ORIGINAL PAPER

Life and death of female gametes during oogenesisand folliculogenesis

Dmitri V. Krysko Æ Araceli Diez-Fraile ÆGodelieve Criel Æ Andrei A. Svistunov ÆPeter Vandenabeele Æ Katharina D’Herde

Published online: 14 July 2008

� Springer Science+Business Media, LLC 2008

Abstract The vertebrate ovary is an extremely dynamic

organ in which excessive or defective follicles are rapidly

and effectively eliminated early in ontogeny and thereafter

continuously throughout reproductive life. More than 99%

of follicles disappear, primarily due to apoptosis of gran-

ulosa cells, and only a minute fraction of the surviving

follicles successfully complete the path to ovulation. The

balance between signals for cell death and survival deter-

mines the destiny of the follicles. An abnormally high rate

of cell death followed by atresia can negatively affect

fertility and eventually lead irreversibly to premature

ovarian failure. In this review we provide a short overview

of the role of programmed cell death in prenatal differen-

tiation of the primordial germ cells and in postnatal

folliculogenesis. We also discuss the issue of neo-oogen-

esis. Next, we highlight molecules involved in regulation

of granulosa cell apoptosis. We further discuss the potential

use of scores for apoptosis in granulosa cells and

characteristics of follicular fluid as prognostic markers for

predicting the outcome of assisted reproduction. Potential

therapeutic strategies for combating premature ovarian

failure are also addressed.

Keywords Granulosa cell � Follicular fluid �Follicular atresia � Apoptosis � Caspases �Premature ovarian failure � Autophagy � Neo-oogenesis �Gap junctions

Introduction

Oocyte cell death and its regulation has always been one of

the most active research areas in vertebrate reproductive

physiology and developmental biology, and they have been

inspiring scientists for over a century. The occurrence of

cell death in the ovary is by no means a new discovery.

Walther Flemming in 1885 made one of the first observa-

tions. He described a process he named ‘‘chromatolysis’’ as

being responsible for the degeneration of inner epithelial

cells (granulosa cells, GCs) in rabbit ovarian follicles. The

cells dying by chromatolysis were characterized by cellular

and nuclear condensation followed by fragmentation. Now

it is well-known that these morphological characteristics

perfectly match the typical features of apoptosis, which

were described almost nine decades later by Kerr et al. [1].

Two decades after Kerr, Gavrieli et al. [2] identified the

oligonucleosomal DNA fragmentation that is pathogno-

monic for apoptosis in GCs of atretic mouse follicles. Since

then, the instrumental role of granulosa cell apoptosis in

follicular atresia was confirmed for many species, includ-

ing humans. In parallel with the unraveling of the signaling

pathways and the contributing gene products involved in

other model systems, researchers analyzed the involvement

D. V. Krysko (&) � P. Vandenabeele

Department for Molecular Biomedical Research, Molecular

Signaling and Cell Death Unit, VIB, Technologiepark 927,

9052 Ghent, (Zwijnaarde), Belgium

e-mail: dmitri.krysko@ugent.be

D. V. Krysko � P. Vandenabeele

Department of Molecular Biology, Ghent University,

Technologiepark 927, 9052 Ghent, Belgium

A. Diez-Fraile � G. Criel � K. D’Herde

Department of Human Anatomy, Embryology, Histology and

Medical Physics, Ghent University, De Pintelaan 171,

4B3, 9000 Ghent, Belgium

A. A. Svistunov

Department of Pharmacology and Clinical Pharmacology,

Saratov State Medical University, B. Kazashia 112, 410012

Saratov, Russia

123

Apoptosis (2008) 13:1065–1087

DOI 10.1007/s10495-008-0238-1

of caspases, Bcl-2 family members, and death receptors

and ligands in the development of the fetal gonad and in

postnatal folliculogenesis. Independently of cell death

research, more than 50 years ago investigators identified

mouse genetic mutations affecting follicular endowment

and thus fertility [3]. Genes with well-known roles in PCD

are now included in the list of genes affecting fetal

endowment. In 1951 Sir Solomon Zuckermann [4] laun-

ched the central dogma in reproductive medicine on the

finite stock of oocytes in mammalian ovaries and in doing

so closed the discussion on the possibility of neo-oogene-

sis, which started at the beginning of the century [5].

Zuckermann’s dogma stimulated researchers to study

the signaling pathways of programmed cell death in the

ovary, with prospects of developing clinical applications,

such as the development of new contraceptives, the pre-

vention of spontaneous or chemotherapy-induced

premature ovarian failure, and the optimization of in vitro

fertilization (IVF) protocols. This dogma was recently

questioned [6, 7], directing several research groups towards

studying the possibility of oocyte neo-oogenesis in the

adult ovary. In this way the story of the ovary is not only

one of understanding cell death mechanisms, but also

comprehending how new life can be generated. This review

will discuss recent findings on the following subjects: (1)

the role of programmed cell death in prenatal differentia-

tion of the primordial germ cells and in postnatal

folliculogenesis, including a discussion of the functional

homology between invertebrate nurse cells and the verte-

brate GCs; (2) the controversial issue of neo-oogenesis; (3)

the contribution of pro-apoptotic and anti-apoptotic mole-

cules to the regulation of granulosa cell apoptosis as the

main driving force of follicular atresia; (4) the contribution

of cell death associated with autophagy to follicular atresia;

(5) the detection of apoptosis and the analysis of follicular

fluid as tools for predicting the outcome of assisted

reproduction; (6) the therapeutic strategies for combating

premature ovarian failure.

Programmed cell death is instrumental in prenatal

differentiation of primordial germ cells

The process of oocyte formation and follicular endowment

includes seven prenatal steps that start with (i) the gener-

ation of primordial germ cells (PGCs), (ii) their migration

to the future gonads and their concurrent proliferation, (iii)

colonization of the gonads, (iv) differentiation of PGCs

into oogonia, (v) proliferation of the oogonia, (vi) initiation

of meiosis and, finally, (vii) arrest at the diplotene stage of

prophase I of meiosis. A phenomenon that is common to

vertebrates and invertebrates is that PGCs often arise in one

portion of the embryo and migrate relatively long distances

to where the gonad will ultimately be formed [8]. During

this migration, the founder PGC population (originally 45

in mice) proliferates. A growing number of spontaneous

and transgene mouse models of ovarian failure with defects

in one or more of the seven developmental stages serve as

important tools in the analysis of programmed cell death

mechanisms in the fetal ovary (for detailed review see [9,

10] and the databases presented elsewhere [11, 12]). The

interaction of c-kit with its ligand (stem cell factor) has

been well-documented as a prerequisite for migration,

survival and proliferation of PGCs. The tyrosine kinase

receptor, c-kit, is expressed at the germ cell surface and

binds to the somatic cells along the migratory pathway,

where its ligand is expressed. This explains the directed

homing of germ cells [13]. Mutations in either the receptor

(KIT) or the ligand (KL) cause a dramatic reduction in fetal

germ cells, discovered 50 years ago [3], besides mislocal-

ization of germ cells. The process of prenatal germ cell

loss, which basically occurs through apoptosis [14, 15], is

designated oocyte attrition and occurs during each phase of

oogenesis, in both mitotic and postmitotic germ cells. From

a peak number of 6.8 9 106 germ cells about the time of

the mitotic to meiotic transition (fifth month of fetal

development in human) [16], the number of germ cells

sharply decreases, particularly during two main periods,

the pachytene stage of meiosis in oocytes and the formation

of primordial follicles [17]. In agreement with these pio-

neer studies, it was reported that during early meiosis in

humans, the number of viable oocytes declined from seven

million on week 20 of gestation to less than one million at

birth [3]. Thus, the characteristic normal fate of female

germ cells during oogenesis is to commit suicide, but the

rationale for this remains a mystery. The initiation of

meiosis (step vi), whereupon the germ cells become

oocytes, coincides with the formation of primordial folli-

cles and is controlled by retinoic acid [18]. Follicle

formation occurs during the second trimester of human

fetal development, whereas in the mouse ovary it occurs

immediately after birth [19]. The somatic cells involved in

human folliculogenesis are derived from both the surface

epithelium and the mesonephros [20]. As for the molecular

mechanisms underlying oocyte PCD, there is no clear

evidence that a death receptor or extrinsic pathway is

involved during the fetal period [21, 22]. The intrinsic

pathway of apoptosis is triggered by extracellular and

intracellular stresses, such as growth factor withdrawal, the

presence of genotoxicants, and DNA damage.

Two hypotheses have been advanced to explain the basis

of the universal prenatal germ cell death [23, 24]. The first

hypothesis is death by neglect, caused by shortage of sur-

vival factors. Indeed, germ cells depend, like other cell

types, on the availability of certain growth factors, such as

kit ligand (KL) and leukemia inhibiting factor (LIF) [25].

1066 Apoptosis (2008) 13:1065–1087

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Dependence on growth factors could be a way of matching

the number of oocytes to the number of somatic cells

involved in folliculogenesis in the same way that the

number of neurons in the central nervous system has to

match the number of target cells. Though in vitro experi-

mental approaches to study fetal oogenesis indicate that

programmed cell death is due to lack of survival factors

[26], it has not been determined whether this is a normal

aspect of fetal oogenesis in vivo. Of course, the infertility

of mice mutant for KL [3] due to apoptosis of primordial

germ cells is highly suggestive of the death by neglect

hypothesis. Interestingly, it was shown that KL might

prevent germ cell apoptosis by maintaining weak expres-

sion of the pro-apoptotic gene bax [27]. Although the

extrinsic pathway of PCD induction is reported not to be

active during the fetal period [21, 22], the phenotypic

appearance of homozygous KIT-deficient mice (i.e. like

KL mutants, lacking oocytes at birth) can be partly rescued

by simultaneous presence of homozygous Fas deficiency.

This indicates that Fas-mediated apoptotic signals in the

fetal ovary crosstalk with KIT-mediated survival signals

[28]. Another hypothesis proposed to explain the engage-

ment of oocytes in an intrinsic pathway leading to PCD is

the death by defect hypothesis [23, 24], which means the

elimination of oocytes with chromosomal abnormalities or

defective mitochondrial genomes [29]. Indeed, most sex

chromosome aneuploidies are associated with gonadal

dysgenesis owing to absence or near absence of germ cells

in the gonads. Although the idea of a program of oocyte

quality control seems appealing, it has not been ascertained

whether the extensive apoptosis of oocytes in the ovaries of

patients with Turner syndrome (XO) is a cause or a con-

sequence of the meiotic defect [30]. Difficult to reconcile

with this hypothesis is that XO mice are fertile and have

normal ovaries at birth, even though they suffer from

premature ovarian failure [31]. A third hypothesis

explaining germ cell loss in vertebrates is the death by self-

sacrifice hypothesis, which is comparable to what is seen

during invertebrate oogenesis. In lagomorphs, rodents and

humans, the PGCs are initially very closely clustered

together. These groups of cells are comparable to Dro-

sophila germ cell cysts and consist of interconnected,

synchronously dividing germ cells surrounded by a layer of

somatic cells. These cysts begin to break down due to

germline cell death, allowing somatic cells to cocoon

individual surviving oocytes and thereby giving rise to

primordial follicles [32]. This type of altruistic cell death

would permit oocytes destined to form primordial follicles

to acquire mitochondria from neighboring dying germline

cells, thus behaving functionally like invertebrate nurse

cells [33, 34]. Due to the different timing of follicle for-

mation in mice and humans, this altruistic cell death

accounts for fetal germ cell loss in humans but not in mice.

To the best of our knowledge this third hypothesis has not

been linked to a specific intra or extracellular stress initi-

ating the intrinsic pathway.

With regard to the intracellular regulators of germ cell

apoptosis, a diverse spectrum of pro- and anti-apoptotic

susceptibility genes, including the Bcl-2 and caspase

families, have been reported to be involved in prenatal

oocyte attrition (for detailed review see [23]). But it should

be emphasized that, depending on the specific stage of

prenatal oogenesis (i to vii), the various pro- and anti-

apoptotic molecules may either affect or not affect apop-

totic signaling. This issue was revealed by counting germ

cells in the bax-/- mouse. The results illustrated that while

bax contributes to the loss of primordial germ cells and

oogonia, it is not involved in apoptosis of oocytes entering

meiotic prophase. This conversion during fetal oognesis

from a bax-dependent to a bax independent apoptotic sig-

naling cascade explains why the bax-/- mouse is born with

a number of germ cells comparable to the wild type [35]. In

conclusion, as long as the central dogma in reproductive

biology remains valid (that most if not all female mammals

are born with a finite stock of germ cells), the study of

signaling factors that affect fetal follicular endowment and

the rationale of the universal prenatal germ loss remain of

utmost clinical relevance.

Programmed cell death in postnatal folliculogenesis

The quiescent ovarian germ stockpile is decreased from

less than one million viable oocytes at birth to about

300,000 oocytes at puberty, of which only *400 will

ovulate over the fertile lifespan [36–38]. During postnatal

degeneration of follicles, the loss of quiescent follicles can

be distinguished from the loss of growing follicles. The

Greek word ‘‘atresia’’ is used to describe the closure of a

natural opening. In the strictest sense, follicular atresia

refers to antral follicles undergoing degenerative changes

before rupture during ovulation. This term is nowadays

used in a broader sense to describe degenerative changes

taking place during ovarian follicular development. Growth

of the dormant follicles is initiated before and throughout

the female’s reproductive life. In humans, a number of

follicles are recruited for development during each repro-

ductive cycle. A single dominant follicle is usually selected

for ovulation, while the cohort of antral follicles are sac-

rificed [39]. Ovarian follicular development and atresia

are regulated by the interaction of pituitary hormones

(gonadotropins) and intra-ovarian regulators, and this

interaction promotes proliferation, growth, differentiation,

and apoptosis. Follicular atresia is initiated within the

granulosa layer and subsequently in the theca cells [40–42].

Widespread cell loss within the granulosa layer provokes

Apoptosis (2008) 13:1065–1087 1067

123

the death of follicles. Only in primordial and primary fol-

licles it is likely that oocyte loss is responsible for

subsequent follicular degeneration [43, 44]. The basic

mechanism of follicular atresia in mammalian and avian

species is apoptosis. This conclusion is supported by the

identification of apoptotic features, such as DNA frag-

mentation in atretic follicles and by comparison of

expression levels of apoptosis-related genes in atretic ver-

sus healthy follicles; this will be discussed later in this

review [44]. Furthermore, programmed cell death is also

instrumental during ovulation [45] as well as during

regression of the mammalian corpus luteum [46] or its

homolog in birds [47]. Though the main cell death type in

the ovary is apoptosis, autophagic cell death has also been

evidenced during follicular atresia [48] and luteolysis [49].

Thus, programmed cell death serves as the basic mecha-

nism in the ovarian cycle [50, 51], and its instrumental role

in determining postnatal follicular atresia has been clarified

in several well-studied knockout and transgenic animals;

this will be addressed at several places in this review.

Functional homology between the invertebrate

nurse cell and the vertebrate GC

One of the conserved features of postnatal oogenesis is the

intimate relationship between the oocyte and the sur-

rounding cells, the latter of which have a somatic origin in

vertebrates (the follicle cells or GCs) but a mixed origin in

invertebrates. While nurse cells are of germline origin,

follicle cells are of somatic origin, as observed for example

in insects and crustaceans.

Next to PCD’s role in oogenesis as a mechanism for

eliminating abnormal gametes (death by defect hypothe-

sis), in invertebrates PCD of both nurse and follicle cells is

an intrinsic part of the production of fully functional

mature eggs [24]. Indeed, nurse cells of germline origin

transfer vital cellular material to the growing oocytes and

undergo programmed cell death by self-sacrifice with

apoptotic hallmarks (Fig. 1). Apoptotic nurse cells are then

phagocytosed by somatic follicle cells, which subsequently

undergo programmed cell death in Drosophila but not in

Artemia [52]. As reported above, a similar mechanism of

germline death exists in prenatal mouse oogenesis with the

breakdown of germline cysts. During avian postnatal fol-

liculogenesis, the somatic cells surrounding the oocyte,

namely GCs, also transfer vital cellular material to the

enclosed oocyte during a limited period of folliculogenesis

without undergoing programmed cell death. This cellular

material consists of the so-called lining bodies (bags of

ribosomes), which are incorporated by the growing oocyte

and are destined for deposition within yolk granules [53–

58]. During mammalian oogenesis, transzonal projections

originating from GCs and terminating at the oocyte plasma

membrane provide a polarized means to orient the secre-

tory organelles of somatic cells. The cumulus GCs allow

the transfer of about 85% of the oocyte metabolic needs via

heterologous gap junctions present at the tip of transzonal

projections [59], and at the same time they mediate an

oocytal effect on the cumulus cells by preventing corpus

luteum formation. In mammals, mural and cumulus GCs,

together with the oocyte, form a gap junction-mediated

syncytium. Bidirectional communication of the oocyte-

cumulus complex is also mediated by a paracrine mecha-

nism. Large molecules are taken up by the oocyte through

receptor-mediated endocytosis in coated vesicles. Although

it is clear that GCs supply the enclosed oocyte with

nutrients and paracrine factors [59], transfer of vital cel-

lular material of somatic origin towards the oocyte during

mammalian postnatal folliculogenesis has not been docu-

mented. In conclusion, for both invertebrates and

vertebrates, the development of the germ cell is tightly

linked to and dependent on the interaction with

Fig. 1 (a) DAPI-stained 2-lm LX section of ovarian tissue of the

invertebrate Artemia franciscana. Germ cells with round nuclei

intermingle with somatic cells possessing elongated nuclei. Apoptotic

condensed chromatin masses of dying nurse cells (arrowheads) are

apparent, example of death by self-sacrifice. (b) Electron microscopic

image showing apoptotic nurse cell with condensed cytoplasm and

fragmentation of nucleus into several condensed chromatin masses

(asterisks). The apoptotic bodies are engulfed by neighboring somatic

cells. Scale bar: 1 lm

1068 Apoptosis (2008) 13:1065–1087

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surrounding somatic cells or other germ cells. The precise

time after which this support is no longer needed during

oogenesis, however, is different for invertebrates and ver-

tebrates. Indeed, the mammalian oocyte is still enclosed

within a cumulus oophorus at ovulation, whereas the

invertebrate egg is isolated before ovulation, i.e. shortly

before germinal vesicle breakdown.

Follicular atresia in the mammalian ovary

and the neo-oogenesis debate

A basic doctrine in reproductive biology is that mammalian

ovaries are endowed with a fixed number of quiescent

primordial follicles during early life [4], while male

mammals can reproduce throughout most of their adult

lives by continuously generating sperm precursors from

germline stem cells maintained within the testis. Females

of the few invertebrate species that remain fertile

throughout life, such as the fruit fly Drosophila melano-

gaster or Artemia francicana, a brine shrimp, contain

germline stem cells like those of males, and use them to

replenish oocyte precursors [60, 61]. Mitotically active

germ cells were documented in some species of prosimian

primates. However, there was no evidence of folliculo-

genesis and ovulation from the proliferating germ cells

[62].

Contrary to long-held views, arguments for the existence

of proliferative germ cells that sustain oogenesis and fol-

liculogenesis in postnatal mouse ovary have been reported

[6, 7, 63]. Based on calculations of the quantity of atretic

follicles at a given moment and the duration of the visible

stages of atretic follicles, it was concluded that postnatal

neo-oogenesis is actually necessary to compensate for the

prepubertal loss. Another team’s calculations showed that

follicle numbers in the primordial follicle pool of C57BL/6

mice remain stable from day 7 to 100. In addition, follic-

ular recruitment into the population of growing follicles

was not paralleled by a decrease in the total number of

healthy follicles, indicating that follicles are renewed

postnatally [64]. Positive staining for germ cell markers

and early meiosis markers was detected in adult mouse

ovaries by two groups [7]. The most convincing experi-

ments in favor of postnatal neo-oogenesis was the

restoration of oocyte production by bone marrow trans-

plantation in wild-type mice sterilized by chemotherapy as

well as in telelangiectasia-mutated gene-deficient mice,

which are otherwise incapable of producing oocytes.

Donor-derived oocytes have also been observed in female

mice following peripheral blood transplantation [7]. Based

on these data, Spradling [60] suggested that menopause in

humans is due to depletion of germline stem cells and to

the age-dependent incidence of follicular atresia.

Three independent laboratories have shown that

embryonic stem (ES) cells can differentiate in culture into

primordial germ cells and thereafter into either oocyte-like

or spermatid-like cells [65–67]. However, the full potential

of these ES cell-derived gametes has not been demon-

strated so far [68], and furthermore, an intense debate

continues on whether this type of oogenesis from germ

cells occurs in vivo in the adult ovary [69–71]. Shortly

after the first paper from Tilly’s group on neo-oogenesis in

the adult human ovary [6], another research team reported

similar data [72]. They stated that the only source of new

primordial follicles would be the surface epithelium of the

ovary rather than an extragonadal source. However, their

immunohistochemically stained cryosections are far from

convincing. Moreover, old experimental data do not sup-

port the hypothesis of Bukovsky et al. because destruction

of surface epithelium did not cause any reduction in the

number of follicles compared to control mice [73]. Another

research team [74] used a mathematical model for assess-

ing follicle progression dynamics to calculate whether the

initial follicle pool was sufficient for adult fertility. The

results of this analysis support the old dogma that the initial

endowment of ovarian follicles is not supplemented by a

significant number of stem cells. A recent study by Eggan

et al. [75] dealing with the issue of neo-oogenesis addres-

sed the physiological relevance of circulating cells for

female fertility. By using a parabiotic and transplantation

mouse model, they assessed the capacity of circulating

bone marrow cells to generate ovulated oocytes. Their

findings do not support the existence of postnatal oogenesis

from an extra-ovarian origin. Furthermore, they claim that

cells of bone marrow origin traveling to the ovary via the

bloodstream exhibit properties characteristic of committed

blood leucocytes. It was recently shown that bone marrow

transplantation in mouse, though it does not result in

pregnancies from donor-derived oocytes, generates imma-

ture oocytes of recipient origin [76]. However, it remains to

be shown whether bone marrow transplantation indeed

reactivates host oogenesis and does not simply rescue

fertility by rescuing a sufficient number of existing oocytes.

In this context, it was reported that a young hypofertile

woman suffering from Fanconi anemia and treated by

chemotherapy, irradiation and bone marrow transplantation

gave birth to a child genetically related to her and not to the

donor of the bone marrow [77]. Although this case report

illustrates that women are not equally sensitive to chemo-

therapy and irradiation, it does not answer the question

whether bone marrow transplantation reactivates host

oogenesis or whether a sufficient number of existing

oocytes survive the treatment.

As most of these data concern the mouse ovary, Liu

et al. [78] investigated the presence of germline stem cells

(GSCs) and neo-oogenesis in adult human ovaries. As

Apoptosis (2008) 13:1065–1087 1069

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genetic manipulation is unethical in humans, they analyzed

the expression of meiotic marker genes and genes for germ

cell proliferation required for neo-oogenesis and compared

them to testis and fetal ovaries as positive controls. No

early meiotic-specific or oogenesis-associated mRNAs

were detectable in adult human ovaries, compared to fetal

ovary and adult testis, suggesting that neo-oogenesis does

not occur in the adult human ovary. Indirect arguments

against the concept of neo-oogenesis were presented in an

ultrastructural study comparing non-atretic resting follicles

from young and older females [79]. That study showed an

age related change that was independent from follicular

atresia in the ultrastructure of the human resting follicular

pool. This would not be observed if it were neo-oogenesis

that supplied the resting follicle pool. So, irrefutable

arguments for neo-oogenesis remain absent until today

(Table 1). The benefit of the work of Jonathan Tilly’s

group was to stimulate other research groups worldwide to

investigate the validity of the reproductive medicine’s

central dogma on the finite stock of oocytes in adult

females. As stem cells are rare and morphologically diffi-

cult to identify, some new experimental designs are needed

to determine whether germ stem cells in the adult ovary

can develop and mature into ovulating eggs.

Apoptosis of granulosa cells in regulation

of follicular atresia

Development and atresia of vertebrate ovarian follicles are

tightly regulated by crosstalk between signals of cell death

and survival. Apoptosis of GCs represents one of the pri-

mary pathways by which defective or excessive follicles

are rapidly and effectively eliminated. Many apoptosis-

related factors have been implicated in follicular atresia,

including death ligands and receptors, caspases, pro- and

anti-apoptotic Bcl-2 family members, gonadotropins, cal-

cium, and gap junctional intercellular communication. All

these will be discussed in this section. In addition, we will

describe several genetically null mouse lines that lack

various apoptosis regulatory proteins. Evaluation of female

reproductive function in these knockout mice yielded

interesting phenotypes and provided a possible means for

distinguishing gene products essential for the execution of

apoptosis from correlated gene products, i.e. gene products

expressed during programmed cell death.

Death receptors

Pathways for induction of apoptosis in mammalian cells

are mediated by cell-surface receptors known as death

domain-containing receptors, which are members of the

tumor necrosis factor (TNF) receptor family. The members

of the TNF receptor family, including Fas receptor, TNF

receptor, and tumor necrosis factor-related apoptosis

inducing ligand (TRAIL), have been implicated in follic-

ular atresia in mammalian ovaries. These membrane-

anchored receptors, which are activated following ligand

binding, are coupled to caspase activation by adaptor

proteins such as Fas-associated death domain (FADD) and

TNF receptor-associated death domain (TRADD) [86].

FasL and Fas are the best characterized apoptotic signaling

machinery in the GCs of many species, including humans

[87, 88], mice [89], rats [90], pigs [91], and cattle [92]. One

line of evidence for the role of Fas in follicular atresia is

provided by transgenic mouse models, including the lpr

mutant mouse (lymphoproliferation), which contains a

mutation in the gene encoding the Fas antigen. Young lpr

Table 1 Comparison of the experimental findings in human and non-human mammalian ovaries that support the old dogma on the finite

stockpile of oocytes at birth or conversely support the concept of neo-oogenesis

Experimental arguments Human

ovary

Mammalian

ovary

Pro neo-oogenesis

Meiotic, mitotic and germline markers [6, 7, 80–82]

Inferential evidence histomorphometric assessment of follicle numbers versus death rates [6, 64, 83]

BMT and peripheral blood generate donor derived oocytes in the recipient ovary, but offspring is of recipient

origin

[7, 76]

Grafting labeled ovary into unlabeled host ovary creates chimaeric follicles [6]

Clinical experiments: BMT after chemotherapy rescues fertility in cancer patients [84]

Contra neo-oogenesis

Absence of meiotic markers, mitotic and germline markers [78] [74]

Inferential evidence histomorphometric assessment of follicle numbers versus death rates do not indicate neo-

oogenesis

[4, 74, 85]

BMT in a parabiotic mouse model does not generate donor derived ovulating eggs [75]

Clinical experiments: BMT after chemotherapy does not rescue fertility in cancer patients [77]

1070 Apoptosis (2008) 13:1065–1087

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mutant mice have morphologically normal ovaries, while

adult mice have a significantly larger number of growing

follicles than wild-type mice [93]. These adult mice have

larger ovaries because they have more follicles, indicating

a crucial role for Fas in follicular atresia in the ovarian

physiology of adult mice.

Activation of the Fas-FasL system can initiate apoptosis

in GCs of rat, mouse [93, 94] and humans [95]. Fas and Fas

ligand have been localized to the granulosa layer in rat

ovarian follicles that have been induced to undergo atresia

in vivo by gonadotropin withdrawal [90]. In human

females, Fas is expressed in GCs of atretic antral follicles,

and its level increases as atresia progresses [88]. Treatment

of cultured bovine GCs with estradiol E2 decreased the

susceptibility to FasL-induced apoptosis [96]. GCs from

rats treated with equine chorionic gonadotropin (eGC)

show reduced Fas and FasL content [90], whereas Fas

content is increased in GCs from atretic mouse follicles

[97]. Nitric oxide, by suppressing activation of the casp-

ases, inhibits apoptosis induced by the Fas/FasL system in

rat GCs, pointing to a cross-talk between the Fas/FasL

system-induced apoptosis pathway and NO-mediated anti-

apoptotic pathway in ovarian follicle atresia [98]. These

findings strongly indicate that Fas/FasL is a key regulator

of granulosa cell apoptosis and follicle atresia.

TNF, which is produced by several ovarian cell types,

including GCs and the oocyte, is another important regu-

lator of follicular development and atresia [99]. This factor

can induce either cell death or proliferation. TNF exerts its

effects by binding to TNF receptor-1 (TNFR) or TNFR-2.

TNFR-1 stimulates apoptotic signaling via its DD; TNFR-2,

which lacks a DD, acts as a survival factor [100]. TNF

induces apoptosis in GCs isolated from rat preantral folli-

cles and hen large white follicles [101, 102]. In addition to

its pro-apoptotic function, TNF can promote survival and

maintenance of follicular growth by inducing intracellular

survival factors. For example, TNF induces NF-jB acti-

vation in rat GCs, resulting in expression of flice-like

inhibitory protein (FLIP) and X-linked inhibitor of apop-

tosis (XIAP) [103, 104], which leads to survival of GCs

isolated from follicles in the antral stage of development.

These data indicate that TNF is an intraovarian modulator

of granulosa cell function. Its pro-survival or pro-apoptotic

effects on GCs depend on the stage of follicular develop-

ment, which in turn determines the relative abundance of

TNFR subtypes and the expression of various intracellular

death and survival factors, such as Fas/FasL, XIAP and

FLIP [99].

TVB is an avian death domain-containing receptor

belonging to the TNF receptor family and is proposed to be

the ortholog of mammalian DR5. The TVB receptor was

originally identified by its ability to bind envelope coat

proteins from cytopathic avian leukosis-sarcoma viruses

(ALV). Treatment of cultured chick embryo fibroblasts

with a recombinant ALV envelope fusion protein induces

apoptosis that requires the presence of a functional TVB

death domain [105]. TVB is more abundantly expressed in

hen GCs of atretic follicles than in healthy follicles [105].

However, increased TVB expression does not precede

induced follicle death in vitro. In addition, its expression in

GCs was strongest during the final stages of follicle

development, when follicles are highly resistant to apop-

tosis. These observations provide evidence that TVB

receptor signaling in the ovary may also have functions

other than mediating granulosa cell death and follicle

atresia.

Increased mRNA expression of TNFR-associated DD

protein (TRADD), which transmits the death signal from

death receptor-4 (DR-4) and/or DR-5 to intracellular

apoptosis-signal transduction components in GCs was

demonstrated only in atretic follicles, indicating that the

TRAIL-receptor system induces apoptosis in GCs during

atresia in porcrine ovaries [106]. It is noteworthy that

mRNA of TRAIL, another member of the TNF family, is

elevated in hen prehierarchal follicles undergoing sponta-

neous or induced atresia [107], indicating that at least four

members of the TNF family may be involved in the process

of follicle atresia.

Caspases

Caspases constitute a family of intracellular cysteine pro-

teases involved in both the initial and final stages of

apoptosis in almost all types of vertebrate cells. Unpro-

cessed caspase-3 can be found in GCs from healthy

follicles, whereas GCs from atretic follicles contain larger

amounts of activated caspase-3 [108]. An antibody raised

against the activated form of caspase-3 reacted strongly

with GCs of degenerating antral follicles in both mouse and

human [109]. Analysis of caspase-3-deficient mice

revealed that caspase-3 is required for granulosa cell

apoptosis and thereby instrumental in follicular atresia, but

dispensable for oocyte apoptosis [109]. Activation of

caspase-3 has been associated with cleavage of Poly-

(ADPiribose)-polymerase (PARP) and actin, and the for-

mation of oligonucleosomes [110]. Caspase-9-deficient

mice contain numerous developing follicles that fail to

complete the process of atresia, apparently due to failure of

granulosa cell apoptosis [108]. Proteolytic activity of cas-

pase-9 in pig GCs increased during follicular atresia, while

the inactive zymogen (procaspase-9 protein) decreased

[111]. Caspase-9 and Apaf1 in murine GCs have also been

shown to contribute to follicular atresia [112]. In that

context we have given evidence that in avian granulosa

cytochrome c release, which is necessary to activate cas-

pase 3 via the apoptosome complex, is confined to a

Apoptosis (2008) 13:1065–1087 1071

123

subpopulation of mitochondria, while other mitochondria

continue to respire [113].

On the other hand, caspase-2-deficient mice have excess

follicles in neonatal ovaries due to attenuation of fetal germ

cell loss [114]. In addition, their oocytes were found to be

resistant to cell death following exposure to chemothera-

peutic drugs [114]. These observations were substantiated

by Morita et al. [115], who showed that germ cells in

caspase-2-deficient fetal ovaries were also resistant to

death caused by complete cytokine starvation in vitro,

supporting the hypothesis that caspase-2 is central to the

execution of PCD in oocytes.

Bcl-2 family members

As expected, the Bcl-2 family proteins are also implicated

in granulosa cell apoptosis. One of the most studied

members of this family is Bax. The survival of rat GCs

mediated by gonadotropin correlates with decreased levels

of bax expression in the absence of any change in Bcl-2 or

Bcl-xL levels [116]. Moreover, in man and other species,

increased bax expression at the mRNA and protein levels

was associated with granulosa cell apoptosis and follicular

atresia. Microinjection of recombinant Bax protein into

isolated oocytes induced apoptosis, thereby supporting the

notion that increased cytoplasmic Bax levels are sufficient

to induce apoptosis in female germ lines [117]. Bax protein

was abundantly expressed in GCs of early atretic follicles

but was scarce or undetectable in healthy follicles [118].

The rate of primordial and primary follicle atresia is sub-

stantially reduced in Bax-deficient mice due to defective

postnatal oocyte apoptosis [119]. Greenfeld et al. [120]

studied antral follicles and showed that Bax deletion did

not affect the extent of antral follicle atresia in vivo. The

authors demonstrated similar numbers of atretic antral

follicles in Bax-/- ovaries and wild type ovaries. In

addition, the extent of apoptosis in Bax-/- ovaries was not

different from that in wild type ovaries [120]. Since atresia

of immature follicles is initiated by oocyte death, whereas

degeneration of antral follicles is controlled by death of

GCs [15], it is conceivable that apoptosis in oocytes and

GCs is regulated by different mechanisms. Importantly, as

atresia of immature follicles can be reduced by Bax

depletion [119], it is possible that oocyte death proceeds

via a Bax-dependent pathway, whereas GCs die via a Bax-

independent pathway [120].

Importantly, targeted expression of Bcl-2 in mouse

oocytes during either fetal development [121] or postnatal

life [40] suppresses apoptotic cell death. Moreover, mice

overexpressing Bcl-2 show decreased apoptosis of ovarian

somatic cells, enhanced folliculogenesis, and increased

susceptibility to germ cell tumorigenesis [122]. In contrast,

ablation of Bcl-2 gene expression is accompanied by a

decrease in the numbers of oocytes and primordial follicles

in the postnatal ovary [123]. These data provide in vivo

proof that Bcl-2 family members regulate oocyte fate

during prenatal development and during adult life. In

addition to Bax and Bcl-2, other pro-apoptotic (e.g. Bcl-xs,

Mtd/Bok, Diva/Boo, Bad, Bim and Bod) and anti-apoptotic

(e.g. Mcl-1) proteins of the Bcl-2 family are known to be

expressed in ovarian germ cells and/or GCs of various

species [117, 124]. For example, ectopic overexpression of

Mtd/Bok or Bad can induce granulosa cell apoptosis [125,

126].

These data indicate that the decision of any given fol-

licle to either undergo atresia or to survive and

subsequently ovulate is controlled by a balance between

multiple pro- and anti-apoptotic Bcl-2 family members.

Further elucidation of the role of Bcl-2 members in the

tissue-specific regulation of apoptosis could facilitate an

understanding of normal physiology and allow develop-

ment of new therapeutic approaches for pathological states

(discussed in more detail in section ‘‘Therapeutic options

for combating premature ovarian failure’’). For example,

oocytes obtained from Bcl-2 transgenic mice and cultured

in vitro were found to be resistant to spontaneous and

anticancer drug-induced apoptosis providing a possible

means to obtain resistance of the female germ line to

naturally occurring and chemotherapy-induced apoptosis

[40]. These observations suggest a possible way to acquire

resistance of the female germ line to naturally occurring

and chemotherapy-induced apoptosis by targeted expres-

sion of Bcl-2 only in oocytes.

Gonadotropins and intraovarian regulators

The decision of the developing follicles to continue to grow

and eventually to ovulate or to undergo atresia depends

mainly on the coordinated action and interaction of cell

survival and cell death factors within the GCs. It is likely

that multiple molecules are involved in regulation of

granulosa cell apoptosis, including follicle-stimulating

hormone (FSH), GDF-9, Nodal, prohibin, TNF, IGF-I and

p53. The response of follicles to these survival factors

depends on the growth stage. For example, follicular

development of primordial to secondary follicles does not

require gonadotropin support. Intraovarian factors, such as

GDF-9, control transition of the follicle from the preantral

to the early antral stage. FSH is required for follicle growth

from the time of past antrum formation until ovulation

[127]. In this section we will briefly review the role of

these factors in follicle atresia and granulosa cell apoptosis.

For a more detailed picture we recommend consulting

other published reviews [39, 127, 128].

Gonadotrophin-mediated inhibition of apoptosis in

ovarian GCs is partially related to changes in the

1072 Apoptosis (2008) 13:1065–1087

123

expression of several cell death-related factors (see section

‘‘Death receptors’’). Gonadotrophin withdrawal by anti-

body neutralization [129, 130], hypophysectomy on the day

of proestrus, or metabolic clearance after a single hormonal

injection induces granulosa cell apoptosis and follicular

atresia [131–133]. Gonadotrophins can induce the expres-

sion of pro-survival molecules, including bcl-2, GATA-4,

FLIP and XIAP, and decrease the expression of pro-

apoptotic molecules, such Bax, Apaf1, Fas/FasL and p53

[112, 132, 134]. It has been shown that FSH can mediate

both progesterone-dependent and progesterone-indepen-

dent survival pathways in pre-ovulatory avian GCs [135].

Besides gonadotropins, other factors that are synthesized

and secreted within the follicle have a direct impact on

granulosa cell apoptosis. Down regulation of growth dif-

ferentiation factor 9 (GDF-9) by intraoocyte injection of a

GDF-9 antisense morpholino increased caspase-3 activa-

tion and granulosa cell apoptosis, but this response was

attenuated by exogenous GDF-9 [127]. In addition, GDF-9

suppressed ceramide-induced apoptosis in primary GCs

from early antral, but not from large/preovulatory follicles

[136]. The phosphatidylinositol 3-kinase inhibitor,

LY294002, and a dominant negative form of Akt prevented

the protective effect of GDF-9. These data suggest that

GDF-9 is antiapoptotic in preantral follicles and protects

GCs from undergoing apoptosis by activating the phos-

phatidylinositol 3-kinase/Akt pathway.

Nodal, a member of the TGF-b family, exerts its bio-

logical effects by signaling through a cell surface serine/

threonine kinase receptor complex composed of types I and

II receptors and intracellular Smad proteins. Nodal can

induce apoptosis and inhibit cell growth. Nodal and its type

I receptor, Alk7, are expressed in the theca layer and in

GCs, respectively. However, they are co-localized in the

GCs when follicular atresia is induced by gonadotropin

withdrawal [137]. Addition of recombinant Nodal to GCs

from large antral follicles, forced overexpression of Nodal,

and expression of a constitutive active form of Alk7 (Alk7-

ca) result in the induction of apoptosis [127, 137]. Over-

expression of either Nodal- or Alk7-ca-activated caspase-3

and -9 increased apoptosis [127]. Moreover, addition of

recombinant Nodal or forced expression of Nodal or Alk7-

ca in primary GCs induces phosphorylation and nuclear

accumulation of Smad2, as well as down-regulation of

phospho-Akt and Xiap content [127]. All these data sup-

port the role of the Nodal/Alk7 signaling pathway in

promoting follicular atresia.

The intracellular protein prohibin also contributes to

granulosa cell apoptosis, and its effect is stage-dependent.

Overexpression of prohibin in undifferentiated GCs from

preantral follicles markedly attenuated apoptosis induced

by ceramide, staurosporine, or serum withdrawal [127].

However, over-expression of prohibin in differentiated

GCs from antral follicles induces apoptosis [127]. Further

studies are required to identify factors regulating its

expression and function in the follicle.

The p53 protein is an anti-proliferative transcription

factor that regulates the rate of transcription of various

genes involved in mitosis and apoptosis. The expression of

p53 is increased during gonadotropin withdrawal, and

overexpression of p53 resulted in extensive granulosa cell

apoptosis [132, 138]. These data suggest that induction of

atresia is a p53-dependent process.

IGFs and IGF-binding proteins (IGFBPs) are thought to

play a critical role in ovarian follicle selection and follic-

ular growth. Indeed, knockout of IGF-I in mice results in

arrested follicular development at the preantral and early

antral stages, leading to ovulation failure [139, 140]. In

addition, IGF-I seems to play a crucial role in the

responsiveness of the ovary to FSH action, because FSH

receptor expression was severely reduced in preantral IGF-

I null follicles, and restored to wild type levels after two

weeks of exogenous IGF-I supplementation [140]. Treat-

ment of the rat pre-ovulatory follicles with IGF-I prevents

spontaneous onset of apoptosis [141]. Proliferation is pro-

moted and apoptosis is suppressed in primary cultured

porcine GCs by IGF-I and in avian GC explants by a

combination of IGF-I and LH [142, 143]. In rat and bovine

GCs, IGF-I activates PI3-K and Akt, with IGF-I driving

phosphorylation, indicating that IGF-I plays an anti-apop-

totic role in GCs by sustaining PI3-K-Akt signaling [100].

Although IGF-I has an essential role in the development of

ovarian follicles in many species, IGF-II is more abundant

and likely more important in the human and non-human

primate ovary, where it appears to act similarly to IGF-I

[144–146]. All these observations led to the suggestion that

the ultimate fate of the follicle depends on intrafollicular

synthesis and bioavailability of IGF, and ultimately to its

interaction with its cognate receptor [147]. Actually, IGF’s

bioavailability rather than its concentration dramatically

changes during growth and atresia of ovarian follicles.

IGFBPs have been proposed to play an essential role in

IGF bioavailability by sequestering IGFs. In particular,

transgenic female mice overexpressing IGFBP-1 were

shown to present a decrease in serum IGF-I levels and

bioavailability, and a reduction in natural and PMSG-

induced ovulation rate [148, 149]. Large differences

between the IGF/IGFBP systems of different mammalian

species have been described. One exception to this rule is

the degradation of IGFBP-2 and -4 that can be found in

follicles undergoing terminal follicular growth, while these

IGFBPs have been described to increase in atretic follicles

[150]. In response to the gonadotropin surge, the compact

cumulus-oocyte complex undergoes expansion by synthe-

sis and deposit of an intercellular matrix enriched in the

mucopolysaccharide, hyaluronan [151]. Hyaluronan in

Apoptosis (2008) 13:1065–1087 1073

123

granulosa cell layers may be involved in cell locomotion

[152], and in the prevention of fragmentation or segmen-

tation of oocytes in vitro [153]. In addition, hyaluronan

decreases the occurrence of degenerated oocytes [154].

Although little is known about regulation of the hyaluronan

receptors in reproductive tissue, it has been suggested that

the most common hyaluronan receptor, i.e. CD44, plays an

important role during human oocyte maturation [155] and

prevents apoptosis in human granulosa cells in a hyaluro-

nan-dependent way [156].

Gap junctional intercellular communication

GCs can communicate either through the local production

of intraovarian factors such as cytokines [157] and growth

factors [158] that act as paracrine and/or autocrine modu-

lators, or through gap junctions [159]. Gap junctions

between mammalian oocytes in follicles and GCs serve to

transfer nutrients from the GCs to the oocyte, sustaining its

growth, and to regulate oocyte meiosis. The gap junctions

connecting mouse oocytes and GCs during oocyte growth

consist of homomeric, homotypic channels composed of

Cx37 [160]. The cumulus and mural GCs are themselves

connected by gap junctions composed of Cx43 [161, 162].

Mouse GCs express both Cx37 and Cx43 but target them to

different cell-surface-membrane domains: Cx37 to contacts

with the oocyte and Cx43 to contacts with each other [160,

163]. It has been shown that gap junctions play an

important role in granulosa cell development, differentia-

tion, and luteinization [164]. The developmental importance

of the gap junctions that couple growing oocytes with GCs

was clearly demonstrated when the gene encoding Cx37

was knocked out [165]. Although viable and without overt

abnormalities, female mice lacking Cx37 are sterile due to

disruption of folliculogenesis in the ovaries. In the absence

of the coupling between oocyte and granulosa cell provided

by Cx37, null mutant oocytes suffer growth retardation and

do not survive to become meiotically competent [166]. The

growth of follicles in these mice is also interrupted. The

mutant GCs form structures resembling corpora lutea,

which would normally develop only after the mature

oocyte has been expelled from the follicle during ovula-

tion. Therefore, Cx37 gap junctions are essential for

maintenance of oocyte growth and survival, which in turn

is necessary for maintaining proper granulosa cell function

[163, 167].

However, whether the modulation of gap junction for-

mation and permeability is a primary or secondary event in

controlling apoptosis is not yet clear. It was found that

glucocorticoids enhanced Cx-43 expression, formation of

gap junctions, and appearance of intact gap junctions in a

rat pre-ovulatory granulosa cell line (RGSP53-10), indi-

cating that gap junctional intracellular communication is an

important mediator in glucocorticoid protection against

apoptosis in GCs [168]. In contrast, the same group

reported earlier that induction of apoptosis by LH and

forskolin was accompanied by increased expression of

Cx43 in human luteinized GCs [169]. This discrepancy

might be related to the use of different cell models and

apoptosis-inducing stimuli. We have established an avian

model (Japanese quail, Coturnix coturnix japonica) of

granulosa cell apoptosis in which granulosa explants are

isolated from the largest ovarian follicle belonging to the

follicle hierarchy [170]. Since these granulosa cell explants

consist of single-layered GCs sandwiched between vitelline

and basement membranes, this model system is suitable for

studying cell-cell contacts in vitro. We reported that

induction of apoptosis was accompanied by decreased

Cx43 immunoreactivity in immunocytochemical and

immunoblotting procedures, suggesting that Cx43 expres-

sion per se may play a role in the survival process [171]. A

similar observation was made by Cheng et al. [172], who

observed in porcine tertiary follicles that Cx43 was

expressed most strongly in GCs of healthy follicles, with

only trace levels in cells of early atretic and progressed

atretic follicles, an indication that the expression levels of

Cx43 protein decrease during follicular atresia [172].

However, we also observed that initiation of apoptosis was

accompanied by an overall increase in the level of gap

junctional coupling, and apoptosis was dose-dependently

inhibited by the gap junction blocker a-glycyrrhetinic acid

[171]. Therefore, these studies indicate that the functional

state of gap junctional communication rather than their

physical integrity may contribute to the resistance of GCs

to apoptotic signals.

Role of calcium as a first and second messenger

in survival and apoptosis of GCs

Calcium, one of the most versatile and universal signaling

agents involved in cell growth and differentiation, has

attracted interest as a potential second messenger in

apoptosis since Kaiser and Edelman [173] demonstrated in

1977 that glucocorticoid-stimulated thymocyte apoptosis is

associated with enhanced Ca2+ influx. Calcium coupling to

the apoptotic effector pathway has been documented for a

myriad of targets, including activation of proteases, trans-

glutaminases, endonuclease(s), and Ca-dependent protein

kinases and phosphatases, leading to alterations in gene

transcription and affecting the enzymes involved in the

maintenance of phospholipid asymmetry in the plasma

membrane [174]. Furthermore, it was shown that bcl-2

suppresses apoptosis by a mechanism that is linked to

intracellular Ca2+ compartmentalization, and it appears

that Ca2+ alterations in some cases of apoptosis occur as

the result of changes within the mitochondria [175]. We

1074 Apoptosis (2008) 13:1065–1087

123

have shown for quail granulosa explants that a transient

intracellular calcium rise due to influx of calcium is

causally related to apoptosis induction [170, 176]. Similar

data supporting the notion that calcium acts as a second

messenger were reported for rat GCs in serum-free

cultures.

Moreover, we suspected that Ca2+ also acts as a first

messenger, because elevation of the extracellular calcium

load in the absence of an intracellular calcium rise pre-

vented apoptosis from occurring [177]. The role of the

extracellular calcium-sensing receptor (CaR) in maintain-

ing Ca2+ homeostasis has been extensively studied,

particularly in tissues contributing to calcium homeostasis,

such as the parathyroids. We have shown for the quail

ovary that CaR is expressed in GCs of the follicles selected

for ovulation, but not in follicles that are lost during fol-

licular atresia, which indicates that CaR might play a

regulatory role in follicle selection. When granulosa

explants are cultured in serum-free media for 24 h and then

the CaR is challenged with different agonists, apoptosis is

inhibited (unpublished results, D’Herde K). As far as we

know, the CaR has not been linked to a survival pathway in

the ovary.

Follicular atresia is not under exclusive control

of apoptosis: autophagic cell death in GCs

Apoptosis is not the exclusive mode of active cell death in

follicular atresia. We have documented by cytochemistry

and electron microscopy (Fig. 2) that a second type of cell

death, called autophagic cell death, may be observed in the

granulosa of starvation-induced atretic follicles of Japanese

quail (Coturnix coturnix japonica) [48]. In that study we

showed that apoptotic and autophagic cell death were

associated with different patterns of acid phosphatase

activity (lysosomal vs. cytoplasmic). Recent observations

[178] indicated that both types of cell death occur in the

ovarian nurse cells during middle and late oogenesis of

Drosophila virilis. It was reported that during mid-oogen-

esis, the spontaneously degenerated egg chambers exhibit

characteristics typical of apoptotic cell death, whereas at

the late stages of D. virilis oogenesis, apoptosis and

autophagy coexist. The authors proposed that apoptosis and

autophagy operate synergistically during D. virilis oogen-

esis to efficiently eliminate the degenerated nurse cells.

Recent studies by Duerrschmidt et al. [179] demonstrated

that autophagy occurs in lectin-like oxidized low-density

lipoprotein receptor-1 positive GCs from IVF patients. All

these reports illustrate that in in vivo models, as opposed to

in vitro models, more than one cell death type often

coexist, complicating the unraveling of the signaling

pathways.

Apoptosis of GCs and follicular fluid (FF) features

as predictive markers for the outcome of assisted

reproduction

Traditional methods for evaluating oocyte quality rely on

the morphological assessment of the follicle, cumulus-

oocyte complex, polar body and/or meiotic spindle [180].

These methods are undoubtedly inexpensive; however, the

use of morphological characteristics to predict oocyte

developmental competence cannot be supported [181].

Therefore, the need for more objective and accurate pre-

dictors has prompted the study of additional forecasters of

oocyte quality associated with the oocyte itself, i.e. the

surrounding follicular cells and the FF.

It is now widely recognized that oocyte-follicle com-

munication is bidirectional and essential for their correct

function and development. GCs are essential in ovarian

folliculogenesis, because they produce factors that are

necessary in normal follicular maturation, such as steroids

and growth factors [182]. Moreover, for an adequate fol-

licular response to gonadotropins, it is necessary to have a

suitable number of GCs, which is determined by the rates

of proliferation and of apoptotic cell death [183]. Taking

the latter observations into consideration, it seems logical

to propose GC apoptosis and FF characteristics as predic-

tive markers for the developmental potential of the oocyte.

Factors in GCs and those secreted in FF would, indeed, be

an ideal source of non-invasive predictors of oocyte com-

petence. Studies have been conducted in diverse species,

physiopathological conditions, and methodologies, and

Fig. 2 Autophagic cell death as one of the three cell death modes

during follicular atresia in the quail ovary. This mode of death is

identified by numerous double membrane vacuoles containing

recognizable cytoplasmic material (arrowheads) with initially no

apparent nuclear changes. Remark also the presence of an elaborate

Golgi apparatus (asterisk), as well as the presence of numerous single

membrane vacuoles suggestive for autophagolysosomes. Scale Bar:

1 lm

Apoptosis (2008) 13:1065–1087 1075

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variable results have been reported. The current section

exclusively covers apoptosis-related prognostic markers for

the outcome of assisted reproduction. Additional informa-

tion on oocyte and embryo viability markers falls outside

the scope of this review.

Apoptosis in follicular aspirates of GCs

It has been claimed that a key aspect in achieving high rates

of pregnancy after IVF is selection of oocytes derived from

women with low incidence of apoptotic cells on mural and/

or cumulus GC masses derived from follicular aspirates

during conventional IVF [184–187]. Conversely, various

studies reported that GC apoptosis cannot be used to predict

the result of assisted reproduction techniques (ARTs) [188,

189]. Some major variations in the experimental setup of

research trials that could lead to seemingly inconsistent

results are discussed thoroughly later in this review.

Different techniques used to detect apoptosis

In the pursuit of markers of oocyte quality, several tech-

niques have been employed to assess apoptosis of GCs.

These include TUNEL staining, nuclear chromatin staining

with fluorescent probes, and annexin V and/or PI labeling.

Evaluation has been carried out mainly by microscopy,

immunoblot and flow cytometry.

A pioneer study [188] examined the correlation between

the occurrence of GC apoptosis, detected by Southern

blotting following the TUNEL assay, and compromised

oocyte quality, in terms of fertilization rates or in vitro

development of embryos to the four-cell stage, on a per

follicle basis. No correlation could be found based on

apoptosis as a categorical variable, namely whether or not

apoptosis could be detected. Later studies considered

apoptosis as a continuous variable, and thus assessed the

incidence or extent of apoptotic bodies in GCs in follicular

aspirates. These studies found an association between

apoptosis and IVF outcome when apoptosis was assessed

by fluorescence microscopy upon chromatin staining [185]

or by flow cytometry, and using the TUNEL assay in both

instances [185, 190]. Flow cytometry has the extremely

important advantage of counting large numbers of cells in a

short time [186]. However, flow cytometry may not be

suitable for determining oocyte quality by evaluating

individual follicles and measuring apoptosis because of the

large number of GCs needed. Alternatively, apoptosis may

be estimated by fluorescence microscopy. Although many

studies morphologically evaluated the percentage of

apoptotic cells upon nuclear staining, Lee et al. [191]

reported that the fragmented, condensed nuclei of cumulus

GCs could not be observed clearly upon staining with

H33258. Hence, these authors recommended use of the

TUNEL assay. As each type of technique has inherent

advantages and disadvantages, it is recommended that

more than one technique be used in order to achieve reli-

able results and to characterize a specific form of cell death

[192, 193].

Apoptosis at the mural region and at the cumulus

region

A space or antrum containing FF physically separates

mural and cumulus granulosa components in a preovula-

tory follicle [194–196]. When a woman ovulates, virtually

all of the granulosa components are obtained by aspiration.

The mural GCs can be found at the FF while the cumulus

GCs are those attached to the oocyte. Differences in the

apoptotic percentage have been reported between mural

and cumulus compartments. Indeed, the mural granulosa

cell region had a higher incidence of apoptotic bodies than

did the cumulus cell region in each follicle [184] and on a

per patient basis [185, 197, 198]. These observations imply

that, although both components seem to be predictive of

oocyte quality and pregnancy outcome, the uniformity of

the sample being evaluated should be taken into consid-

eration to increase the accuracy and validity of the result.

Apoptosis in individual follicles or per patient

When the general capacity of folliculogenesis of each

patient is to be assessed, it is important to analyze the

incidence of apoptotic bodies on a per patient basis.

However, if multiple follicles are aspirated per patient per

cycle, no direct conclusions can be drawn concerning the

relationship between the incidence of GC apoptosis and

oocyte competence on a per follicle basis [199].

The intensity of apoptosis on a per follicle basis during

the same superovulatory period clearly indicates that a

higher incidence of apoptotic bodies is associated with

empty follicles, poor oocyte fertilization, and poor embryo

quality [184]. Therefore, the occurrence of high-order

pregnancies may be avoided by selectively transferring

considerably fewer high quality embryos. Although inves-

tigation of apoptosis on the follicular basis seems to be the

proper way, it is difficult to perform in the clinical setting.

Moreover, even after observation on the follicular basis, it is

still not certain which embryo is implanted after transferring

two or three embryos. Therefore, more refined studies are

required through investigation of apoptosis on a follicular

basis and with single embryo transfer.

The assisted reproduction technique employed

The most frequently used assisted reproduction techniques

are IVF and intracytoplasmic sperm injection (ICSI).

1076 Apoptosis (2008) 13:1065–1087

123

While there is a fairly broad consensus that the incidence

of apoptosis in GCs is indicative of ovarian function and

is a prognostic factor in IVF program [184, 185, 200], it

remains controversial for ICSI. Indeed, two research

groups could not relate the percentage of apoptosis in

GCs to oocyte maturity and fertilizability by ICSI or to

follicular quality in stimulated cycles of normal women

[189, 200]. The authors suggested that ICSI does not need

high quality oocytes, defined as low apoptosis of GCs,

because this technique bypasses the natural barriers by its

invasive nature. Oocytes of relatively low quality,

including those of older women and those with poor

ovarian fecundity, have been proposed to have a greater

chance of being fertilized with ICSI than with IVF.

However, in another study, maturity and fertilization of

the oocyte were found to be correlated with apoptosis in

the cumulus cells during ICSI [201]. In addition, apop-

tosis of GCs induced by oxidative stress, measured by the

8-hydroxy-20-deoxyguanosine index, was correlated with

the fertilization rate and embryo rate independently of the

insemination method, i.e. IVF or ICSI [202]. Therefore,

the latter research groups [185, 201, 202] pointed to the

fact that the quality of the oocyte is more relevant than

the insemination method in determining the outcome.

Oocyte acquisition upon ovarian stimulation

Fresh, mature oocytes in metaphase II are usually

obtained after pituitary down-regulation using gonado-

tropin releasing hormone agonists (GnRHa) followed by

FSH administration. Ovulation is then stimulated by

human chorionic gonadotropin, and GnRH antagonists

may be simultaneously administered to avoid early ovu-

lation [180, 203]. It was shown that GC apoptosis tends to

be low in follicles stimulated with the survival factor FSH

[204, 205]. Therefore, it is understandable not to find

consistent results between GC apoptosis and parameters

of oocyte quality for oocytes obtained after ovarian

stimulation with FSH [206, 207].

Superovulation with GnRHa may raise the probability of

fertilization and pregnancy by increasing the number

of fertilizable oocytes ovulated per cycle, the chances of

ovum pickup, and the density of gametes in the female

reproductive tract [208, 209]. However, GnRHa super-

ovulation may result in oocytes of poor quality. Indeed,

GnRHa potentially increase the incidence of human GC

apoptosis in vitro as well as in vivo [210, 211]. Hence,

protocols excluding GnRHa may increase fecundity by

improving oocyte quality. Due to the decrease in oocyte

quality observed upon ovarian stimulation, the trend is now

to harvest a single oocyte in the natural cycle with minimal

stimulation.

Studies during IVF under various pathophysiological

conditions

Endometriosis patients have a higher incidence of apop-

totic GCs compared to normal women [184, 212].

Moreover, the incidence of GC apoptotic bodies increased

as the severity of the endometriosis increased [198].

Patients with deficit of the ovarian reserve also have a high

percentage of apoptosis compared with patients with other

causes of infertility and with normal women [213].

Accelerated GC apoptosis has been proposed to play a

pivotal role in the etiology of unexplained infertility [187].

Patients from whom a large number of oocytes were

retrieved had a lower incidence of apoptosis in GCs com-

pared to those from whom fewer oocytes were retrieved

[184, 197]. No difference was found in the first study [185]

on the incidence of apoptotic cells among different age

groups in which endometriosis patients were included.

However, when evaluating normo-ovulatory women

undergoing IVF due to infertility of their partners, those

who were over 40 years old had significantly more apop-

tosis, lower fertilization rate, and fewer oocytes retrieved

and mature than younger patients [191, 214]. Thus in order

to obtain conclusive results, care should be taken when

selecting the population to be included in an experiment.

Basal vs. induced GC apoptosis

Apoptosis of the cells retrieved from the stimulated follicle

is a rare event during ART. Indeed, when evaluating

apoptosis in unstimulated GCs, the average apoptotic index

is low, ranging from 0.43% in a group with a good IVF-

embryo transfer (IVF-ET) prognosis up to 1.81% in women

with bad IVF-ET prognosis [191]. On the other hand, upon

stimulation with IFN-c and triggering apoptosis with anti-

Fas antibody, the percentage of apoptotic GCs drastically

increased [95]. In the latter study, mean apoptotic indices

for good and bad IVF-ET outcome varied between 11.6%

and 59.5%, respectively. The subtle differences obtained in

unstimulated GCs could easily be missed compared with

stimulated samples when counting less than *1,000 cells

per sample. Therefore, induction of apoptosis in GCs may

more precisely predict oocyte quality and subsequently

outcome of IVF-ET.

Biochemical features of apoptotic GCs

As previously discussed in this review, many pro- and anti-

apoptotic proteins are involved in GC regulation. However,

few have been correlated with the oocyte developmental

potential in ART. Expression of the anti-apoptotic protein

Bcl-2 was found to be significantly higher in the pregnant

group upon IVF [186]. However, the test was not sensitive

Apoptosis (2008) 13:1065–1087 1077

123

enough to enable the use of Bcl-2 to predict pregnancy

outcome [186]. It is known that apoptosis is induced by a

variety of stimuli, including oxidative stress. Eight-

hydroxy-20-deoxyguanosine (8-OHdG) is a sensitive indi-

cator of DNA damage due to oxidative stress. A negative

correlation was found between 8-OHdG and the fertiliza-

tion rate or production of good embryos [202]. Further

studies regarding the relative expression of biochemical

markers of apoptosis are required before they can be used

as a screening method. The use of novel molecular biology

techniques, such as cDNA expression array, will help to

elucidate the activation of clusters of genes involved in the

regulation of follicular atresia, and especially the regula-

tion of apoptosis in GC.

Apoptosis-related metabolites present in FF

The composition of the FF could create a suitable micro-

environment for oocyte development and maturation [215].

Interestingly, the concentration of factors potentially

influencing apoptosis in FF have been demonstrated to

correlate with the developmental potential of the human

oocyte [216, 217].

Intrafollicular steroid concentrations have been analyzed

to detect the oocytes or embryos that have the highest

potential to proceed to each stage of development. The

results are often inconsistent because various stimulation

protocols have been employed in the different IVF institutes,

and IVF protocols have not been standardized yet [218, 219].

Estradiol and progesterone concentrations in large follicles

were significantly higher while the percentage of GCs with

nuclear fragmentation was significantly lower compared to

those in medium and small follicles. On the other hand,

testosterone concentration in small follicles containing the

highest percentage of apoptotic bodies was significantly

higher than in large follicles [220]. Another study reported

that the incidence of apoptotic bodies and the free testos-

terone level were significantly higher in the group of follicles

from which no oocytes were retrieved, while estradiol and

progesterone levels showed no difference between the two

groups. Each of the hormones could be linked with oocyte

maturity, quality or high IVF rate [185]. In two other reports,

no correlations were found between levels of steroid hor-

mones (E2, progesterone and testosterone) in FF and the

number and proportion of GCs undergoing apoptosis derived

from healthy women [189, 214]. These authors argued that

the results could be explained by the maintenance of ste-

roidogenesis observed in apoptotic GCs [221], due to the fact

that steroidogenic organelles, i.e. mitochondria and smooth

endoplasmic reticulum, remain intact during the initial

stages of apoptosis.

The expression of the insulin growth factor binding

proteins (IGFBP) has been linked to follicular atresia in

humans [222]. Accordingly, IGF ligands serve to regulate

GCs and theca cell growth and differentiation, and they are

also thought to act as antiapoptotic factors [223–225]. The

IGFBP expression profile of FF can be used to better pre-

dict oocyte developmental competence in bovines [226].

Therefore, the evaluation of IGFBP in human FF could also

be predictive of IVF-ET outcome.

Hyaluronic acid is an antiapoptotic component of the

extracellular matrix and is produced by cumulus GCs after

induction of ovulation by luteinizing hormone [227]. The

hyaluronic acid concentration in FF is affected by the

regimens of assisted reproduction stimulation [228]. A

significantly lower content of hyaluronan was found in FF

harvested from patients treated with GnRHa in combina-

tion with human menopausal gonadotropin (hMG) in

comparison to those receiving clomiphene citrate in com-

bination with hMG or with hMG alone. The hyaluronan

level in FF containing subsequently normally fertilized

oocytes was significantly lower than that in FF with

unfertilized oocytes [200]. Hence, the concentration of

hyaluronan in FF has been pointed to as an indicator for the

estimation of oocyte viability for fertilization.

Further, hyaluronic acid inhibits apoptosis of granulosa

cells via CD44 [156]. This molecule is a ubiquitous mul-

tistructural and multifunctional cell surface adhesion

molecule involved in cell-to-matrix interactions. Soluble

CD44 (sCD44) was expressed more strongly in follicles

containing oocytes that were not fertilized than in those

containing fertilized oocytes. On the other hand, shedding

of CD44 from the fertilized oocytes resulted in their

development into good-quality embryos, while the oocytes

in the follicles containing a small amount of sCD44 turned

into poor-quality embryos. A balance of CD44 on GCs and

sCD44 in FF appears, therefore, to influence the quality of

the embryos [229]. These authors suggested that levels of

sCD44 in human FF may be useful in assessing the prog-

nosis of different IVF programs.

Soluble components of the Fas-FasL system are abun-

dantly expressed in FF. Levels of the anti-apoptotic

molecule, soluble Fas, have been demonstrated to be sig-

nificantly higher in FF samples containing immature

oocytes compared with those containing atretic oocytes.

However, the similarity of the levels of soluble Fas and

FasL detected in FF among patients with or without clinical

pregnancy indicates that these soluble apoptotic factors

may not be predictive of the success of IVF [230].

Many members of the transforming growth factor (TGF-

b) superfamily are involved in controlling cellular growth

and differentiation [231]. Different research groups have

evaluated the predictive value of the inhibin subfamily in

pregnancy outcome. While one study has shown that

inhibin levels in FF are positively associated with fertil-

ization outcome and embryo quality [232], other reports

1078 Apoptosis (2008) 13:1065–1087

123

have evidenced that this compound could not predict

oocyte quality [233, 234].

In summary, oocyte quality assessment is important for

successful pregnancy during assisted reproduction proce-

dures. For now, the most convenient evaluation system for

oocyte quality is based on oocyte morphology and status of

oocytes-cumulus complexes. However, morphological

assessment could be imprecise and subjective because there

is no clear correlation between oocyte morphology and rate

of fertilization or pregnancy. Several studies have linked the

use of oocyte quality markers to the clinical outcome of

patients undergoing ART. Among these investigational

prognostic markers is the direct or indirect measurement of

apoptosis in GCs. This appears to be a valuable tool that can

be applied to more precisely predict the success of IVF/

ICSI-ET. However, results are not definitive yet and much

remains to be understood before a standard method can be

introduced in assisted reproduction.

Therapeutic options for combating premature

ovarian failure

Premature ovarian failure (POF; also known as premature

menopause) is a well-known adverse effect of cancer treat-

ment. It involves an accelerated decline in the number of

oocyte-granulosa cell units to a critical threshold. Additional

consequences associated with premature menopause are

vasomotor, psychosocial, skeletal and cardiovascular prob-

lems [235, 236]. Ovarian damage induced by cancer

treatment depends on the patient’s age, specific chemother-

apeutic agents used, irradiation field, and total doses

administered. Older women have a higher incidence of

complete ovarian failure and permanent infertility in com-

parison with younger women [237–239]. This can be

explained by the age-related decline of primordial follicle

reserve [240]. However, POF is mainly a long-term conse-

quence of successful treatment of young cancer patients

[241]. Rapidly dividing cells, such as bone marrow, gastro-

intestinal tract and thymus, do recover, but it seems that

damage to the postnatal oocyte pool, a cell-lineage arrested

in meiosis-I and thus postmitotic, is irreversible. Alkylating

agents were found to cause the highest risk of ovarian failure

[242, 243]. Patients who received both alkylating agents and

abdominal-pelvic radiation were more likely to suffer from

POF than those who did not receive combined therapy [244].

In addition, the risk of POF increases with increasing dose of

abdominal-pelvic radiation and alkylating agent’s dose [244,

245].

Therapeutic options for combating POF include assis-

ted-reproduction techniques, such as ovarian transposition

(oophoropexy) and embryo, oocyte or ovarian tissue

cryopreservation [246, 247]. Additionally, recent evidence

indicates that a major mechanism of genotoxicity induced

by cancer therapy is mediated by triggering apoptosis of

the germ stockpile [248, 249]. Therefore, one of the main

avenues of research to protect the germline from anticancer

treatments is controlled manipulation of oocyte apoptosis

[250]. Indeed, ovarian tissue can potentially be protected

in situ by administration of GnRHa in parallel with che-

motherapy. It has been shown that GnRHa administration

before cyclophosphamide treatment in rats significantly

increased the pregnancy/mating rate and the number of

implantations/mated animals [251]. Moreover, 65% of the

primordial follicle population in Rhesus monkeys was lost

following cyclophosphamide treatment, in contrast to 29%

loss in GnRHa-treated monkeys [252]. This preliminary

experience in rats and Rhesus monkeys is in line with

recent clinical results. Only 7% of patients developed POF

after co-treatment with GnRH agonist and chemotherapy,

compared to more than half of the patients in the chemo-

therapy control group [253]. Ongoing clinical trials further

reinforce the potential use of GnRHa in minimizing che-

motherapy-associated gonadotoxicity [254]. The protective

effect of GnRHa to minimize chemotherapy-associated

gonadotoxicity could be explained by their ability to

upregulate intragonadal anti-apoptotic molecules such as

sphingosine-1-phosphate (S1P) [254]. Because disruption

of the gene encoding acid sphingomyelinase in female

mice results in birth of female mice with twice as many

oocytes as wild type animals, it was hypothesized that

ceramide functions as a critical second messenger in

female germ cell apoptosis [116]. This hypothesis could be

confirmed by experiments in which the ceramide antago-

nist S1P could enhance germ cell survival in wild type

animals [255].

Notwithstanding all the above, several studies have

reported that disruption of the Bax gene or enhanced

expression of the Bax antagonist Bcl-2 in mice protects

oocytes against the effects of doxorubicin [40, 248] and

environmental toxicants [109, 256]. Additionally, oocytes

from caspase-2 and caspase-3 double knockout female

mice were more susceptible to apoptosis induced by DNA

damaging agents and, conversely, more resistant to meth-

otrexate-induced apoptosis compared to wild type oocytes

[257]. A recent study demonstrated that inactivation of the

pro-apoptotic Bax gene in mice sustains ovarian lifespan

into advanced age, extends fertile potential, and minimizes

postmenopausal related diseases [258]. Importantly, and

contrary to popular belief, in this study prolongation of

ovarian function into advanced age by Bax deficiency did

not lead to an increase in tumor incidence.

Such etiological strategies for restoring apoptotic bal-

ance might provide an elegant approach to improving

oocyte quality when undergoing cancer therapy. A better

understanding of the genes responsible for regulating

Apoptosis (2008) 13:1065–1087 1079

123

apoptosis is crucial for elucidating further the mechanisms

involved and the reasons for its occurrence. Additional

studies are needed to demonstrate the safety and efficacy in

controlled manipulation of oocyte apoptosis.

Conclusions

Precise knowledge of the signals, receptors and intracel-

lular signaling pathways leading to apoptosis of GCs is

limited. It is likely that multiple molecules are involved,

and here we sought to briefly overview these potential

actors. Many more interesting and challenging findings

concerning the molecular basis of ovarian life and death are

expected to emerge, with consequent resolution of the

controversial issue of neo-oogenesis. Further, this knowl-

edge will stimulate the development of new treatment

strategies for infertility, the improvement of strategies for

preventing chemotherapy-induced infertility, and the

understanding of normal and pathological processes lead-

ing to reproductive senescence.

Acknowledgements Dmitri V. Krysko is supported by a postdoc-

toral fellowship from the BOF (Bijzonder Onderzoeksfonds

01P05807), Ghent University. We thank Dr. Amin Bredan for editing

the manuscript.

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