BIOLOGY OF REPRODUCTION 60, 1087–1092 (1999)
Accumulation of Caspase-3 Messenger Ribonucleic Acid and Induction of Caspase
Activity in the Ovine Corpus Luteum Following Prostaglandin F2a Treatment In Vivo 1
Bo R. Rueda,2,4,5 Isabel R. Hendry,4 Jonathan L. Tilly,6 and Debora L. Hamernik3,7
The Women’s Research Institute,4 Wichita, Kansas 67214-3199
Department of Obstetrics and Gynecology,5 University of Kansas School of Medicine-Wichita,
Wichita, Kansas 67214-3199
Vincent Center for Reproductive Biology,6 Department of Obstetrics and Gynecology, Massachusetts
General Hospital/Harvard Medical School, Boston, Massachusetts 02114
Department of Physiology,7 University of Arizona, Tucson, Arizona 85724-5051
ABSTRACT
Caspase-3, a vertebrate homologue of the protein encoded
by the Caenorhabditis elegans cell death gene, ced-3, induces
apoptosis when overexpressed in eukaryotic cells. Since apoptosis occurs during corpus luteum (CL) regression in many species,
including the ewe, these studies were conducted to 1) isolate a
cDNA encoding ovine caspase-3, 2) measure steady state
amounts of caspase-3 mRNA in the CL during luteolysis induced
by prostaglandin F2a (PGF2a) and during the time of maternal
recognition of pregnancy, and 3) measure changes in caspase
activity during PGF2a-initiated luteal regression. Oligonucleotide
primers corresponding to a human caspase-3 cDNA sequence
were combined with total RNA from ovine CL in a reverse transcription-polymerase chain reaction-based procedure to amplify
a 640-base pair partial cDNA with a nucleotide sequence 86%
and 81% identical to the human and rat caspase-3 cDNAs, respectively. CL were collected from ewes at 0, 12, or 24 h after
treatment with PGF2a on Day 10 of the estrous cycle and from
nonpregnant and pregnant ewes on Day 12 or Day 14 of the
cycle. Northern blot analysis of total cellular RNA from ovine
CL and a radiolabeled ovine caspase-3 cRNA probe indicated
the presence of a single mRNA transcript of approximately 2.5
kilobases. Levels of caspase-3 mRNA were approximately 3-fold
higher ( p , 0.05) in CL at 12 h and 24 h after PGF2a in comparison to those levels measured in matched CL from untreated
ewes. There were no differences ( p . 0.05) in amounts of caspase-3 mRNA in CL on Day 12 or Day 14 of the estrous cycle
compared to Day 12 or Day 14 of pregnancy, respectively. Caspase activity in CL (measured by the ability of CL lysates to cleave
an artificial caspase substrate) was also significantly ( p , 0.05)
increased in CL collected after treatment with PGF2a compared
to CL collected from nontreated ewes. We conclude that physiological cell death during PGF2a-induced luteal regression in
the ewe is mediated, at least in part, via increased expression
and activity of the caspase family of pro-apoptotic proteases.
INTRODUCTION
In mammals, the corpus luteum (CL) synthesizes and
secretes progesterone to provide uterine quiescence for the
establishment and maintenance of pregnancy. In the absence of a conceptus, however, the CL regresses and the
Accepted December 4, 1998.
Received July 28, 1998.
1
Supported in part by the Wesley Medical Research Institute (B.R.R.),
NIH R01-HD34226 (J.L.T.), NIH R01-AG12279 (J.L.T.), and USDA-9537203-2032 (D.L.H.).
2
Correspondence: Bo R. Rueda, The Women’s Research Institute, 1010
N. Kansas, Wichita, KS 67214-3199. FAX: 316 293 1881;
e-mail: [email protected]
3
Current address: USDA-CSREES-NRI, 1400 Independence Ave., SW,
Stop 2241, Washington, DC 20250-2241.
estrous or menstrual cycle resumes. Thus, premature disruption of normal CL function could result in the loss of
pregnancy, irregular estrous or menstrual cycles, and an
overall reduction in reproductive efficiency. Prostaglandin
F2a (PGF2a), a primary luteolysin in domestic animals [1],
is known to reduce luteal progesterone production (functional luteal regression) and to disrupt luteal cell integrity
(structural luteal regression). Although the cellular mechanisms leading to the demise of the CL in the absence of a
viable embryo, or those mechanisms involved in maintaining the function of the CL around the time of maternal
recognition of pregnancy, are not fully understood, these
events likely involve regulated expression of various genes
associated with the inhibition or acceleration of the physiological cell death process (also referred to as apoptosis).
Many reports have now shown that apoptosis occurs during
luteolysis, and it is generally accepted that controlled cell
death plays a central role in the physical removal of the CL
from the ovary at the end of the estrous or menstrual cycle
[2, 3].
Recently, specific genes believed important in the regulation of apoptosis have been identified, characterized, and
shown to be expressed in the CL. For example, several
genes belonging to the bcl-2 family, including both antiapoptotic (bcl-2, bcl-xL, mcl-1) and pro-apoptotic (bax)
family members, are known to be expressed in luteal cells
of various species [2, 4–8]. These data are in agreement
with the general concept that members of the Bcl-2 protein
family serve as an evolutionarily conserved checkpoint in
the cell death pathway [9–11], and more specifically with
the hypothesis that Bcl-2 family members are central to
apoptosis regulation in diverse ovarian cell lineages [12,
13]. In addition to their proposed roles in modulating the
intracellular reduction-oxidation state [14, 15], mitochondrial stability [16, 17], and ion flux [17], recent data indicate that Bcl-2 family members, via dimeric interactions
with other cell death-regulatory molecules [18–20], are important for regulating activation of a family of enzymes,
referred to as caspases [21], that serve as regulators and
effectors of apoptosis [22–25].
Analogous to the functional and structural homology between Ced-9 in the worm and Bcl-2 family members in
vertebrates, caspases were originally identified as central to
the cell death pathway in vertebrates by homology with a
gene in C. elegans, termed ced-3, whose expression is indispensable for cell death [26, 27]. When cloned, the ced3 gene product was found to be structurally related to a
cytokine-processing cysteine protease in vertebrates referred to as interleukin-1b-converting enzyme (ICE) [28].
Despite the fact that ICE (now referred to as caspase-1) had
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RUEDA ET AL.
been isolated and characterized as a mediator of the inflammatory response, reflective of its role in pro-interleukin-1b
processing, Yuan et al. [28] provided evidence for a possible novel function of this protease in cell death committal.
Since this landmark report, 11 Ced-3 homologues have
been identified in vertebrate species, and these enzymes
have now been, for the sake of clarity, collectively referred
to as caspases (cysteine aspartic acid-specific proteases
[21, 29]).
All caspases are synthesized as zymogens that require
cleavage to form the active enzyme [25, 30]. Initially it was
believed, based on genetic studies of cell death in C. elegans [26, 27], that members of the caspase family in vertebrate species were involved in the final steps of the cell
death cascade. However, more recent evidence, as well as
the growing number of caspase family members, suggests
that this is not entirely true. With minor exceptions, the
caspase family has now been roughly separated into two
categories: regulators and effectors [31]. The regulators,
recognized as those caspases with long pro-domains, are
believed to play either no role or a more upstream role in
the cell death cascade, the latter of which includes activation of the effector caspases. In contrast, the effector caspases, which possess short pro-domains, are primarily responsible for cleavage of key substrates that facilitate
execution of the apoptotic program, including signaling
molecules, DNA repair enzymes, mRNA processing components, cytoskeletal and nuclear scaffold proteins, and nuclease-activating factors [25, 32, 33]. Among the 11 caspases presently known, the vast majority of data support a
fundamental role for caspase-3 (originally referred to as
CPP32; [34]) in proteolytic disruption of cellular homeostasis and the ultimate dismantling of the cell destined for
apoptosis [35–37].
Many members of the caspase family are known to be
expressed in the ovary [6, 38–42]. Data from peptide inhibition studies and from substrate cleavage assays have
documented an induction of caspase activity during apoptosis in granulosa cells [43]. Unfortunately, however, little is
known regarding the expression or function of caspases in
the CL. In the cow, increased levels of caspase-1 mRNA
have been detected in regressing CL on Day 21 of the estrous cycle as compared to functional CL on Day 21 of
pregnancy [6]. Moreover, Krajewska et al. [39] have reported abundant expression of caspase-3 in human CL, and
thus speculated that this protease is important for luteal
regression. On the basis of these observations, we conducted the present studies to investigate the expression and
regulation of caspase-3 mRNA and protein activity in the
ovine CL during luteal regression.
MATERIALS AND METHODS
Isolation of Ovine Caspase-3 cDNA
Total cellular RNA from ovine CL was reverse-transcribed into cDNA using random hexamer primers and avian myeloblastosis virus reverse transcriptase (Promega,
Madison, WI). The resultant first-strand cDNA was then
subjected to 35 cycles of amplification using oligonucleotide primers corresponding to bases 62–85 (forward primer)
and bases 725–748 (reverse primer) of the human caspase3 cDNA coding sequence [34], as described previously for
similar generation of human and rat partial cDNAs [38,
40]. The resultant polymerase chain reaction was separated
through a 1.5% agarose gel, and the primary product (665
base pair [bp]) was isolated, purified, and subcloned into
the pCRII vector (Invitrogen, Carlsbad, CA). Nucleotide
sequence analysis of the cloned cDNA was performed by
the dideoxy chain termination reaction using Sequenase 2.0
(Amersham, Arlington Heights, IL).
Animals
To synchronize estrous cycles, two injections of PGF2a
(Lutalyse, Kalamazoo, MI; 7.5 mg/i.m. injection) were administered at 10-day intervals to mature ewes of mixed
breeds common to the western United States. The first day
of estrus was detected with a vasectomized ram (for studies
with nonpregnant ewes) or a fertile ram (for studies with
pregnant ewes) and was designated Day 0. In nonpregnant
ewes, ovaries were surgically removed on Day 10 of the
estrous cycle and at 12 or 24 h after i.m. injection (7.5 mg)
of PGF2a (n 5 5 ewes per group). In other experiments,
ovaries were surgically removed during the luteal phase
(Day 12 or Day 14) from pregnant or nonpregnant ewes (n
5 5 ewes per group). The presence of a conceptus in uterine flushes was used to confirm pregnancy. Blood samples
were drawn by jugular venipuncture immediately prior to
tissue collection; from these, serum samples were prepared
and stored for subsequent analysis of progesterone concentrations (DPC Coat-A-Count Kit; Diagnostic Products, Los
Angeles, CA) [43].
Isolation of DNA
Genomic DNA was extracted from individual CL, precipitated, and stored as described previously for internucleosomal cleavage analysis [44, 45]. After spectrophotometric (A260) quantitation, 1 mg of genomic DNA from
each sample was labeled on 39-ends with [a-32P]dideoxyATP (ddATP, 3000 Ci/mmol; Amersham) using 25 U of
terminal transferase enzyme (Boehringer-Mannheim, Indianapolis, IN) as described previously [46]. Samples were
separated by agarose gel electrophoresis and analyzed for
the occurrence of internucleosomal DNA cleavage by autoradiography. Low molecular weight DNA fractions (, 15
kilobases [kb]) were excised from the gel, mixed with 3 ml
of scintillation fluid (Scintiverse BD; Fisher Scientific,
Pittsburgh, PA), and counted in a beta counter to provide
a quantitative estimate of the degree of internucleosomal
DNA cleavage among samples [44, 46].
Isolation of RNA and Northern Blot Analysis
Total cellular RNA from individual CL was isolated using a modification of the one-step procedure with Trizol
(Gibco-BRL; Gaithersburg, MD). Samples of RNA (5 mg/
lane) were separated through a 1.5% agarose denaturing
gel, transferred to nylon membranes (Zeta Probe GT; BioRad, Hercules, CA), and hybridized to a radiolabeled cRNA
(caspase-3) or cDNA (18S ribosomal RNA) probe. An antisense RNA probe complementary to the ovine caspase-3
mRNA coding sequence was synthesized in vitro using SP6
polymerase (Promega) and [a-32P]CTP (3000 Ci/mmol;
NEN, Boston, MA) [47, 48]. To control for equal loading
of RNA on the Northern gels, a cDNA probe complementary to 18S rRNA was prepared by random priming [45]
using [a-32P]cATP (3000 Ci/mmol; NEN) and hybridized
to the blots after the caspase-3 probe was removed by highstringency washing. Amounts of radioactivity in heteroduplexes were visualized and quantitated with an Instant Imager (Packard, Meriden, CT).
CASPASES IN THE OVINE CL
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FIG. 1. Changes in serum concentrations of progesterone (ng/ml) at 0,
12, and 24 h after PGF2a treatment on Day 10 of the estrous cycle. Levels
of progesterone were determined by RIA as described in Materials and
Methods (data points represent mean 6 SE). * Significantly different from
control.
Caspase Activity
A colorimetric assay kit (Clontech, Palo Alto, CA),
which relies on the caspase-mediated cleavage of a chromophore (p-nitroanilide) from a synthetic caspase substrate
peptide (DEVD), was used to evaluate caspase activity in
ovine CL lysates according to the manufacturer’s guidelines. Frozen luteal tissue was homogenized in lysis buffer,
and concentration of protein was determined. The protein
lysate (250 mg) was mixed with double-strength reaction
buffer in a 96-well plate. The increase in protease activity
was determined by colorimetric detection and comparison
to a standard curve. A control was performed by treating
the induced lysates with a caspase-3 inhibitor before incubation with the substrate. This control reaction confirms
that the signal detected is a result of protease activity.
Data Presentation and Analysis
The data presented for each experiment are representative of 3–5 individual CL from different ewes. Representative autoradiograms are presented where appropriate for
qualitative analysis, whereas quantitative data (combined
results from the replicate experiments) were analyzed by
one-way ANOVA followed by Duncan’s New Multiple
Range test. A value of p , 0.05 was considered statistically
significant.
RESULTS
Characterization of Ovine Caspase-3 Partial cDNA
A 665-bp fragment of the ovine caspase-3 cDNA was
isolated (accession no. AF068837), and nucleotide sequence analysis revealed that it shared an 86% and 81%
identity to the corresponding sequences in the human [34]
and rat caspase-3 cDNAs [38], respectively.
PGF2a Induced Expression of Caspase-3 in the Ovine CL
Serum concentrations of progesterone were lower (p ,
0.05) at 12 and 24 h after PGF2a administration compared
to values in untreated controls (Fig. 1). The genomic DNA
isolated from Day 10 CL did not exhibit visible DNA laddering (a characteristic of apoptosis) as evidenced by the
FIG. 2. Qualitative biochemical analysis of DNA integrity in functional
and regressing CL. Genomic DNA was isolated from individual CL on
Day 10 of the estrous cycle (lanes 1–2) and from CL collected 12 h (lanes
3–4) or 24 h (lanes 5–6) after injection of PGF 2a. Oligonucleosomal DNA
fragmentation was evaluated by [32P]ddATP 39 end-labeling with terminal
transferase enzyme. Approximately 250 ng of labeled DNA sample was
loaded in each well.
[32P]ddATP labeling on the 39 end (Fig. 2). Furthermore,
based on the biochemical analysis of 32P labeling of low
molecular weight DNA, DNA laddering was minimal (241
6 43 cpm; mean 6 SEM of samples) relative to that observed after administration of PGF2a (p , 0.002, n 5 3).
Consistent with the loss of progesterone production after
administration of PGF2a, there were significant increases in
low molecular weight DNA labeling at the 12- and 24-h
time points (3- and 5-fold, respectively; 860 6 67 and 1311
6 166 cpm). Steady state amounts of mRNA for caspase3 were elevated (p , 0.05) in CL collected at 12 and 24
h after administration of PGF2a compared to those values
obtained on Day 10 of the estrous cycle (prior to PGF2a
injection) (Fig. 3). Increased expression of caspase-3
mRNA occurred simultaneously with the drop in serum
concentrations of progesterone and internucleosomal fragmentation of DNA.
Consistent with PGF2a-mediated increase in caspase-3
mRNA levels (Fig. 3), protein lysates isolated from CL of
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RUEDA ET AL.
FIG. 3. A) Autoradiograph of Northern blot analysis of caspase-3 mRNA in ovine CL collected on Day 10 (n 5 3) of the estrous cycle (Day 0 5 estrus)
and 12 (n 5 5) or 24 h (n 5 4) after injection of PGF2a on Day 10. Each lane contains 5 mg of total cellular RNA from individual CL. B) Quantitative
assessment of caspase-3 after phospho-image analysis of hybridization signal intensities and normalization of data against 18S RNA (mean 6 SE).
Injection of PGF2a on Day 10 of the estrous cycle resulted in increased ( p , 0.05) amounts of mRNA for caspase-3. * Significantly different from
control.
nonpregnant ewes 24 h after PGF2a exhibited significantly
increased levels of caspase activity compared with lysates
prepared from CL of untreated ewes (p , 0.05) (Fig. 4).
The caspase activity present in CL extracts was ameliorated
by inclusion of a specific caspase-3 inhibitor, DEVD-CHO,
in the reaction assay (p , 0.05; data not shown), suggesting
that PGF2a treatment specifically induced activity of caspase-3 in the CL.
Caspase-3 Expression in the Ovine CL Did Not
Change during Maternal Recognition of Pregnancy
Serum concentrations of progesterone were similar (p .
0.05) on Day 12 (3.0 6 0.86) and Day 14 (2.5 6 0.33) of
FIG. 4. Comparison of caspase-3 activity in crude protein lysates isolated from CL collected from ewes 0, 12, 24 h after treatment with PGF2a
on Day 10 of the estrous cycle. Caspase activity was measured with a
colorimetric assay kit that relies on caspase-mediated cleavage of p-nitroanilide (pNA) from a synthetic caspase substrate peptide (DEVD). The
kit was used in accordance with manufacturer’s guidelines (data points
represent mean 6 SE). * Significantly different from control.
the estrous cycle compared to Day 12 (3.08 6 0.90) and
Day 14 (3.0 6 0.58) of pregnancy. There was no evidence
of internucleosomal DNA fragmentation in genomic DNA
in CL on Day 12 and Day 14 of the estrous cycle or Day
12 and Day 14 of pregnancy (data not shown). In addition,
there were no differences (p . 0.05) in steady state
amounts of mRNA for caspase-3 in CL collected on Day
12 (3.48 6 0.65) or Day 14 (4.2 6 0.29) of the estrous
cycle compared to Day 12 (4.37 6 1.44) and Day 14 (5.4
6 1.01) of pregnancy.
DISCUSSION
In our ongoing attempts to characterize the downstream
effectors of PGF2a-induced apoptosis during luteal regression, we focused on the possible role of a key proteolytic
enzyme, caspase-3. Without question, the most well-characterized death effector enzyme is caspase-3 [35, 36]. Many
reports have substantiated a central role for this specific
caspase family member in apoptosis, responsible for producing many features attributed to apoptotic cells, including
cell shrinkage, membrane budding, and internucleosomal
DNA cleavage [49]. Surprisingly, however, despite the accumulating evidence that caspase-3 is central to the proper
execution of apoptosis in many cell types, genetically manipulated mice that lack expression of functional caspase3 show defects in apoptosis in a relatively restricted set of
cell lineages [50]. Although these latter findings suggest
that caspase-3 is in fact dispensable for many paradigms of
apoptosis in the body, it is also highly plausible that many
cell types have simply recruited other caspase family members to act in place of the ‘‘knocked-out’’ caspase-3, serving as a prime example of the so-called ‘‘redundancy’’ hypothesis.
In studies of ovarian function, caspase-3 is known to be
expressed and hormonally regulated [38]. The report that
presented those findings, the first to implicate caspases as
important components of apoptotic cell death in the female
CASPASES IN THE OVINE CL
gonad, has since been confirmed and extended by a number
of studies from our laboratories [13, 40, 42, 51] and others
[39, 41]. Of direct relevance to luteolysis, one of our previous studies identified a dramatic increase in levels of
caspase-1 mRNA in regressing CL collected from nonpregnant cows on Day 21 of the cycle as compared with those
levels present in functional CL of cows on Day 21 of pregnancy [2]. Although this would suggest a role for caspase1 in luteal regression, similar analyses of caspases in a related paradigm of ovarian cell death, e.g., follicular atresia,
have indicated that caspase-2 and caspase-3, but not caspase-1, are probably important components of granulosa cell
demise [38, 42]. On the basis of these findings, recent observations that caspase-3 is expressed in human granulosaluteal cells [40] and rat luteal cells [41], and the fact that
caspase-3 is more abundant in luteal cells as opposed to
follicular granulosa cells in the adult human ovary [39], we
sought to further examine the role of this specific caspase
in PGF2a-initiated luteal regression.
In the present studies, we observed that a single injection
of PGF2a given to nonpregnant ewes during the midluteal
phase of the estrous cycle (Day 10) rapidly initiated features associated with both functional (loss of progesterone)
and structural (apoptosis) luteolysis. We noted, in association with the decrease in circulating levels of progesterone
and the appearance of internucleosomal DNA cleavage in
the CL, a marked increase in the levels of caspase-3 mRNA
in luteal tissue from prostaglandin-treated ewes relative to
amounts of caspase-3 mRNA present in CL of untreated
ewes. These data, which provide the first evidence for acute
prostaglandin regulation of a critical cell death-regulatory
gene in the CL, support the hypothesis that induction of
caspase-3 is a component of PGF2a-mediated luteolysis in
the ewe.
To further examine a possible temporal relationship between caspase-3 induction and luteal regression, we next
examined whether nonpregnant ewes in the mid-to-late luteal phase of the cycle (Days 12–14) showed any differences in levels of caspase-3 mRNA in preparation for luteolysis. As controls, CL were collected from pregnant
ewes on the same days of the cycle, serving as a model of
luteal rescue during the time of maternal recognition of
pregnancy. On the basis of comparable levels of serum progesterone and the absence of apoptosis, CL from both nonpregnant and pregnant ewes on Days 12–14 were considered fully functional. Moreover, in contrast to the changes
observed in caspase-3 mRNA levels following an irreversible luteolytic stimulus (e.g., administration of PGF2a),
there were no differences in the levels of caspase-3 mRNA
in CL on Day 12 or Day 14 of the estrous cycle or pregnancy. Thus, if induction of caspase-3 expression is an important factor in luteolysis, changes in caspase-3 mRNA
levels appear to tightly coincide with the actual initiation
of luteolysis as opposed to being the result of a gradual
process of accumulation throughout the luteal phase.
It is well recognized that the changes observed in the
levels of a given mRNA transcript do not always equate to
similar changes in the protein product. This point is particularly important in the study of caspases, since these enzymes are synthesized and stored in cells as inactive zymogens. Taking advantage of the unique specificity of caspases for cleavage at aspartate residues, and the fact that
caspase-3 prefers the amino acid sequence DEVD for
cleavage activity [30, 35], cellular lysates can be assessed
for changes in caspase activity by monitoring the ability of
the lysates to catalyze release of a chromophore (pNA)
1091
from an artificial caspase-3 substrate, DEVD. Using a commercially available caspase-3 activity kit based on these
concepts, we showed that the PGF2a-initiated increase in
caspase-3 mRNA levels in the CL was followed by a significant elevation in DEVD cleavage activity in CL lysates.
Although we presume from the mRNA data that this cleavage activity reflects changes in caspase-3 activity, recent
reports have suggested that other caspases are capable of
recognizing the DEVD site in target proteins [52]. Therefore, as a means to corroborate the data obtained from the
DEVD cleavage experiments, future studies will be needed
to quantitate amounts of caspase-3 protein in the ovine CL
when antisera useful for this protein become available.
Nonetheless, the findings presented herein support the hypothesis that PGF2a-mediated luteal regression in the ewe
involves increased expression and activity of caspases
needed for apoptosis.
ACKNOWLEDGMENTS
We thank Dr. Paula Gentry, Ms. Bridgette Kirkpatrick, Mr. Derron
Wahlen, and Mr. Eric Harmon for assisting with collection of CL. We also
thank Ms. Bridgette Kirkpatrick and Mr. Eric Harmon for conducting RIAs
for progesterone. These studies were initiated while Dr. Bo Rueda was at
the Department of Physiology, University of Arizona (Tucson, AZ).
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