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Treatment In Vivo1
a The Women's Research Institute, Wichita, Kansas 67214-3199
b Department of Obstetrics and Gynecology, University of Kansas School of Medicine-Wichita, Wichita, Kansas 67214-3199
c Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114
d Department of Physiology, University of Arizona, Tucson, Arizona 85724-5051
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2) and during the time of maternal recognition of pregnancy, and 3) measure changes in caspase activity during PGF2-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 PGF2 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 PGF2 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 PGF2 compared to CL collected from nontreated ewes. We conclude that physiological cell death during PGF2-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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2), 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 anti-apoptotic (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 ced-3 gene product was found to be structurally related to a cytokine-processing cysteine protease in vertebrates referred to as interleukin-1ß-converting enzyme (ICE) [28]. Despite the fact that ICE (now referred to as caspase-1) had been isolated and characterized as a mediator of the inflammatory response, reflective of its role in pro-interleukin-1ß 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 caspase-3 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 PGF2 (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 PGF2 (n = 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 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 µg of genomic DNA from each sample was labeled on 3'-ends with [-32P]dideoxy-ATP (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 µg/lane) were separated through a 1.5% agarose denaturing gel, transferred to nylon membranes (Zeta Probe GT; Bio-Rad, 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 [-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 [-32P]cATP (3000 Ci/mmol; NEN) and hybridized to the blots after the caspase-3 probe was removed by high-stringency washing. Amounts of radioactivity in heteroduplexes were visualized and quantitated with an Instant Imager (Packard, Meriden, CT).
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 µg) 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
PGF2 Induced Expression of Caspase-3 in the Ovine CL
Serum concentrations of progesterone were lower (p < 0.05) at 12 and 24 h after PGF2 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 [32P]ddATP labeling on the 3' end (Fig. 2). Furthermore, based on the biochemical analysis of 32P labeling of low molecular weight DNA, DNA laddering was minimal (241 ± 43 cpm; mean ± SEM of samples) relative to that observed after administration of PGF2 (p < 0.002, n = 3). Consistent with the loss of progesterone production after administration of PGF2, there were significant increases in low molecular weight DNA labeling at the 12- and 24-h time points (3- and 5-fold, respectively; 860 ± 67 and 1311 ± 166 cpm). Steady state amounts of mRNA for caspase-3 were elevated (p < 0.05) in CL collected at 12 and 24 h after administration of PGF2 compared to those values obtained on Day 10 of the estrous cycle (prior to PGF2 injection) (Fig. 3). Increased expression of caspase-3 mRNA occurred simultaneously with the drop in serum concentrations of progesterone and internucleosomal fragmentation of DNA.
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Consistent with PGF2-mediated increase in caspase-3 mRNA levels (Fig. 3), protein lysates isolated from CL of nonpregnant ewes 24 h after PGF2 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 PGF2 treatment specifically induced activity of caspase-3 in the CL.
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Caspase-3 Expression in the Ovine CL Did NotChange during Maternal Recognition of Pregnancy
Serum concentrations of progesterone were similar (p > 0.05) on Day 12 (3.0 ± 0.86) and Day 14 (2.5 ± 0.33) of the estrous cycle compared to Day 12 (3.08 ± 0.90) and Day 14 (3.0 ± 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 ± 0.65) or Day 14 (4.2 ± 0.29) of the estrous cycle compared to Day 12 (4.37 ± 1.44) and Day 14 (5.4 ± 1.01) of pregnancy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-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 caspase-3 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 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 caspase-1 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 granulosa-luteal 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 PGF2-initiated luteal regression.
In the present studies, we observed that a single injection of PGF2 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 PGF2-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 PGF2), 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) from an artificial caspase-3 substrate, DEVD. Using a commercially available caspase-3 activity kit based on these concepts, we showed that the PGF2-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 PGF2-mediated luteal regression in the ewe involves increased expression and activity of caspases needed for apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Bo R. Rueda, The Women's Research Institute, 1010 N. Kansas, Wichita, KS 67214-3199. FAX: 316 293 1881; brueda{at}kumc.edu
3 Current address: USDA-CSREES-NRI, 1400 Independence Ave., SW, Stop 2241, Washington, DC 20250-2241.
Accepted: December 4, 1998.
Received: July 28, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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identified as a luteolytic hormone in sheep. Nat New Biol 1972; 83:527–536.This article has been cited by other articles:
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