| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Medicine, Flinders University of South Australia (I.L.V.W., T.C.L., R.J.R.), South Australia 5042; and the Department of Anatomy and Human Biology, University of Western Australia (A.M.D.), Nedlands, Western Australia 6907, Australia
Address all correspondence and requests for reprints to: Dr. Raymond J. Rodgers, Department of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5042, Australia. E-mail: ray.rodgers{at}flinders.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cells generally die by one of three mechanisms: apoptosis, necrosis, or terminal differentiation leading to cell death, as occurs in the skin during keratinization or in erythropoiesis. In apoptosis, the nucleus typically condenses and forms buds, frequently with a crescent shape of condensed chromatin. These, or the fragments of cells containing them, are often referred to as apoptotic bodies and are the morphological hallmark of apoptosis. The apoptotic bodies are either phagocytosed by macrophages or, in epithelia, phagocytosed by neighboring cells or extruded into a lumen (4). In many apoptotic cells, the DNA is degraded by specific endonucleases, producing DNA fragments that are multiples of nucleosome size (180 bp) in length. This partially degraded DNA can be detected as a ladder by gel polyacrylamide electrophoresis (PAGE). It can also be detected in tissue sections by end labeling the multitude of DNA ends by TUNEL (terminal deoxy-UTP nick end labeling) or related methods (5, 6, 7). During necrosis, the nucleus shrinks initially, but in contrast to apoptosis, it does not bud into apoptotic bodies nor is its DNA degraded into multiples of nucleosome lengths. Instead, in the first instance the DNA is randomly nicked, which by gel electrophoresis produces a smear of random sizes of DNA. Necrosis often involves more than one cell, and the cellular debris is removed by phagocytic macrophages (4). Terminal differentiation is an alternative mechanism that in selected cell types, such as red blood cells (8) and the outer squamous layers of skin (9), involves expulsion or destruction of the nucleus before the cessation of cellular function leads ultimately to cell death. The question of which of these three forms of cell death is involved in the death of granulosa cells in healthy and atretic follicles has yet to be answered.
Much of the current literature emphasizes the a role of apoptosis in granulosa cell death during atresia (10, 11). Thus, a ladder-like pattern typical of apoptotic cells was produced by gel electrophoresis using DNA from granulosa cells (10, 12), and TUNEL and related techniques have labeled granulosa cells in many species [human (13, 14), cow (15), sheep (16), pig (17), rat (18), and rabbit (19)], all of which suggests that granulosa cells undergo apoptosis. However, little attempt has been made to determine where apoptosis occurs in the membrana granulosa, and there are a number of ambiguities and inconsistencies in this literature. Opposing the idea that granulosa cells die by apoptosis, the granulosa cells from neither dominant nor nondominant follicles in one study of human ovaries were labeled by TUNEL (20), and ultrastructural studies have reported granulosa cells with necrotic rather than apoptotic features (21, 22). Inexplicably, in one study, only a portion of all of the pyknotic nuclei in the membrana granulosa of atretic follicles were labeled by TUNEL (23). Furthermore, although pyknotic nuclei have been observed in the membrana granulosa and the antrum of ovarian follicles using light microscopy (10, 24, 25), there have been no studies directly determining whether these are the result of apoptosis or necrosis.
Therefore, in the current study of healthy and atretic bovine antral follicles we have sought to examine the modes of granulosa cell death. We initially addressed this issue by examining the ultrastructure of granulosa cells in healthy and slightly atretic follicles. To further examine the integrity of DNA in dying granulosa cells of healthy and atretic follicles, we then used a range of techniques, including propidium iodide staining, TUNEL, an adaptation of the comet assay of cell death (26, 27), and PAGE. On the basis of all of our observations, we propose that granulosa cell death occurs by more than one pathway, dependent upon the location within the membrana granulosa.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Classification of follicular health
One section from each of the Carnoy’s-fixed, paraffin-embedded
ovaries was stained with hematoxylin, and each antral follicle that had
a membrana granulosa (as opposed to extremely atretic follicles in
which the membrana granulosa was absent) was examined. Pyknotic nuclei
(round or crescent shaped) were identified according to standard
descriptions and stained intensely with hematoxylin. The health of
antral follicles was initially determined according to the integrity of
the membrana granulosa and the general quantity of pyknotic nuclei in
this layer. Thus, healthy follicles were those with an intact membrana
granulosa and few if any pyknotic nuclei in this layer; atretic
follicles had a tattered-looking membrana granulosa and a moderate
number of pyknotic nuclei; very atretic follicles had numerous pyknotic
nuclei, and very few nuclei in the membrana granulosa appeared
healthy.
Electron microscopy
The method of processing tissue for electron microscopic
examination has been described in detail previously (28). Briefly, one
ovarian artery per reproductive tract was cannulated, and blood was
flushed from the ovary with Earle’s balanced-salt solution. The ovary
was then perfused with 2.5% glutaraldehyde in 0.1 M
MOPS (pH 7.3). Incisions were subsequently made in regions close
to antral follicles to allow greater penetration of the fixative, and
the ovary was then placed in 2.5% glutaraldehyde in 0.1 M
MOPS (4-morpholino-propanesulfonic acid) buffer (pH 7.3) for a minimum
of 24 h. Small wedges of tissue containing antral fluid as well as
granulosa and thecal layers were cut from the follicles and further
processed, involving postfixation in osmium tetroxide, dehydration in
acetone, and embedding in epoxy resin. Thick sections were stained with
methylene blue in sodium borate. Thin sections were stained with uranyl
acetate and lead citrate and observed and photographed using a JEOL CS
1200 electron microscope (Peabody, MA).
Nuclear staining observed by light microscopy
Tissue sections from paraffin-embedded ovaries that had been
stained with hematoxylin were counterstained with propidium iodide,
which intercalates with DNA. This involved pretreatment of
deparaffinized tissue sections with 0.1% Triton X-100 (catalogue no.
T-6878, Sigma Chemical Co., St. Louis, MO; 7 min),
followed by rinses in PBS (10 mM sodium/potassium phosphate
in 0.137 M NaCl and 5 mM KCl solution, pH 7.3;
three times, 5 min each time), then incubation with 50 µg/ml
propidium iodide (catalogue no. P-4170, Sigma Chemical Co.; 45 min, room temperature) in PBS, and a further rinse in
PBS (5 min) before mounting in buffered glycerol (0.167 M
Na2CO3 in 67% glycerol, pH 8.6). Propidium
iodide staining was visualized with an Olympus Corp. Vanox
AHBT3 fluorescence microscope, using the G filter. Sections that had
been stained with both hematoxylin and propidium iodide were in some
cases observed using the U filter on the same microscope, which enabled
visualization of both brightfield (i.e. hematoxylin
staining) and fluorescence (i.e. propidium iodide)
concurrently. Nuclei that stained with both hematoxylin and propidium
iodide appeared white, whereas those stained with hematoxylin only or
stained only faintly with propidium iodide appeared blue.
One randomly chosen follicle from each of five sections (one section per ovary) that had been stained with hematoxylin and propidium iodide was examined in detail. Pyknotic nuclei in each follicle were identified under brightfield conditions, as nuclei with intense hematoxylin staining. Each of these nuclei was then examined under the conditions showing both hematoxylin and propidium iodide staining (described above), and a count was made of the number of pyknotic nuclei that appeared white, indicating essentially intact DNA, and those that appeared blue, indicating degraded DNA.
TUNEL
Deparaffinized sections from each of the six ovaries fixed in
Carnoy’s solution were incubated in 3% H2O2
in methanol (30 min, room temperature) to block endogenous peroxidase
activity. All sections were subsequently rinsed in PBS (three times, 5
min each time), then incubated in 5 µg/ml proteinase K (catalogue no.
745723, Boehringer Mannheim, Mannheim, Germany) in PBS (45
min, 37 C) and rinsed again in PBS (three times, 5 min each time).
Sections were subsequently incubated with 0.5 nM
digoxygenin-11–2'-deoxy-uridine-5'-triphosphate (dig-11-dUTP;
catalogue no. 1093088, Boehringer Mannheim), 50 U/ml
terminal transferase (catalogue no. 220582, Boehringer Mannheim), 1.5 mM CoCl2 in buffer (30
mM Tris-Cl, pH 7.2, and 140 mM Na-cacodylate;
1 h, 37 C). The dig-11-dUTP was omitted from negative control
sections. Sections were rinsed in PBS (three times, 5 min each time),
then incubated with a 1:100 dilution of mouse monoclonal
anti-digoxygenin (catalogue no. 1333062, Boehringer Mannheim) in PBS (overnight, room temperature). After rinsing
again in PBS (three times, 5 min each time), sections were incubated
with 1:100 biotinylated horse antimouse IgG (catalogue no. BA-2000,
Vector Laboratories, Inc., Burlingame, CA; 3 h, room
temperature), rinsed in PBS (three times, 5 min each time), incubated
with 1:100 avidin-biotin complex from the Vectastain ABC
kit (catalogue no. PK-4001, Vector Laboratories, Inc.) in
PBS, rinsed in PBS (three times, 5 min each time), incubated with 0.7
mg/ml diaminobenzine hydrotetrachloride (10 min, room temperature),
then with 0.7 mg/ml diaminobenzine hydrotetrachloride in 0.06 mg/ml
urea H2O2 in 0.06 M Tris buffer
(SigmaFast, D-4418, Sigma Chemical Co.; 10 min, room
temperature), and finally rinsed in PBS (twice, 5 min each time) and
mounted with buffered glycerol. After observation and in some cases
photography, coverslips were floated off in PBS, and sections were
counterstained with hematoxylin.
Comet assay of cell death
Sections (10 µm) were cut from frozen ovaries and collected on
fully frosted slides (Fisher Scientific, Pittsburgh, PA)
that had been coated with a layer of 0.8% agarose (catalogue no.
200-0010, Progen Industries, Inc., Darra, Australia) in PBS, and a
second layer of agarose was prepared over the section. Slides were then
placed in cell lysis solution (0.25 M NaCl, 0.01
M EDTA, 1 mM Tris, 0.1% sodium lauryl
sarcosinate, and 0.1% Triton X-100, pH 10.0) for 3 h at 4 C. They
were subsequently immersed in either alkaline electrophoresis buffer
(0.1% 8-hydroxyquinolone, 0.01 M EDTA, 0.30 M
NaOH, and 0.02% dimethylsulfoxide, pH 10.0) or nondenaturing
electrophoresis buffer (0.1 M Tris buffer, pH 8.0,
containing 0.09 M sodium borate and 1 mM EDTA)
for 20 min at room temperature, electrophoresed in fresh
electrophoresis buffer for 15 min (24 V), then washed in a solution of
0.4 M Tris (pH 7.5). Sections were fixed in ethanol (20
min), then stained with propidium iodide (50 µg/ml in PBS; 45 min),
washed in PBS, and mounted with buffered glycerol for observation by
fluorescence microscopy.
Ceramide-treated 293 cells (American Type Culture Collection, Manassas, VA) were used as an apoptotic control. These cells were cultured in DMEM-Ham’s F-12 medium (catalogue no. 50–327-PA, Trace Biosciences, Castle Hill, Australia) with 10% FCS and antibiotics (100 µg/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone; CSL, Melbourne, Australia) in 5% CO2 in air, with or without addition of the apoptosis-inducing agent C6-ceramide (N-hexanoyl-D-sphingosine; catalogue no. H-6524, Sigma Chemical Co.) to the culture medium (10 µM, 12 h). At the end of the culture period, cells were centrifuged to form a pellet and were snap-frozen in OCT for use in the comet assay of cell death, as described above.
Collection and identification of globules from antral fluid
In preliminary observations using an Olympus Corp.
CK2 inverted microscope, it was observed that fluid that had been
aspirated from antral follicles frequently contained spherical
globules, which were often several times larger than individual cells
and apparently had no outer limiting membranes. Therefore, the fluid
was aspirated from all follicles of each of three ovaries from
nonpregnant cows and three ovaries from pregnant cows, using an
18-gauge needle and sterile 10-ml syringe. For each ovary, the fluid of
all of its follicles was expelled into a 35-mm petri dish and examined
by inverted microscope and grid eyepiece. The number and size of
globules in the region of the grid were counted in 10 random areas of
each dish, and the number of globules per ml fluid was calculated. The
size of the globules was also recorded.
The fluid from an additional three ovaries was pooled, and globules were transferred by mouth pipette onto glass slides coated with poly-L-ornithine (Sigma Chemical Co.) and Fro-Tissuer (Probing and Structure, Thuringowa Central, Australia) and air-dried overnight. The slides were subsequently immersed first in Carnoy’s solution (30 min) and then in absolute ethanol (5 min), and brief rinses in decreasing concentrations of ethanol (95% and 70%) were performed before a final rinse in PBS (5 min). They were then incubated with propidium iodide (50 µg/ml in PBS, 45 min) and examined by fluorescence microscopy.
To confirm that globules were composed of DNA and to determine the integrity of this DNA, we collected the globules from an additional six ovaries (four from pregnant, two from nonpregnant cows) and examined the DNA by PAGE. The globules were transferred by mouth pipette into microfuge tubes. As a small number of other ovarian cells were inadvertently transferred at the same time, equivalent quantities of ovarian cells were collected into separate microfuge tubes as control samples. Cells were lysed, and protein was digested by incubation in the presence of 200 µg/ml proteinase K (Boehringer Mannheim) in NET buffer (200 mM NaCl, 20 mM Tris-HCl, and 2 mM EDTA, pH 7.4) for 2 h at 37 C. The cell lysate was extracted with phenol-chloroform (29). The DNA was precipitated with ethanol (2–3 vol) with (20 µg, -20 C overnight) or without (-20 C, 1 h) the addition of carrier transfer RNA. The final DNA pellet was dried under vacuum (15 min, Speed-Vac, Savant Instrument Co., Farmingdale, NY) and dissolved in 20 µl sterile water. Aliquots were mixed with loading buffer and electrophoresed on a 6% polyacrylamide gel in TBE buffer (0.09 M Tris, 0.09 M borate, and 2 mM EDTA) (29). DNA fragment sizes were estimated using a 100-bp ladder (Pharmacia Biotech). After electrophoresis, the gel was stained with ethidium bromide (1 µg/ml) and destained in water, then visualized under UV and photographed using Polaroid film under transillumi- nation.
Photography
All photographs were taken using Kodak T-Max 400 black and white
film (Eastman Kodak Co., Rochester, NY). An Olympus
C35AD-4 camera attachment was used with the Olympus Vanox AHBT3
fluorescence microscope, and an Olympus SC35 camera attachment was used
with the Olympus BX50 microscope and the Olympus CK2 inverted
microscope (Olympus Corp., New Hyde Park, NY).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). From our studies it is clear that
healthy or atretic antral follicles contained degraded nuclei of two
types. Those more prevalent in the middle layers of the membrana
granulosa were pyknotic. Near the antral surface of the membrana
granulosa and in the antral follicular fluid there were both pyknotic
nuclei and large globular DNA-containing structures. Each of these
types is described separately below.
|
Electron-dense structures in the membrana granulosa, interpreted as the
pyknotic nuclei evident by light microscopy, were clearly visualized by
electron microscopy (Figs. 1
and 2). The
usual form of these structures was a crescent or rounded shape, which
stained homogeneously, being more electron dense than the nuclei of
adjacent healthy granulosa cells, but accompanied by small clumps of
more electron-dense granular material (Fig. 2, a–c). In most cases,
these pyknotic nuclei were located within the cytoplasm of a cell that
had its own apparently healthy nucleus and intact organelles including
mitochondria, endoplasmic reticulum, and lipid (Fig. 2, a–c). This
description fits the idea that one cell that had (or went on to
develop) a pyknotic nucleus was phagocytosed by a healthy granulosa
cell. The pyknotic nuclei were approximately the same diameter as the
healthy nuclei, and often retained some semblance of a nuclear
membrane. Surrounding the nuclear membrane were numerous closely packed
organelles that resembled mitochondria in size; some of these had
cristae. Endoplasmic reticulum was also evident in this region. The
pyknotic nucleus and closely packed organelles were all separated from
the cytoplasm of the healthy cell by a membrane system, probably the
remains of the original cell membrane.
|
Pyknotic nuclei in the antral layers of the membrana granulosa and
in the antrum
Using light microscopy, structures that stained with hematoxylin
and resembled pyknotic nuclei were also present in the antrum, often
loosely associated with the antral layer of granulosa cells (see Fig. 3, a and c, and Fig. 4, c and e). These were usually spherical
and ranged in size up to approximately 5 times the diameter of a normal
granulosa cell nucleus. However, the largest of these frequently
contained vacuoles that did not stain with hematoxylin. Crescent-shaped
pyknotic nuclei were occasionally seen in association with the membrana
granulosa, but rarely in the antrum proper.
|
|
). Occasionally, electron-dense
spheres resembling those just described, but without the surrounding
organelles and membranous envelope, were observed (Figs. 1 and 2d). It
is likely that this second type resulted from destruction of the first
type during tissue processing, as membranous matter and organelles were
seen in close association with some type 2 structures (Fig. 2d).
Thirdly, structures were seen that contained no electron-dense sphere
in the plane of section, but were composed of swirled, branching
membranes (Fig. 2e). Although granulosa cells of preantral follicles and antral follicles that were very atretic did display ultrastructural features of necrosis (not shown), in the current study of healthy or slightly atretic antral follicles, granulosa cells displaying lysed organelles or clumped chromatin were not seen in the membrana granulosa.
Light microscopy
The six Carnoy’s solution-fixed ovaries contained a total of 17
healthy antral follicles and 8 atretic antral follicles with remnants
of a membrana granulosa. Additionally, there were 12 follicles in later
stages of atresia that did not contain granulosa cells and were
therefore not further considered in this study.
Dual staining with hematoxylin and propidium iodide
Dual staining with hematoxylin and propidium iodide
was carried out to determine whether all of the structures that stained
intensely with hematoxylin were indeed rich in DNA and thus derived
from nuclei (Fig. 3). All nuclei of healthy granulosa cells that
stained with hematoxylin also stained with propidium iodide, including
mitotic nuclei. Pyknotic nuclei in the membrana granulosa stained
with propidium iodide, but this staining was usually less intense than
staining of nuclei from healthy cells, in contrast to the hematoxylin
staining of pyknotic nuclei, which often appeared more intense in
pyknotic than in healthy nuclei (Fig. 3). When the filters in the
fluorescence microscope were adjusted to enable visualization of both
hematoxylin staining and propidium iodide concurrently (Fig. 3d), 18%
of the pyknotic nuclei in the membrana granulosa appeared blue
(hematoxylin staining stronger than propidium iodide staining) rather
than white (both). In most cases, return to the fluorescence filter
alone revealed that these nuclei did stain with propidium iodide, but
very weakly. In only a few cases, was no propidium iodide staining
evident.
In contrast to the pyknotic nuclei in the membrana granulosa, the
nuclei in the antrum and those loosely associated with the membrana
granulosa stained very intensely with propidium iodide (Fig. 3, b and
e) and with the same pattern as that of staining by hematoxylin.
TUNEL in the membrana granulosa
Punctate brown staining was observed in the membrana granulosa of
follicles in each of the treated sections, but was absent from
negative-control parallel sections in which the dig-11-dUTP was omitted
from the TUNEL protocol (Fig. 4). Little if any staining was observed
in healthy follicles (Fig. 4a) compared with atretic follicles (Fig. 4b). Stained granulosa cells were located throughout the membrana
granulosa of atretic follicles, but predominantly in the central
region, consistent with our observations of the location of pyknotic
nuclei. However, the pyknotic nuclei loosely associated with the
membrana granulosa or more freely distributed in the follicular antrum
rarely stained, except at the periphery of vacuole-like structures in
the largest of these structures (Fig. 4d). Counterstaining with
hematoxylin revealed that not all of the structures identified as
pyknotic nuclei by hematoxylin staining were TUNEL positive (Fig. 4, compare b with c and d with e).
The comet assay of cell death
293 cells were used as control cells. In either alkaline
denaturing buffer or in nondenaturing buffer, untreated cells produced
no tails (not shown); cells treated with ceramide produced the
full plume tails of apoptosis (Fig. 5a).
Antral follicles in the bovine ovaries were unable to be sectioned at
less than 10 µm due to the fragility of the frozen follicular fluid.
The nuclei of granulosa cells are located very close to each other, and
when 10-µm sections were used in the assay of cell death, it was
impossible to distinguish between the nuclei and the tails. However,
the globules in the antrum were more dispersed than the nuclei in the
membrana granulosa, and their tails were streak-like (Fig. 5b) even in
alkaline denaturing buffer. This indicates that the DNA was of high mol
wt, with very little nicked DNA, and that DNA was not degraded by the
process of apoptosis.
|
) and appearing bubbly. When the
globules were air-dried onto glass slides, the vacuoles were still
evident (Fig. 6b). The globules ranged in size, with the largest
observed being 620 µm. Their mean size was 40 ± 8 µm
(mean ± SEM). The number of globules was estimated at
1200/ml follicular fluid. The appearance of the globules did not differ
between nonpregnant and pregnant animals.
|
, c and d). The globules stained much more intensely than did
oocytes; zona pellucidae were not stained by propidium iodide.
On PAGE (Fig. 6e), the DNA globules from ovaries of nonpregnant or
pregnant cows appeared as large smears indicative of necrotic DNA, with
only a faint ladder-like pattern indicative of apoptotic DNA. Thus, the
vast majority of the DNA in these globules, derived purely from dead
granulosa cells, was not degraded by the process of apoptosis. The
control samples, containing small numbers of ovarian cells, produced no
bands (Fig. 6a). We estimate from the approximate yields of DNA that
the globules are aggregates derived from large numbers (up to
thousands) of dead granulosa cells.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The study of apoptosis of granulosa cells has become a very popular topic in recent years (11). Surprisingly, though, none of the landmark papers in this area have considered the ultrastructure of these cells in situ, given the importance of morphology for defining apoptosis (4). TUNEL and related techniques have been used in previous studies by Billig et al. (30) and Chun et al. (31), and although these techniques preferentially label apoptotic cells, they also label necrotic cells (32, 33). Hence, they should not be used alone to differentiate between these two forms of cell death. The production of a ladder-like pattern by gel electrophoresis (10, 12, 30, 31, 34, 35). Also, this method requires a pool of cells. By making ultrastructural observations of condensed nuclei of crescent or circular shape, we now have very conclusive evidence for a form of apoptosis in the membrana granulosa of bovine follicles. This was observed to occur most frequently in the middle layers of the membrana granulosa, the region also observed to contain dividing cells (van Wezel, I. L., and R. J. Rodgers, unpublished observations).
In the classic process of apoptosis, nuclear condensation precedes budding of the nucleus and of the cell itself into several small apoptotic bodies (4). In epithelia (4), these apoptotic bodies are rapidly phagocytosed by neighboring epithelial cells, and thus, free apoptotic bodies are rarely seen. However, it is common to observe these small vesicles once they have been phagocytosed. Phagocytosis has now been observed in ovine (36) and bovine follicles. The observations we have made suggest that cells of the membrana granulosa of bovine follicles do not follow all of these classic processes precisely. Apoptotic nuclei found in the middle layers of the membrana granulosa were similar in size to normal nuclei, and thus had presumably not budded. However, these were commonly seen within a granulosa cell that contained its own apparently healthy nucleus, indicating that the healthy cell had phagocytosed the apoptotic cell. We thus hypothesize that either the apoptotic nuclei do not have the capacity to bud or, more likely, that phagocytosis is an early event in the apoptosis of bovine granulosa cells, preceding any potential budding. Thus, the endonuclease system of the apoptotic nuclei, if activated, would potentially be destroyed in the lysosomes of the phagocytosing cell. This could explain why only a proportion of apoptotic nuclei was labeled by TUNEL as observed here and previously (23).
During follicular atresia, the atrophy of granulosa cells has been reported to involve features of necrosis (21, 22). We have observed in advanced stages of atresia of antral follicles and in preantral follicles the presence of free organelles from lysed cells and granulosa cells with lysed organelles and clumped chromatin typical of necrosis (van Wezel, I. L., and R. J. Rodgers, unpublished observations). The current studies do not rule out a role for necrosis in advanced stages of follicular atresia, perhaps as a widespread mechanism by which the membrana granulosa is ultimately destroyed. However in the present ultrastructural study of healthy follicles and follicles that were only slightly atretic, no necrotic cells were observed in the membrana granulosa.
In the antrum we observed globules of DNA and pyknotic nuclei, frequently in membrane-bound bodies. Collectively, these structures have previously been called atretic bodies. They were originally described as arising from the swelling of cells at the surface of the membrana granulosa, followed by the dissolution of the cell membrane and later the nuclear membrane (24). It is likely that the DNA globules, as we have called them, are derived from aggregates of variable numbers of pyknotic nuclei after dissolution of the cell membrane. In the sheep, Hay et al. (36) reached a similar conclusion. However, the description of these atretic bodies (24) is far from consistent with apoptosis, yet in one of the landmark papers investigating apoptosis by molecular means (10), the atretic bodies were renamed apoptotic bodies. Are these atretic bodies really apoptotic? In support of the idea that they are, release of apoptotic bodies into a nearby lumen has been observed in other organs (4), and in the current study, we observed that nuclei in this region were uniformly electron dense, consistent with apoptosis, rather than exhibiting clumping of the nuclear chromatin typical of necrosis. However, we obtained evidence that they do not undergo the high degree of DNA fragmentation typical of apoptosis. Propidium iodide staining of nuclei in the antrum was intense, and the globules or pyknotic nuclei in the antrum were not TUNEL positive, suggesting that the DNA was not excessively degraded, especially compared with that of pyknotic nuclei observed within the membrana granulosa. In the comet assay of cell death, the globules in the antrum produced a streak tail of randomly cut DNA rather than a plume of apoptotic DNA, and globules of DNA that were present in follicular fluid isolated from fresh ovaries were shown by PAGE to be mostly high mol wt rather than producing a ladder-like pattern of apoptotic DNA. It is possible that apoptosis of these cells had taken place without fragmentation of the DNA into multiples of nucleosome length. However, as no crescent-shaped nuclei were observed, and there was no evidence of DNA fragmentation to the degree commonly expected with apoptosis, more evidence is required before concluding that these atretic bodies are apoptotic bodies.
If the antral atretic bodies are not formed by apoptosis, then two alternative mechanisms for the formation of pyknotic nuclei or atretic bodies remain: necrosis or terminal differentiation preceding cell death. The ultrastructure of the atretic bodies was not consistent with necrosis, as none of the usual features, such as dissolution of the cell membranes and lysis of organelles, was observed here. The remaining alternative is terminal differentiation, comparable to the formation of mature erythrocytes (8) or mature keratinocytes in skin (9). The changes in DNA during terminal differentiation that ultimately result in cell death are not well understood and may subsequently be shown to share some features in common with apoptosis. The formation of the reticulocytes requires the progressive condensation of nuclear material to form electron-dense pyknotic nuclei, similar to the granulosa nuclei observed in the follicular antrum. These nuclei become polarized in the cell and are subsequently extruded, leaving the mature reticulocyte (8). The extruded nucleus is subsequently phagocytosed (37). We are not aware of any studies examining the nature of DNA degradation during terminal differentiation in other organs. However, in contrast to apoptosis, terminal differentiation is associated with cellular loss of the ability to divide (9, 38), and in a related study we observed that mitotic figures were less frequently observed in the antral portion of the membrana granulosa than in the middle portions (van Wezel, I. L., and R. J. Rodgers, unpublished observations). Thus, the features of the atretic bodies are highly consistent with terminal differentiation. The concept of terminal differentiation leading ultimately to cell death has not previously been considered in the context of ovarian follicles.
Both the current results and other unpublished observations support the
concept that the membrana granulosa is dynamic and highly structured
and has a number of features in common with the skin epidermis. This is
illustrated in Fig. 7. The basal/antral
structure of some small antral follicles is similar to that of skin;
the granulosa cells closest to the basal lamina are columnar, those
cells slightly further from the basal lamina are more rounded, and
those cells furthest from the basal lamina are often flattened (24).
This profile might not be static, as the columnar basal cells might
become rounded as the follicle enlarges. Like skin, cell division is
predominantly within a zone of the epithelium, but whereas this zone is
basal in skin, in the membrana granulosa it is found in the middle
region. Cells closer to the antrum are older and become progressively
flattened in shape, as occurs in skin. The cells at the surface slough
off, as occurs in skin, and this process leads to death of these cells.
Our examination suggests that the mode of death of these cells closest
to the antrum does not follow the process of apoptosis or necrosis but,
rather, follows that of terminal differentiation. Skin also has a
population of stem cells, and we have postulated that granulosa cells
arise from stem cells (38, 39, 40). In support of this, we have
demonstrated that a small proportion of granulosa cells have at least
one property of stem cells, namely the ability to divide without
anchorage in vitro (38, 39, 40).
|
In conclusion, the pyknotic nuclei evident in hematoxylin-stained tissue sections of healthy and slightly atretic ovarian follicles represent two distinct pathways of cell death. Those in the middle layers of the membrana granulosa undergo apoptosis, but with phagocytosis apparently preceding any potential budding, whereas atretic bodies in the antrum are apparently derived from the sloughing off of cells from the granulosa layers closest to the antrum. These bodies share features in common with other cell types undergoing a terminal differentiation that results in cell death. Necrosis only appears to play a greater part in advanced atresia. These findings explain many of the conflicting observations and conclusions made previously about follicular atresia. Our findings also raise the questions of why the membrana granulosa is structured the way it is, and why it undergoes the changes it does. These changes occur as the follicular fluid accumulates in the expanding antrum, a process about which we know very little. It remains to be seen whether these processes are all functionally related.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received April 1, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  C. M. Tingen, S. K. Bristol-Gould, S. E. Kiesewetter, J. T. Wellington, L. Shea, and T. K. Woodruff Prepubertal Primordial Follicle Loss in Mice Is Not Due to Classical Apoptotic Pathways Biol Reprod, July 1, 2009; 81(1): 16 - 25. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Zachut, A Arieli, H Lehrer, N Argov, and U Moallem Dietary unsaturated fatty acids influence preovulatory follicle characteristics in dairy cows Reproduction, May 1, 2008; 135(5): 683 - 692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Duerrschmidt, O. Zabirnyk, M. Nowicki, A. Ricken, F. A. Hmeidan, V. Blumenauer, J. Borlak, and K. Spanel-Borowski Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1-Mediated Autophagy in Human Granulosa Cells as an Alternative of Programmed Cell Death Endocrinology, August 1, 2006; 147(8): 3851 - 3860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. G Clarke, S. A Hope, S. Byers, and R. J Rodgers Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans Reproduction, July 1, 2006; 132(1): 119 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. S. Hussein, D. A. Froiland, F. Amato, J. G. Thompson, and R. B. Gilchrist Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins J. Cell Sci., November 15, 2005; 118(22): 5257 - 5268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E Valdez, S P. Cuneo, and A. M Turzillo Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation Reproduction, July 1, 2005; 130(1): 71 - 81. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R Braw-Tal and Z Roth Gene expression for LH receptor, 17{alpha}-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary Reproduction, April 1, 2005; 129(4): 453 - 461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Clark, H. F. Irving-Rodgers, A. M. Dharmarajan, and R. J. Rodgers Theca Interna: The Other Side of Bovine Follicular Atresia Biol Reprod, October 1, 2004; 71(4): 1071 - 1078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.J.M. Hendriksen, B.M. Gadella, P.L.A.M. Vos, E. Mullaart, T.A.M. Kruip, and S.J. Dieleman Follicular Dynamics Around the Recruitment of the First Follicular Wave in the Cow Biol Reprod, December 1, 2003; 69(6): 2036 - 2044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. E. Hoyer, O. B.F. Terkelsen, A. Grete Byskov, and H. Nielsen Fetuin and Fetuin Messenger RNA in Granulosa Cells of the Rat Ovary Biol Reprod, December 1, 2001; 65(6): 1655 - 1662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Baker, V. Srsen, R. Lapping, and N. Spears Combined Effect of Follicle-Follicle Interactions and Declining Follicle-Stimulating Hormone on Murine Follicle Health In Vitro Biol Reprod, October 1, 2001; 65(4): 1304 - 1310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.J. Devine, C.M. Payne, M.K. McCuskey, and P.B. Hoyer Ultrastructural Evaluation of Oocytes During Atresia in Rat Ovarian Follicles Biol Reprod, November 1, 2000; 63(5): 1245 - 1252. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
