K. Reynaud, M.A.

Driancourt * INRA-URA CNRS 1291,

PRMD, 37380 Nouzilly, France

Molecular and Cellular Endocrinology 163 (2000) 101–108

www.elsevier.com/locate/mce
Oocyte attrition

Received 25 June 1999; received in revised form 6 August 1999; accepted 29 September 1999

Abstract

During oogenesis, germ cell numbers sharply decrease when meiosis is initiated. There is solid evidence (DNA ladders, in situ detection)
that this loss is through apoptosis. Oocyte apoptosis appears to hit mitotic primordial germ cells (PGC), pachytene oocytes and early
primordial follicles. The control of oocyte apoptosis is not fully understood, although survival factors (LIF, kit ligand and FGF), as well as
death inducing factors (fas ligand, TGFb), have been identified. Fas ligand binding on oocytic fas may result in caspase 8 activation. Two
pathways inducing oocyte apoptosis may then be operating. In the first one, activated caspase 8 will induce activation of executioner
caspases. In the second one, activated caspase 8 will trigger the cleavage of the bcl2 family member Bid, which will act on mitochondria,
resulting in cytochrome c release, caspase 9 activation and finally, activation of all executioner caspases. As a consequence of caspase
activation, alterations in the cell nucleus (DNAse activation, PARP fragmentation), in the cell cytoskeleton (lamin) and cell metabolism will
occur, producing cell death. During folliculogenesis, germ cell loss, owing to oocyte apoptosis, has been postulated within primordial and
preantral follicles. Its regulatory mechanisms may be even more complex than those operating in foetal oocytes since additional control
factors include EGF/TGFa and bcl2 (survival) and activin (death inducer). In contrast, oocytes from antral follicles appear to be very
unsensitive to death inducing stimuli. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Oocyte; Apoptosis; Follicle

Driancourt).
1. Introduction
Females of all domestic species and primates have a
finite store of germ cells, which is established during
oogenesis and used later for folliculogenesis. Formation of
this store (i.e. the pool of primordial follicles) gener ally
occurs during foetal life as a result of a number of
overlapping events, migration of the primordial germ cells
(PGC) to the gonadal ridge, proliferation of these PGC,
initiation of meiotic prophase followed by its block to the
diplotene stage, and finally, formation of the primordial
follicles, when cells originating from the rete ovarii
surround the oocytes. Germ cell loss appears however to
occur at each of these steps. The first
* Corresponding author. Tel.: +33-2-41228265; fax: +33-2- 41228251.
E-mail address: marcantone.driancourt@intervet.okzonobel.nl (M.A.
section of this review will attempt to clarify how and why
germ cell loss occurs throughout oogenesis. Ewes and
women ovulate every 17 and 28 days, respectively. This
infrequent occurrence of ovulation contrasts with the
continuous exit from the reserve of primordial follicles (2
and 10–30 follicles initiate growth) every day in sheep and
in young women, respectively, according to Driancourt et
al. (1985), Faddy and Gosden (1995). This suggests that a
very strong tuning of the number of growing follicles
occurs throughout folliculogenesis. The respective
contribution of oocyte apoptosis versus apoptosis of
somatic cells to generate a single ovulatory follicle from a
continuous flux of growing follicles will be detailed in the
second section of this review. For clarity, only three species
which differ in the timings of their phases of oogenesis
(mouse versus sheep and humans) or in their strategies to
use their pool of primordial follicles for folliculogen esis
(mouse and sheep versus humans who display menopause)
will be considered.
0303-7207/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII:
S0303-7207(99)00246-4
102 K. Reynaud, M.A. Driancourt / Molecular and Cellular Endocrinology 163 (2000) 101–108
2.2. During folliculogenesis
2. Cell death in the ovary
In all species, the store of primordial follicles de creases
2.1. During oogenesis with time. In women, menopause is the conse quence of the
exhaustion of the pool of such follicles. Gougeon et al.
In all species, peak numbers of germ cells are ob served (1994), using mathematical modeling of the decrease in the
around the time of the mitotic to meiotic transi tion size of the primordial pool with time, have demonstrated
(Gondos, 1978). At this stage, the mean germ cell store of a that in women younger than 38-years-old, this decrease is
mouse, cow or woman ovary is 2.5×105, 2.1×106 and produced by growth initia tion and oocyte loss while, in
6.8×106 germ cells, respectively (Baker, 1963; Erickson, older women, it is solely generated by growth initiation
occurring at a faster pace.
1966; Tam and Snow, 1981). In vivo results (i.e.
visualization of ‘atretic divisions’, Baker, 1963) suggest In sheep, comparison of the population of primordial
follicles in ovaries of 2 and 8-year-old ewes has demon
that the rapid proliferation of oogonia may be associated
strated that eight primordial follicles disappear from the
with germ cell loss. Furthermore, strong evidence for germ
reserve pool every day (Driancourt et al., 1985). This
cell loss amongst the popula tion of proliferative oogonia
contrasts with the estimated rate of initiation of growth
has been obtained in vitro (Coucouvanis et al., 1993; Pesce
from the pool (two and three follicles per day) and strongly
et al., 1993). Distinc tive features of the degenerating cells
suggests that five to six follicles die every day within the
are condensed nuclei with clumps of dense chromatin
primordial pool. Since granulosa cell apoptosis is never
located at the nuclear periphery. From their peak number
visualized in such follicles, it may be assumed that oocyte
observed at E13, E80–110 and fifth month of pregnancy
death is the cause of this high death rate within primordial
(mouse, cattle and women, respectively), the number of
follicles (Fig. 1). A similar reasoning demonstrates that
germ cells sharply decreases with two main periods of high
50% of the preantral folli cles disappear through the
germ cell loss, the pachytene stage of meiosis in oocytes
preantral stage (Driancourt et al., 1985; Fig. 1).
and the formation of the primordial follicles (Gondos,
1978). As a consequence, the number of germ cells Although these mathematical considerations strongly
support the idea that oocyte death occurs within pri mordial
enclosed in primordial follicles at birth is less than 20%
and preantral follicles until recently, there were few data
(human, Baker, 1963) or less than 5% (cow, Erickson,
firmly demonstrating it. This is mainly because pycnotic
1966) of its peak number. This is solid evidence demon
bodies, the most common marker used to
strating that the normal fate for a female germ cell during
oogenesis is death.

Fig. 1. Importance of germ cell loss within the pool of primordial follicles and the preantral follicles. These data derive from the counts of follicles in sheep
ovaries at two ages (Driancourt et al., 1985).
K. Reynaud, M.A. Driancourt / Molecular and Cellular Endocrinology 163 (2000) 101–108 103

identify a dying cell, are seldom visualized in oocytes. In
addition, because the process of oocyte death ap pears to be
fast (see below), the chances of picking up a small fraction
of dying oocytes within several thou sand and several
hundred of primordial and preantral follicles, respectively,
are rather limited. However, re cent experiments describing
the consequences on the ovary of targeted expression of
bcl2 in mouse oocytes (increased number of healthy
preantral follicles and decreased number of atretic preantral
follicles, Morita et al., 1999a), have provided the first solid
evidence demonstrating this event. Furthermore, the
observation that bax -/- mice have increased numbers of
primary/ preantral follicles when mature (Perez et al.,
1999) also demonstrates that apoptosis is a key regulatory
process of primary/preantral numbers.
Interestingly, once an antrum has appeared within a
follicle, there is a clear shift in the respective sensitivi ties
to cell death of germ cells and somatic cells. This coincides
with the disappearance of DNAse I in the oocyte (Boone
and Tsang, 1997). Hence, the final tun ing of the number of
growing follicles to a single ovulatory follicle (cow,
women) is primarily produced by somatic cell death
(reviews, Hsueh et al., 1994; Tilly, 1996). At the large
antral stage, it is quite puzzling to note that, at least in
cattle, the oocyte is very resistant to death inducing factors
as demonstrated by the ability of oocytes contained in
atretic follicles to develop to the blastocyst stage following
in vitro maturation, fertiliza tion and culture
(IVM/IVF/IVC, Blondin and Sirard, 1995).
3. Oocyte apoptosis during oogenesis
3.1. E6idence that germ cell loss is through apoptosis
Solid evidence supporting this claim has been ob tained
in mice using three approaches. Firstly, Coucou vanis et al.
(1993) used flow cytometry to identify (by their small size
and reduced DNA content) a popula tion of apoptotic cells
within foetal mouse ovaries. Some apoptotic cells were
identified at E13 (4.3% of the total cells) and their
frequency increased to reach 9– 11% of the total cells at
E15 and E17. Secondly, in a more recent study (Ratts et al.,
1995), detection of DNA ladders was achieved on mouse
foetal ovaries, starting at E13.5. Apoptosis is associated
with activa tion of endonuclease activity with specifically
cleaves cellular DNA between regularly spaced
nucleosomal units (180–200 bp). Such oligonucleosomal
fragments, when separated by agarose gel electrophoresis,
form a distinctive DNA ladder pattern. While absent in
E13.5 ovaries, obvious DNA ladders, assumed to be
produced by oocyte apoptosis were visualized at E15.5,
E18.5 and birth (Ratts et al., 1995). A major drawback of
this
method is that it does not allow the in situ determina tion of
104 K. Reynaud, M.A. Driancourt / Molecular and Cellular Endocrinology 163 (2000) 101–108
Table 1
What can be learned from survival of PGC and gonocytes PGC
which cell type is apoptotic. This is why in situ
identification of apoptotic cells was achieved in recent
studies (De Pol et al., 1997; Driancourt et al., 1997) using
terminal deoxynucleotidyl transferase (TdT) me diated
dUTP nick end labeling (TUNEL) method. This procedure
uses TdT to catalyze addition of residues of
digoxigenin-nucleotide to 3% OH ends of fragmented DNA
generated by internucleosomal cleavage. The apoptotic
cells are then visualized by an anti-digoxi genin antibody
labeled with peroxydase.
Using this approach, De Pol et al. (1997) have shown
that apoptosis in human foetuses 18-weeks-old is re stricted
to germ cells, and that around 8% of them are apoptotic at
this stage. Furthermore, Driancourt et al. (1997), using
observations of TUNEL stained sections of foetal mouse
ovaries (E15.5, E17.5 and birth), have characterized
changes in apoptosis throughout foetal life in mice and
demonstrated a steady decrease in the frequency of
apoptotic oocytes from 3.691.0 at E15.5, to 2.490.7 and
1.790.6% at E17.5 and at birth, respectively.
Since Morita and Tilly (1999) have shown that germ cell
apoptosis is absent in E13.5 foetal gonads, the above
changes in the frequency of apoptotic germ cells suggest
that the highest risk of death for female germ cells is
throughout the meiotic prophase (E13–E16, according to
Borum, 1961), with a more limited risk occurring at the
time of primordial follicle formation (E18 to birth,
according to Borum, 1961).
The rather limited frequency of apoptotic oocytes
estimated in these studies sharply contrasts with the
magnitude of the germ cell loss observed during oogen esis
(see above). Two hypotheses can be put forward to
reconcile these two findings. Either germ cell apoptosis is
not the only way contributing to the decrease in the number
of germ cells. Some authors (Gondos, 1978) have claimed
that germ cells may leave the ovary by traveling through the
ovarian epithelium. Alternatively, germ cell apoptosis may
be a very rapid process, with the TUNEL positive oocytes
being rapidly destroyed. At least two lines of evidence
support this last hypothe sis. Firstly, induction of apoptosis
by oxidative stress in vitro results in the appearance of an
increased propor tion of TUNEL positive oocytes as soon
as 6 h follow ing treatment (Reynaud and Driancourt,
unpublished). Secondly, the pioneering studies of Morita et
al. (1999a,b) have demonstrated that in vitro culture of
foetal ovaries in the absence of specific growth factors
results in massive oocyte loss with a 50% drop in oocyte
numbers at 48 h and a 90% drop at 72 h of culture. Finally,
it has been reported (Gumienny et al., 1999) that germ cells
of Coenorhabditis elegans die by apop tosis and disappear
within 1 h.
Gonocytes
Sur6i6al factors
SCF, acts on survival (Dolci et SCF, survival factor al., 1991;
 Godin et al., 1991; (Yoshinaga et al., 1991) Pesce et al.,
1993) and its
receptor c-kit is present
LIF, increases survival (Dolci et LIF, survival factor (De al.,
1993; Pesce et al., 1993) Miguel et al., 1996) and its receptor
is present
(Cheng et al., 1994)
FGF2 FGF mitogenic and/or survival , survival factor (Van factor
(Resnick et al., 1992) Dissel-Emiliani et al., 1996) and FGF
receptors are
present on PGC’s (Resnick et
al., 1998)
IL4, survival factor (Cooke et
al., 1996)
Death factors
? Fas/fas L, death inducing factors (Lee et al., 1997)
? TGFb1 and TGFb2, death inducing factors (Olaso et al.,
1998)
3.2. Candidates for the control of oocyte apoptosis in
oogonia/PGC
Two approaches have been used to provide informa tion
in this field. Most of the data reported below have been
obtained in vitro after addition of compounds present in
young foetal gonads to cultured PGC. These data were
partly extended by observing the extent of apoptosis in
foetuses harboring specific gene manipulations.
Extensive data on apoptosis of PGC/mitotic oogonia
(mostly obtained in vitro) and some useful information on
apoptosis of testicular gonocytes are available (Table 1).
Strong evidence identifying three families (kit/kit ligand;
LIF and its receptor; FGF and its receptor) as survival
factors have been obtained both from the studies on PGC
and testicular gonocytes. Whether expression of the
receptor binding a specific compound and its ligand occur
on the same cell (in tracrine regulation) or involves
interactions between cells (juxtacrine regulation) is not yet
known. Further more, the partial additivity of LIF and kit
ligand effects on young PGC cultured for 3 days (Dolci et
al., 1993) may indicate that survival signals may differ from
cell-to-cell.
Interestingly, testicular gonocytes have also provided
information on death inducing compounds (Table 1). Both
TGFb and fas ligand, through the interaction with their
respective receptors, appear to be inimical to gonocyte
survival (Lee et al., 1997; Olaso et al., 1998).
It is generally agreed that, within a specific cell, the
balance between members of two families present within
mitochondria (survival, bcl2+bclXL; death, bax+bclXS) is a
key intracellular modulator of death or survival factors.
K. Reynaud, M.A. Driancourt / Molecular and Cellular Endocrinology 163 (2000) 101–108 105
late oogenesis (Reynaud and Driancourt, unpublished),
although evidence that these oocytes are TUNEL posi tive
is still missing. Reports on the detection of other caspases
in foetal ovaries are not yet available. Only limited data
support a possible modulating effect of bcl2 family members
on oocyte apoptosis. Bax positive oocytes can be detected
in foetal gonads during late oogenesis (Reynaud and
Driancourt, unpublished) but it is quite puzzling to observe
Support for a positive role of bclXL is suggested by the
observations showing that transfec tion of PGC with a bclXL
containing construction pro motes their survival (Watanabe
et al., 1997). Since bax KO mice (Perez et al., 1999) and
bcl2 KO mice (Ratts et al., 1995) have been generated, the
observation of their gonads, when at the PGC stage, will
provide informa tion on the effect of these two compounds
on PGC apoptosis. This has yet to be performed.
3.3. Candidates for the control of apoptosis throughout
late oogenesis (E14 to birth)
The same approaches as before (in vitro culture with
compounds known to be present at this stage, gene
knock-out) have been used to identify relevant candi dates.
Extensive information suggesting a role of the kit–kit ligand
interaction is available. With the excep tion of E16 when
neither the mRNA nor the kit protein are present, numerous
data (Horie et al., 1991; Keshet et al., 1991; Manova et al.,
1993; Yoshida et al., 1997) demonstrate that the kit–kit
ligand interaction may be functional. In addition, the recent
results of Morita et al. (1999b), who cultured E13 foetal
ovaries for 3 days, have provided evidence that kit ligand,
in the presence of LIF, is a survival factor for oocytes.
Positive effects of LIF, retinoic acid and Insulin like growth
factor-1 have been reported on oocyte survival by the same
group (Morita and Tilly, 1999; Morita et al., 1999a,b), but
these data should be considered preliminary since it is
unclear whether the concentrations used in vitro are
physiologically relevant. For example, it is unknown
whether foetal ovaries have the ability to produce IGF
binding proteins regulating IGF-1 bioavailability. On the
same line, negative effects of TGFb on oocyte survival have
been reported (Morita et al., 1999b) but it is unknown
whether activation of TGFb from its latent form may occur
in foetal gonads. As regards the fas/fas ligand interaction,
we (Reynaud and Driancourt, un published) have recently
immunolocalized fas and fas ligand in foetal gonads
between E15 and birth, the proportion of fas ligand positive
cells being similar to the proportion of TUNEL positive
cells. Furthermore, addition of 50 ng/ml fas ligand to
cultured E16 ovaries produced an increased proportion of
apoptotic germ cells.
The intracellular mechanisms modulating life and death
have been partly unraveled through the findings obtained
following gene knock-out. For example, cas pase 2
knock-out results in increased numbers of pri mordial
follicles present at birth (Bergeron et al., 1998). Caspase 3
is also present in some oocytes throughout
that bax knock-out does not affect the size of the primordial
was
follicle pool at birth (Perez et al., 1999). Bcl2
undetectable by immuno
histochemistry on foetal gonads,
while the presence of both forms of bclX (bclXL and bclXS) is
not yet documented.
3.4. Oocyte apoptosis during late oogenesis: which
mechanisms are operating?
Since apoptosis appears to be a well conserved mech
anism (Gumienny et al., 1999), the data summarized above,
together with data available on fas-mediated apopotosis (Li
et al., 1998; Luo et al., 1998), have been used to generate a
working hypothesis (Fig. 2). Apopto sis in oocytes would
occur as an interaction between fas and fas ligand. This
interaction produces activation of caspase 8, which is the
apical caspase in the pathway. Active caspase 8 results in
activation of Bid, a bcl2 family member, and its
translocation from the cytosol to the mitochondria. Active
Bid has the ability to induce complete release of
cytochrome c from mito chondria. In addition, Bid may
inactivate anti apop
totic members of the bcl2 family (particularly bclXL) by
dimerization. The release of mitochondrial cytochrome c
may produce caspase 9 activation, followed by cas pase 3
activation, an event resulting in morphological and
functional changes typical of apoptosis. This in cludes: (1)
alterations within the nucleus occurring as a consequence
of DNAse I activation, PARP fragmenta tion and U1-70 k
inhibition; (2) disassembly of cell structure by cleavage of
Fig. 2. Schematic drawing of the apoptotic cascade in foetal oocytes. For details, see text.
106 K. Reynaud, M.A. Driancourt / Molecular and Cellular Endocrinology 163 (2000) 101–108
al., 1999). Amongst the compounds appearing in pri
mordial oocytes, around growth initiation, GDF-9, fol
listatin and activin A can be listed. In addition, at least in
hamsters and pig, EGF/TGFa has been localized to oocytes
of primordial/primary follicles (McNatty et al., 1999).
Inhibin A becomes detectable in oocytes at the preantral
stage.
Most of the compounds which were already operat ing in
foetal oocytes are still present in the postnatal ovary. Solid
evidence demonstrating the presence of kit and kit ligand
(Manova et al., 1993), FGF and its receptor (McNatty et al.,
cytoskeleton proteins and (3) disruption of cell energy
metabolism. Heat shock proteins, particularly hsp90 which
is present in large amounts in oocytes (Ohsako et al., 1995),
through their chaperone effects, may be involved at this
stage to control the magnitude of the proteolytic effects of
caspases.
This summary figure should be considered prelimi nary
since there is no information at present on Bid or caspase 8
in foetal oocytes. The identification of the site(s) of action
of survival factors (block in caspase activation, increased
amounts of mitochondrial bclXL…) also requires further
investigations.
4. Oocyte apoptosis during folliculogenesis
4.1. Follicle/oocyte function in primordial, primary and
preantral follicles
Changes in gene expression associated with initiation of
follicular growth up to the preantral stage have been
summarized recently in an excellent review (McNatty et
1999) and TGFb and its receptor in primordial to preantral
follicles is available. Furthermore, fas mediated oocyte
apoptosis is also likely to occur since there are reports of
fas ligand (Hakuno et al., 1996) and possibly fas (Kondo et
al., 1996) expression in oocytes (by
immunohistochemistry). Furthermore, Lpr mice that
express little fas antigen due to a mutation have increased
numbers of secondary follicles (Sakamaki et al., 1997; Xu
et al., 1998). Com ponents of the TNFa signaling pathway
also appear to be present in oocytes of preantral follicles
(Cheng et al., 1993). Caspase 3 appears in contrast to be
present in oocytes of immature gonadotrophin treated rats
(Boone and Tsang, 1998)
There is also solid evidence that members of the bcl2
family (bcl2, bclXL, bax…) play a key role in oocyte
apoptosis in primordial to preantral follicles. Such a claim
is supported by the increased numbers of such follicles
following bax knock-out (Perez et al., 1999) and by the
decreased numbers of primordial follicles in bcl2 knock-out
mice (Ratts et al., 1995). Caspase 1 and bclXS may not be
important as their expression was not detectable in fully
grown oocytes (Jurisicova et al., 1998).
4.2. Which of these new compounds are rele6ant to
oocyte sur6i6al/death?
At least two main strategies (i.e. knock-out of specific
genes and in vitro culture) have been fruitful to identify
relevant compounds amongst those identified above.
Firstly, the generation of transgenic mice which dis
played defects/blocks in follicular growth demonstrated that
GDF-9 (Dong et al., 1996) and follistatin (Guo et al., 1998)
were likely to be involved in the control of survival of
primary/preantral follicles or in the control of cell
proliferation in such follicles. In vitro culture systems were
used to check which of these two hypothe ses was valid.
They demonstrated that the primary role of GDF-9 was
mostly on cell proliferation (Hayashi et al., 1999). As
regards follistatin, there are no in vitro data analyzing its
effect on preantral follicles. But one of the proteins bound
by follistatin, activin, has detri mental effects on follicle
survival in vitro (Smitz et al., 1998).
Secondly, the development of culture systems for
preantral follicles of a range of species (Eppig and O’Brien,
1996; Wandji et al., 1996; Smitz and Cortvrindt, 1998) has
been useful to check which growth factors/proteins present
in preantral follicles could modulate their survival. The
most comprehensive study (Wandji et al., 1996) established
that EGF and FGF were survival factors while TGFb had a
marked detrimental effect on survival. Good evidence
support ing the relevance of the kit–kit ligand interaction
for the survival of the preantral follicles has been reported
by Yoshida et al. (1997) who demonstrated a large and
quick drop in the population of preantral follicles fol
K. Reynaud, M.A. Driancourt / Molecular and Cellular Endocrinology 163 (2000) 101–108 107
systems for primordial, preantral follicles from primates
and domestic animals; and possibly (3) development of new
strategies to manage the pool of primordial folli cles to
postpone menopause.
Acknowledgements
We kindly acknowledge J. Smitz and R. Cortvrindt for D.L., Tsang, B.K., 1998. Caspase 3 in the rat ovary: localiza tion and
exciting discussions and efficient collaboration and J.J. possible role in follicular atresia and luteal regression. Biol. Reprod. 58,
Panthier and F. Bernex for provision of the WlacZ/ +mice.
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