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Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology (T.M., G.I.P., B.R.R., J.L.T.), Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114; Section of Immunobiology, Yale University School of Medicine (T.S.Z., R.A.F.), New Haven, Connecticut 06510; and Department of Obstetrics and Gynecology, University of Kansas School of Medicine, and Wesley Medical Center (T.R.K.), Wichita, Kansas 67214
Address all correspondence and requests for reprints to: Jonathan L. Tilly, Ph.D., Massachusetts General Hospital, VBK137C-GYN, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: jtilly{at}partners.org
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Introduction |
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Current evidence from studies of a number of cell types indicates that the release of cytochrome c from mitochondria (8, 9), a key intracellular event in the PCD pathway controlled by bcl-2 gene family members (10, 11), engages the downstream machinery needed for the execution of apoptosis. This machinery is comprised of an adapter protein termed Apaf-1 (12) that, when bound with cytochrome c, forms a complex with the proform of caspase-9 (13). In many cell types, caspase-9 is believed to be the most proximal enzyme in a cascade of proteolytic activity required for dismantling the cell during PCD (9, 14, 15, 16). Formation of the Apaf-1/procaspase-9 complex permits activation of the enzyme through an induced proximity model (17). This biochemical step heralds the execution phase of PCD, characterized by the cleavage-activation of downstream caspase family members, such as caspase-2, -3, -6, or -7, that produce the morphological (e.g. cellular condensation, budding, and fragmentation, and nuclear pyknosis) and biochemical (e.g. DNA cleavage through nuclease activation) hallmarks of apoptosis. In addition, caspases irrevocably commit cells to death through disablement of key structural proteins, signaling factors, and nuclear enzymes required for cellular homeostasis (9, 14, 15, 16).
In the mammalian ovary, a case has been made for the existence of this
PCD pathway in both oocytes and granulosa cells (1, 2).
For example, a prominent role for the proapoptotic Bcl-2 family member,
Bax, in activating PCD in female germ cells and follicular granulosa
cells has been reported based on endogenous gene expression studies
(18, 19, 20, 21) and in-depth analyses of striking ovarian
phenotypes in Bax-deficient female mice (22, 23, 24).
Mitochondria, the principal intracellular targets for the actions of
Bcl-2 family members (11), have been identified as direct
participants in controlling oocyte apoptosis (25). In
addition, mitochondrial release of cytochrome c has been
shown to occur in murine granulosa cells during apoptosis, coincident
with the processing of procaspase-3 to the active enzyme
(26). Although the requirement for Apaf-1 in connecting
mitochondrial cytochrome c release to caspase activation in
ovarian cells remains to be directly established, studies documenting
the accumulation of Apaf-1 protein in granulosa cells during the early
stages of follicle atresia suggest that this adapter protein is indeed
involved (26). Further evidence implicating caspase-3 in
atresia comes from studies of gonadotropin-regulated caspase-3
messenger RNA (27) and protein (28) levels in
rat granulosa cells and expression of caspase-3 in human luteinizing
granulosa cells (19). We have also reported that the
caspase-3-specific intracellular substrate, 
-fodrin
(29), is cleaved in granulosa cells during atresia
(30), and that caspase inhibitors attenuate the occurrence
of PCD in cultured antral follicles (30). Lastly, cleavage
of a rhodamine-conjugated DEVD (Asp-Glu-Val-Asp) peptide [the
preferred cleavage recognition site for caspase-3 (31)]
has been shown to occur in murine granulosa cells during apoptosis
in vitro (26).
Preliminary observations from our laboratory indicate that caspase-9, the most proximal enzyme in the execution phase of PCD, is required for apoptosis in both oocytes and granulosa cells (32). Although caspase-3 is known to be expressed in female germ cells (33), recent gene knockout studies have shown that caspase-2 [which is also expressed in oocytes (34);] is functionally required for developmental and anticancer drug-induced oocyte apoptosis (34). Nevertheless, based on observations that inhibitors of caspase-3-like enzymes prevent oocyte fragmentation induced by anticancer drugs in vitro (23), and that cleavage of a rhodamine-conjugated DEVD peptide occurs in oocytes during apoptosis (35), it may be that both caspase family members are needed to execute PCD in female germ cells. Herein we used caspase-3-null mice (36) to directly test for the functional requirement of this executioner protease in ovarian germ cell vs. granulosa cell demise in vivo and in vitro. The results presented herein indicate that oocytes and granulosa cells undergo PCD via genetically distinct pathways, one reliant on caspase-2 and the other dependent on caspase-3. Furthermore, we provide evidence for evolutionary conservation of caspase-3 function in the apoptosis of human granulosa cells during atresia of maturing antral follicles in vivo and show that an additional executioner caspase is also activated during granulosa cell demise.
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Materials and Methods |
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Human ovarian tissues
Ovarian biopsies were collected from four patients (25–35 yr of
age; tissue from each patient serving as an experimental replicate)
undergoing gynecological surgery for various benign conditions after
informed consent and protocol approval by the Wesley Medical Center
institutional review board. Ovarian tissues were fixed in 4%
paraformaldehyde, embedded, sectioned (8 µm), and mounted on glass
slides for histological or histochemical assessments (see below).
Mouse ovarian collection and oocyte counts
Ovaries were collected from wild-type and caspase-3-deficient
female mice on day 4 postpartum and at 2 and 6 months of age. The
ovaries were then fixed (0.34 N glacial acetic acid, 10%
formalin, and 28% ethanol for histology, 4% paraformaldehyde for
histochemistry), paraffin-embedded, serially sectioned (8 µm), and
mounted in order on glass microscope slides. For histomorphometrics,
the sections were stained with hematoxylin-picric acid methyl blue and
analyzed for the number of healthy (nonatretic) primordial, primary,
and small preantral follicles per section in every fifth section
through the entire ovary, as previously described (37).
Primordial follicles were identified as having a compact oocyte
surrounded by a single layer of flattened (fusiform) granulosa cells,
whereas primary follicles were identified as having an enlarged oocyte
surrounded by a single layer of cuboidal granulosa cells. Intermediate
stage follicles (compact or enlarged oocyte with a single layer of
mixed fusiform and cuboidal granulosa cells) were scored as primary,
because the change in granulosa cell morphology from fusiform to
cuboidal is a sign that the primordial follicle is no longer quiescent.
Small preantral follicles were identified as having an enlarged oocyte
surrounded by at least a partial or complete second layer of cuboidal
granulosa cells but no more than four layers of cuboidal granulosa
cells. All oocyte-containing follicles were counted in each ovarian
section scored, and each ovary was given a numerical code so that all
follicle counts were conducted without knowledge of genetic background.
Slides were then decoded, and the total number of healthy follicles per
ovary was calculated. Follicles at the primordial, primary, and small
preantral stages of development were deemed atretic if the oocyte was
degenerating (convoluted and condensed, or fragmented) or absent
(24, 37).
In vitro follicle cultures
Prepubertal (day 24–26 postpartum) female mice (wild-type or
caspase-3 mutant) were given a single ip injection of 10 IU
equine CG (eCG; Professional Compounding Centers of America, Houston,
TX), and the 8–12 largest antral (preovulatory) follicles were
nonenzymatically dissected from ovaries 42 h after injection, as
previously described (18, 30). Follicles were then either
fixed (histology, histochemistry) or frozen (electrophoretic DNA
analysis) immediately or were cultured for 12 or 24 h under
serum-free conditions, as detailed previously (18, 30),
before fixation or freezing.
In vitro oocyte cultures
Oocytes were isolated, cultured, and assessed for apoptosis as
detailed previously (23, 34, 35, 37, 38). Briefly,
wild-type or caspase-3-deficient female mice were injected with 10 IU
eCG followed by 10 IU hCG (Serono Laboratories, Inc.,
Randolph, MA) 46 h later to induce superovulation. Mature
(metaphase II) oocytes were collected from the oviducts 16 h after
hCG injection and denuded of cumulus cells by a 1-min incubation in 80
IU/ml hyaluronidase (Sigma, St. Louis, MO). The oocytes
were washed three times with culture medium (human tubal fluid; Irvine
Scientific, Santa Ana, CA) supplemented with 0.5% (wt/vol) BSA
(fraction V; Life Technologies, Inc., Grand Island, NY).
Pools of 10–20 oocytes were then cultured in 0.1-ml drops of culture
medium under paraffin oil in the absence or presence of 200
nM doxorubicin (Sigma). After culture, oocytes
were checked for changes characteristic of apoptosis (condensation,
budding, cellular fragmentation, DNA fragmentation), and the percentage
of oocytes that underwent apoptosis of the total number of oocytes
cultured per drop in each experiment was determined.
In vitro granulosa cell cultures
Granulosa cell cultures were carried out essentially as
described previously (26). Briefly, immature (21–24 days
postpartum) wild-type and caspase-3-deficient female mice were injected
with 10 IU eCG. Forty-two hours later, ovaries were dissected out and
decapsulated, and the stimulated follicles were punctured with fine
needles to collect the granulosa cells into Waymouth’s MB752/1 medium
(Life Technologies, Inc.) supplemented with 100 U/ml
penicillin, 100 µg/ml streptomycin, and 0.29 mg/ml
L-glutamine. After trypan blue staining analysis,
approximately 3 x 105 viable cells were
cultured in 35 x 10-mm dishes containing 2 ml culture medium for
24 or 48 h with 10% FBS (HyClone Laboratories, Inc.,
Logan, UT). The medium and nonadherent cells were removed, fresh
serum-free culture medium (2 ml) was added, and the cells were
maintained for an additional 48 or 24 h, respectively (total
length of culture was 72 h in both cases), to induce apoptosis. At
the termination of culture, granulosa cells were fixed with 4%
paraformaldehyde for 30 min at 20 C, stained for 10 min with
4',6-diamidino-2-phenylindole (DAPI; Sigma) at a final
concentration of 30 µg/ml, and then stored at 4 C until examined by
fluorescence microscopy using an UV light filter to detect nuclear
chromatin condensation and fragmentation characteristic of apoptosis
(26). In each experiment, over 1 x
103 granulosa cells in randomly chosen fields
were scored for the occurrence of nuclear condensation vs.
fragmentation (of the total number of cells present).
DNA isolation, radiolabeling, and gel electrophoretic
analysis
Genomic DNA was extracted, quantitated, and 3'-end labeled with
[-32P]dideoxy-ATP (3000 Ci/mmol;
Amersham Pharmacia Biotech, Piscataway, NJ) using terminal
deoxynucleotidyl transferase (Roche Molecular Biochemicals, Indianapolis, IN), as previously described
(39, 40). Radiolabeled samples were resolved through 2%
agarose gels, and the extent of internucleosomal cleavage was assessed
by autoradiography.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL)
The occurrence of apoptosis in ovarian and follicle sections was
assessed, as described previously (40), by monitoring the
presence of DNA fragmentation in situ. Slides were analyzed
by conventional light microscopy after light counterstaining with
hematoxylin. Cells exhibiting dark brown staining from the colorimetric
reaction were considered positive for DNA fragmentation. Negative
controls, conducted by omitting the labeling enzyme, yielded no
reaction product (data not shown).
Immunohistochemistry
Paraffin-embedded sections (6 µm for follicles or 8 µm for
ovaries) were analyzed by immunohistochemistry without (follicle
sections) or with (mouse and human ovarian sections) high temperature
antigen unmasking, as previously described (26, 37, 41, 42). Briefly, after peroxidase quenching [and microwave
treatment if employed (42)], sections were incubated with
a 1:3000 dilution of the cleaved caspase-3 antibody (CM1)
(43) for 1 h at room temperature or with a 1:100
dilution of the cleaved caspase-7 antibody (Cell Signaling
Technology/New England Biolabs, Inc., Beverly, MA)
overnight at 4 C. Sections were washed and then incubated with a 1:200
dilution of biotinylated goat antirabbit Ig antibody
(Calbiochem-Novabiochem, La Jolla, CA) for 1 h at
room temperature. Sections were washed, incubated for 45 min at room
temperature with horseradish peroxidase-conjugated streptavidin,
washed, and reacted with ice-cold 0.5 mg/ml 3,3'-diaminobenzidine with
0.03% hydrogen peroxide at room temperature for antigen detection.
Sections were then counterstained with hematoxylin and analyzed by
light microscopy.
Immunocytochemistry
Granulosa cells harvested from gonadotropin-primed mice were
cultured for 24 h with 10% FBS as described above (see In
vitro granulosa cell cultures). The medium and
nonadherent cells were removed, and 2 ml fresh serum-free culture
medium were added. The cells were then maintained for an additional
48 h to induce apoptosis. At the termination of the culture,
medium and nonadherent cells were removed, after which 1 ml 3%
neutral-buffered paraformaldehyde was added to the dish. Following
fixation for 10 min at 4 C, immunocytochemical staining was carried out
using the CM1 antibody essentially as previously described
(43) or the cleaved caspase-7 antibody (Cell Signaling
Technology/New England Biolabs, Inc.) according to the
manufacturer’s instructions. In brief, the cells were permeabilized
for 10 min at -20 C in 100% methanol and blocked with buffer
containing 5.5% normal goat serum. The cells were then incubated with
a 1:1000 (CM1) or 1:100 (cleaved caspase-7) dilution of primary
antibody for 1 h at room temperature (CM1) or 15 h at 4 C
(cleaved caspase-7). The cells were washed and incubated with a 1:200
dilution of biotinylated goat antirabbit Ig antibody for 1 h at
room temperature. The cells were washed again and incubated for 45 min
at room temperature with horseradish peroxidase-conjugated
streptavidin. After washing, the cells were finally reacted with an
ice-cold preparation (0.5 mg/ml) of 3,3'-diaminobenzidine in the
presence of 0.03% hydrogen peroxide at room temperature for antigen
detection. Cells were then lightly counterstained with hematoxylin and
analyzed by light microscopy.
Data presentation and statistical analysis
Each experiment was independently replicated at least three
times with different mice in each experiment. All graphs represent the
mean ± SEM of combined data from the replicate
experiments, whereas representative photomicrographs are presented for
histology, histochemistry, cytochemistry, and autoradiography (see
figures and tables for more details). For quantitative analyses,
Student’s t test was used for comparison of mean values
obtained with wild-type vs. caspase-3-null mice,
and a P < 0.05 was chosen to indicate a statistically
significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; data not shown for 12
or 48 h points). In addition, the low rate of spontaneous oocyte
apoptosis (no drug treatment) in vitro was unaffected by the
absence of caspase-3 (Fig. 1B).
Histomorphometric evaluation of follicle numbers in adult caspase-3
mutants
Previous studies have shown that Bax-deficient female mice are
born with a normal endowment of primordial follicles at birth, but
possess 3-fold more primordial follicles in young adult life. This
phenotype was determined to be the result of defects in postnatal
oocyte death leading to attenuated atresia of primordial and primary
follicles (24). Based on these observations, we next
tested whether a similar situation exists in caspase-3
mutant females. At 2 months of age, however, young adult wild-type and
caspase-3-null females were found to possess comparable
numbers of nonatretic primordial, primary, and small preantral
follicles in their ovaries (Fig. 1C).
Immunolocalization of cleaved (activated) caspase-3 in the mouse
ovary
To determine whether active caspase-3 was present and correlated
with somatic cell apoptosis in the ovary in vivo,
immunohistochemistry was performed using a rabbit polyclonal antiserum
(CM1) that recognizes the p18 subunit of cleaved caspase-3 but not the
zymogen form of the enzyme (43). As a model, wild-type
prepubertal female mice were killed immediately (0 h; moderate level of
early antral follicle atresia) or were given a single injection of eCG,
then killed 48 h later (preovulatory follicle development, no
atresia) or 96 h later (atresia of the preovulatory cohort
developed 48 h earlier) (44, 45). In mice before
gonadotropin injection, ovaries contained seven to nine maturing
follicles per section with granulosa cells labeled by the CM1 antibody
(Fig. 2A) and three to five maturing
follicles per section with granulosa cells labeled by TUNEL (to detect
DNA cleavage associated with apoptosis; Fig. 2B). At 48 h post-eCG
injection, we could not detect CM1 immunoreactivity (Fig. 2C) or TUNEL
staining in any of the antral follicles (Fig. 2D), consistent with the
ability of eCG priming to suppress caspase-3 expression (26, 27) and antral follicle atresia (18, 43, 44) in the
prepubertal rodent ovary. At 96 h post-eCG, granulosa cells of the
now atretic cohort of antral follicles induced to undergo preovulatory
development 48 h earlier by eCG priming were again characterized
by CM1 immunoreactivity (Fig. 2E) and TUNEL staining (Fig. 2F).
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, A and B), as
well as those just initiating (Fig. 3, C and D), cellular condensation
associated with apoptosis.
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, A–C), ovaries from all adult
caspase-3 gene knockout mice were characterized by the
presence of aberrant atretic follicles exhibiting a shrunken or
disorganized appearance without or with blood cell infiltration (Fig. 4, D–F). In many instances, the oocytes had either spontaneously
activated (resumed meiosis) or degenerated. Histochemical assays of
caspase-3 mutant ovarian sections revealed the persistence
of granulosa cells in these follicles that failed to be eliminated by
apoptosis. This was concluded based on the absence of TUNEL-positive
staining for the occurrence of DNA cleavage (Fig. 5, E and F; compare with wild-type in
Fig. 5, A and B) and on the absence of nuclear pyknosis as determined
by DAPI staining of nuclear morphology (Fig. 5G; compare with wild-type
in Fig. 5C). Quantitative analysis of randomly selected sections
prepared from ovaries of three mice per genotype revealed 3.6 ±
0.2 TUNEL-positive follicles/ovarian section in wild-type mice (118
TUNEL-positive follicles of 33 sections scored). By comparison, we
detected only 0.8 ± 0.1 TUNEL-positive follicles per ovarian
section in caspase-3-null mice (29 TUNEL-positive follicles
of 36 sections scored; P < 0.001). When analyzed using
the CM1 antibody, granulosa cells within atretic follicles of
caspase-3-deficient mice showed light cytoplasmic staining
with only a single granulosa cell, on occasion, being labeled more
intensely (Fig. 5H). This pattern contrasted with the immunoreaction
observed in the majority of granulosa cells within atretic follicles of
wild-type mice (Figs. 2 and 5D).
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, A and B), histochemical assays
(Fig. 6, E, F, I, and J), and gel electrophoretic analysis of DNA
integrity (Fig. 7). After in
vitro culture without hormonal support for 12 h, granulosa
cells within wild-type follicles displayed all of the expected features
of apoptosis, including cellular condensation and detachment (Fig. 6C),
DNA cleavage (Figs. 6K and 7), and formation of large numbers of
apoptotic bodies (Fig. 6C). By comparison, all of these events were
either markedly attenuated or absent in granulosa cells of
caspase-3-null follicles cultured in parallel (Fig. 6, D and
L, and Fig. 7). Furthermore, the pattern of CM1 immunoreactivity in
follicles induced to undergo atresia in vitro was similar to
that observed for atresia in vivo (Figs. 2 and 5). The
intense CM1 immunoreactivity detected in granulosa cells of wild-type
follicles cultured in vitro (Fig. 6G) was markedly reduced,
but not entirely absent, in granulosa cells of caspase-3-deficient
follicles incubated in parallel (Fig. 6H). By 24 h of culture, it
became increasingly difficult to distinguish differences in granulosa
cells of wild-type vs. caspase-3-deficient follicles by
morphology and TUNEL (data not shown) and by gel electrophoretic
analysis of DNA (Fig. 7).
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, A and B). By
comparison, caspase-3-deficient granulosa cells cultured in parallel
frequently exhibited abnormal clumping of DNA into a single mass of
partially condensed nuclear material (Fig. 8, C and D). By scoring over
1 x 103 granulosa cells in randomly chosen
fields for the occurrence of nuclear condensation vs.
fragmentation, we determined that caspase-3-deficient granulosa cells
were defective in their capacity to complete the process of nuclear
fragmentation after the initial condensation of DNA (Table 1).
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), caspase-3 processing was the
first sign of pending granulosa cell death (Fig. 10B) with low or no
staining of granulosa cells by TUNEL (Fig. 10D). Additionally,
processed caspase-7 was not detected in granulosa cells of follicles at
these earliest stages of atresia (Fig. 10C). As atresia progressed
(granulosa cell disorganization, increased numbers of pyknotic cells;
Fig. 10E), CM1 immunoreactivity and TUNEL staining became much more
pronounced (Fig. 10, F and H), and processed caspase-7 became
detectable (Fig. 10G). In grossly atretic follicles (nearly complete
dissolution of the granulosa cell layers, shedding of apoptotic bodies
into the antral space; Fig. 10I), CM1 and cleaved caspase-7
immunoreactivity remained high in dying/dead granulosa cells, and CM1
immunoreactivity became detectable in the adjacent theca-interstitial
cells (Fig. 10J).
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One possibility, supported in part by the scientific literature, is that caspase-2 is required for oocyte apoptosis (34), but is dispensable for granulosa cell demise. Indeed, we have recently observed that atresia of maturing antral follicles, a process driven by granulosa cell apoptosis, proceeds normally in caspase-2 mutant female mice (unpublished observations). As caspase-7 gene knockout is lethal (16), and generation of caspase-6 gene knockout mice remains as yet reported (16), we focused our attention on mice lacking the principal executioner caspase activated by caspase-9, that being caspase-3 (36). This particular enzyme was a logical candidate for investigation in the context of granulosa cell apoptosis based on previous findings of an inverse correlation between caspase-3 expression (messenger RNA and protein) and apoptosis in granulosa cells of the rat ovary (27, 28). Furthermore, procaspase-3 processing and caspase-3-like enzymatic activity have been detected in murine granulosa cells during apoptosis (26), and cell-permeant peptide inhibitors selective for caspase-3 (e.g., zDEVD-fmk) suppress granulosa cell death in cultured ovarian follicles of the mouse (30). However, the fact that oocytes express both caspase-2 (34) and caspase-3 (33) coupled with reports that caspase-3-selective inhibitors repress oocyte death (23) and that caspase-3-like enzymatic activity is detectable in dying oocytes (35) raised the possibility that oocytes use both executioner caspases to complete PCD.
Our first series of experiments failed to provide evidence supporting a functional requirement for caspase-3 in germ cell apoptosis. Contrasting the increased number of primordial and primary follicles endowed in caspase-2 mutant females due to defective apoptosis in oocytes during prenatal ovarian development (34), caspase-3-deficient female mice were born with comparable numbers of oocyte-containing follicles as their wild-type female siblings. Other studies from our laboratory have shown that bax mutant female mice are not born with more oocytes, but possess an excess number of primordial and primary follicles in young adult life due to defects in oocyte apoptosis associated with atresia at these early stages of follicle development (24). Thus, defects in oocyte death due to a gene knockout can become apparent in either prenatal (caspase-2 mutant) or postnatal (bax mutant) life. Nonetheless, this latter possibility was not the case with caspase-3-deficient females based on our observations that follicle numbers in wild-type and mutant females were not different in young adult life. Moreover, and again in contrast to caspase-2-null oocytes (34), caspase-3-deficient oocytes failed to show resistance to anticancer drug-induced apoptosis in vitro. In further support of these findings, microinjection of active recombinant caspase-3 (30) into murine oocytes does not trigger apoptosis (G. I. Perez and J. L. Tilly, unpublished data) despite the fact that microinjection of recombinant Bax, an upstream activator of the caspase cascade, induces oocyte death (38). These latter observations are consistent with recent data derived from studies of neurons microinjected with various recombinant caspases in which some, but not all, of the caspases tested induced apoptosis (48). Collectively, these data, which show that expression of gene product (i.e., caspase-3 in oocytes) (33) does not always equate to functional importance in a given cellular response (i.e., apoptosis), underscore the importance of evaluating gene mutant mice.
Our next set of experiments using an antibody (CM1) that recognizes the cleaved form of caspase-3 (43) demonstrated a positive correlation between CM1 immunoreactivity and apoptosis in granulosa cells both in vivo and in vitro. Such findings are in accordance with published observations implicating caspase-3 in granulosa cell death (19, 26, 27, 28, 30). That the active caspase-3 enzyme recognized by the CM1 antibody is a key functional participant in the granulosa cell death program was supported by the presence of aberrant atretic follicles in caspase-3-deficient mouse ovaries. These follicles were characterized by the persistence of granulosa cells that failed to be properly eliminated by apoptosis, as confirmed by assessments of cellular morphology, TUNEL for DNA cleavage and DAPI staining for nuclear condensation. To further examine this phenotype under controlled experimental conditions that resemble atresia in vivo, we studied the progression of granulosa cell apoptosis in antral follicles isolated from gonadotropin-primed immature mice and placed in serum-free cultures. These experiments underscored the abnormal or attenuated progression of many apoptotic events in caspase-3-null granulosa cells, including defective nuclear collapse (an end point further confirmed with the granulosa cell cultures), the absence of cellular shrinkage and budding to generate apoptotic bodies, and delayed genome fragmentation. The inability of caspase-3-deficient granulosa cells to fully execute PCD is in agreement with the reported role of caspase-3 in degradation of cytoskeletal proteins leading to cellular shrinkage as well as in cleaving nuclear scaffold proteins and activating nucleases required for chromatin condensation (14, 15, 16, 29, 49).
We would point out that the lack of functional caspase-3 in granulosa cells, although causative of many defects in the temporal execution or progression of events normally associated with apoptosis, did not prevent the eventual death of granulosa cells in vitro or in vivo. This point is most clearly shown by results from the follicle culture studies, in which the striking differences in dying granulosa cells of wild-type vs. caspase-3-null follicles cultured for 12 h became difficult to discern when the cultures were allowed to progress for 24 h. Moreover, the nearly complete absence of nuclear fragmentation in caspase-3-deficient granulosa cells starved of hormonal support for 24 h (compared with the significantly higher incidence of nuclear fragmentation in the wild-type cells cultured in parallel) became less pronounced as the cultures were continued for up to 48 h. These findings are in keeping with recent data derived from studies of hepatocytes and thymocytes lacking caspase-3. These cells were reported exhibit abnormal morphological changes and delayed DNA fragmentation after Fas activation, but the cells still eventually died (50). Nonetheless, the in vivo significance of delayed granulosa cell death in follicles attempting to undergo atresia may be reflected by the greater number of oocytes ovulated in the caspase-3-null females, compared with their wild-type sisters, after treatment with exogenous gonadotropins. Although the basis of the 2-fold higher ovulatory response in the mutant mice remains to be established, the delay in atresia of maturing antral follicles in caspase-3-null females may allow for more follicles to be available for rescue from atresia by gonadotropins.
Although these data indicate that caspase-3 is functionally required for the normal execution of PCD in granulosa cells, the findings that granulosa cells lacking caspase-3 do eventually die suggest that caspase-independent mechanisms of cell death become activated (51) or that other caspase family members are involved. Regarding the latter, the low level of CM1 immunostaining observed in dying caspase-3-deficient granulosa cells provided one hint at the identity of at least one of these other family members. Previous work has shown that CM1, although highly selective for the cleaved form of caspase-3, exhibits a weak cross-reactivity to the large catalytic subunit of caspase-7 (43). These findings are not entirely unexpected, because 10 of the 13 amino acids in the cleaved caspase-3 peptide immunogen used to produce the CM1 antibody are conserved in caspase-7 (43). In addition, studies of hierarchical caspase activation after various apoptotic stimuli have shown that procaspase-7 is processed in parallel with procaspase-3 (36, 52, 53), and that caspase-3 is not needed for the activation of caspase-7 (52). Moreover, recent evidence indicates that a compensatory induction of caspase-7 activation occurs in caspase-3-deficient hepatocytes after exposure to a lethal stimulus (54).
Based on this information, we used a recently developed antibody that recognizes the cleaved form of caspase-7 to study its expression, in the absence or presence of functional caspase-3, in granulosa cells during apoptosis. Consistent with the idea that caspase-7 may compensate for the loss of caspase-3 function to eventually ensure granulosa cell death, we observed a positive correlation between the presence of cleaved caspase-7 and the occurrence of apoptosis. As this observation held true for both wild-type and caspase-3 mutant granulosa cells, it may be that in wild-type cells caspase-7 normally functions in tandem with caspase-3 during the execution phase of granulosa cell death. Similar results were obtained from a parallel analysis of caspases and apoptosis in granulosa cells of human ovarian follicles during atresia. This set of experiments revealed that intense CM1 immunoreactivity in granulosa cells appeared to herald their pending death, whereas cleaved caspase-7 was only detected in dying granulosa cells of moderately and grossly atretic antral follicles. Although these findings collectively support an evolutionarily conserved role for caspase-3 and caspase-7 in mediating granulosa cell death, a recent study has identified cleavage activation of procaspase-6, in addition to procaspase-3, in avian granulosa cells during apoptosis (55). Future analysis of ovaries in caspase-6-deficient female mice (16) should clarify whether this executioner caspase is involved in mammalian granulosa cell death.
In conclusion, this study has demonstrated the functional importance of caspase-3 in the execution of PCD in ovarian follicular somatic, but not germ, cells of the mouse ovary. We have also shown from studies of the human ovary that caspase-3 activation is probably an evolutionarily conserved feature of granulosa cell demise. In addition, we have provided evidence for the activation of caspase-7 in mouse and human granulosa cells during apoptosis, indicating that caspase-9 may serve to process at least two executioner caspases in somatic cells of the ovary during apoptosis. Interestingly, recent investigations have reported that caspase-3 is also activated in bovine (41, 56) and mouse (Rueda, B. R., and J. L. Tilly, unpublished observations) luteal cells during apoptosis. Such findings suggest that the requirement for caspase-3 in the normal execution of granulosa cell death shown herein may also be maintained as these cells transform into luteal cells after ovulation. Our current efforts are aimed at addressing this possibility as well as using a number of other gene knockout mouse lines to elucidate a molecular blueprint of how PCD is executed in oocytes, granulosa cells, and luteal cells via shared or cell lineage-specific signaling.
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Acknowledgments |
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Footnotes |
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2 Research Fellow supported by the Finnish Foundation for Pediatric
Research and the Finnish Cultural Foundation.
3 Present address: Biogen, Inc., 14 Cambridge Center, Cambridge,
Massachusetts 02142.
4 Investigator with the Howard Hughes Medical Institute.
Received October 9, 2000.
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-fodrin cleavage but dispensable for
cleavage of other death substrates in apoptosis. J Biol Chem 273:15540–15545 treatment in vivo.
Biol Reprod 60:1087–1092This article has been cited by other articles:
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