Human Reproduction vol.12 no.10 pp.2235–2241, 1997
Apoptosis of germ cells during human prenatal
oogenesis
A.De Pol1, F.Vaccina, A.Forabosco, E.Cavazzuti and
L.Marzona
Dipartimento di Scienze Morfologiche e Medico Legali, Sezione di
Istologia – Embriologia e Genetica, Università di Modena,
via del Pozzo 71, 41100 Modena, Italy
1To
whom correspondence should be addressed
During human oogenesis two contrasting processes can be
observed: germ cell proliferation and differentiation, and
contemporaneous germ cell death. It is well known that
apoptosis is a type of physiological cell death that occurs
in proliferating and differentiating tissues. The aim of this
study is to demonstrate, through ultrastructural and insitu 39 end labelling observations in intact sections of
human fetal ovaries, that germ cell loss during fetal life is
due to a phenomenon of apoptosis. We evaluated the
presence of programmed cell death in female germ cells in
fetal ovaries at 18–20 weeks of postconceptional age. The
alterations that occur during apoptosis were detected by the
electron microscope and include cytoplasmic condensation,
organelle relocalization and compaction, chromatin condensation, and finally, production of membrane-enclosed
particles containing intracellular material, known as apoptotic bodies, that are phagocytosed. The fragmentation of
DNA, characteristic of apoptotic cells, was detected by the
use of the in-situ 39 end labelling procedure on histological
sections of ovaries where only some nuclei of germ cells
were positively stained. The parallel use of these two
methods on human ovaries of 18–20 weeks postconceptional
age has allowed us to show that the numerical decrease of
human female germ cells during the fetal period is due to
an apoptotic phenomenon.
Key words: apoptosis/germ cells/human oogenesis/ultrastructure
Introduction
During oogenesis, two contrasting processes can be observed:
the first consists of intense proliferative and differentiative
activity of the oogonia; the second consists of cellular loss.
This antithetic phenomenon concerns the germ cells at various
stages of oogenesis with species-specific mechanisms and
modalities (Gondos, 1978). At the end of this process in human
there is, however, a large reduction of the number of germ
cells during the reproductive life and an even greater reduction
during the fetal period.
In the postpubertal period the loss of germ cells appears to
be due mostly to regressive phenomena induced by cell death
© European Society for Human Reproduction and Embryology
through apoptosis of the follicular cells (follicular atresia)
(Tilly et al., 1991; Billig et al., 1993; Chun et al., 1994;
Palumbo and Yeh, 1994; Billig et al., 1996; D’Herde et al.,
1996; Hakuno et al., 1996).
In the fetal period, on the other hand, the oogonia proceed
to an active proliferation, which brings the germinal population
of both ovaries up to 5–7 3106 at around the 5th month of
fetal life (Baker, 1963). Subsequently, the germinal component
itself begins to decrease so sharply that the number drops to
about 700 000 oocytes in follicle at birth (Block, 1953; Baker,
1963; Forabosco et al., 1991). This loss occurs by direct germ
cell death (attrition).
How this drastic reduction comes about has been a subject
for study for a long time (Baker, 1963; Gondos, 1978; Makabe
et al., 1991). During embryonic and postnatal development,
the elimination of single cells or groups of cells seems to be
a phenomenon of vital importance (Hinchliffe, 1981; Raff,
1992; Raff et al., 1993). This elimination occurs by the
activation of a specific programme of self-destruction, or
‘programmed cell death’, which is connected with morphological and biochemical changes and has been called apoptosis
(Wyllie et al., 1980; Arends and Wyllie, 1991).
Apoptosis was originally defined on the basis of the morphological changes undergone by the cells concerned with this
phenomenon (Kerr et al., 1972). In the initial stages the cell
contracts, the nuclear chromatin condenses and may break
into smaller masses. The plasma membrane maintains its
physiological characteristics, as do the majority of the cellular
organelles. Later the cell breaks up into fragments surrounded
by a membrane which is then phagocytosed and eliminated
(Duvall et al., 1985; Clarke, 1990). During the apoptotic
process it seems that the endonucleases play a fundamental
role by cutting the DNA into oligonucleosomes of 180–200
base pairs or multiples (Wyllie, 1980; Arends et al., 1990;
Peitsch et al., 1993).
In the light of this knowledge we wanted to verify whether
the apoptotic process exists and plays a fundamental role in
the great numerical loss of human female germ cells. For this
purpose we used human ovaries at around 18–20 weeks of
postconceptional life. At this time there is the greatest number
of germ cells at the various stages of meiotic prophase I,
together with oogonia in active mitotic multiplication, and at
the same time a fairly high percentage of germ cells in
degeneration (Baker, 1963; Kurilo, 1981).
Materials and methods
The study was carried out on ten ovaries from fetuses at 18–20 weeks
of postconceptional age with normal karyotype. The fetuses were
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A.De Pol et al.
Figure 1. Semithin sections of fetal human ovary. (A) Ovarian cortex with sex cords (sc) and stroma (st). At the bottom the ovarian medulla
(m) begins (original magnification 3125). (B) Sex cords with some degenerating oocytes (arrows). G 5 germ cells; S 5 somatic cells.
Bar 5 40 µm (original magnification 3200).
obtained by spontaneous and therapeutic abortions for maternal
diseases. The postconceptional age of the fetuses was estimated by
measuring their crown–heel length, their foot length and their weight;
the length of the maternal menstrual cycle and the date of the last
menstrual period were also taken into account. The specimens were
obtained for the study of the karyotype and in conformity with Italian
law (with reference to DPR 10/9/1990 N. 285).
Histological analysis
Seven ovaries were fixed in 2.5% glutaraldehyde in Tyrode’s buffer,
pH 7.4 for 24 h at 4°C, postfixed in 1% osmium tetroxide in the
same buffer for 2 h at 4°C, dehydrated through graded concentrations
of ethanol and embedded in toto in epoxide resin. For light microscopy,
semithin sections (1 µm) were stained with Toluidine Blue. For
electron microscopy, thin sections (98 nm), mounted on grids, were
stained with uranyl acetate and lead citrate and examined with a
Zeiss 109 electron microscope.
In-situ end labelling analysis
The remaining ovaries were fixed in 4% buffered formaldehyde at
4°C, embedded in paraffin by the standard method, cut by microtome
into 5-mm-thick sections, and mounted on silanized slides. After
being deparaffinized and rehydrated, they were then treated in
a humidified chamber with proteinase K (Boehringer, Mannheim,
Germany) (20 µg/ml) for 15 min at room temperature, washed in
double-distilled water, treated with 2% H2O2 in methanol for 10 min
at room temperature and then washed in double-distilled water.
The slides were preincubated with terminal deoxynucleotidyl transferase (TdT) buffer and 1 mM CoCl2 for 5 min at room temperature
and then incubated for 60 min in a humidified chamber at 37°C with
50 µl TdT and biotinylated deoxyuridine triphosphate (Bio dUTP)
(Boehringer) (TdT 0.3 U/µl and Bio dUTP 8 µM in TdT buffer and
CoCl2 1 mM). Sections were washed in bidistilled apyrogen water
(four times for 2 min), phosphate-buffered saline (PBS) (twice for
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5 min), human serum albumin 2% for 5 min, and PBS for 5
min then covered with streptavidin–biotinylated peroxidase complex
(Boehringer) diluted 1:100 in a humidified chamber for 45 min at
room temperature, washed in PBS and stained with diaminobenzidine
50 mM (0.05%). The slides were then washed in water and counterstained with haematoxylin for 1 min and mounted.
Positive and negative controls were included in each experiment. For
the positive controls, sections were treated with DNase I (1 µg/ml)
(Boehringer) in DNase buffer for 10 min at room temperature before
exposure to biotinylated dUTP and TdT. For negative controls, the
sections were incubated without the TdT enzyme.
Results
In the ovaries at 18–20 weeks of postconceptional age the
cortex is composed of sex cords and clusters made up of
somatic and germ cells, oogonia and oocytes in the different
stages of the first meiotic prophase. At the boundary with the
medulla the primordial follicles, formed of diplotene oocytes
and surrounded by a layer of flattened somatic cells, begin to
break away from the clusters. Sex cords, clusters and follicles
are surrounded and separated one from another by the stroma
composed of mesenchymal tissue.
The medulla is formed of mesenchymal tissue, richly
vascularized, which is continuous with the cortical stroma
(Figure 1A).
The germ cells can be distinguished from the somatic cells
on account of their larger size, their shape (which is roughly
rounded compared with the more flattened shape of the somatic
elements) and the morphology of the mitochondria, which are
always globular in the germ cells but lengthened in the somatic
cells (see below). The morphology of the nucleus is different
Apoptosis of germ cells during oogenesis
too: in the germ cells it is rounded, with chromatin that is
thickened and distributed in different ways according to the
meiotic phase, while in the somatic cells the nucleus is oval
with an irregular outline. Among the germ cells, some elements
that present nuclear or cytoplasmic alterations are recognizable,
but with the light microscope only instances of germ cells
with evident and widespread alteration can be defined.
These germ cells in degeneration can be found in different
parts of the ovary, from the superficial epithelium down to the
deepest cortical layers, mainly in sex cords outside of the
primordial follicles. Folliculogenesis has just started.
The alterations that can be seen with the light microscope
concern mainly the nucleus. At first some irregularities can be
observed in the outline of the nucleus, such as indentations
that are more or less pronounced. Subsequently the chromatin
thickens considerably, and the nucleus decreases in size or
breaks into fragments composed of irregular highly heterochromatic masses (Figure 1B). Cytoplasmic alterations do not
appear to be particularly evident with the light microscope.
Examination of sections treated with the technique of insitu end labelling showed positivity for the reaction only in
9% of germ cell nuclei (Figure 2A). The positive controls
showed a uniform staining of all the nuclei (Figure 2B). The
negative controls carried out by omitting the TdT showed a
completely negative reaction (Figure 2C).
Examination at the ultrastructural level of the degenerating
germ cells allows us to point out alterations mainly of the
nucleus, even if in many cases cytoplasmic structures were
also involved. Different stages of condensation and alteration
of the chromatin can also be observed.
In Figure 3A the germ cell preserves its rounded form. The
cytoplasm presents the typical appearance of a cell in meiotic
prophase with some elements of rough endoplasmic reticulum
and mitochondria; the nuclear envelope has a very irregular
contour with protrusions and indentations that give the nucleus
a corolla-like appearance. Chromatin masses are enveloped in
the protrusions of the nucleus, and two central masses, probably
corresponding to the nucleoli, can be clearly identified.
The cell in Figure 3B has also undergone a degenerative
process that seems more advanced. The morphology of the
nucleus, shows a body composed of electron-dense masses
that have moved slightly closer to one another and that have
become even thicker.
In a more advanced stage of alteration (Figure 3C) the
nucleus has very thick chromatin with masses that are very
close to one another, and it maintains a rounded form. In
the cytoplasm, it is possible to observe rough endoplasmic
reticulum, mitochondria and some big vacuoles.
The morphology of these cells and of the chromatin masses,
however, is variable. It is possible to go from a high degree
of condensation and fragmentation to a much lower one. The
cytoplasm can also show considerable preservation of the
organelles in some cells, whereas in others it is possible
to note that it is already greatly altered with considerable
vacuolization. What allows us to identify with certainty these
elements as germ cells is the form and the dimension of the
cells and also the roundish appearance of the mitochondria,
which is specific to germ cells.
Figure 2. (A) In situ 39 end-labelling of germ cells from human
fetal ovary. Labelled cells exhibit a brownish color. (B) Positive
control. (C) Negative control obtained by omitting the enzyme
terminal deoxynucleotidyl transferase. Bar 5 50 µm (original
magnification 3240).
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A.De Pol et al.
Figure 3. Transmission electron microscope appearance of germ cells during early and late phases of apoptosis. O 5 normal oocytes;
S 5 somatic cells; nu 5 nucleolus; m 5 mitochondria; V 5 vacuoles. (A and B) Degenerating germ cells with typical features of the initial
stages of apoptosis: rounded cellular shape, morphologically intact cytoplasmic organelles, aggregates of compacted chromatin along the
nuclear membrane (original magnification 37200). (C) The nucleus exhibits a complete condensation while the cytoplasmic organelles are
still preserved (original magnification 37200). (D) Germ cell in advanced apoptosis which shows progressing degeneration of its
cytoplasmic elements. Bar 5 1 mm (original magnification 38000).
The next step in cellular degeneration might be represented
in Figure 3D, in which the chromatin appears highly condensed
in a single mass while the cytoplasm is completely altered.
In Figure 4A a cellular element of very small dimensions
and extremely electron-dense can be observed. The particular
arrangement of the somatic cells around this cell makes us
suppose that it is a germ cell at the beginning of the primordial
follicle stage.
Sometimes inside the ovarian parenchyma some macrophages can be found, situated on the inside of follicle-like
formations. A macrophage cell is identified by the nuclear shape
and the evident apoptotic masses that have been phagocytosed:
probably the macrophage has migrated from the surrounding
vessels and has replaced an oocyte in advanced apoptosis,
phagocytosing it (Figure 4B).
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Discussion
Multiplication, migration, differentiation and cellular death
constitute the most important stages in the developmental
processes and in the turnover of tissues and organs. Cellular
death can happen in two ways: necrosis or apoptosis (Wyllie
et al., 1980; Kressel and Groscurth, 1994). Necrosis is the
result of injuries or environmental pathological influences, and
produces a series of cellular alterations that begin with the
alteration of permeability of the cellular membrane, consequent
disruption of cytoplasmic structures and ensuing nuclear
degeneration. Apoptosis on the other hand is a process of
cellular self-destruction which also involves active processes
of intracellular synthesis (Wyllie et al., 1980; Cohen and Duke,
1984), and it is controlled by cellular genes. In this kind of
cell death the primary event is the nuclear compaction: the
Apoptosis of germ cells during oogenesis
Figure 4. (A) Apoptotic germ cell (G), displaying an increase in
condensation, partially enveloped by somatic cells (S) (original
magnification 34800). (B) Apoptotic bodies (A) phagocytosed by a
macrophagic cell (arrow) recognizable from the nuclear shape.
Bar 5 2 µm (original magnification 34800).
chromatin collapses into large irregular masses surrounded by
the nuclear envelope (Kerr et al., 1972), and the plasma
membrane introflexes forming deep incisions that confer to
the cell a very irregular appearance. Despite this, the cellular
permeability remains unaltered at the beginning and the organelles maintain their morpho-functional integrity. Subsequently
the cell fragments into spheroidal sub-unities surrounded
by membranes (apoptotic bodies) that contain portions of
cytoplasm and nucleus that are finally phagocytosed by neighbouring cells or by macrophages (Duvall et al., 1985; Williams
and Smith, 1993).
The two kinds of cellular death are therefore distinguishable
mainly on account of different ultrastructural morphology and,
at the biochemical level, on account of different behaviour of
the chromatin: that in necrosis is altered at the end and in a
disorderly fashion, during apoptosis it is precociously cut in a
regular sequence.
The existence of an apoptotic process in the germinal
component of the gonads has been described in the primordial
germ cells of the mouse by Pesce and De Felici (1994).
Recently the phenomenon of germ cell death by apoptosis in
the prespermatogenesis of the golden hamster (Miething, 1992)
and in the spermatogenesis of different vertebrates has been
described (Allan et al., 1992; Henriksen et al., 1995; BlancoRodriguez and Martinez-Garcia, 1996). As far as the germinal
female population is concerned, however, the phenomenon of
apoptosis has been described only in the mouse during prenatal
life (Coucouvanis et al., 1993) and more recently in postnatal
life in ovulated oocytes (Fujino et al., 1996).
It has already been reported by those who have investigated
the process of human oogenesis that the female germ cells
undergo a process of great reduction during the course of
prenatal and postnatal oogenesis (Baker, 1963; Baker and
Franchi, 1967). In their study on the number of germ cells
during prenatal development, they have noticed the presence
of numerous oogonia and oocytes in degeneration. Our preliminary observations indicate that the percentage of germ cell
loss is between 18 and 20 postconceptional weeks ~10% of
all germ cells, which is near to the value already reported at
this age by Baker (1963) and Baker and Franchi (1967). This
direct germ cell death is the most important mechanism of
prenatal germ cell loss, and is a different phenomenon from
follicular atresia.
The loss of germ cell population by follicular atresia, due
to apoptotic death of follicular somatic cells, begins later,
shortly before birth when follicles start to grow (Van Wagenen
and Simpson, 1965) and in women is probably the commonest
mechanism of postnatal germ cell loss (Billig et al., 1996).
Human germ cells with characteristic degenerating features
have already been seen and described by those investigating
prenatal oogenesis. Baker and Franchi (1967), Gondos et al.
(1971) and Gondos (1973) described some germ cells in
degeneration and showed some images similar to those reported
by us.
The morphological data presented in this paper show how
in the human ovary during the process of prenatal oogenesis
some germ cells have the ultrastructural features of the
apoptotic cells already described for other cells. The germ cells
found by us in the human ovary have the same characteristics as
the apoptotic cells described elsewhere, both on account of
the chromatin condensation and on account of the state of
preservation of the cytoplasmic organelles. The chromatin
condensation along the nuclear envelope, shown in Figure 3A,
is a morphological feature characteristic of the first stages of
apoptosis. Nuclear fragmentation is frequently observed
(Figure 3B) while blebs in germ cells are rarely observed.
More frequent is the presence of a small, very compact nucleus
contained in a cytoplasm that presents well preserved organelles
(Figure 3C). More difficult to define are the morphological
changes in Figure 3D, that on the one hand show a remarkable
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A.De Pol et al.
nuclear condensation whose morphology is identical to the
element of the previous picture, while on the other hand show
pronounced cytoplasmic alterations including disruption of
the mitochondrial cristae. Analogous pictures were found in
prespermatogonia of the golden hamster by Miething (1992)
and were interpreted as a variant of necrosis or as a very
advanced apoptosis. To us the second hypothesis seems more
likely since in human ovaries of this postconceptional age we
have never found germ cells that were clearly in necrosis.
The removal of degenerating cellular material by phagocytosing cells is a finding that is quite frequent in the human
ovary and is considered one of the most diagnostic features
of apoptotic cell death (McConkey et al., 1996). Phagocytosing
cells can be clearly identifiable as macrophages or can be
somatic cells that participate in the formation of the sex cords,
and they are disposed around the altered germ cells with an
arrangement like follicular cells in the primordial follicle.
Another important parameter for the evaluation of apoptosis,
after the morphological one, is DNA cleavage. Wyllie (1980)
has shown that there is a correlation between the characteristic
morphology of apoptosis and fragmentation of the DNA into
oligonucleosomes of 180–200 bp or multiples of these.
With the technique of in-situ 39 end labelling terminal
transferase (Gavrieli et al., 1992) used on histological sections
in the present study, we have pointed out how, in the human
prenatal ovary during oogenesis, there are several germ cells
positively marked. Evidently, in human female germ cells, as
in other cells of the mammalian ovary (Zeleznik et al.,
1989), there is a specific endonuclease responsible for the
fragmentation of the DNA.
However, for a sure identification of apoptotic cell death in
tissues, the demonstration of DNA fragmentation is not sufficient because chromatin dissolution also affects the necrotic
cells (Kressel and Groscurth, 1994; Grazil-Kraupp et al.,
1995). Thus in-situ 39 end labelling technique, in combination
with a morphological demonstration at ultrastructural level,
allows us to distinguish clearly between the two cases.
The parallel use we have made of techniques allowing us
to observe the structural changes taking place in the human
female germ cells that go on to degeneration, together with
methods that use in-situ end labelling, have provided the first
evidence for a direct link between human ovarian germ cell
degeneration and programmed cell death. In human germ cells,
too, apoptosis is present. In the prenatal human ovary apoptosis
is the counterpart of mitosis, regulating the number of female
germ cells.
Acknowledgement
This study was supported by MURST 60% grants.
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Received on April 23, 1997; accepted on July 29, 1997
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