Prostaglandin F2�-mediated Activation of Apoptotic SignalingCascades in the Corpus Luteum during ApoptosisINVOLVEMENT OF CASPASE-ACTIVATED DNase*□S

Received for publication, August 20, 2004, and in revised form, December 20, 2004Published, JBC Papers in Press, December 28, 2004, DOI 10.1074/jbc.M409596200

Vijay K. Yadav‡, Garimella Lakshmi, and Rudraiah Medhamurthy§

From the Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science,Bangalore 560 012, India

Prostaglandin F2� (PGF2�) acting via a G protein-cou-pled receptor has been shown to induce apoptosis in thecorpus luteum of many species. Studies were carried outto characterize changes in the apoptotic signaling cas-cade(s) culminating in luteal tissue apoptosis duringPGF2�-induced luteolysis in the bovine species in whichinitiation of apoptosis was demonstrable at 18 h afterexogenous PGF2� treatment. An analysis of intrinsicarm of apoptotic signaling cascade elements revealedthat PGF2� injection triggered increased ratio of Bax toBcl-2 in the luteal tissue as early as 4 h posttreatmentthat remained elevated until 18 h. This increase wasassociated with the elevation in the active caspase-9 and-3 protein levels and activity (p < 0.05) at 4–12 h, but aspurt in the activity was seen only at 18 h posttreatmentthat could not be accounted for by the changes in theBax/Bcl-2 ratio or changes in translocation of Bax tomitochondria. Examination of luteal tissue for FasL/Fasdeath receptor cascade revealed increased expressionof FasL and Fas at 18 h accompanied by a significant(p < 0.05) induction in the caspase-8 activity and trun-cated Bid levels. Furthermore, intrabursal administra-tion of specific caspase inhibitors, downstream to theextrinsic and intrinsic apoptotic signaling cascades, in apseudopregnant rat model revealed a greater impor-tance of extrinsic apoptotic signaling cascade in medi-ating luteal tissue apoptosis during PGF2� treatment.The DNase responsible for PGF2�-induced apoptoticDNA fragmentation was found to be Ca2�/Mg2�-depend-ent, temperature-sensitive DNase, and optimally activeat neutral pH conditions. This putative DNase was in-hibited by the recombinant inhibitor of caspase-acti-vated DNase, and immunodepletion of caspase-acti-vated DNase from luteal lysates abolished the observedDNA fragmentation activity. Together, these data dem-onstrate for the first time temporal and spatial changesin the apoptotic signaling cascades during PGF2�-in-duced apoptosis in the corpus luteum.

Although PGF2�1 was discovered as a physiological luteoly-

sin nearly three decades ago (1), cellular events associated withluteolysis remain poorly characterized, in part because of thelack of availability of a suitable in vitro model system thatmimics all of the cellular events that occur in vivo in responseto spontaneous or PGF2�-induced luteolysis (2). ProstaglandinF2� interacts with its G protein-coupled receptor, present pre-dominantly on large luteal cells, but are also present on smallluteal and endothelial cells of the corpus luteum (3) and acti-vates Gq/phospholipase C/protein kinase C pathway (4, 5), re-sulting in decreased steroidogenesis; The intracellular signal-ing events that lead to structural regression of luteal tissue arepoorly characterized; however, it is now well established thatapoptosis or programmed cell death plays a central role in thestructural regression of luteal tissue during PGF2�-induced orspontaneous luteolysis of several species (6–10).

Apoptosis or programmed cell death is an evolutionarilyconserved mechanism orchestrated by the genome-encodedproteins of the host that form part of two distinct (intrinsic andextrinsic) signaling cascades. The intrinsic apoptotic signalingcascade is generally thought to be activated by apoptotic stim-uli that originate within a cell in response to certain drugs,radiation, or growth factor withdrawal and primarily causechanges in mitochondrial permeability through alterations inthe ratio of pro-apoptotic to anti-apoptotic Bcl-2 family mem-bers (11). On the other hand, the extrinsic apoptotic signalingcascade is activated by extracellular signals (viz. FasL) thatinteract with cell surface receptors (viz. Fas) to induce celldeath (12). Changes in the mitochondrial permeability or deathreceptor activation lead to activation of a cascade of intracel-lular proteases known as caspases (13). Once activated,caspases cleave various cellular substrates including actin,poly(ADP-ribose) polymerase (PARP), DFF45/inhibitor ofcaspase-activated DNase (ICAD), fodrin, and lamin that con-tribute to the morphological changes seen in apoptoticcells (13).

Fragmentation of DNA constitutes the final cellular eventduring apoptosis. It is mediated by the internucleosomal cleav-age of DNA by endonucleases resulting in the formation of a180-bp DNA ladder, which is considered as one of the hall-marks of cellular apoptosis. Candidates for such endonucleases* This work was supported in part by grants from the Council of

Scientific and Industrial Research, Indian Council of Medical Research,and University Grants Commission (New Delhi, India). The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains Supplemental Figs. 1–5.

‡ Supported by fellowship from the Council of Scientific and Indus-trial Research (New Delhi, India).

§ To whom correspondence should be addressed: Dept. of MolecularReproduction, Development and Genetics, Indian Institute of Science,Bangalore 560012, India. E-mail: rmm@mrdg.iisc.ernet.in.

1 The abbreviations used are: PGFF2�, prostaglandin F2�; ICAD, in-hibitor of caspase-activated DNase; CAD, caspase-activated DNase;PARP, poly(ADP-ribose) polymerase; PVDF, polyvinylidene difluoride;Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; GST, glutathione S-transferase; RT, reverse transcription; DTT, dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonicacid; ANOVA, analysis of variance; tBid, truncated Bid; JNK, c-JunN-terminal kinase; MAPK, mitogen-activated protein kinase; AFC, 7-amino-4-trifluoromethylcoumarin; Ac, acetyl; CHO, aldehyde.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 11, Issue of March 18, pp. 10357–10367, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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include the caspase-activated enzymes, such as DFF40/caspase-activated DNase (CAD) (14, 15) and Nuc70 (16), diva-lent cation-dependent neutral (17) or acidic endonucleases (18),and Ca2�/Mg2�-dependent endonucleases (19–21). Recently,however, another DNase, endonuclease G, released from mito-chondria during apoptosis has also been shown to mediateapoptotic DNA fragmentation (22).

Unlike the classical cell surface receptors that induce celldeath, PGF2� receptor is a G protein-coupled receptor thatlacks any intracytoplasmic region with an identity/similarity tothe death domain/caspase activation and recruitment domainthat recruits and/or activates caspases. However, caspase-8,one of the initiator caspases acting downstream of the Fasreceptor pathway, has recently been reported to be activatedduring PGF2�-induced luteal tissue apoptosis in the corpusluteum of murine species (23) and the authors hypothesizedthat some level of cross-talk exists between PGF2� receptorsignal transduction and the classical apoptotic signaling cas-cades. This hypothesis is further strengthened by the observa-tion that increased Bax and Fas expressions have been re-ported to be associated with the apoptosis in spontaneouslyregressing luteal tissue of the non-fertile cycles, whereas a sig-nificant attenuation in expression in these genes are seen in thecorpus luteum during fertile cycle in the bovine species (24, 25).Also, several in vitro studies indicate that apoptotic stimuli suchas soluble FasL (25, 26), serum withdrawal (6), interferon-� (27),and tumor necrosis factor-� (25, 28) that activate classical apo-ptotic signaling cascades are capable of inducing apoptosis in theluteal cells. The final phase of cellular demise, i.e. fragmentationof DNA, is very well characterized in the luteal tissue and isfrequently used as an index of structural luteal regression. How-ever, the nature of the DNase that executes DNA fragmentationduring PGF2�-induced apoptosis in the luteal tissue has not beenidentified,althoughseveralDNase-likeactivities,viz.Ca2�/Mg2�-dependent DNase (29), Zn2�-inhibitable DNase (6), and DNaseI-like enzymes (30), have been observed to be active in the lutealcells under different conditions. These studies indicate that ele-ments of apoptotic signaling cascades are present in the lutealcells, but the sequence of events that commit luteal cells toapoptosis in response to PGF2� remains to be determined.

The purpose of this study was to systematically analyze theinvolvement of classical apoptotic signaling cascades duringPGF2�-mediated apoptotic cell death in the corpus luteum.Moreover, the biochemical nature of the endonuclease respon-sible for apoptotic DNA fragmentation in the corpus luteum inresponse to PGF2� has not been previously studied and repre-sents the major focus of our investigation. We chose to studythese pathways in the corpus luteum of buffalo cows (Bubalusbubalis), because the characterization of apoptosis followingspontaneous or PGF2�-induced luteolysis in this species wasreported recently (7). Our results show for the first time thatPGF2�-mediated apoptosis in the corpus luteum involves theactivation of mitochondrial apoptotic signaling cascade thatprecedes the activation of FasL/Fas cascade during PGF2�-induced apoptotic cell death in the bovine corpus luteum. Apseudopregnant rat model was standardized to investigate therelative roles of intrinsic and extrinsic apoptotic signaling cas-cades in PGF2�-induced luteal tissue apoptosis. Using the pseu-dopregnant rat model, we demonstrate that intrabursal injec-tion of a peptide-based cell-permeable caspase-8 inhibitor (Z-IETD-fmk) prior to PGF2� injection abolished the caspase-3activity and DNA fragmentation during PGF2�-induced lutealtissue apoptosis, whereas intrabursal injection of caspase-9inhibitor (Z-LEHD-fmk) only attenuated the apoptosis. More-over, our results point to an important role played by CAD in

mediating apoptotic DNA fragmentation during PGF2�-in-duced luteolysis.

EXPERIMENTAL PROCEDURES

Reagents

The polyclonal antibodies specific to Bcl-2 (number 197207), Bax(number 196820), phospho-BAD (number 9291), BAD (number 9292),cleaved caspase-9 (number 9501S), cleaved caspase-3 (number 9661S),Bid (number 2002), and ICAD (number PC366T) were purchased fromCalbiochem (numbers 197207, 196820, and PC366T) and Cell SignalingTechnology (Beverly, MA) (numbers 9291, 9292, 9501S, 9661S, and2002). PARP antibody (AM30) was purchased from Oncogene ResearchProducts (Boston, MA). Caspase-activated DNase antibody (Ab8406)was purchased from Abcam Inc. (Cambridge, MA). �-Actin antibody(A5441) was purchased from Sigma. Caspase substrates and inhibitors(Z-DEVD-AFC (caspase-3 substrate 264150), Z-IETD-AFC (caspase-8substrate 368059), Z-LEHD-AFC (caspase-9 substrate 218765), Ac-DEVD-CHO (caspase-3 inhibitor 235420), Z-IE(OMe)TD(OMe)-fmk(caspase-8 inhibitor 218759), Z-LE(OMe)HD(OMe)-fmk (caspase-9 in-hibitor 218761), and Z-VAD(OMe)-fmk (General executionary caspase(caspase-1, -3, -4, and -7) inhibitor 627610) were purchased from Cal-biochem. In situ apoptosis analysis kit (number 1684809) was pur-chased from Roche Diagnostics (Mannheim, Germany). The expressionplasmid for glutathione S-transferase (GST)-ICAD-L (see Ref. 14 fordetails of the vector) was a kind gift from Professor S. Nagata (Depart-ment of Genetics, Osaka University Medical School, Osaka, Japan).Glutathione-Sepharose 4B and pGEX-5X3 (GST expression plasmid)were obtained from Amersham Biosciences. GeneScreen Plus andPVDF membranes were purchased from PerkinElmer Life Sciences. Allof the other reagents were purchased from Sigma or Invitrogen orsourced locally.

Animal Models and Methods

A. PGF2�-induced Luteal Tissue Apoptosis in the Buffalo Cows(B. bubalis)—All of the procedures in animals were approved by theInstitutional Animal Ethics Committee (Indian Institute of Science).Buffalo cows (B. bubalis) on day 11 of the estrous cycle were injectedintramuscular with 750 �g of Tiaprost (Iliren®, Intervet InternationalB.V., Boxmeer, Holland), a synthetic analog of PGF2� (n � 3–4 animals/time point). At 4, 12, and 18 h following PGF2� injection, ovaries werecollected in cold phosphate-buffered saline and washed in phosphate-buffered saline prior to processing. Under sterile conditions, the corpusluteum from the ovary was extirpated, cut into 6–8 pieces, transferredto labeled cryovials, flash-frozen in liquid nitrogen, and stored at�70 °C until analysis. Also, a small portion of corpus luteum tissue wasfixed in Bouin’s solution for histological examination.

B. PGF2�-induced Luteal Tissue Apoptosis in the PseudopregnantRats: Effect of Caspase Inhibitors—Adult rats (Wistar strain) werehoused in a controlled environment (22–24 °C) and kept under a pho-toperiod of 14-h light and 10 h of darkness with ad libitum access to foodand water. Pseudopregnancy was induced in the female rats by cohab-itation with vasectomized male rats on the afternoon of pro-estrus.Following cohabitation, female rats were examined for the presence ofvaginal plug and/or subjected to screening of vaginal smears daily forthe extension of the di-estrus period. The presence of vaginal plug/day1 of continuous di-estrus (at least for 3 days) following cohabitation withthe vasectomized male rats was considered as day 1 of pseudopreg-nancy. In addition, pseudopregnancy was also confirmed by serumprogesterone analysis on day 5 of pseudopregnancy. Only the femalesshowing serum progesterone, �50 ng/ml were used in the study. On day8 of pseudopregnancy, female rats were anesthetized with pentobarbi-tal sodium (7.5 mg/rat, intraperitoneal) and abdominal cavity was ac-cessed through two dorsolateral subrenal incisions. The ovary with thesurrounding tissues was exposed, and a 30-gauge needle attached to a1-ml tuberculin syringe was gently entered into the ovarian bursa.Different caspase inhibitors (dissolved in Me2SO) diluted in 0.15 M

NaCl solution or vehicle solution for the respective inhibitors (15 �g/ovary) were injected into the ovarian bursal cavity in a volume of 60 �l.Only animals with the swelling of bursa and no visible leakage of theinjected fluids were included in the study (n � 3 rats/treatment group).The intrabursal route of administration has been utilized in various invivo studies to deliver different drugs to the ovary (31–33). After thecompletion of the injection procedure, ovaries were returned to theabdominal cavity and the abdominal wall was sutured with absorbablesuture and the skin with the surgical clips. Immediately after surgery(15 min), rats received (subcutaneous) either PGF2� (15 �g of clopro-stenol, Estrumat, Pitman-Moore Ltd., Harefield, United Kingdom) di-

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luted in 0.3 ml of 0.15 M NaCl or 0.3 ml of 0.15 M NaCl (vehicle)treatment. The animals were killed 24 h later, and the ovaries werevisualized under a dissecting microscope and processed for corporalutea collection. Corpora lutea were snap-frozen in liquid nitrogen andstored at �70 °C until analysis. The doses of peptide-based cell-perme-able caspase inhibitors used in this study were prepared based on theprevious studies where as few as 300 ng of caspase inhibitors/rat havebeen found to effectively block apoptosis in several in vivo models ofapoptosis when injected directly into the tissue of interest (34–38). Theluteolytic dose of PGF2� employed in this study was based on a previousstudy in the pseudopregnant rat model (39).

In Situ Apoptosis Analysis, DNA fragmentation Analysis,and Immunohistochemistry

Corpora lutea were processed for in situ apoptosis analysis accordingto the manufacturer’s recommendations. DNA fragmentation analysisand immunohistochemistry using Bax polyclonal antibody were carriedout as described previously by us (7).

RNA Extraction and Semi-quantitative RT-PCR Analysis

Total RNA was extracted from luteal tissue using TRIzol reagentaccording to the manufacturer’s recommendations. The quality andquantity of each RNA sample were assessed spectrophotometrically andon a 1% formaldehyde-agarose gel. Semi-quantitative RT-PCR wascarried out essentially as described previously (40). Oligonucleotideprimers were designed for Bax, FasL, Fas, and L-19 genes based on theconserved regions present in mRNA sequences of Homo sapiens, Bostaurus, and Mus musculus. The primers (forward and reverse) and PCRconditions (annealing temperature and PCR cycle number) used wereas follows: 5�-TCATCCAGGATCGAGCAGGG-3� and 5�-CGGCCCCAG-TTGAAGTTGCC-3� for 237-bp Bax (56 °C and 35 cycles); 5�-CCTGTG-GATGACTGAGTACC-3� and 5�-GAGACAGCCAGGAGAAATCA-3� for128-bp Bcl-2 (53 °C and 35 cycles); 5�-AATAGGTCACCCCAGTCCAC-CC-3� and 5�-CAGCCCAGTTTCATTGATCACAAGGC-3� for 174-bpFasL (55 °C and 35 cycles); 5�-AGGGGAACGAGTACACAGAC-3� and5�-GCAAGGGTTACAGTGTTCAC-3� for 178-bp Fas (50 °C and 33 cyc-les); and 5�-GAAATCGCCAATGCCAACTC-3� and 5�-TCTTAGACCTG-CGAGCCTCA-3� for 406-bp L-19 (58 °C and 25 cycles). Ethidium bromi-de-stained agarose gels displaying PCR products were scanned usingUVI-Tech gel documentation system and quantitated using UVI-BandMap software.

Western Blot Analysis

Corpus luteum tissue lysate was prepared as per the previouslypublished procedures (7). Equal amount of luteal tissue lysate (200 �gof protein/lane) was resolved by 10 or 12% SDS-PAGE and electro-blotted onto PVDF membrane, and Western blot analysis was per-formed as per the published procedures. Autoradiographs were scannedusing UVI-Tech gel documentation system and quantitated using UVI-Band Map software.

Caspase-8/9 Activity Assays

Caspase-8/9 activity assays were performed using fluorogenic sub-strates Z-IETD-AFC (caspase-8) and Z-LEHD-AFC (caspase-9). Lutealtissues collected before and after PGF2� treatment and after coinjection(15 min apart) of caspase inhibitors and PGF2� were homogenized inlysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM

EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM �-glyc-erophosphate, and 1 mM Na3VO4). After determining the protein con-centration, an equal amount (200 �g) of luteal lysate protein (at non-saturating concentration of the assay, for details see Supplemental Fig.1) was mixed in 0.5 ml of caspase-8/9 activity assay buffer (100 mM

HEPES, pH 7.5, 20% glycerol, 5 mM DTT, and 0.5 mM EDTA) followedby the addition of 20 �M Z-IETD-AFC/10 �M Z-LEHD-AFC substrate.Reaction mixture was incubated at 37 °C for 4–5 h, and liberated AFCwas measured in a spectrofluorometer with an excitation wavelength of400 nm and an emission wavelength of 420–520 nm. The specificity ofthe caspase-8/9 assay was determined by the addition of caspase-8/9-specific inhibitor Z-IETD-fmk/Z-LEHD-fmk).

Caspase-3 Activity Assays

Caspase-3 activity assays were performed using a fluorogenic sub-strate, Z-DEVD-AFC. Equal amounts (100 �g) of luteal lysate protein(at non-saturating concentration of the assay, for details see Supple-mental Fig. 1) from different time points post-PGF2� treatment wereadded to 1 ml of caspase-3 activity assay buffer (20 mM HEPES, pH 7.5,10% glycerol, and 2 mM DTT) followed by the addition of 20 �M Z-

DEVD-AFC substrate. Reaction mixtures were incubated at 37 °C for1 h, and liberated AFC was measured in a spectrofluorometer with anexcitation wavelength of 400 nm and an emission wavelength of 420–520 nm. The specificity of the caspase-3 assay was determined by usingcaspase-3-specific inhibitor Ac-DEVD-CHO.

Deoxyribonuclease Substrates

Rat liver nuclei were prepared as described previously (41, 42). Ratliver was homogenized in homogenization buffer (15 mM HEPES-NaOH, pH 7.4, 80 mM KCl, 15 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.5mM spermidine, 0.2 mM spermine, and 1 mM phenylmethylsulfonylfluoride) containing 250 mM of sucrose in a Dounce homogenizer. Thehomogenate was filtered through four layers of cheese cloth, and anequal volume of homogenization buffer containing 2.3 M sucrose wasadded and mixed thoroughly. The homogenate then was layered over 5ml of homogenization buffer containing 2.3 M sucrose in a BeckmanSW28 centrifuge tube and centrifuged at 22,000 � rpm for 90 min at4 °C. The pellet was resuspended in homogenization buffer at a concen-tration of �3 � 106 nuclei/�l and stored at �70 °C. Purified rat nucleiwere used as a chromatin substrate for DNase activity assays, whereasthe plasmid DNA obtained from pGEMT empty vector was used as anon-chromatin (naked DNA) substrate for DNase activity assays.

Deoxyribonuclease Activity Assays

For DNase assays crude cytosolic extracts were prepared from theluteal tissue essentially as described previously (43, 44). Luteal tissuecollected before and at 18 h post-PGF2� treatment was swollen in anequal volume of extraction buffer (50 mM PIPES, pH 7.4, 50 mM KCl, 5mM EGTA, 2 mM MgCl2, 1 mM DTT, 20 �M cytochalasin B, 1 �g/mlleupeptin, 1 �g/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluo-ride) for 10 min followed by five cycles of freezing and thawing, whichwas accompanied by grinding with a pestle each time. The resultantlysate was then centrifuged at 12,000 � g for 15 min at 4 °C. Thesupernatant was aspirated, aliquoted, and frozen at �70 °C until usefor protein estimation by Bradford (microassay) method. This superna-tant was used as crude luteal cytosolic extract for DNase assays. DNaseassays were performed according to the previously published proce-dures (43, 44) with some modifications. For DNase assay, using non-chromatin substrate, 10 �g of plasmid DNA was incubated with 50 �gof crude luteal cytosolic extract at 37 °C for 30 min (with the exceptionof the time course analysis of DNase activity where assay was termi-nated at different time points) in a DNase assay buffer (20 mM HEPES,pH 7.5, 1 mM CaCl2, 5 mM MgCl2, 1 mM DTT, and 0.1 mM phenylmeth-ylsulfonyl fluoride if not stated otherwise). For analysis of pH depend-ence of the DNase activity, using naked DNA as a substrate, differentbuffers were used that included acetate NaOH, pH 4.0 and 5.0, phos-phate buffer, pH 6.0, MOPS-NaOH, pH 6.5, HEPES-NaOH, pH 7.5, andTris-HCl, pH 8.0 and 9.0, with the other components of the assay bufferremaining the same. Assay was terminated by the addition of 6�loading dye, and samples were analyzed in a 2% agarose gel containing0.5 �g/ml ethidium bromide.

For DNase assays using chromatin substrate, �3 � 106 nuclei wereincubated with crude luteal cytosolic extract in different pH assaybuffers as indicated above for 30 min at 37 °C. Assay was terminated byadding the lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.2 M

NaCl, 0.2% SDS, and 0.1 mg/ml proteinase K), and DNA was precipi-tated after overnight incubation at 37 °C by the addition of an equalvolume of isopropyl alcohol. The resultant nucleic acid pellet was dis-solved in Tris-HCl, pH 8.0, containing 1 mM EDTA and 0.1 mg/mlRNase A and incubated at 37 °C for 30 min. The DNA was extractedand analyzed by gel electrophoresis using a 2% agarose gel in thepresence of 0.5 �g/ml ethidium bromide.

Immunodepletion of Caspase-activated DNase

Immunoprecipitation was carried out by incubating 400 �g of re-gressing (18 h after PGF2� treatment) luteal lysate protein with poly-clonal anti-CAD antibody or rabbit IgG and protein A-agarose at 4 °Covernight. The pellet containing immune complexes was obtained bycentrifugation at 15,000 � g for 10 min at 4 °C. The pellet was dilutedin 50 �l of immunoprecipitation buffer, and an aliquot was used in thein vitro DNase activity assay, whereas supernatant was concentrated toa volume of 50 �l and then used in the DNase activity assay. Similarprocedures were followed for control IgG supernatantand pellet.

Statistical Analyses

Wherever applicable, the data were expressed as mean � S.E. Thearbitrary densitometric units were represented as the percentage rela-

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tive to control, which was set at 100%. The data were analyzed byone-way ANOVA followed by Tukey’s multiple comparison test (PRISMGraphPad, version 2, GraphPad Software Inc., San Diego, CA). A valueof p 0.05 was considered statistically significant.

RESULTS

Prostaglandin F2� Injection into Buffalo Cows InducesLuteal Cell Apoptosis in the Corpus Luteum

In situ apoptosis analysis of the luteal tissue collected atdifferent time points after PGF2� injection showed an increasednumber of apoptotic cells (small and large luteal cells) 18 hafter PGF2� injection into buffalo cows (Fig. 1B). Although noincidence of apoptosis was observed in the corpus luteum at 4 h,some evidence of apoptosis was present in the corpus luteum at12 h after PGF2� injection. These data confirm our previousobservation of the time of onset of apoptotic DNA fragmenta-tion during PGF2�-induced luteolysis (7) and further validateour model for the analysis of apoptotic signaling cascades thatculminate in the apoptotic cell death in the corpus luteum.

Activation of Mitochondrial Apoptotic Signaling Cascadein the Corpus Luteum during PGF2�-induced Apoptosis

Fig. 2 illustrates changes in steady-state mRNA and proteinlevels of pro- and anti-apoptotic Bcl-2 family members duringPGF2�-induced luteal tissue apoptosis. Bax (a pro-apoptoticgene) mRNA (Fig. 2A) and protein levels (Fig. 2B) increasedwithin 4 h and remained high at 18 h, the last time pointobserved, but mRNA and protein levels of Bcl-2 (an anti-apop-totic gene) did not change in the luteal tissue post-PGF2�

treatment (Fig. 2, A and B). Changes in Bax and Bcl-2 mRNAand protein levels were also expressed as Bax/Bcl-2 ratio, anindex that is used for the activation or inhibition of the mito-chondrial permeability to apoptogenic molecules (Fig. 2C). Ascan be seen in Fig. 2C, Bax/Bcl-2 ratio, both mRNA and proteinlevels, increased with a more profound effect on the Bax/Bcl-2protein ratio as early as 4 h, which remained high until 18 h,suggesting an increase in the mitochondrial permeability toapoptogenic molecules. Immunohistochemical analysis of Bax(Fig. 2D) revealed increased punctate appearance of cells (inthe perinuclear region) during PGF2�-induced luteal tissue ap-optosis, a parameter that confirms increased localization ofproteins to mitochondria (45). However, IgG control did notshow any staining (shown in inset). Bad, another pro-apoptoticBcl-2 family member that gets activated upon hypophospho-rylation, was also examined in the luteal tissue that showed adecrease in the phosphorylation of Bad within 4 h, and thelevels remained low at 18 h (Supplemental Fig. 2A). We nextdetermined changes in the active caspase-9 protein levels andactivity during PGF2�-induced luteal tissue apoptosis. As seenin Fig. 3A, active caspase-9 protein levels increased in the

luteal tissue 4–12 h after PGF2� treatment and were highest at18 h. In vitro caspase activity assays using caspase-9-specificsubstrate Z-LEHD-AFC essentially corroborated the Westernblot analysis results (Fig. 3B). Z-LEHD-fmk (a specific inhibi-tor of caspase-9) completely abolished the caspase activity ob-served in the luteal tissue lysates, confirming the specificity ofthe assay (Fig. 3B).

Activation of Caspase-3 in the Corpus Luteum duringPGF2�-induced Apoptosis

To address whether caspase-9 activation (initiator caspase)was associated with an increase in the activation of down-stream target effector caspase, caspase-3, we measured theactive form of caspase-3 by Western blot analysis and caspase-3activity by in vitro caspase-3 activity assay. As shown in Fig.4A, a dramatic increase in the active caspase-3 protein levelswas seen at 18 h. An increase in the active caspase-3 proteinlevels was associated with an increase in the cleaved form ofPARP, an endogenous substrate of caspase-3, and a significantincrease (p 0.05) in the caspase-3 activity in the corpusluteum at 18 h post-PGF2� treatment (Fig. 4B). The addition ofAc-DEVD-CHO (a specific inhibitor of caspase-3) completelyabolished the caspase activity observed in the luteal tissuelysates, confirming the specificity of the assay (Fig. 4B).

Analysis of Extrinsic Apoptotic Signaling CascadeElements in the Corpus Luteum

Fig. 5 illustrates changes in steady-state mRNA levels of Fasand FasL in the corpus luteum before and at different timepoints after PGF2� treatment. As can be seen in Fig. 5A, FasLmRNA expression levels were unchanged in the corpus luteumat 4 and 12 h but the expression was high at 18 h. The Fasreceptor mRNA levels were up-regulated in the corpus luteumwithin 4 h (4.5-fold compared with 0 h) and became low at 12 h(1.4-fold compared with 0 h) but increased again at 18 h (2.3-fold compared with 0 h). An analysis of caspase-8 activationrevealed no change at 4–12 h posttreatment, but the activityincreased significantly (p 0.05) at 18 h (Fig. 5C). Z-IETD-fmk(a specific inhibitor of caspase-8) completely abolished thecaspase activity observed in the luteal tissue lysates, confirm-ing the specificity of the assay (Fig. 5C). We next determinedchanges in the cleavage of Bid, a downstream target ofcaspase-8, that can interact with mitochondria and lead toamplification in the mitochondrial cascade. Immunoblot anal-ysis of truncated Bid (tBid) levels in the corpus luteum isshown in Fig. 5B. Whereas tBid was undetectable at 0, 4 and12 h, the signal was high at 18 h post-PGF2� treatment.

Relative Contribution of Intrinsic and Extrinsic ApoptoticSignaling Cascades in PGF2�-induced Apoptosis

We next investigated the relative contribution of intrinsicand extrinsic apoptotic signaling cascades in mediating PGF2�-induced luteal tissue apoptosis. For this purpose, we standard-ized a pseudopregnant rat model for PGF2�-induced luteolysisand employed a pharmacological blockade of specific caspasesassociated with intrinsic and extrinsic apoptotic signaling cas-cades as well as general caspases using peptide-based, cell-permeable, and irreversible caspase inhibitors. Before analyz-ing the effects of caspase inhibition on PGF2�-induced lutealtissue apoptosis in pseudopregnant rats, it was necessary todetermine whether PGF2� treatment activated similar apop-totic signaling cascades in the rat corpus luteum. The experi-mental design employed in rats is presented in Fig. 6A, andDNA fragmentation analysis, semi-quantitative RT-PCR anal-ysis of upstream activators of intrinsic and extrinsic apoptoticsignaling cascades (Bax and FasL, respectively), and changes

FIG. 1. Prostaglandin F2�-induced apoptosis in the bovine cor-pus luteum. A, schematic representation of the experimental proce-dure for PGF2� treatment and corpus luteum collection in buffalo cows(B. bubalis). Asterisk indicates time of collection of corpus luteum. B,corpora lutea collected from the buffalo cows at different time pointsafter PGF2� treatment were processed for in situ apoptosis analysisusing in situ cell death detection kit (Roche Diagnostics). Arrows indi-cate apoptotic nuclei. Magnification, �100.

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in caspase-9, -8, and -3 activities during PGF2�-induced lutealtissue apoptosis are presented in Fig. 6, B–F. As can be seen inFig. 6B, increased DNA oligonucleosome formation is discern-

ible in the DNA isolated from luteal tissues collected 24 h afterPGF2� injection, indicative of cell death through apoptosis.Semi-quantitative RT-PCR analysis revealed up-regulation inthe steady-state mRNA levels of Bax and FasL during PGF2�-induced apoptosis in the rat luteal tissue (Fig. 6C). We nextdetermined whether increases in the expression of these genesthat act upstream of apoptotic signaling cascades are associ-ated with the activation of downstream caspases. The results ofcaspase activation analysis are presented in Fig. 6, D–F. As canbe seen from the figures, a significant (p 0.05) up-regulationin caspase-9 (1.9 � 0.04-fold versus 0 h), caspase-8 (3.5 �0.5-fold versus 0 h), and caspase-3 (4.8 � 0.3-fold versus 0 h)activities occurred in the luteal tissue following PGF2� injec-tion. These results suggest striking similarities in the mecha-nisms of apoptosis initiation in the cow and rat luteal tissueand validate our choice of use of pseudopregnant rat model forexamining the relative role of intrinsic and extrinsic apoptoticsignaling cascades during PGF2�-induced luteal tissue apopto-sis in vivo.

Fig. 7A represents the experimental design utilized to exam-ine the effect of peptide-based cell-permeable caspase inhibi-tors (caspase-9 inhibitor (Z-LEHD-fmk), caspase-8 inhibitor(Z-IETD-fmk), and general caspase inhibitor (Z-VAD-fmk)) onPGF2�-induced luteal tissue apoptosis, and changes incaspase-3 activities and oligonucleosome formation in the lu-teal tissue are presented in Fig. 7, B–D. Although caspase-9inhibitor significantly (p 0.05) decreased PGF2�-inducedcaspase-3 activity, DNA oligonucleosome formation was onlymarginally decreased in the luteal tissue. In contrast, the ad-ministration of caspase-8 inhibitor or general caspase inhibitorresulted in dramatic reduction in PGF2�-induced caspase-3activity and oligonucleosome formation. These results suggestthe requirement of activation of extrinsic apoptotic signalingcascade, which is necessary and sufficient for PGF2�-induced

FIG. 2. Prostaglandin F2�-induced changes in Bcl-2 and Bax mRNA and protein levels in the bovine corpus luteum. A, total RNAisolated from the corpora lutea collected before and at different time points after PGF2� treatment was subjected to semi-quantitative RT-PCRanalysis for Bcl-2 and Bax mRNA levels. L-19 mRNA expression was utilized as an internal control. The estimated sizes of PCR products (in bp)are indicated on the left. B, equal amounts of total protein lysates (200 �g/lane) were resolved on a 12% SDS-PAGE and transferred onto PVDFmembrane for immunoblotting with Bax and Bcl-2 antibodies. As a control for protein loading, the blot was stripped of the bound antibodies andreprobed with �-actin antibody. The estimated sizes (in kDa) of the protein bands are indicated on the left. C, quantitative changes in the ratio ofBcl-2 and Bax mRNA (A) and Bcl-2 and Bax protein levels (B) in corpora lutea collected before and at different time points after PGF2� treatment.Mean � S.E. of three experiments is shown. Bars a and b are significantly different (p 0.05). D, representative corpora lutea sections ofimmunohistochemical staining for Bax. Corpora lutea sections from the luteal tissue collected before and at different time points after PGF2�

treatment were rehydrated and incubated with Bax antibody followed by incubation with fluorescein isothiocyanate-conjugated anti-rabbitantibody raised in goat. Sections were mounted in glycerol and visualized under a confocal microscope at �100 (Zoom 1.5). Inset showsimmunostaining observed with rabbit IgG (negative control).

FIG. 3. PGF2�-induced regulation of processed caspase-9 pro-tein levels and activity in the bovine corpus luteum. A, equalamounts of total protein lysate (200 �g/lane) prepared from corporalutea collected before and at different time points after PGF2� treat-ment were resolved on a 12% SDS-PAGE and transferred on to PVDFmembrane for immunoblotting. Membrane was probed with antibody thatdetects endogenous levels of large fragment (37 kDa) of cleaved caspase-9,and the blot was stripped of the bound cleaved caspase-9 antibody andreprobed with �-actin antibody to confirm equal loading of protein acrosslanes. Mean � S.E. of cleaved caspase-9 protein levels relative to �-actinare shown below the Western blot image. B, caspase-9 activity assay onluteal tissue lysates using Z-LEHD-AFC as a substrate indicatingchanges in caspase-9 activity in corpora lutea collected before and atdifferent time points after PGF2� treatment. Specificity of the assay waschecked using caspase-9-specific inhibitor Z-LEHD-fmk (see “Experimen-tal Procedures” for details of the assay). Mean � S.E. of three experimentsis shown. Bars a–c are significantly different (p 0.05).

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apoptosis in the luteal tissue, whereas the activation of intrin-sic apoptotic signaling cascade alone is insufficient.

Induction of a Ca2�/Mg2�-dependent DNase Activity inCorpus Luteum during PGF2�-induced Apoptosis

To examine the DNase activity responsible for apoptoticDNA ladder formation during PGF2�-induced luteal tissue apo-ptosis, we analyzed cellular DNase activity in the corpus lu-teum collected 18 h post-PGF2�. Before subjecting the PGF2�-treated tissues for characterization of DNase activity, tissueswere analyzed for DNA oligonucleosome formation (Supple-mental Fig. 3) to confirm for the presence of DNase activity incorpus luteum collected from the buffalo cows 18 h post-PGF2�

injection. Biochemical characterization of the DNase activity inthe crude cytosolic extract of corpus luteum collected 18 hpost-PGF2� treatment indicated that maximum DNase activitywas observed under neutral pH conditions (pH 6.5–7.5) (Fig.8A). DNase activity was found to be dose-dependent, maximumat 50 �g of cytosolic extract (Fig. 8B), and maximum activitywas observed at 60 min (Fig. 8C) of the assay under the con-ditions tested. As shown in the Fig. 8D, the observed DNaseactivity was inhibited by the addition of EDTA/EGTA, suggest-ing the requirement of divalent cations Ca2�/Mg2� by theDNase. Heat treatment inactivated the DNase activity, butRNase was ineffective, suggesting proteinaceous nature of theDNase activity.

Inhibition of DNase Activity by ICAD

We next examined changes in the ICAD fragmentation dur-ing PGF2�-induced luteal tissue apoptosis. Increased low mo-lecular weight ICAD fragmentation products that corre-sponded to previously published partial ICAD fragments (46,47) were observed following PGF2�-induced apoptosis in thecorpus luteum (Supplemental Fig. 4). The chemical propertiesof DNase in association with the degradation of ICAD sug-gested that it could be CAD. To examine this possibility,ICAD-L, a specific inhibitor of CAD, was expressed in Esche-richia coli as a GST fusion protein and purified as describedpreviously by Nagata and co-workers (14) (Supplemental Fig.5). This GST-ICAD-L fusion protein was then used in an invitro DNase assay to examine whether it can inhibit the DNaseactivity. As shown in Fig. 9, GST-ICAD-L was able to inhibitthe DNase activity (oligonucleosome formation in nuclei-basedassay and DNA smear formation in plasmid DNA-based assay)observed in the crude cytosolic extract prepared from the lutealtissue collected at 18 h post-PGF2� treatment in a dose-depend-ent fashion. Complete inhibition was observed at a dose of 100ng, whereas GST even at a concentration of 200 ng did notinhibit the DNase activity. GST-ICAD-L was not observed toinhibit DNase I or II activities (data not presented).

FIG. 4. Prostaglandin F2�-induced regulation of processedcaspase-3 protein levels and activity in the bovine corpus lu-teum. A, equal amounts of total protein lysate (200 �g/lane) preparedfrom corpora lutea collected before and at different time points afterPGF2� treatment were resolved on a 12% SDS-PAGE and transferredon to PVDF membrane for immunoblotting. Membrane was probed withantibody that detects endogenous levels of large fragment (19/17 kDa)of cleaved caspase-3. The blot was stripped of the bound caspase-3antibody and reprobed with �-actin antibody to confirm equal loading ofprotein across lanes. Cleaved caspase-3 protein levels relative to �-actinare indicated below the Western blot image. Changes in the PARPcleavage as determined by Western blotting before and at different timeafter PGF2� treatment is shown. B, caspase-3 activity assay on lutealtissue lysates using Z-DEVD-AFC as a substrate indicating changes incaspase-3 activity in corpora lutea collected before and at different timepoints after PGF2� treatment. Specificity of the assay was checkedusing caspase-3-specific inhibitor Ac-DEVD-CHO (see “ExperimentalProcedures” for details of the assay). Mean � S.E. of three experimentsis shown. Bars a–c are significantly (p 0.05) different.

FIG. 5. PGF2�-induced changes in the extrinsic apoptotic sig-naling cascade elements in the bovine corpus luteum. A, totalRNA isolated from the corpora lutea collected before and at differenttime points after PGF2� treatment was subjected to semi-quantitativeRT-PCR analysis for FasL and Fas mRNA levels. As a control for RNAamount variation in samples, same cDNA preparation was analyzed forL-19 mRNA levels. The estimated sizes of PCR products (in bp) areindicated on the left. B, Western blot analysis using Bid antibody showstBid levels in the corpora lutea collected before and at different timepoints after PGF2� treatment. Blot was stripped of the bound Bidantibody and reprobed with �-actin antibody for confirmation of equalloading of protein across lanes. C, caspase-8 activity assay on lutealtissue lysates using Z-IETD-AFC as a substrate indicating changes incaspase-8 activity in corpora lutea collected before and at different timepoints after PGF2� treatment. Specificity of the assay was checkedusing caspase-8-specific inhibitor Z-IETD-fmk (see “Experimental Pro-cedures” for details of the assay). Mean � S.E. of three experiments isshown. Bars a and b are significantly different (p 0.05).

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Induction of Caspase-activated DNase in the CorpusLuteum following PGF2� Administration

We next addressed the question of whether the ICAD-sensi-tive DNase activity with a potential to cleave DNA into oligo-nucleosomes under in vitro conditions was indeed induced dur-ing PGF2�-induced luteal tissue apoptosis. As shown in Fig.10A, this ICAD-sensitive DNase activity was induced in thecorpus luteum by PGF2� treatment associated with apoptoticDNA fragmentation. To further confirm the identity of theDNase as CAD, we carried out immunodepletion studies withthe CAD antibody. These results are presented in Fig. 10B. Ascan be seen in Fig. 10B, depletion of CAD from the corpusluteum tissue lysate collected 18 h post-PGF2� treatment abol-ished the DNA fragmentation activity in the lysate superna-tant (Fig. 10, S). When the CAD immune complex pellet (P) wasused in the DNase assay, it induced DNA fragmentation andthe activity in the pellet was abrogated by the addition of 100ng of GST-ICAD-L. Although IgG control did not show anyactivity in the immune complex, the activity was still present inthe supernatant, demonstrating the specificity of the immu-nodepletion experiment. These results confirm the identity ofthe apoptotic endonuclease, CAD, in mediating the PGF2�-induced DNA fragmentation in the luteal tissue.

DISCUSSION

Prostaglandin F2� actions on the corpus luteum can be di-vided into two distinct but interrelated processes, functionaland structural luteolysis. The functional luteolysis, which is

characterized by rapid reduction in serum/luteal tissue proges-terone levels, may be viewed as the commencement of initiationof a new reproductive cycle due to the withdrawal of negativefeedback effect of progesterone on the gonadotropin secretion,whereas structural luteolysis that occurs as a consequence ofapoptotic cell death is required for resorption of luteal struc-ture in the ovarian stroma (48). Prostaglandin F2� affects pro-gesterone production either by interfering with the cholesteroltransport to the inner mitochondrial membrane (49) or byenhancing catabolism of progesterone (50). In the rat, PGF2�

has been reported to enhance catabolism of progesterone ratherthan inhibit the synthesis and this action is brought about bythe stimulatory effects of PGF2� on Nur77-mediated increasedexpression of 20�-hydroxy steroid dehydrogenase, a key en-zyme involved in progesterone catabolism (50). However, in thebovine corpus luteum, PGF2� does not seem to have a signifi-cant effect on the stimulation of 20�-hydroxy steroid dehydro-genase and it is likely that the PGF2�-mediated decreasedtransport of cholesterol to inner mitochondrial membrane isthe key mechanism resulting in decreased steroidogenesis (2).It is interesting to note that mitochondria might be playing animportant role in PGF2�-activated signal transduction path-ways responsible for decreased steroidogenesis as well as struc-tural luteolysis. In this study, we investigated spatial andtemporal changes in the apoptotic signaling cascades in theluteal tissue of the buffalo cow, a bovine species, in whichPGF2� is recognized as the physiological luteolysin. In addition,a pseudopregnant rat model was utilized to investigate the

FIG. 6. Characterization of apoptotic signaling events during PGF2�-induced luteal tissue apoptosis in the pseudopregnant rats.A, schematic representation of the experimental procedure for induction of luteal tissue apoptosis in the pseudopregnant rat model. Asteriskindicates time of collection of corpora lutea. B, genomic DNA (30 �g) isolated from corpora lutea obtained 24 h after vehicle or PGF2� analoginjection on day 8 of pseudopregnancy was separated on 2% agarose gel and stained with ethidium bromide. Note the presence of DNAoligonucleosomes in the DNA isolated from PGF2� treated-animals. C, semi-quantitative RT-PCR analysis of Bax and FasL mRNA levels forvehicle/PGF2�-treated luteal tissue. L-19 mRNA expression was utilized as an internal control. Bar diagram represents quantitative changes inthe mRNA levels relative to L-19. D–F, caspase-9, -8, and -3 activity assays on luteal tissue lysates using substrates specific to respective caspases.For details, see legends to Figs. 3–5.

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relative contribution of intrinsic (mitochondrial) versus extrin-sic (death receptor-mediated) apoptotic signaling cascades inPGF2�-mediated luteal tissue apoptosis.

Studies carried out in several species have clearly estab-lished that apoptosis plays an important role during structuralluteal regression (6–10). To elucidate the molecular eventsassociated with the onset of apoptosis, it is necessary to selectsuitable time points with clear earliest evidence for onset ofapoptosis. For this purpose, we employed two methods to de-termine initiation of apoptosis. The DNA fragmentation anal-ysis reported earlier by us (7) and the in situ apoptosis analysisresults reported in this study indicated the onset of apoptosis inthe luteal tissue at 18 h post-PGF2� treatment. To characterizechanges leading to the initiation of apoptosis, we included twoadditional time points (4 and 12 h) earlier to 18 h in which noevidence for apoptosis was evident but, at these time points,the effect of PGF2� on steroidogenesis was clearly discernible.These results broadly corroborate with the observation made(6–10) by others with regard to the initiation of apoptosis incattle and other species.

Great strides have been made in the recent years toward abetter understanding of the intracellular signaling cascadesthat regulate the initiation of apoptosis in the cells exposed toapoptotic stimuli. One of the major intracellular signalingpathways that regulate apoptosis during cellular stress ismediated by changes in the Bcl-2 family of proteins that areclassified either as anti-apoptotic (Bcl-2, Bcl-XL, and so on) or

pro-apoptotic (Bax, Bad, and so on), and it has been proposedthat the fate of a cell at any given time is decided by the ratioor balance between the pro-apoptotic and anti-apoptoticmembers (11). A luteolytic dose of PGF2� brought about arapid increase in the Bax levels (mRNA and protein) butwithout having much effect on the Bcl-2 levels (mRNA andprotein), resulting in an increase in the Bax/Bcl-2 ratio.Higher Bax mRNA levels have been observed in the sponta-neously regressing bovine corpus luteum by Rueda et al. (24),and the results of this study also confirms the importance ofBax in the luteal regression process and provides evidence forthe regulation of Bax expression by PGF2� in the corpusluteum. Apart from Bcl-2 and Bax, another Bcl-2 familymember; Bad, plays an important role during apoptosis and adecrease in the phosphorylation status of Bad has beenshown to be associated with the apoptosis in the cells (51). Ananalysis of luteal tissue revealed hypophosphorylation of Badas early as 4 h that remained lower thereafter, suggesting itsincreased pro-apoptotic activity.

We next examined changes in the caspase-9 and -3, theactivation of which has been linked to the initiation (caspase-9)and execution (caspase-3) of apoptosis downstream to mito-chondria (52). Although the involvement of caspase-9 duringluteal tissue apoptosis has not been reported in corpus luteumof any species thus far, several lines of evidence suggest that itplays an important role in the cellular apoptosis via cleavage-mediated activation of its downstream target, caspase-3 (53).

FIG. 7. Effects of caspase inhibitors on PGF2�-induced changes in the apoptotic signaling in the corpus luteum. A, schematicrepresentation of the experimental procedure to investigate the effect of caspase inhibitors on PGF2�-induced luteal tissue apoptosis in thepseudopregnant rat model. Asterisk indicates time of collection of corpora lutea. B, determination of caspase-3 activity. DEVDase enzyme activitycorresponding to caspase-3 activity was measured in the luteal tissue 24 h after in vivo treatments. 15 min prior to subcutaneous injection of vehicleor PGF2� treatment, rats received intrabursal injection of vehicle or specific caspase inhibitors (Z-LEHD-fmk, a caspase-9 inhibitor, or Z-IETD-fmk,a caspase-8 inhibitor, or Z-VAD-fmk, a general caspase inhibitor) as described under “Experimental Procedures.” Results are presented asarbitrary fluorescence units (AFU). Mean � S.E. of three experiments is shown. Bars a–c are significantly different (p 0.05). Different treatmentsare indicated at the bottom of panel D. C, qualitative changes in DNA fragmentation. DNA (30 �g) isolated from luteal tissue before and 24 h afterdifferent treatments (for details, see B) was subjected to agarose gel electrophoresis followed by ethidium bromide staining. Different treatmentsare indicated at the bottom of the gel. D, quantitative changes in DNA fragmentation. Low molecular weight DNA fragments (panel C) werequantified by densitometric analysis. Results (fold change from vehicle treatment) are presented as arbitrary densitometric units. Mean � S.E. ofthree experiments is shown. Bars a–c are significantly different (p 0.05). Different treatments are indicated at the bottom of the bars.

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Caspase-3 in turn cleaves several downstream targets involvedin cellular homeostasis, viz. PARP, ICAD, and so on. In thisstudy, although the total form of PARP increased in expressionat 18 h, the cleaved form of PARP was only found to be up-regulated at 18 h following PGF2� injection compared withlevels in the corpus luteum of control animals, further confirm-ing the activation of caspase-3 in the process. In the murinecorpus luteum, Wang et al. (54) have reported increased ex-pression of PARP during PGF2�-induced luteal tissue apoptosisand this could be due to the increased stability of PARP mRNAor higher protein expression during luteolysis. The involve-ment of caspase-3 in mediating apoptosis in the mammaliancells is well established (52). Rueda et al. (55) found an increasein the caspase-3 activation in the ovine luteal tissue duringPGF2�-induced luteolysis. Carambula et al. (23) reported thatcorpus luteum of caspase-3-deficient mice is resistant toPGF2�-mediated apoptosis that further confirms the involve-ment of caspase-3 in luteal cell apoptosis. Surprisingly, al-though the increase in the caspase-9 and -3 activation observedat 4–12 h time points correlated with the expression changes inthe Bcl-2 family members (increased Bax levels and decreasedphospho-Bad levels), the dramatic induction in their activitiesat 18 h, particularly that of caspase-3, could not be explained bychanges in the expression or localization of the mitochon-

drial Bcl-2 family members, suggesting the involvement ofother pathways in the activation of caspase-3 in addition tocaspase-9.

Apart from the intrinsic apoptotic signaling cascade, extrin-sic (or death receptor-mediated) apoptotic signaling cascadehas been reported to play an important role in the regulation ofapoptosis in various cell types exposed to a variety of apoptoticstimuli (12). The finding that Fas expression was increased atdifferent time points post-PGF2� treatment suggested a role for

FIG. 8. DNA fragmentation induced by PGF2�-activated lutealcytosolic extract. A, pH dependence of DNA fragmentation. Equalamount of cytosolic extract protein (50 �g) prepared from the corpusluteum collected at 18 h after PGF2� treatment was added to the nuclei(3 � 106) from normal rat liver and incubated at 37 °C for 30 min in theDNase assay buffer at the indicated pH values. DNA was extracted andanalyzed by agarose gel electrophoresis. The position of standard DNAfragments (in bp) are indicated on the left. B, dose-dependent DNasefragmentation. The cytosolic extract prepared from the corpus luteumcollected at 18 h after PGF2� treatment at the indicated proteinamounts was added to the isolated nuclei (3 � 106) from normal rat liverfollowed by incubation at 37 °C for 30 min in the DNase assay buffer atpH 7.5. DNA was extracted and analyzed by agarose gel electrophore-sis. C, time course of DNA fragmentation. Cytosolic extract protein (50�g) prepared from the corpus luteum collected at 18 h after PGF2�

treatment was added to the isolated nuclei (3 � 106) from normal ratliver and incubated at 37 °C for indicated time periods in DNase assaybuffer at pH 7.5. DNA was extracted and analyzed by agarose gelelectrophoresis. The standard DNA fragments (lane M) are indicated onthe left (in bp). D, Ca2�/Mg2�-dependent protein in the luteal cytosolicextract is responsible for inducing DNA fragmentation. Cytosolic ex-tract protein (50 �g) prepared from the corpus luteum collected at 18 hafter PGF2� treatment was preincubated at 37 °C for 60 min with 100�g/ml RNase A or 68 °C for 10 min (heat) or 5 mM EGTA � 5 mM EDTAat 37 °C for 10 min. Isolated nuclei (3 � 106) from normal rat liver wereadded to the treated cytosolic extracts and incubated at 37 °C for 30 minin DNase assay buffer, pH 7.5. DNA was extracted and analyzed byagarose gel electrophoresis. As a positive control for DNase activity,cytosolic extract (50 �g) from luteal tissue was incubated with theisolated nuclei (3 � 106) without any preincubation (lane 1).

FIG. 9. Inhibition of DNase activity by recombinant ICAD.Equal amounts (50 �g) of cytosolic protein extract prepared from thecorpus luteum collected 18 h after PGF2� treatment were preincubatedwith the indicated amounts of GST-ICAD-L (lanes 4–13), and theDNase activity was determined with isolated nuclei (3 � 106) fromnormal rat liver (top panel) or 10 �g of plasmid DNA (bottom panel). Theeffect of control GST protein on the DNase activity present in thecytosolic protein extract is also shown (lane 14). Lane 2 shows theintegrity of the nuclei/plasmid DNA used for the assay, and lane 3shows DNA fragmentation activity of the cytosolic extract withoutpreincubation with GST/GST-ICAD-L. The position of standard DNAfragments (in bp) are indicated on the left.

FIG. 10. Changes in DNA fragmentation activity in luteal tis-sue during PGF2�-induced apoptosis. A, equal amount of cytosolicprotein extract (50 �g) prepared from the corpus luteum collected beforeand at 18 h after PGF2� treatment was added to the 10 �g of plasmidDNA (used as naked DNA substrate; left panel) or to the isolated nuclei(3 � 106) from normal rat liver (used as a chromatin substrate; rightpanel) and incubated at 37 °C for 30 min in DNase assay buffer, pH 7.5.DNA was extracted and analyzed by agarose gel electrophoresis. Thestandard DNA fragments (in bp, lane M) are indicated on the left. B,immunodepletion of CAD from luteal tissue lysates. CAD was immu-noprecipitated from 400 �g of luteal tissue lysates, and the resultantsupernatant (S) and the CAD immune complex pellet (P) were used inthe DNase assay as described under “Experimental Procedures.” IgGsupernatant and pellet were used as a control for the assay. Please notethe presence of DNA fragmentation activity in the CAD pellet andabolition of DNA fragmentation activity in the CAD pellet with theaddition of 100 ng of GST-ICAD-L.

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Fas in luteal tissue apoptosis. Taniguchi et al. (25) also ob-served increased expression of Fas in the corpus luteum duringthe late luteal phase, a period in which spontaneous luteolysiswould have occurred. However, it has been reported that theexpression of Fas alone is not sufficient for inducing cell deathand that FasL expression is the key determinant in regulatingthe activity of Fas in mediating apoptosis (56). The observationthat FasL expression was induced in the luteal tissue at 18 hpost-PGF2� treatment suggests the involvement of Fas-FasL inapoptosis of the luteal tissue, further confirmed by the obser-vation of caspase-8 activation, which acts as an initiatorcaspase downstream to the Fas receptor. In a recent study,Carambula et al. (23) using induced multiple follicular growthand ovulation model reported that PGF2� can induce the acti-vation of caspase-8 associated with the apoptosis in the murinecorpus luteum; however, the upstream death receptor coupledto the activation of caspase-8 was not reported in their study.Our results in rat and bovine corpus luteum provide support forthe importance of caspase-8 signaling in the luteal tissue apo-ptosis and strongly suggest that this is more probable becauseof the induction of FasL and Fas expression by PGF2�. Inter-estingly, the activation of extrinsic apoptotic signaling cascadewas observed only at 18 h and this might provide an explana-tion for the induction in the caspase-3 activity at the 18 h timepoint because caspase-3 is a downstream target of caspase-8.However, it does not explain the amplification in the mitochon-drial cascade observed at 18 h. In this regard, it has beenproposed that activated caspase-8 cleaves Bid and that thisform (tBid) can amplify the mitochondrial cascade by modulat-ing the ratio of pro-apoptotic to anti-apoptotic members at thelevel of mitochondria (57). Indeed, the examination of the cor-pus luteum for tBid levels at different time points post-PGF2�

treatment revealed an induction in the tBid levels only at 18 h,providing possible explanation for amplification in thecaspase-9 activity observed at the 18-h time point.

Because it was impractical due to the very large body size(�400 kg), difficulty in accessing ovaries in vivo, and quantityof inhibitors required to mechanistically address the involve-ment of intrinsic and extrinsic apoptotic signaling cascades inthe PGF2�-induced luteal tissue apoptosis in the bovine model,we investigated the relative contribution of these cascades inPGF2�-induced luteal tissue apoptosis using a well establishedpseudopregnant rat model (39, 58). Although PGF2� has beenreported to induce luteolysis in this model (39), the involve-

ment of apoptotic signaling cascades in this model remainspoorly characterized. Therefore, we first examined the involve-ment of intrinsic and extrinsic apoptotic signaling cascadesduring PGF2�-induced luteal tissue apoptosis in the pseudo-pregnant rats. Our results of increased expression of Bax andFasL and activation of caspase-9, -8, and -3 demonstrate for thefirst time PGF2�-induced activation of apoptotic signaling cas-cades in the rat corpus luteum and that this model can be usedto study the effect of pharmacological inhibition of activities ofdifferent caspases on PGF2�-induced luteal tissue apoptosis.Two peptide-based, cell-permeable, and irreversible caspaseinhibitors were used to inhibit initiator caspase in intrinsic(caspase-9 (Z-LEHD-fmk)) or extrinsic (caspase-8 (Z-IETD-fmk)) apoptotic signaling cascades. In addition, a broad spec-trum caspase inhibitor (Z-VAD-fmk) was used to demonstratethe effect of executionary caspase (caspase-1, -3, -4, and -7)inhibition on luteal tissue apoptosis. These peptide-basedcaspase inhibitors have been demonstrated to inhibit caspase-dependent cell death in various in vivo models of apoptosis, viz.cerebral ischemia (34, 35), myocardial dysfunction in sepsis(36), liver injury (37), and thymic dysfunction in sepsis (38). Inthis study, the broad spectrum caspase inhibitor inhibited bothcaspase-3 activation and DNA fragmentation following PGF2�

injection in the luteal tissue. Caspase-8 inhibitor also couldreduce PGF2�-induced caspase-3 activity and DNA fragmenta-tion in the luteal tissue. The levels were similar to those ob-served in the luteal tissue of vehicle-treated control animals,but caspase-9 inhibitor only had a marginal effect on theseparameters. These results suggest that extrinsic apoptotic sig-naling cascade plays an indispensable role during PGF2�-in-duced luteal tissue apoptosis. To our knowledge, this is the firstdemonstration of activation of classical apoptotic signaling cas-cades by an atypical death receptor via induction of upstreamgenes that activate these apoptotic signaling cascades under invivo conditions. However, the involvement of other death re-ceptors in this process cannot be ruled out because caspase-8 isalso known to act downstream of many other receptors thatinduce cell death such as tumor necrosis factor-� receptor (56).

DNA fragmentation is an important event in the execution-ary phase of cellular apoptosis (59). Although several endonu-clease-like properties have been reported to be present in theluteal cells (29, 30), the endonuclease responsible for apoptoticDNA fragmentation in response to PGF2� remains to be iden-tified. We demonstrate the presence of a Ca2�/Mg2�-dependenttemperature-sensitive DNase maximally active at neutral pHconditions in the crude cytosolic extract prepared from lutealtissue collected at 18 h post-PGF2� treatment. Moreover, weshow that this putative DNase was induced by PGF2� and wasassociated with the DNA fragmentation in the corpus luteumat the 18 h time point. The optimal DNase activity in the lutealcytosolic extract was observed at pH 6.5–7.5, ruling out thepossible involvement of alkaline or acidic endonucleases(DNase I/II) or endonucleases that show two pH optima, viz.endonuclease G during PGF2�-induced apoptotic DNA degra-dation in the corpus luteum. The biochemical nature of theDNase observed in the regressing corpus luteum was verysimilar to the properties of CAD reported by Nagata and co-workers (43). Caspase-activated DNase has been reported to beactivated during apoptosis by caspase-3-dependent cleavage ofits inhibitor (ICAD). The examination of luteal tissue lysatesfor ICAD by Western blotting revealed increased presence of a�30-kDa ICAD-reactive product at 12 h that apparently re-flects a partially processed intermediate observed by others(43, 47). The partially cleaved ICAD further decreased at 18 h,suggesting that degradation of ICAD at 18 h post-PGF2� mightbe responsible for the activation of the caspase-activated

FIG. 11. A schematic representation of PGF2�-activated apo-ptotic signaling cascades in the bovine corpus luteum. Dashedlines represent the results reported by others, and solid lines representthe results reported in this study. The detailed explanation is providedunder “Discussion.”

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DNase in the luteal tissue, resulting in the fragmentation ofchromosomal DNA into a 180-bp ladder. We reasoned that, ifthis were to be true, the addition of exogenously expressedICAD should be able to abolish the DNase activity observed inthe cytosolic extract prepared from PGF2�-treated luteal tissue.Indeed, the addition of GST-ICAD-L to the DNase assay abol-ished the DNase activity present in the cytosolic extract. Inaddition, immunodepletion of CAD from regressing luteal ly-sates abolished the DNA fragmentation activity observed in theregressing luteal lysates and the DNase activity observed inthe immune complex was abolished by the addition of GST-ICAD-L in the DNase assay. Together, these data provideconvincing evidence for the involvement of CAD in apoptosisassociated with PGF2�-induced luteal tissue apoptosis.

Based on the findings in this study and studies by others, wepropose a model for the PGF2�-induced apoptotic signalingpathways in the corpus luteum (Fig. 11). Prostaglandin F2�-induced apoptotic signaling in the corpus luteum involves theactivation of stress-activated protein kinases such as JNK andp38 MAPK (7, 60) that have been shown to result in theincreased expression of genes that are primary initiators ofapoptotic signaling machinery, viz. Bax and FasL/Fas (61).Ligation of the FasL to Fas receptor or changes in the mito-chondrial permeability results in the activation of the initiator(caspase-8 and -9) and executionary (caspase-3) caspases. Theimportance of caspase signaling in mediating luteal tissue apo-ptosis is further demonstrated in the present study by the useof caspase inhibitors and in a previous study (23) by the ab-sence of luteal apoptosis in response to PGF2� in caspase-3knock-out mice. These caspases then cleave key cellular pro-teins that include ICAD, resulting in the release of CAD fol-lowed by the translocation of CAD to the nucleus that executesthe DNA fragmentation. These apoptotic luteal cells then arerecognized and cleared from the ovarian stroma by immunecells that have been shown to infiltrate in the ovary duringluteolysis. In conclusion, the results from this study suggestactivation of both intrinsic and extrinsic apoptotic signalingcascades in the corpus luteum in response to PGF2� treatmentand there appears to be distinct phases in which each cascadeis initiated but eventually both converge to induce apoptosis.

Acknowledgments—We are grateful to Professor S. Nagata (Depart-ment of Genetics, Osaka University Medical School, Osaka, Japan) forkindly providing us the GST-ICAD-L construct and for discussionsduring the course of DNase characterization. We thank ProfessorKumarvel Somasundaram for the generous gift of general caspase in-hibitor (Z-VAD-fmk), Dr. B. R. Srinath and K. R. Shiva Kumar for helpwith experiments in rats, and T. N. Vivek for help with expression andpurification of the recombinant GST fusion proteins.

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PGF2�-activated Apoptotic Signaling Cascades in Corpus Luteum 10367

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Vijay K. Yadav, Garimella Lakshmi and Rudraiah MedhamurthyDNase

Corpus Luteum during Apoptosis: INVOLVEMENT OF CASPASE-ACTIVATED -mediated Activation of Apoptotic Signaling Cascades in theα2Prostaglandin F

doi: 10.1074/jbc.M409596200 originally published online December 28, 20042005, 280:10357-10367.J. Biol. Chem. 

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