endometrial hyperplasias are divided into
four subcategories: 1) Simplex type hyperplasia that includes cystic glandular
hyperplasia, 2) simplex type hyperplasia with cellular atypia (this subtype is
exceptionally infrequent), 3) complex type hyperplasia without atypia, and 4) complex
type hyperplasia with atypia. Hyperplasias without cellular atypia very seldom develop
into cancer, i.e. in less than 2% of cases, while complex hyperplasia with atypia leads to
cancer diagnosis in 23% of the patients (WHO 1994, Kurman & Norris 1982).
According to World Health Organization, endometrial carcinomas can be divided into
three grades. Grade I carcinoma consists of well differentiated carcinoma, grade II

41
represents moderately differentiated disease and grade III carcinoma comprises poorly
differentiated cancer (WHO 1994).

3 Aims of the study

When the present study was started, evidence concerning the crucial role of apoptosis in
the regulation of reproductive systems in animal models was accumulating. Therefore, we
decided to elucidate the role of apoptosis in the human female reproductive system (i.e
ovary and endometrium), which was virtually not studied at all, and to determine the
factors that control this process.
The specific aims were to study:
1. Apoptosis and its regulation in human fetal and adult ovary
2. The role of apoptosis during follicular development and corpus luteum regression
3. The role of apoptosis in the endometrium during normal endometrial cycles and in
endometrial hyperplasias and cancer.

4 Materials and methods

This study was approved by the University of Oulu, University of Helsinki and University
of Ume Ethics Committees. A summary of the material and the experimental procedures
used is presented in Table 3.

4.1 Tissue samples

4.1.1 Fetal and neonatal ovaries

Ovarian tissue was obtained from 8 fetuses (fetal age 1322 and 33 weeks) and 6
neonates (fetal age 2339 weeks at birth and died within 48 h after birth) after
spontaneous and therapeutic abortions because of maternal disease. All subjects had a
normal karyotype. Fetal samples with detectable autolysis were excluded from the study.
All of the ovarian samples were fixed in 4% buffered formaldehyde for 24 hours and
embedded in paraffin. Ovaries from three fetuses were snap-frozen in liquid nitrogen and
stored at -70 C for RNA extraction and Northern blot analysis.

4.1.2 Adult ovaries

Adult normal ovarian tissue was obtained from patients (aged 2245) undergoing
ovariectomy as a result of uterine myomas (n = 4) or focal endometrial cancer (n = 8,
grades I and II).

44

Table 3. Materials and methods of the studies.

Study
I

Material

Methods used

Fetal ovaries

In situ 3-end labelling;

Adult ovaries

Bcl-2 and Bax IHC;

GATA-4 ISH, IHC and Northern blotting

II

III

Ovarian biopsies

from patients

In situ 3-end labelling;

homozygous for C566T mutation of the

Aromatase P450 IHC;

FSHR

GATA-4 IHC;

Adult ovaries

rFSH treatment;

Fetal ovaries

Hormone assays

Corpus luteum

In situ 3-end labelling

Cultured granulosa-luteal cells

DNA; fragmentation analysis;

Bcl-2 and Bax ISH and IHC;
TNF- and NF- B IHC;
17HSD type 1 and 2 ISH

IV

Endometrial biopsies

In situ 3-end labelling

Uterine sections from normal cycling

DNA; fragmentation analysis;

endometrium

Bcl-2 and Bax IHC and Western blotting;

Ki-67 IHC;
Hormone assays

Uterine sections from normal cycling

In situ 3-end labelling;

endometrium, endometrial hyperplasia and

Bcl-2 and Bax ISH, IHC;

endometrial carcinoma

TNF- , NF- B and Caspase-3 IHC

IHC = immunohistochemistry
ISH = in situ hybridisation

4.1.3 Patients with inactivating FSHR mutation

Ovarian biopsies from four women were obtained at the time of primary diagnosis of
ovarian dysgenesis. Later on they were confirmed to be homozygotes for the inactivating
C566T FSHR mutation.

45

4.1.4 The corpus luteum

Human corpora lutea were obtained from twenty-nine patients (age 2543) undergoing
surgery due to benign reasons (uterine myoma or fibroma) at the Department of
Obstetrics and Gynaecology, Ume University Hospital. The patients were otherwise
healthy and had not received any hormonal therapy during the preceding 6 months. All
patients had proven fertility and had a history of regular menstrual cycles ranging
between 24 and 30 days. The age of the CL was determined in days, where day 1
(ovulatory day) was defined as the first day after a positive urinary LH test results
(Clearplan One Step, Unipath Ltd., Bedford, UK). Patients were scheduled for surgery in
the early (days 25), mid- (days 610) or late (days 1114) luteal phase. Immediately
after laparotomy, the CL was extirpated and the tissue was immersion-fixed in Bouins
solution for 4 hours, embedded in paraffin and used later for immunohistochemical
studies and for 17 HSD type 1 and 2 in situ hybridisation.
Corpora lutea for Bcl-2 and Bax in situ hybridisation analysis were obtained from five
premenopausal patients (aged 2245) undergoing ovariectomy as a result of local
endometrial cancer. Tissues were fixed in 4% buffered formaldehyde for 24 hours and
embedded in paraffin.

4.1.5 Cycling endometrium

Endometrial biopsies were collected from 39 healthy, premenopausal women undergoing
laparoscopic sterilisation. To confirm spatial localisation of findings in the curettage
samples, uterine sections were obtained from 9 premenopausal women who underwent
hysterectomy as a result of benign conditions: myomas, endometriosis and ovarian cysts.
The cycle phase was determined histologically and subjects with abnormal serum
estradiol or progesterone concentrations were excluded from the study. For
immunohistochemical and in situ analysis, endometrial tissue was fixed in 4% buffered
formaldehyde for 24 hours and embedded in paraffin. For electrophoretic DNA
fragmentation and Western blot analysis, endometrial tissue was snap-frozen in liquid
nitrogen and stored at -70 C.

4.1.6 Endometrial hyperplasias and carcinomas

A total of 54 hysterectomy specimens that included normal endometrium, endometrial
hyperplasia or adenocarcinoma were examined. The age of the subjects varied from 25 to
77 years (mean 56 years). The uterine tissue was examined and suitable material was
investigated by using routine histopathological methods. All specimens were fixed with
formaldehyde for 24 h and embedded in paraffin for in situ 3-end labelling, in situ
hybridisation and immunohistochemical analysis. Histopathological classification was
carried out using haematoxylin-eosin-stained specimens and standard criteria, and the

46
specimens were classified into atrophic and proliferating endometrium, simplex, complex
and atypical hyperplasia and grade I, II and III adenocarcinoma.

4.2 Cell culture

Human luteinized granulosa cells were obtained from 12 patients undergoing in vitro
fertilization (IVF). The patients were stimulated using a routine long GnRH agonist
protocol, and after transvaginal puncture of follicles, oocytes were collected and the
remaining granulosa cells were washed twice with phosphate-buffered saline (PBS). After
centrifugation at 250 x g for 3 min, the granulosa cells were snap-frozen and stored at -70
C for electrophoretic DNA fragmentation analysis. For in situ 3-end labelling of
apoptotic cells, cells were placed in 2-well flat bottom culture plates (Becton Dickinson),
suspended in culture medium (Modified Eagles Medium) and incubated for 1, 2, 4 or 8
hours. Before analysis, the cells were fixed with 4% neutral buffered formalin.

4.3 In situ 3 end labelling of apoptotic cells

Paraffin-embedded tissue sections were dewaxed with xylene and rehydrated for 3'-end
labelling. Tissue sections were incubated with Proteinase K (20 g/ml) (Boehringer
Mannheim GmbH, Mannheim, Germany) at room temperature for 15 minutes. The 3'-end
labelling of apoptotic cell DNA was performed by using an ApopTag in situ apoptosis
detection kit (Oncor, Gaithesburg, MD, USA), following the instructions of the
manufacturer. Endogenous peroxidase activity was quenched with 2% hydrogen peroxide
in phosphate-buffered saline, pH 7.2 (PBS). Terminal transferase was used to catalyse the
addition of digoxigenin-labelled nucleotides to the 3'-ends of fragmented DNA.
Thereafter, anti-digoxigenin-peroxidase solution was applied to the slides. Diluted DABhydrogen peroxide was used to develop the colour reaction. Specimens were lightly
counterstained with haematoxylin.
The rate of apoptosis in fetal ovaries was calculated as the number of apoptotic
oocytes/total number of oocytes. In the CL the proportion of apoptotic cells was
calculated as the number of apoptotic cells per field of vision using 500 x magnification.
Apoptosis in normal and pathological endometrium is presented as apoptotic ratio
(apoptotic cells/all glandular cells).

47

4.4 Gel electrophoretic DNA fragmentation analysis

The specificity of the in situ 3'-end labelling technique was further validated by Southern
blot analysis of fragmentation of genomic DNA. Radioactive and non-radioactive
labelling methods were used.

4.4.1 Radioactive DNA fragmentation analysis

Genomic DNA was extracted from the frozen luteinized granulosa cell samples and
quantified spectrophotometrically at 260 nm. Aliquots of DNA (500 ng) from each
sample were labelled at their 3-ends with [P32]dideoxy-ATP (ddATP; 300 Ci/mmol,
Amersham, Arlington Heights, IL), using terminal transferase (25 U/sample; BoehringerMannheim, Indianapolis, IN). Labelled DNA preparations were fractionated through 2%
agarose gels by electrophoresis, dried in a slab gel dryer without heat and exposed to
Kodak X-Omat film (Eastman Kodak, Rochester, NY) at -70 C.

4.4.2 Non-radioactive DNA fragmentation analysis

Total DNA was isolated from frozen endometrial tissue and quantified by absorbance at
260 nm. One microgram of DNA from each sample was labelled at 3'-ends with DIG-11ddUTP (Boeringer-Mannheim), using 25 U of terminal transferase (BoehringerMannheim). The labelled DNA samples were loaded onto 2% agarose gels and separated
by electrophoresis for 3 h at 50 V. After resolving, the gels were blotted on positively
charged nylon membranes (Boehringer-Mannheim) and DNA was immobilized by UV
irradiation. After blocking non-specific binding, digoxigenin-bound nucleotides were
identified by using sheep antidigoxigenin conjugated to alkaline phosphatase
(Boehringer-Mannheim). The membranes were incubated in sealed hybridisation bags for
5 minutes in 2 ml CSPD solution and exposed to Kodak Scientific Imaging Film.

4.5 In situ hybridisation analysis

4.5.1 Bcl-2 and Bax

Formaldehyde-fixed tissue sections were subjected to in situ hybridisation using biotinlabelled human Bcl-2 and Bax oligoprobes (R&D systems, Minneapolis, MN, USA) and
a commercial in situ hybridisation kit, according to the instructions of the manufacturer

48
(Zymed, San Fransico, CA). Briefly, the sections were deparaffinised through a graded
alcohol series and air-dried. The sections were digested with pepsin solution and treated
with 0.1 N HCL before hybridisation. The probe was applied to the specimens and they
were denatured for 5 minutes at 95 C and hybridised for 2 h at 37 C. A mixture of six
30-mer oligonucleotides complementary to ALU repeats was used as a positive control
and a DNA probe for pSP 3.0 kb vector as a negative control. The hybridised sections
were treated with alkaline phosphatase conjugate and a colour reaction was developed
with DAB. The sections were counterstained with methyl green.

4.5.2 GATA-4
Sections of human fetal ovaries fixed in 4% paraformaldehyde and embedded in paraffin
were subjected to in situ hybridisation using human GATA-4 riboprobes transcribed from
a 575-bp GATA-4 cDNA. Tissue sections were deparaffinised through graded alcohols
and subjected to Proteinase K treatment. Thereafter the sections were dehydrated and airdried. Tissue sections were hybridised overnight with 1 x 106 cpm of [33P]-labelled (1000
3000 Ci/mmol, Amersham, Arlington Heights, IL) antisense or sense riboprobe in a total
volume of 80 l at 60 C. Hybrisation signals were detected after 14-day exposure to
NTB2 emulsion (Eastman Kodak, Rochester, NY) at 4 C. The sections were developed
using D-19 developer and unifix solution (Eastman Kodak), following the instructions of
the manufacturer.

4.5.3 17HSD type 1 and 2

Probes for in situ hybridisation were prepared from a 376-bp fragment (nucleotides 1
376) of human 17 HSD type 1 cDNA (Peltoketo et al. 1998) and a 380-bp fragment
(nucleotides 191570) of human 17 HSD type 2 cDNA (Wu et al. 1993), cloned in
pGEM-4Z plasmids (Promega, Madison, W1), and used as a template for in vitro
transcription. Sense and antisense [35S]CTP-labelled RNA probes were transcribed with
T7 or SP6 RNA polymerases using linearized plasmids as templates. Specific activities of
the synthesized RNA probes were approximately 6 x 106 cpm/l. Hybridisation signals
were detected after 15-day exposure to autoradiographic NTB2 emulsion (Eastman
Kodak, Rochester, NY) at 4 C. The slides were developed using D-19 developer and
unifix solution (Eastman Kodak), following the instructions of the manufacturer. Nuclei
were further stained with Hoechst 33258 (Sigma), after which the slides were mounted
with glycergel (DAKO A/S, Glostrup, Denmark).

49

4.6 Northern blotting

Total RNA from human fetal ovaries was extracted by using the guanidine isothiocyanatecaesium chloride method (Chirgwin et al. 1979) and RNA was quantified by absorbance
at 260 nm. For Northern blots 11 g RNA was size-fractionated in 1.5% agarose gels and
transferred to Biodyne Transfer membrane (Pall Europe Ltd., Portsmouth, England). The
filter was UV-cross-linked (Stratalinker 1800, Stratagene, La Jolla, CA, USA) prior to
hybridisation.
As probes for filter hybridisation we used human GATA-4 cDNA. Human cyclophilin
cDNA was used as control for even loading in filter hybridisation. All the cDNA inserts
were labelled with [32P]- -deoxy-CTP (3000 Ci/mmol; Amersham, Arlington Heights,
IL) and a Prime-a-gene kit (Promega, Madison, WI, USA) was used. The probes were
purified with Nuck Trap columns (Stratagene, La Jolla, CA, USA) and used at 13 x 106
dpm/ml in hybridisation solution containing 50% formamide, 6 x SSC, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, 0.1% BSA, 100 g salmon sperm DNA/ml, 100 g yeast RNA/ml
and 0.5% sodium dodecyl sulphate (SDS). Northern blots were hybridised for 16 h at 42
C and washed once for 20 min at RT and once at 50 C with 1 x SSC-0.1% SDS. Filters
were exposed to Agfa Curix Ortho X-ray film with Trimax T4/T8 intensifying screens
(3M, Ferrania, Italy) at -70 C.

4.7 Immunohistochemistry
Immunohistochemical
analyses
were
performed
according
to
standard
immunohistochemical techniques. Paraffin sections were deparaffinised in xylene and
hydrated gradually through a series of graded alcohols. Endogenous peroxidase activity
was blocked with 3% hydrogen peroxide in methanol. Non-specific binding was
prevented by using 1 ml of fetal calf serum in 4 ml PBS. Primary antibodies are presented
in Table 4. Commercially available avidin-biotin immunoperoxidase systems (Vectastain
Elite anti-mouse/rabbit/goat ABC Kit, Vector Laboratories, Burlingame, CA, USA) were
used to visualize bound antibody. The colour reaction was produced with DAB in the
presence of 0.03% hydrogen peroxide. Counterstaining was carried out with
haematoxylin.
Immunohistochemical staining was evaluated by using an arbitrary staining index
based on preliminary tests. Staining intensity was determined on the scale: 0 = none, 1 =
low or indistinct, 2 = moderate, occurring in some cells, and 3 = strong, found in most or
all cells.

4.8 Western blotting

Frozen endometrial samples were sonicated in ice-cold 50 mM Tris-SO4, 150 mM NaCl,
5 mM EDTA buffer, pH 8.7 containing 1 mM phenylmethylsuphonylfluoride (PMSF), 1

50
mM benzamidine, and 1 mM o-phenanthroline as protease inhibitors. 25 g aliquots of
protein extract were separated by SDS-polyacrylamide gel electrophoresis using 13.5%
acrylamide separating gel and transferred to PVDF membrane (Millipore Corporation,
Bedford, MA, USA). Primary antibody concentrations of 1:200 for Bcl-2 and 1:1000 for
Bax were used. Alkaline phosphatase-conjugated rabbit anti-mouse and goat anti-rabbit
immunoglobulins (1:3000) were used as secondary antibodies to Bcl-2 and Bax
respectively. Visualization was carried out by using chemiluminescence substrate (BioRad Laboratories).
Table 4. Antibodies.
Antigen

Clonality

Dilution

Bcl-2

Mouse

MC

1:25

Source
DAKO

Bax

Rabbit

PC

1:500

Pharmingen

TNF-

Mouse

MC

1:300

Santa Cruz Biotechnology

NF- B

Rabbit

MC

1:1000

Santa Cruz Biotechnology

GATA-4

Goat

PC

1:500

Santa Cruz Biotechnology

Caspase-3

Mouse

MC

1:500

Santa Cruz Biotechnology

Aromatase

Rabbit

PC

1:200

Dr. Yoshio Osawa

Ki-67

Mouse

MC

1:100

DAKO

PC = polyclonal, MC = monoclonal

4.9 FSH and hCG stimulation

Two women (A, B) and one man (C) homozygous for inactivating C566T mutation of the
FSHR were treated with daily subcutaneus injections of recombinant human FSH (rFHS)
(Ares-Serono, Geneva, Switzerland). The starting dose was 300 IU of rFHS/day. On the
third day dose was increased to 600 IU and on the fifth day to 900 IU. Follicular growth
was evaluated using trans-vaginal ultrasonography during FSH treatment. Furthermore
female subject A was given a single intramuscular injection of hCG only (5000 IU,
Pregnyl, Organon, Oss, The Netherlands). Subject B was given 5000 IU of hCG after 6
days of FSH treatment. The hormonal values of Subjects were followed up to 14 from the
beginning of treatment (II).

4.10 Hormone measurements

Serum progesterone and estradiol concentrations were measured using commercial RIA
kits obtained from Wallac OY (Turku, Finland). Serum inhibin A and B dimer
concentrations were separately determined by commercial enzyme-linked
immunosorbent assays (ELISAs) using monoclonal antibodies to the common subunit
and the specific A and B subunits of inhibin (Serotec Limited, Oxford, U.K.). Serum

51
concentrations of T and PRL were determined by a chemiluminescence system (ACSL80, Medfield, MA, USA). LH and FSH concentrations were determined by
fluoroimmunoassays (Wallac Ltd., Turku, Finland) as were those of E2 (Orion
Diagnostica, Oulunsalo, Finland), and androstenedione (A) (Diagnostic Products
Corporation, Los Angeles, CA, USA) following the instructions of the manufacturers. All
hormones were assayed in one batch. The intra-assay variation was 5.5% for inhibin A,
5.2% for inhibin B, 4.0% for T, 3.8% for PRL, 4.9% for LH, 3.8% for FSH, 5.7% for E2
and 5.0% for A.

4.11 Histopathological analysis

The classification of endometrial pathologies was carried out according to standard
criteria (Kurman et al. 1994, Longacre et al. 1995).

4.12 Statistics
Statistical analyses were performed on the basis of two independent experiments. The
data was analysed by ANOVA, followed by students t-test. P < 0.05 was considered
statistically significant.

5 Results
The main results of the studies are characterized in Table 5.
Table 5. Main results of the studies.
Study
I

Main results
Apoptosis was abundant in oocytes of fetal ovaries. In adult ovaries apoptosis was observed
mainly in granulosa cells of growing follicles.

II

C566T mutation presented a complete block for FSH action in vivo and no apoptosis or
expression of aromatase or GATA-4 was detected.

III

An increase in apoptosis was detected at the end of the corpus luteum life-span, together with
a small increase in TNF- expression. This was preceded by an increase in 17SHD type 1
expression.

IV

A low rate of apoptosis was observed in proliferating endometrium. Apoptosis increased in

secretory and reached its peak in menstruating endometrium with a simultaneous decrease in
Bcl-2/Bax ratio.

Glandular apoptosis in endometrial hyperplasia was similar to that seen in normal

proliferating endometrium. In endometrial adenocarcinoma an increase in the rate of
apoptosis was observed.

5.1 Apoptosis in the fetal ovary

Apoptosis was already present in fetal ovaries at the age of 13 weeks. At this point, the
follicular structures were not evident, but most of the apoptotic cells were recognized as
oocytes (I: Fig. 1). Although occasional non-identified cells were apoptotic, the oocytes
still represented the overwhelming majority of the labelled cells. After the 14th week

53

Apoptotic oocytes (%)

some follicular structures started to emerge, and a few apoptotic granulosa cells were
identified. After a slight increase in the number of apoptotic oocytes between weeks 13
and 14, the extent of apoptosis remained stable, and it started to decrease during the last
quarter of fetal life. At term, only occasional apoptotic oocytes were present in the ovaries
(I: Table 1) (Fig. 12).
20
15
10
5
0

13

16

19

22

25

28

31

34

37

40

Age in weeks

Fig. 12. Presentation of apoptosis in fetal ovaries. Apoptosis was mainly detected in oocytes
and only rarely were apoptotic stromal or granulosa cells seen. The rate of apoptosis
remained high from the 13th week to the 20th week and decreased thereafter. At term no
apoptotic oocytes were detected.

5.1.1 Regulation of apoptosis in fetal ovary

Bcl-2 was detected only in the two youngest fetuses, aged 13 and 14 weeks. Bax protein
was also detected in the ovaries at the 13th week of development, but Bax expression
persisted until term. Before the 14th week pregranulosa cells were stained, but no
significant Bax staining was detected in the oocytes. Later on in fetal life, Bax expression
was localized both in the oocytes and granulosa cells. During the second half of fetal life
Bax immunostaining remained relatively stable in oocytes and granulosa cells, but was
negligible in the stroma (I: Table 1).
High GATA-4 expression was found in the ovaries of the youngest fetuses (weeks 13
14). As soon as the first follicular structures became evident, GATA-4 protein was
localized mainly in the granulosa cells and to a lesser extent in stromal/pregranulosa
cells. No GATA-4-positive oogonia or oocytes were detected at any stage of development.
From gestational week 24-25 onwards the number of GATA-4-positive granulosa cells as
well as the intensity of the immunostaining were lower than in ovaries obtained from
earlier fetal ages (I: Table 1). However, a few growing follicles observed in the ovaries of
fetuses older than 27 weeks showed high GATA-4 expression. To confirm the results of
immunohistochemistry, in situ hybridisation and Northern blot analysis were performed.
GATA-4 mRNA was localized to the stromal and granulosa cells in fetal ovaries but not in

54
oocytes. Northern blot analysis of GATA-4 in fetal ovaries showed a single mRNA band
of approximately 4.4 kb at both ages studied (I: Fig. 2).

5.2 Apoptosis in adult ovary

During adult life, apoptotic cells were mainly seen in the innermost layer of granulosa
cells of large growing follicles, i.e. antral follicles. Furthermore, apoptotic granulosa cells
were detected in atretic follicles. In the earlier stages of folliculogenesis, apoptotic cells
were rarely observed. Only occasional apoptotic cells were detected in the secondary
follicles and no sign of apoptosis was found in primordial or primary follicles (I: Fig. 4,
Table 2) (Fig. 13). Oocyte apoptosis was not detected in any of the adult ovaries studied.
This may be due to the fact that only a few oocytes were present in the follicles of
analysed sections. The number of follicles was markedly lower than in fetal ovaries and
consequently the chances of finding positive oocytes were considerably lower.
Autoradiographic analysis demonstrated high levels of DNA fragmentation in granulosaluteal cells from IVF patients. Apoptosis was present in all of the follicles studied, and
there was great variation in the extent of apoptosis between cells from different follicles
(III: Fig. 2) (Fig. 14).

Follicles containing apoptotic

granulosa cells (%)

75 %

50 %

25 %

0%
Primordial

Primary

Secondary

Antral

Follicle type
Fig. 13. Apoptosis in adult ovaries. Apoptosis was mainly observed in the granulosa cells of
growing follicles, but not in oocytes. The figure displays the percentage of follicles that
contained apoptotic granulosa cells.

55

5.2.1 Apoptosis-regulating factors in adult ovary

In adult ovaries, Bcl-2 immunostaining was observed mainly in the granulosa cells of
secondary and antral follicles. No significant staining was observed at earlier stages of
folliculogenesis. Bax immunostaining was found in the granulosa and theca cells of
primary, secondary and antral follicles, and the number of positive follicles increased
with follicular maturation (I: Fig. 4, Table 2).
GATA-4 expression was negligible in primordial follicles but clearly detectable in
primary, secondary and antral follicles, confirming our earlier results. GATA-4 protein
was detected in granulosa and theca cells, and to a lesser degree also in stromal cells (I:
Fig. 4, Table 2).

Fig. 14. DNA fragmentation analysis of apoptotic granulosa cells from three different IVF
patients. Each lane represents granulosa cells gathered from a single follicle.

5.3 Role of FSH in ovarian apoptosis

The ovaries of FSHR mutation homozygotes contained numerous primordial follicles. In
the biopsies taken from five subjects, only two follicles which had developed beyond the
primary follicle stage were observed. Furthermore, all of the resting follicles and the few
growing follicles were negative for apoptosis. These follicles showed only very low or
negligible GATA-4 expression (II: Fig. 1). Histologically the ovarian architecture of
subjects with ALA189VAL FSHR mutation resembled fetal tissue more than adult ovary:
a large number of primordial follicles were present in the ovaries of FSHR mutation
subjects as well as in fetal ovaries, and only a few follicles showed further development.
In normal adult ovaries aromatase expression was observed in granulosa cells of growing
follicles, but in FSHR mutation subjects ovarian aromatase expression was absent.

56
Similarly to FSHR mutation subjects, fetal ovaries showed only minimal aromatase
staining (II: Fig. 1).
Treatment of subjects with inactivating mutation of FSHR with recombinant FSH or
LH did not result in gonadal stimulation as measured by serum inhibin A or B
concentrations. Furthermore, there were no changes in serum E2, T, A or PRL
concentrations in any of the subjects during FSH treatment. No ovarian follicular
development was observed using transvaginal ultrasonography during the therapy (II:
Table 2).

5.4 Apoptosis in the corpus luteum

Apoptotic cells per field of

vision

In situ analysis of DNA fragmentation showed that apoptotic luteal cells were already
observed in early and mid-luteal phases. While occasional samples at these two phases
showed an increased number of apoptotic luteal cells, the rate of apoptosis increased
clearly in the late luteal phase. A clear peak in the number of apoptotic cells occurred at
day 1012 of the luteal phase; all of the sections showed a remarkably high number of
apoptotic cells (III: Fig. 1, Table 1) (Fig. 15).
In situ analysis of luteinized granulosa cells showed DNA cleavage similar to that seen
in CL cells. Autoradiographic analysis of DNA fragmentation showed that luteinized
granulosa cells exhibited the appearance of DNA cleavage into low molecular weight
ladders characteristic of apoptosis, and confirmed the results of in situ labelling analyses
in the same cells and in corpora lutea (III: Fig. 2).

80
60
40
20
0
1

10 11 12 13 14

CL age
Fig. 15. Graphical presentation of apoptosis in the corpus luteum during luteal regression.
CL age is presented as days after the LH surge.

57

5.4.1 Regulation of apoptosis in the corpus luteum

Expression of the apoptosis-regulating proteins Bcl-2 and Bax was studied by
immunohistochemistry and in situ hybridisation. Bcl-2 immunostaining was observed in
the CL throughout the luteal phase. Staining was slightly more prominent during the early
and mid-luteal phase than in the late luteal phase, during which some samples were found
to be negative for Bcl-2 immunostaining. However, due to variations between the
samples, firm conclusions cannot be drawn. Bax protein was present in the CL from the
early to the late luteal phase. Immunostaining was generally strong, with a few
exceptions. In situ hybridisation analysis indicated that mRNA for both Bcl-2 and Bax
was present in human CL, thus confirming the results of immunohistochemical analyses
(III: Fig. 3, Table 2).
Positive staining for TNF- was found in CL in all luteal phases. In the early and midluteal phases the staining intensity was weak and there was no difference between these
phases. In the late luteal phase some samples showed moderately increased
immunostaining that coincided with an increased number of apoptotic cells in the late
luteal phase. Moderate NF- B immunostaining was observed in every sample, indicating
the presence of this apoptosis-regulating transcription factor in the human CL (III: Fig. 3,
Table 2).
Immunohistochemical analysis indicated that Caspase-3 was uniformly expressed in
the CL throughout the luteal phase. Strong staining was observed in all specimens
examined and there were no marked differences in the intensity of staining between the
different phases, although individual specimens with a high apoptotic rate showed intense
Caspase-3 staining (III: Fig. 3, Table 2).

5.4.2 17HSD type 1 and 2 expression in the corpus luteum

The mRNA for the 17HSD type 1 enzyme was expressed in CL. Its expression increased
gradually from day 2 of the luteal phase, and reached a maximum around day 9,
whereafter it started to decrease. 17HSD type 2 mRNA expression was not detected in
any of the samples studied (III: Fig. 4, Table 2).

5.5 Apoptosis in the endometrium during the menstrual cycle

Apoptotic cells were mainly detected in the glandular epithelium of the endometrium.
Only very few positive cells were detected in the stroma at any stage of the cycle. In the
late proliferative phase of the cycle apoptosis in the glands was scarce (Fig. 16). During
the secretory phase, the abundance of apoptotic cells increased and reached a maximum
at menstruation. At early proliferation apoptosis was still present but the number of
apoptotic cells decreased towards late proliferation (IV: Fig. 3).

58
DNA fragmentation analysis confirmed the results of in situ 3'-end labelling. Due to
the abundance of blood cells present in the curettage samples the quality of analysis was
not entirely satisfactory, but it clearly showed that fragmentation was highest in the
menstrual phase (IV: Fig. 2, 3, 4).

Apoptotic cells (%)

6
5
4
3
2
1
0
EP

LP

ES

MS

LS

ME

Fig. 16. Presentation of apoptosis in the endometrium during the menstrual cycle in
endometrial glands. EP = early proliferative (cycle days 39), LP = late proliferative
endometrium (1014), ES = early secretory (1519), MS = middle secretory (2026), LS = late
secretory endometrium (2729), ME = menstruating endometrium (15)

5.5.1 Regulation of apoptosis in cycling endometrium

The intensity of Bcl-2 staining in glandular epithelial cells was highest in the late
proliferative phase, although there was considerable variation in staining intensity
between the samples. In the mid- and late secretory phase and in the menstruating phase,
it was absent or scarce. In the stroma, Bcl-2 immunostaining was negligible throughout
the cycle. Only a small decrease in the intensity of staining was detected during the
secretory phase. Western analysis of Bcl-2 protein revealed the expected 26 kDa protein
in endometrium and a similar expression pattern as seen in immunohistochemistry was
observed (IV: Fig. 5, 6, Table 1) (Fig. 17).
Bax was expressed at all phases of the cycle. In glandular epithelium staining was
most intense, but the protein was also expressed in the stroma. In the glandular
epithelium, the expression of Bax was high during the late proliferative and early
secretory phase. In secretory and menstruating endometrium, immunostaining of Bax
decreased, but the relative decrease seemed to be less pronounced than that of Bcl-2. In
the stroma Bax was also found at all phases of the cycle. In proliferative endometrium,
stromal Bax immunostaining was more intense than in the secretory or menstruating
phases. In Western analysis of Bax the expected 21 kDa protein was observed, and there

59
was a decreasing tendency in the amount of protein detected towards menstruation (IV:
Fig. 5, 6, Table 1) (Fig. 17).

Bcl
Bax

3
2
1
0
EP

LP

ES

MS

LS

ME

Fig. 17. Relationship of Bcl-2 and Bax proteins in the endometrium during the menstrual
cycle. EP = early proliferative, LP = late proliferative endometrium, ES = early secretory,
MS = mid- secretory, LS = late secretory endometrium.

5.5.2 Proliferation during normal endometrial cycles

Cell proliferation in the endometrium was studied by immunostaining the endometrial
samples for antigen Ki-67, which is a nuclear protein present in cells at the replicating
phase of the cell cycle. As expected, proliferative endometrium revealed intense staining
of Ki-67. Positive cells were found mostly in the glandular epithelium, but also in the
stroma. The number of immunopositive cells diminished towards the secretory phase, and
almost disappeared in the late secretory phase. During menstruation, the expression of
Ki-67 was also low. The sections showing high intensity of Ki-67 staining clearly showed
very little apoptosis (IV: Fig. 3).

5.6 Apoptosis in endometrial hyperplasias and carcinomas

5.6.1 Apoptosis in endometrial hyperplasia

Apoptosis in endometrial glands in simplex, complex and atypical hyperplasia was
similar to the rate of apoptosis observed in proliferating endometrium (IV: Fig. 1, 2) (Fig.
18). However, stromal apoptosis was clearly decreased in comparison with proliferating
endometrium and it was at a low level in all cases of endometrial hyperplasia.

60
Bax expression reached a maximum in simplex hyperplasia, while peak Bcl-2
expression was seen in proliferating endometrium. Thereafter, decreasing trends in
mRNA expression and protein levels of both Bcl-2 and Bax were observed (IV: Fig. 3, 5).
In simplex and complex hyperplasia TNF- immunostaining was similar to that seen in
proliferating endometrium, but it was down-regulated in atypical hyperplasia. Moderate
NF- B expression was observed in endometrial hyperplasia (IV: Fig. 4, 5).

Apoptotic index (%)

2,0
1,5
1,0
0,5

III

II
a
C

I
a
C

ic
a
yp

pl
om
C

At

ex

ex
pl
m
Si

At
ro
ph
y
Pr
ol
ife
ra
tio
n

0,0

Fig. 18. Apoptotic cells in endometrial glands in normal endometrium, endometrial

hyperplasia and cancer.

5.6.2 Apoptosis in endometrial adenocarcinoma

The rate of glandular cell apoptosis in grade I adenocarcinoma was similar to that in
proliferating endometrium and hyperplasia. However, the number of apoptotic cells was
markedly increased in grade II carcinoma. In grade III carcinoma the number of apoptotic
cells was lower than in grade II carcinoma but still higher than in grade I carcinoma,
endometrial hyperplasia or proliferating endometrium (V: Fig. 1, 2) (Fig. 18).
Low levels of Bcl-2 mRNA and protein were present in grade I carcinoma. In grade II
and III carcinomas, very low expression of Bcl-2 mRNA was observed, but Bcl-2 protein
was absent, as analysed by immunohistochemistry. Although a decreasing pattern was
also seen in Bax expression, both Bax mRNA and protein levels were higher than those of
Bcl-2 (V: Fig. 2, 3, 5). In grade I carcinoma a low level of TNF- immunostaining was
observed in some specimens, but in grade II and III carcinoma, TNF- immunostaining
was negligible. In grade I carcinoma moderate NF- B immunostaining, similar to that
seen in proliferating endometrium and hyperplasia, was observed. NF- B expression was
decreased in grade II carcinoma and negligible in grade III carcinoma (V: Fig. 4, 5).

61

5.7 Functional study of C566T FSHR mutation

Treatment of homozygous C566T FSHR mutation patients with FSH effectively raised
the serum FSH concentration in the female (48.0 58.5 IU/l to 98.8 104.4 IU/l) and
male (47.4 to 65.3 IU/l) subjects. The increase in serum FSH concentration did not result
in measurable increase in serum inhibin A or B levels. Furthermore there were no changes
in serum E2, T, A or PRL concentrations and no follicular growth could be detected by
ultrasonography (II: Table 2) (Table 6).
Table 6. Effects of treatment with FSH and/or hCG on serum inhibin B (ng/l), T
(nmol/l) and E2 (nmol/l) concentrations in subjects A, B and C (sex/treatment)
homozygous for C566T FSHR mutation.
Day

(Female/ FSH)

(Female / FSH + hCG*)

(Male / FSH)

Inhibin B

E2

Inhibin B

E2

Inhibin B

E2

< 10.0

1.6

0.07

< 10.0

0.8

0.03

41.4

8.8

0.06

< 10.0

1.9

0.02

< 10.0

0.9

0.03

< 10.0

2.8

0.01

< 10.0

0.8

0.04

38.9

9.2

0.06

< 10.0

2.5

0.06

< 10.0

0.8

0.02

33.3

8.4

0.07

< 10.0

2.4

0.04

< 10.0

0.9 *

0.03

< 10.0

2.2

0.02

< 10.0

1.1

0.04

11

< 10.0

3.1

0.02

< 10.0

0.7

0.04

24.7

9.3

0.07

14

< 10.0

1.0

0.05

< 10.0

0.9

0.03

* 5000 IU of hCG i.m. was given on day 7

** Inhibin B (ng/l), T (nmol/l), E2 (nmol/l)

6 Discussion
6.1 Germ cell attrition
During ovarian development, over half of the oocyte population dies prior to birth,
reducing the ovarian reserve of resting follicles to a fraction of its peak number of oocytes
(Sadler 1990). The underlying processes have been studied in animals and strong
evidence indicates that apoptosis is the mechanism of germ cell attrition. Furthermore,
knockout and transgenic animal models demonstrate that the genes involved in apoptosis
also control the event of cell death in the ovaries and the size of the follicle reserve
(Morita & Tilly 1999, Hsu & Hsueh 2000). The present results show for the first time,
that oocyte apoptosis is also abundant during the human fetal development, suggesting
that the same fundamental mechanism of oocyte depletion also takes place in the human
ovary, as previously observed in other mammals (I) (Fig. 19). This suggests that human
oocytes are deleted according to a pre-orchestrated genetic programme.
Indeed, apoptosis-regulating factors, anti-apoptotic Bcl-2 and pro-apoptotic Bax, were
also detected in human ovaries. Bcl-2 protein was detected at the early stages of fetal
development, from week 13 to 14 (I). Another research group has recently reported their
findings on studies with ovaries aged 6 to 12 weeks. They found out that Bcl-2 was
present in early fetal development (De Pol et al. 1997). It looks as if Bcl-2 expression is
evident in the ovary around the time when germ cells migrate to the developing gonad.
After downregulation of Bcl-2, an increase in oocyte apoptosis was observed (II),
implying that Bcl-2 might have a role in protecting oocytes during early development.
Animal studies confirm the role of Bcl-2 in the ovary. Bcl-2 knockout mice have lowered
numbers of oocytes when compared with age matched wild type animals (Ratts et al.
1995). Bcl-2 expression has also been detected in mouse oocytes during fetal
development, at E12.5 and E15.5 (Pesce & De Felici 1994), and during postnatal
development (Jurisicova et al. 1998). Concomitantly, transgenic mice with an inhibinpromoter-driven Bcl-2 gene, resulting in ovarian Bcl-2 overexpression, had lowered
follicular cell apoptosis and enhanced folliculogenesis (Hsu et al. 1996).
Bax protein was detected throughout fetal development from gestational week 13 to
40. While Bax expression was clearly detectable in oocytes in late gestation, the rate of

63
oocyte apoptosis declined towards term (I). It has been shown that intracellular
localization of Bax plays a major part in the proteins ability to induce apoptosis. After an
apoptotic stimulus, Bax is relocated from the cytoplasm to the outer mitochondrial
membrane, where it can activate apoptosis (Zamzami et al. 1998, Putcha et al. 1999). It is
possible that Bax protein seen in oocytes in late pregnancy is located in the cytoplasm.
Bax knockout mice have an equal number of oocytes at the time of birth, when compared
with wild type mice (Knudson et al. 1995, Perez et al. 1999). However, around the time
of puberty, the oocyte reserve of Bax knockout mice has shrunk less than in wild type
animals, being three-fold compared with age-matched mice (Perez et al. 1999). It is
obvious that Bax is not essential for deletion of oocytes during fetal life in mice, but it has
a role in postnatal oocyte demise. One explanation is that Bax protein may be controlled
by inhibiting factors or that it is waiting for a proper signal to transfer to a mitochondrion
where it can trigger apoptosis. Polycyclic aromatic hydrocarbons (PAHs) or cytotoxic
drugs might provide apoptosis-inducing signals, since they both increase Bax expression
and apoptosis in mouse oocytes (Perez et al. 1997b, Matikainen et al. 2001a).
Transcription factor GATA-4 was expressed in granulosa cells during fetal life. No
expression was seen in oocytes. Strong GATA-4 expression could already be detected at
the 13th week. Thereafter the expression decreased, but was still detectable during the
last trimester of pregnancy (I). This expression pattern of GATA-4 in human fetal ovary is
in partial contrast with earlier findings in mice. Viger et al. (1998) have previously
reported that GATA-4 is absent in mouse fetal ovary after development of follicular
structures. However, supporting to our results, GATA-4 expression has also been
observed in porcine fetal ovary (McCoard et al. 2001). Previously, GATA-4 expression
has been localized to granulosa cells of growing follicles in mouse ovaries and in cultured
granulosa cells GATA-4 downregulation was observed before follicular atresia, i.e.
apoptosis (Heikinheimo et al. 1997). Another member of the family of GATA binding
proteins, GATA-1, is expressed in haematopoietic cell lineages and has been suggested to
have an apoptosis-repressing function in erythroid cells (De Maria et al. 1999). GATA-4
might have a similar function in human granulosa cells, resulting in survival of the
primary follicle.
Mitochondria seems to be at the very centre of oocyte decision making when it
comes to life and death. Krakauer and Mira (1999) have proposed that oocyte apoptosis
serves an evolutionary cause known as Mllers ratchet. Mitochondrial DNA is much
more prone to mutations than genomic DNA and since all of the mitochondria are
inherited from the oocyte, the selection of a follicle that contains low-grade
mitochondria would provide poor chances for the conceived offspring and lead to further
problems due to accumulation of mutations, especially in such species that produce only
a small number of offspring. Providing offspring with good quality mitochondria requires
a method for their selection during ovarian development and folliculogenesis. A low
number of mitochondria contained in oocytes (high clonality) has been observed to be
associated with a low number of offspring and a high proportion of germ cell attrition
during development (Krakauer & Mira 1999). These results suggest that in species that
produce only a small number of offspring, each oocyte that reaches ovulation goes
through rigorous selection to ensure the quality of mitochondrial DNA. This is supported
by the observation, that microinjection of mitochondria purified from non-apoptotic

64
granulosa cells into oocytes decreases the occurrence of apoptosis in these oocytes (Perez
et al. 2000).

Fig. 19. Representation of ovarian apoptosis in fetal and adult life. In fetal ovaries apoptosis is
mainly observed in the oocytes and the majority of them are deleted before birth. It is likely
that a low rate of oocyte apoptosis also continues in postnatal life. However, in adult ovaries,
apoptosis in only detected in the granulosa cells of growing follicles.

6.2 Follicular atresia

During adult life, resting follicles continuously enter the growth phase. However, in every
menstrual cycle, only one of the follicles is selected for ovulation. All the rest of this
follicle batch become atretic and are deleted. Apoptosis was detected mainly in granulosa
cells of large growing follicles, i.e. antral follicles. Only occasional apoptotic granulosa
cells were detected at earlier stages of folliculogenesis. In contrast to fetal ovaries, in
adult ovaries oocyte apoptosis was not observed, which may be due to a lowered number
of oocytes and/or a decreased rate of apoptosis. Work with rodent ovaries has provided
information concerning the regulation of granulosa cell apoptosis. Gonadotrophins and
estrogens seem to act as survival factors for follicular cells, while testosterone increases
follicular apoptosis in mice (Chun et al. 1994, Tilly et al. 1995a). Evidence of hormonal
control of follicle atresia has also been reported in humans. Follicles with a high
androgen/estrogen ratio are more susceptible to undergo apoptosis than follicles with low
androgen/estrogen ratio (Yuan & Giudice 1997). In rodents, the Bcl-2 family is intimately
involved in the hormonal regulation of follicular atresia (Tilly et al. 1995a, Hsu & Hsueh
2000). In humans Bcl-2 and Bax are also expressed in the granulosa cells of growing
follicles (I). Neither of the proteins was detected in primordial follicles, but Bax
expression was evident from the primary follicle stage onwards. Bcl-2 expression was
seen in some of the secondary follicles, but granulosa cells in all antral follicles exhibited
expression of both genes. Interestingly, Bax expression was present in the theca cell layer

65
of antral follicles, but no Bcl-2 expression was detected there. No apoptosis was detected
in the theca cells, while a low Bcl-2/Bax ratio predicts a high rate of cell death. The antiapoptotic actions of Bcl-2 could be replaced by those of another member of the Bcl-2
family in theca cells. One likely candidate is Bcl-XL, which has a role in protecting
follicular cells against apoptosis in rodents (Tilly et al. 1995a, reviewed in Hsu & Hsueh
2000).
In addition to the Bcl-2 family, many other apoptosis-regulating factors have been
proposed to regulate granulosa cell apoptosis. The amount of p53 protein has been
reported to be increased in atretic follicles and in apoptotic granulosa cells in vitro
(Makrigiannakis et al. 2000). Transforming growth factor- , epidermal growth factor and
fibroblast growth factor can suppress apoptosis in granulosa cell cultures (Tilly et al.
1992). Downregulation of transcription factor GATA-4 has been observed prior to
follicular atresia in mice (Heikinheimo et al. 1997). In human ovary GATA-4 is expressed
in granulosa cells of growing follicles, and was already detectable in primary follicles (I).
In haematopoietic cell lineages, transcription factor GATA-1 induces proliferation and
opposes apoptosis (Blobel & Orkin 1996). The present results suggest that GATA-4 might
have a similar function in granulosa cells of the human ovary.

6.3 Two-cell-two-gonadotrophin theory

According to the two-cell-two-gonadotrophin theory, the ovary has two cellular
compartments that are driven independently by LH and FSH to produce ovarian steroids.
LH stimulates the theca cell segment of the follicle and induces production of androgens
from cholesterol, while FSH is responsible for promoting conversion of androgen
precursors to estrogens in the granulosa cell compartment. When women homozygous for
the inactivating C566T mutation of the FSHR, were given high doses of recombinant
FSH or LH in combination or simultaneously to stimulate ovarian function, two
interesting aspects were observed (III). Firstly, even high doses of FSH were unable to
produce any measurable increase in serum inhibin concentrations, providing further proof
that C566T mutation produces a complete blockade to FSH action (Aittomki et al. 1995,
Aittomki et al. 1996). Secondly, administration of LH alone was unable to produce any
response in ovarian steroidogenesis, while the two-cell-two-gonadotrophin theory
suggests that even without FSH action, LH should be able to increase ovarian androgen
production.
Recently, Barnes et al. (2000) reported new evidence against the two-cell-twogonadotrophin theory by treating a woman with isolated FSH deficiency either with hCG
alone or first with recombinant FSH and 24 hours later with hCG. Surprisingly,
administration of FSH resulted in greater serum steroid concentrations than hCG
administration, suggesting that paracrine factors stimulated by FSH are required for
proper ovarian steroid production. Observations in C566T FSHR mutation subjects
support the findings of Barnes et al. (2000) and give further strength to the concept that in
humans both gonadotrophins are required for proper ovarian steroidogenesis.

66

6.4 Corpus luteum regression

A notable increase in the rate of apoptosis in late CL was observed, and a maximum was
reached 12 days after the LH surge (III). This temporal relationship between cell death
and CL regression further confirms the role of apoptosis in this process. Previously,
another research group reported that during CL demise, a decrease in the Bcl-2/Bax ratio
was observed (Sugino et al. 2000). Our results show a slight decrease in the Bcl-2/Bax
ratio, but only after the peak in the rate of apoptosis has been reached. Therefore, the
increase in apoptosis towards the end of the CL lifespan cannot be explained solely by the
altered relationship between Bcl-2 and Bax. The presence of TNF- has been reported in
human CL (Roby et al. 1990). We observed TNF- in CL throughout the CL lifespan,
with a slight increase in immunostaining at the time of increased apoptosis. TNF- might
thus contribute to the process of CL regression in mammals (Fig. 20). It has been
previously demonstrated that apoptosis in cultured granulosa luteal cells utilizes the
caspase cascade, specifically caspase-3 (Khan et al. 2000). The expression of caspase-3
in CL throughout the luteal phase implies that CL apoptosis is caspase-3-mediated.
In rabbits, rats and pigs, E2 has been considered as a luteotrophic hormone, but in
humans and other primates the concept is not solid (Vega et al. 1994, Duffy et al. 1994).
In addition to progesterone, the CL also produces estrogens. Type 1 17HSD is the ratelimiting enzyme in conversion of estrone to E2 (Peltoketo et al. 1999). Estradiol functions
as a survival factor for granulosa-luteal cells at normal circulating concentrations in vitro.
However, at high concentrations E2 has been reported to have an opposite effect (Endo et
al. 1998). In the CL, increased expression of type 1 17 HSD was observed prior to an
increase in apoptosis (III). High 17HSD 1 expression results in a high local concentration
of E2. While the role of E2 in human CL requires further studies, it is possible that E2 has a
role in triggering luteolysis (Fig. 20).

6.5 Endometrial apoptosis during the normal menstrual cycle

The endometrium undergoes continuous cyclic changes of cell death and proliferation.
Apoptosis was seen in the stromal cells throughout the menstrual cycle, but in the
endometrial glands apoptosis was scarce in proliferating endometrium. An increase in
apoptosis was seen when approaching the secretory phase and the maximum was reached
in menstruating endometrium. This increase was preceded by a decrease in the Bcl-2/Bax
ratio, predisposing the cells to apoptosis (Fig. 21). However, this ratio change took place
several days earlier than the actual increase in cell death. Real-time in vitro experiments
suggest that apoptosis takes place very shortly after the cell death signal is received
(Dumont et al. 2001). It is plausible that other members of the Bcl-2 protein family, such
as Bcl-XL or Bak, which have been detected in human endometrium (Tao et al. 1997, Tao
et al. 1998), are present in late secretory endometrium and carry out their roles in
controlling apoptosis. Alternatively, the lowered Bcl-2/Bax ratio alone might not be
enough to change the balance in favour of apoptosis. Other apoptosis-regulating factors,
for instance TNF- are present in the endometrium and could take part in the regulation of

67
endometrial cell death (Hunt et al. 1992, Tabibzadeh et al. 1995, Tabibzadeh et al. 1999)
(Fig. 21).

Fig. 20. Model of corpus luteum regression. High 17HSD1 expression was observed in the
mid-luteal phase. The resulting high local E2 concentration can have a role in predisposing the
CL to apoptosis. In the late luteal phase increase in TNF- expression can function as a
trigger to apoptosis.

6.6 Regulation of endometrial homeostasis

It is generally acknowledged that dysfunction of apoptosis is an important factor in
carcinogenesis (Williams 1991). The endometrium is a highly active tissue, which
undergoes regular periods of cell proliferation and cell death. Apoptosis was observed to
participate in the physiological regulation of the endometrium during normal menstrual
cycles (IV). Endometrial hyperplasias are thought to result from increased cellular
proliferation driven by an unopposed estrogen influence (Kounelis et al. 2000). The rate
of apoptosis was similar in endometrial hyperplasia to that in normal cellular proliferation
(V), suggesting that hyperplasia is indeed triggered by escalated proliferation, rather than
diminished cell death. However, it is possible that abnormalities in the apoptosisregulating pathways partially contribute to the development of hyperplasia, due to
inability of cell death mechanisms to respond to the increased proliferation. Grade I
endometrial cancer exhibited a lower rate of apoptosis than seen in normal endometrium

68
or endometrial hyperplasias, suggesting that the onset of cancer might be related to a low
rate of apoptosis (V). However, apoptosis was significantly increased in grade II
carcinomas. This finding is in agreement with previous reports that have described a high
rate of apoptosis in malignant tumours (Aihara et al. 1995, Trmnen et al. 1995). In
grade III carcinoma, the rate of apoptosis was significantly decreased from that in grade
II, possibly indicating lost control of cell homeostasis, poor differentiation and increased
malignancy.
Bcl-2 and Bax expression were also detected in both endometrial hyperplasia and
carcinoma. Both proteins were present in hyperplasias, but Bcl-2 was downregulated in
grade II carcinoma, while weak Bax expression was detected in grade II and III
carcinomas. A decrease in the Bcl-2/Bax ratio might also predispose cells to apoptosis in
endometrial cancer, similarly to normal cyclic endometrium.

Fig. 21. Regulation of apoptosis in human endometrium. In secretory endometrium Bcl-2/Bax

ratio decreases. This event is likely to be under control of ovarian hormones. The decrease in
Bcl-2/Bax ratio precedes the increase of apoptosis in the menstruating endometrium and it is
possible that other factors play a role in induction of apoptosis during menstruation.

6.7 Future perspectives

The current observations verify the presence of apoptosis in the human ovary and
endometrium. In the ovary apoptosis plays a major role in determining the female

69
reproductive lifespan. The present results show, that the apoptosis-regulating factors Bcl2 and Bax are expressed in fetal ovary, and their exact function requires further study (I).
While the essential role of Bax in inducing oocyte apoptosis has been demonstrated in
mice, such observations have to be confirmed in humans due to species-specific
differences (Perez et al. 1999, Matikainen et al. 2001a). One of these differences
concerns transcription factor p53, which has been shown to be related to the activation of
apoptosis in several cell types. P53 is expressed in both human and mouse ovaries, but the
mouse bax gene lacks a p53 binding site, while human bax promotor has a consensus
binding site (Miyashita & Reed 1995, Tilly et al. 1995, Schmidt et al. 1999,
Makrgiannakis et al. 2000,), suggesting that in humans the activation of p53 may lead to
increased Bax expression and consequently to the activation of apoptosis. To find out the
exact mechanisms of Bcl-2 and Bax in human ovarian apoptosis, more complete
documentation of the whole Bcl-2 family is required.
For obvious reasons, functional experiments with human tissue are complicated.
Immortalized human cell lines are easily available, but they often have little relevance to
actual physiological function. The present work establishes, that apoptosis is the
mechanism of corpus luteum regression and suggests that the local estrogen
concentration might have a role in the regulation of this event (III). However, functional
studies on human CL tissue or luteinized granulosa cells are required to verify this.
Several knockout and transgenic animal models have been used to study ovarian
apoptosis. Similarly, it is possible that careful studies on humans with proven genetic
mutations that affect their reproductive phenotype will provide more information about
the molecular mechanisms behind the regulation of apoptosis. Ultimately, better
understanding of ovarian apoptosis may lead to novel methods to control female
reproductive functions. By preventing oocyte apoptosis, it could be possible to increase
the size of the resting follicle stock, thereby prolonging the reproductive life span and
delaying the onset of menopause, which could be of special importance for women at risk
of premature ovarian failure. In addition, the prevention of oocyte apoptosis could be
important in preventing oocyte loss and sterility caused by environmental insults such as
radio- and chemotherapy or industrial toxins that are known to cause oocyte apoptosis
(Matikainen et al. 2001a, reviewed in Morita & Tilly 1999).
Defects in apoptosis-regulating pathways play a role in the development of cancer
(reviewed in Wyllie 1997a). In endometrial cancer, significant alterations in the rate of
apoptosis were observed, which may be due to changed expression of apoptosisregulating factors (V). Detailed knowledge of the molecular mechanisms of
transformation of normal proliferative tissue to cancer will most likely reveal new targets
for pharmacological intervention in this event.

7 Summary and conclusions

The female reproductive tract undergoes dramatic cycles of growth and degeneration in
which apoptosis plays an important regulatory role. In the ovary, apoptosis is responsible
for the marked germ cell attrition during fetal life, which determines the size of the
resting follicle stock. The number of resting follicles continuously decreases through
follicle atresia and ultimately only about 400 follicles out of 7 million will survive and
ovulate.
In adult ovaries, apoptosis was mainly detected in granulosa cells of large growing
follicles, and no apoptosis was observed in the oocytes. However, a low rate of oocyte
apoptosis cannot be ruled out, and possibly this process continues until menopause. It is
likely that follicular atresia of unselected follicles is primarily caused by granulosa cell
apoptosis and not oocyte demise. Ovaries non-responsive to FSH did not show follicular
development or granulosa cell apoptosis, suggesting that in adult life apoptosis controls
cell/tissue homeostasis, and the rate of apoptosis is related to cell proliferation.
After ovulation, the dominant follicle collapses and forms the CL. If pregnancy does
not occur, CL regression takes place to allow new follicles to be recruited during the next
menstrual cycle. CL regression takes place by an apoptotic mechanism that is possibly
regulated by TNF- and E2.
In the endometrium, superficial layers are shed during menstruation to permit growth
of new endometrium to ensure the highest receptivity at the time of ovulation. A high rate
of apoptosis was detected in menstrual endometrium, indicating a role for programmed
cell death in this process. This increase in apoptosis was preceded by a decrease in the
Bcl-2/Bax ratio. Although direct evidence is missing, it seems likely that in the
endometrium these apoptosis-regulating factors are under the control of ovarian steroids.
In endometrial hyperplasia, the rate of apoptosis was equal to that seen in normal
proliferating endometrium, but in grade II carcinoma it was significantly increased. This
may reflect a mechanism to maintain cell homeostasis, the control of which is probably
disturbed or lost in more advanced carcinomas.

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