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BOR - Papers in Press, published online ahead of print February 5, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013144
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BIOLOGY OF REPRODUCTION 68, 2232–2240 (2003)
DOI: 10.1095/biolreprod.102.013144
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Regulated Expression of Inhibitor of Apoptosis Protein 3 in the Rat Corpus Luteum1

Ricky R. Lareu3, Markus D. Lacher4, Cara K. Bradley3, Rajagopala Sridaran5, Robert R. Friis4, and Arun M. Dharmarajan2,3

School of Anatomy and Human Biology,3 The University of Western Australia, Crawley, Western Australia 6009, Australia, and the West Australian Institute of Medical Research, Sir Charles Gairdner Hospital, Shenton Park, Western Australia 6008, Australia Department of Clinical Research,4 Faculty of Medicine, University of Berne, Berne, Switzerland Department of Physiology,5 Morehouse School of Medicine, Atlanta, Georgia 30310-1495


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We sought to investigate the role inhibitor of apoptosis proteins (IAPs) play in the life cycle of the corpus luteum (CL) of the rat. We isolated two clones with amino acid homology to rat IAP2 (BIRC 3) and three to rat IAP3 (rIAP3; BIRC 4). The expression of rIAP3 mRNA was examined in the rat CL during and after pregnancy, in Day 8 pregnant rats after 24-h treatment of gonadotropin-releasing hormone-agonist (GnRH-Ag), and in a CL organ culture model of spontaneous apoptosis in the absence of tropic support with and without superoxide dismutase. We used real-time RT-PCR to quantitate rIAP3 mRNA expression. Interestingly, a significant reduction in rIAP3 levels was seen at the time of CL regression in the course of natural pregnancy and the GnRH-Ag model. Surprisingly, rIAP3 mRNA levels in the CL organ culture model of spontaneous apoptosis failed to show significant changes, although TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) reaction showed 30%–40% of the cells undergoing DNA fragmentation after 2 h in culture. In situ hybridization revealed that rIAP3 expression was localized to the cytoplasm of luteal and granulosa cells. These data clearly demonstrate both the presence of IAPs in the rat CL and the regulation of rIAP3 during in vivo apoptotic cell death, indicating a role for IAPs in the maintenance of CL function and demise.

apoptosis, corpus luteum, follicle, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The corpus luteum (CL) is a transitory, ovarian structure that arises from the ovulated follicle, with the main function of secreting progesterone, needed for blastocyst implantation and the maintenance of pregnancy [1]. Initiation of luteinization marks the end of proliferation for most granulosa cells and their differentiation into cells characterized by a striking hypertrophy and an increase in the enzyme systems of steroid-producing cells [2]. Progesterone production by the rat CL increases during the first 16 days of pregnancy and then declines to very low levels by Days 20–22 of pregnancy. This change at Day 16 of pregnancy demarcates the onset of functional luteolysis [3] with structural luteolysis then occurring postpartum [4]. Apoptosis, a form of programmed cell death, is instrumental in various events of cell turnover in the ovary [5]. These include perinatal germ cell attrition [6], granulosa and theca-interstitial cell death during follicular atresia [7], and death of the ovarian surface epithelium during ovulation [8]. The signaling events, molecular mechanisms, and genetic controls that determine the timing of luteolysis and its delay during pregnancy remain largely unknown, but accumulating evidence suggests that structural luteolysis correlates with the onset of luteal cell apoptosis [9–13]. Further, we have demonstrated that in vivo administration of gonadotropin-releasing hormone-agonist (GnRH-Ag) during early pregnancy induces functional and structural luteal cell apoptosis [14].

Inhibitor of apoptosis proteins (IAPs), as their name suggests, are a family of proteins that have been shown to inhibit apoptosis. Strictly speaking, the presence of a certain protein motif, the Baculoviral Inhibitor of Apoptosis Repeat (BIR) domain, and the ability to inhibit apoptosis defines membership in this family. However, in the light of the recent discoveries of proteins that contain BIR domains without apparent inhibition of apoptosis, they are now categorized as BIR-containing (or BIRC) proteins. Originally identified in the baculovirus in 1993 [15, 16], IAPs have since been cloned from several organisms including drosophila [17], chicken [18], pig [19], mouse [20–23], rat [24], and human [25–28]; homologues display a high degree of species conservation.

Inhibitor of apoptosis proteins have been shown to protect cells from a plethora of proapoptotic signals and agents, such as serum deprivation, overexpression of interleukin-1 beta-converting enzyme, menadione, tumor necrosis factor alpha (TNF{alpha}), cyclohexamide, ultraviolet light, cisplatin, agonistic antibody to Fas, and caspase 2 and 8 [26, 29, 30]. Their broad-spectrum inhibition can be attributed to their targets, namely the caspases, which are the central effectors of apoptosis. The BIR domain has been shown to facilitate the physical association of several of the IAPs with TNF{alpha} receptor-associated molecules TRAF1/2 [25] and the caspases [31, 32]. Recent structural studies on X-linked IAP (XIAP) have demonstrated that the N-terminal extension of the BIR2 domain physically blocks substrate binding to caspase 3 and 7 [33–35] and that the actual BIR domain has a role in caspase binding and Smac/DIABLO neutralization [36, 37].

In the rat ovary, preantral follicles that are susceptible to apoptosis exhibited a lower expression of XIAP and Hiap-2 than mature follicles [38]. Like the CL, its precursor cellular structure, the follicle readily undergoes atresia through apoptosis [7]. Depending on the species, about 0.1% of follicles proceed to ovulation [7]. A role for several IAPs in maintaining follicle survival has been suggested. In the chicken ovary, hierarchal preovulatory follicles express significantly higher levels of the inhibitor of T-cell apoptosis (ITA; BIRC 3 family member) than the prehierarchal follicles, which readily undergo atresia [18]. Recent studies further confirm the involvement of IAPs in the ovary, specifically XIAP in granulosa cell fate. The presence of the proapoptotic TNF{alpha} pathway and its regulation by XIAP was demonstrated in granulosa cells by in vitro transfection studies [39]. Furthermore, XIAP was shown to activate the phosphatidylinositol 3-kinase (PI 3-K)/Akt survival pathway in granulosa cells [40].

In light of these observations, we hypothesize that IAPs may play a part in the maintenance or survival of the rat CL. In the present study we report the cloning of two IAP family members, namely rat IAP2 (rIAP2; BIRC 3) and rat IAP3 (rIAP3; XIAP/BIRC 4), and the localization and regulation of rIAP3 during the course of natural regression, in vivo induction of apoptosis by GnRH-Ag, and in vitro spontaneous CL regression with and without the presence of superoxide dismutase (SOD).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Sexually mature (10–12-wk old) female Wistar rats were used in this study. They were housed at 21°C with 55% humidity in a 12L:12D cycle. Their feed was autoclaved normal cubes and acidified water, both provided ad libitum. Rats were placed overnight for mating, and the next morning, rats positive for spermatozoa in vaginal smears were designated Day 1 of gestation. Litters were born on Day 23 of pregnancy. All protocols were approved by the University of Western Australia Animal Experimentation Ethics Committee.

Full-Length Screening for Rat IAP Clones

Cloning of full-length rat IAPs was performed on a rat ovarian cDNA library (Lambda Zap II/EcoRI library; Stratagene, La Jolla, CA) at Day 15 of pregnancy. Three rounds of screening were performed using a mixture of two probes generated with the polymerase chain reaction (PCR) employing digoxigenin-11-deoxy-uridine triphosphate (Roche Diagnostics, Castle Hill, NSW, Australia) label. The PCR primers were chosen from conserved regions of several known mouse IAP sequences (as no rat IAPs had yet been cloned). The two probes spanned 1.3 kilobases (kb) of the coding sequence (primer set 1: sense, 5'-TGT GGC CTG ATG TTG GAT AA and anti-sense, 5'-GAA ACC ATT TGG CGT GTT CT; primer set 2: sense, 5'-GAG CAG CTT GCA AGT GCT GGA T and anti-sense, 5'-GCT GCA GCA TTT CCC TTG ACT AA). Plaque lifts were carried out on confluent XL-1 Blue MRF' E. coli (Stratagene) lawns with GeneScreen Plus nylon membrane (NEN Life Science Products, Boston, MA). On the first round, 200 000 phage clones were screened. Rescue of putative clones was carried out after the third round of screening in pBluescript SK+ phagmid with the inclusion of the VCS-M13 helper phage and subsequent infection of SOLR cells. Plasmids were prepared by overnight bacterial culture in LB broth (Sigma, Castle Hill, NSW, Australia) at 37°C followed by extraction using the BRESAspin Plasmid Mini Kit (Roche Diagnostics) and Wizard Plus Midipreps DNA Purification System (Promega Corporation, Annandale, NSW, Australia). Clones were investigated by restriction analysis and automated sequencing.

Automated Sequencing

Automated sequencing was performed with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) on the ABI 373 DNA Sequencer. Ten-microliter reaction volumes were used according to the manufacturer's protocol. The thermal cycling reaction consisted of the terminator mix, 1 µg of template (purified plasmid), and 3 pmol of primer. Routine sequencing primers M13 reverse and M13 forward (-20) were used including several specifically designed primers (data not shown) to sequence the IAP clones; each was sequenced three times.

Sequence Analysis

The homology search was performed with the BLAST v 2.1 program at the National Center for Biotechnology Information. Protein motif identification was performed with the ScanProsite program of the Expert Protein Analysis System. Sequencing primers were designed with Web Primer at the Saccharomyces Genome Database [41].

GnRH Regulation of IAP Expression

Gonadotropin-releasing hormone-agonist (5 µg/day) was administered continuously using osmotic minipumps starting on the morning of Day 8 of pregnancy [14]. Control rats underwent sham surgery without treatment. Briefly, in each rat, an osmotic minipump (model 2001; Alza, Mountain View, CA) was implanted subcutaneously in the dorsal surface of the neck under metofane anesthesia. The pumps were incubated in saline overnight prior to implantation and delivered a GnRH-Ag ([Pyro]-Glu-His-Trp-Ser-Tyr-D-Trp-NmeLeu-Arg-Pro-E thylamide-LHRH; Wyeth-40972). Three rats per group were sacrificed at 0 and 24 h after the commencement of treatment. At autopsy, ovaries were removed. The CL from one ovary of each animal were separated and stored at -70°C for mRNA studies.

In Vitro Organ Culture

Corpora lutea from Day 16 pregnant rats were dissected under sterile conditions and used for in vitro organ culture. In order to induce spontaneous apoptosis, CL were incubated in media without trophic support (no serum) for 2 h with and without SOD as described previously [9, 42]. The 0-h control was snap-frozen without incubation. For each time-point, samples were prepared in triplicate. Each samples consisted of 3 CL per vial in 2 ml of media. Following incubation, the CL were snap-frozen and stored at -80°C.

RNA Extraction

Total RNA was isolated from snap-frozen CL by homogenization and extraction with RNAzol B (Tel-Test Inc., Friendswood, TX) as described by Roughton [12]. RNA samples were resuspended in RNase-free water and stored at -80°C until used. RNA quantitation and purity were determined by spectrophotometry.

Northern Blot Analysis

Northern blot analysis was performed on total RNA from pregnant and postpartum rat CL. For each stage, three individual samples were pooled. Ten micrograms of total RNA per stage were fractionated through a denaturing formaldehyde gel as described in Lehrach and colleagues [43] and capillary transferred onto Nitropure nitrocellulose membrane (Geneworks, Adelaide, SA, Australia) as per manufacturer's protocol. The cDNA probe for rIAP3 message was generated by a random primed reaction (Gigaprime DNA Labeling Kit; Geneworks) with {alpha}-32P dCTP (Geneworks) as per manufacturer's protocol. The complete rIAP3 cDNA clone 1 was used to generate the cDNA probe. Purification of the probe was carried out with ProbeQuant G-50 Micro Columns (Amersham Pharmacia Biotech, Castle Hill, NSW, Australia). Hybridization and washing conditions were as per manufacturer's protocol for the membrane. Briefly, hybridization was carried out overnight at 42°C in a 50% formamide solution. The radioactive cDNA probe was added at 2–4 million cpm per ml of solution. Washes were performed at low stringency at 37°C. After washing, membranes were wrapped in clear wrap and exposed to x-ray film for 6–9 days. Even loading of RNA was assessed by densitometric analysis of RNA stained by Vistra Green (Amersham Life Science, UK).

Quantitative (Real-Time) Reverse Transcription-PCR (RT-PCR) for rIAP3 Message

Complementary DNA was generated from total RNA with SuperScript II RNase H- Reverse Transcriptase (Gibco-BRL, Melbourne, VIC, Australia) as per manufacturer's protocol. Briefly, 5 µg of RNA were incubated with 500 ng of Oligo (dT)15 primer (Promega, Madison, WI) and 0.5 mM of each of the 4 deoxy-nucleotide triphosphates (Promega) at 65°C for 5 min followed by chilling on ice. The reaction was completed by adding 5x first-strand buffer, 10 mM DTT, 40 units of RNase inhibitor (Promega), and 200 units of enzyme and incubated at 42°C for 50 min, followed by enzyme inactivation at 70°C for 15 min. The reverse-transcribed cDNA was purified through spin columns (Ultra Clean GelSpin Kit; GeneWorks) and eluted out in 50 µl 10 mM Tris, pH 8.

Quantitation of rIAP3 mRNA was performed by real-time RT-PCR on the Roche LightCycler using the LightCycler-Fast Start DNA Master SYBR Green I Kit (Roche) with external standards. Specific PCR primers were designed based on the rat rIAP3 sequence published in this paper generating a 144-bp fragment: sense primer 5'-CGC AGG ATG AGT CAA GTC AG, anti-sense primer 5'-TGA CCA GAT GTC CAC AAG GA. Reaction conditions were as per manufacturer's protocol with optimization of primers and magnesium chloride (MgCl2) concentrations and annealing temperature. A 10th volume of cDNA target and master premix were incubated with 3 mM MgCl2 and 0.4 {rho}mol/µl of each primer in a capillary. The reaction run program was as follows: an initial denaturation step at 95°C for 10 min; 40 cycles at 95°C for 15 sec, 55°C for 7 sec, and 72°C for 14 sec; followed by a fluorescence measurement after each cycle. A melt curve was performed with continuous fluorescence measurement between 70°C and 90°C. Heating rate was set at 0.1°C/sec. The external standards were amplified in parallel and were 10-fold serial dilutions of the full-length rIAP3 clone 1 in a pBluescript SK+ plasmid in 10 mM Tris, pH 8. Results were analyzed with the LightCycler software v 3.0 by the "Fit Point Method." To confirm reproducibility, a Day 16 CL sample was replicated five times in a reaction run, and the same reaction was performed on three separate days using different master premixes. The intra-assay variation was calculated by dividing the mean of the five replicates by the standard error (SEM) and expressed as a percentage of the mean and the interassay variation was calculated by dividing the mean of the three runs by the SEM and expressed as a percentage of the mean.

Quantitative (Real-Time) RT-PCR for Ribosomal Protein L19 Assay

To correct for differences in RNA amounts and reverse transcription reactions, the constitutively expressed ribosomal protein L19 was employed [44]. A quantitative (real-time) RT-PCR assay was developed based on previously published rat primers [45]. Reaction component concentrations and conditions were as for the rIAP3 RT-PCR assay with the following differences. The reaction run program consisted of 25 cycles at 95°C for 15 sec, 56°C for 5 sec, and 72°C for 13 sec, producing a PCR product of 194 bp long. The external standards were 10-fold serially diluted, purified reaction products.

In Situ Hybridization

Tissues for mRNA localization were fixed in 4% freshly prepared paraformaldehyde and paraffin embedded. Sections were prepared at 5 µm on silanated glass slides. Tissue sections were dewaxed through two changes of toluene followed by rehydration through graded ethanol to 1x PBS. Sections were pretreated with Proteinase K (10 µg/ml) in 50 mM Tris and 2 mM EDTA (pH 8) for 10 min at 37°C and then incubated in a solution of 0.3% (v/v) acetic anhydride (ICN Biomedicals Inc., OH) and 1.3% (v/v) triethanolamine (ICN) and titrated with HCl to pH 8. Prehybridization was carried out at 55°C for a minimum of 2 h with the following hybridization solution: 50% deionized formamide (Sigma), 300 mM NaCl, 30 mM sodium citrate, 1x Denhardt solution (Sigma), and 50 µg/ml tRNA (Sigma). For hybridization, 200–400 ng/ml of denatured digoxigenin-11-uridine triphosphate-labeled RNA probe (antisense or sense) was added with fresh hybridization solution and incubated overnight at 55°C. Posthybridization washes were as follows: Sections were rinsed in 2x SSC (300 mM sodium chloride/30 mM tri-sodium citrate [pH 7.0]) at room temperature followed by one wash at 65°C and one wash at room temperature in 2x SSC and 50% formamide for 15 min; one wash at room temperature and one at 65°C in 2x SSC and 0.2% SDS for 15 min; finally, two washes in 0.1x SSC and 0.2% SDS for 15 min at 65°C. Immunological detection of signal was carried out using components from the DIG Nucleic Acid Detection Kit (Roche). Essentially, the alkaline phosphatase conjugated antidigoxigenin antibody was diluted 1 in 3000, and the colorimetric reagent NBT/BCIP was used as substrate.

TUNEL (Terminal Deoxynucleotide Transferase-Mediated dUTP Nick End-Labeling)

For the staining of dying cell nuclei, we used the ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit (Intergen Company, New York, NY) as previously published [46]. The procedure was according to the protocol for paraformaldehyde-fixed, paraffin-embedded tissue, using a 10-min incubation at room temperature for proteinase K digestion. Cellular nuclei were counterstained with methyl green. The relative percentage of positive nuclei was determined using light microscopy.

Statistical Analysis

One-way analysis of variance (ANOVA) with least significant difference was used to determine statistical significance between treatment groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Full-Length Rat IAPs

We screened a rat lambda cDNA library from ovarian tissue at Day 15 of pregnancy and isolated a total of five clones that were comprehensively sequenced. Three of the clones had close homology to XIAP/BIRC 4 (i.e., rIAP3), and the two other clones had close homology to the BIRC 3 group (i.e., rIAP2). However, these lacked part of the coding sequence. Two of the rIAP3 clones possessed the full-length coding sequence and were identical (clone 1, 2032 bp, GenBank accession no. AF304333; clone 2, 3032 bp, AF304334). The rIAP3 clones code for a putative protein of 501 amino acids long in a single open reading frame. Computer-based structural analysis identified protein motifs that are characteristic of BIRC 4 proteins. These included three BIR domains at the N-terminus (BIR 1, 26–93; BIR 2, 163–230; BIR 3, 264–329) and one RING zinc finger domain at the C-terminus (449–483). Amino acid sequence homologies to other known BIRC 4 proteins were rat 98% (BAA85304/AAG22969), mouse 95% (Q60989), and human 89% (CAB95312). The only amino acid difference between the rat IAPs was at their carboxyl terminus. The rIAP3 protein presented here possessed a unique carboxyl terminus between amino acids 493 and 496, including an additional five amino acids between 497 and 501.

Validation of Quantitative (Real-Time) RT-PCR Assay

A quantitative (real-time) RT-PCR assay was developed to specifically detect rIAP3 mRNA (Fig. 1). Amplification was seen only in the test and standard samples; both demonstrated identical melt peaks (Fig. 1A). No amplification was detected in the negative control (water). In addition, samples amplified by the previously described assay were separated on an agarose gel (Fig. 1B). A single, discrete band at the predicted size of 144 bp confirmed the specificity of the assay. Sensitivity and linearity of detection were determined by using 10-fold serial dilutions of the standard (Fig. 1C). Linearity of amplification was demonstrated over six orders of magnitude; between 1.0 ng and 1.0 fg of standard template cDNA. The mean intra-assay variation was 4.5%, and the interassay variation was 12.1%. The ribosomal protein L19 RT-PCR assay was also reproducible with an intra-assay variation of 6.9% and interassay variation of 6.0% (data not shown).



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  		FIG. 1. Specificity and sensitivity of amplification by the rIAP3 quantitative (real-time) RT-PCR assay. A) Plot of the negative derivative of the melting curves versus temperature demonstrating peaks with identical Tm obtained from a test sample (Day 16 of pregnancy; solid line) and a control sample (1 fg cDNA standard; dashed line). The negative control (water) did not show any amplification. B) Agarose gel of quantitative rIAP3 RT-PCR assay amplification products showing a single, discrete band at a size of 144 bp. CL samples were from a Day 16 of pregnancy (lane 1) and Day 1 postpartum (lane 2). Included were a standard of 1 fg of cDNA (lane 3), a negative control (water; lane 4), and 400 ng of a molecular weight standard (lane 5; Gene Ruler 100-bp DNA ladder Plus, Frementas). The picture is a color-inverted scan of an ethidium bromide stained, 1.5% agarose, 1x TAE gel. C) The standard curve is a plot of the crossing point (cycle number) versus the logarithmic concentration for each sample. It was generated by 10-fold serial dilutions of the standard (dilution range was 1 ng to 1 fg) and demonstrated linearity over this range

Expression of rIAP3 in the CL During Pregnancy

We demonstrated the presence of specific rIAP3 message in the rat CL by Northern blot analysis and quantitative RT-PCR. Figure 2A shows a Northern blot hybridized with the full-length of rIAP3 (clone 1) during pregnancy at Day 1 (lane 1), Day 16 (lane 2), and Day 22 (lane 3) and postpartum Day 1 (lane 4). Two transcripts were detected, at 7.5 and 3.5 kb. Only the largest transcript demonstrated regulation. Equal loading of total RNA is demonstrated in Figure 2B.



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  		FIG. 2. Quantitative analysis of rIAP3 mRNA expression during pregnancy and postpartum stages. A) Northern blot analysis showing two transcripts at 7.5 and 3.5 Kb for the stages tested: Days 1 (lane 1), 16 (lane 2), and 22 (lane 3) of pregnancy and postpartum Day 1 (lane 4). Only the larger demonstrated regulation. Three individual samples were pooled per stage. B) RNA gel corresponding to above Northern blot showing the ribosomal 28S and 18S bands to demonstrate even loading. Ten micrograms per lane were loaded, stained with Vistra Green, and scanned by a fluorescence scanner. C) Quantitation by real-time RT-PCR. The stages tested were Days 1, 16, and 22 of pregnancy and Day 1 postpartum. Statistically significant increased levels of rIAP3 mRNA were detected at Days 16 and 22 of pregnancy, especially when compared to Day 1 postpartum (difference between a and b, P < 0.05). Results were expressed as the mean of three samples per stage ± SEM. Test samples were quantitated by serially diluted, external standards run in parallel and plotted as percentage values relative to Day 1 of pregnancy. Abbreviations: D, days; P, postpartum

Real-time quantitation of relative mRNA levels for rIAP3 for the four time points shown in Figure 2A were determined according to the method described previously (Fig. 2C). A significant increase in the level of rIAP3 mRNA was seen from early to late pregnancy followed by a rapid decrease in expression from Day 22 of pregnancy to Day 1 postpartum. A 2-fold increase in expression seen from Day 1 to Day 22 of pregnancy (P < 0.05) was followed by an equal decrease from Day 22 to Day 1 postpartum (P < 0.05). The expression of rIAP3 mRNA appeared to be associated with the growth and structural integrity of the CL, which showed a rapid decline during the approximate 72 h from Day 22 of pregnancy to Day 1 postpartum, at the time when the CL begins to structurally regress.

Localization of rIAP3 mRNA in the ovary was performed by in situ hybridization. Luteal cells of the CL, as well as granulosa and thecal cells of developing follicles, demonstrated expression, localized to the cytoplasm. Figure 3, A and B, shows the presence of rIAP3 in the cytoplasm of luteal cells at Day 16 of pregnancy and Day 1 postpartum, respectively. No apparent signal was detected in endothelial cells of the CL; however, because of the absence of counterstain, this was difficult to determine. Figure 3C illustrates a CL neighboring a follicle, demonstrating the relative expression. The luteal cells have a large cytoplasm, whereas the granulosa cells have a very narrow cytoplasm compressed between the nuclei of adjacent cells, concentrating the message and the resultant signal into a narrow band in each cell. Many but not all follicles were positive for rIAP3 expression. Figure 3D shows several preantral follicles at Day 8 of pregnancy. However, most follicles that demonstrated histological signs of atresia had little to no rIAP3 expression; pyknotic nuclei, cell detachment, membrane blebbing, and vacuoles were observed in granulosa cells of these follicles. Figure 3E shows structurally healthy antral follicle, and Figure 3F shows antral follicles with early signs of atresia such as detachment of granulosa cells and small apoptotic bodies. Both were from the same Day 16 slide. Figure 3G illustrates a CL at Day 16 of pregnancy hybridized with sense probe as a negative control, and Figure 3H presents a hematoxylin and eosin stained section to illustrate the detailed morphology of the CL, also at Day 16 of pregnancy.



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  		FIG. 3. Localization of rIAP3 mRNA expression in the rat ovary during pregnancy and postpartum by in situ hybridization. A and B) Expression of rIAP3 in the CL at Day 16 of pregnancy and Day 1 postpartum, respectively, demonstrating cytoplasmic expression. C) Relative expression of rIAP3 in a Day 16 CL and a neighboring follicle and (D) in pregnancy Day 8 follicles. E and F) Expression of rIAP3 in structurally healthy and early atretic follicles at Day 16 of pregnancy, respectively. G) A section of Day 16 CL was hybridized with sense probe to show specificity, and (H) a hematoxylin and eosin stained section illustrates the histology of the CL at Day 16 of pregnancy. Abbreviations: cl, corpus luteum; ec, endothelial cell; f, follicle; gc, granulosa cell; lc, luteal cell

In Vivo mRNA Expression of rIAP3 in the GnRH-Induced Luteal Regression Model

This experiment was carried out in order to determine whether there was a change in rIAP3 mRNA expression in the CL of Day 8 pregnant rats in response GnRH-Ag treatment. Quantitative RT-PCR analysis of rIAP3 mRNA clearly demonstrated a specific reduction in levels following administration of GnRH-Ag (Fig. 4). A 9-fold reduction in rIAP3 mRNA levels was seen compared to controls (P < 0.05).



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  		FIG. 4. Quantitation of rIAP3 mRNA expression in CL from pregnant rats treated with GnRH-Ag or left as controls. The real-time RT-PCR assay for rIAP3 mRNA was used to quantitate expression in the test and control samples that had been treated with and without GnRH-Ag for 24 h, respectively. A statistically significant decrease in rIAP3 mRNA expression levels was seen between the control and GnRH-Ag-treated samples (a and b, P < 0.05). CL from three rats per group were quantitated by comparison to serially diluted, external standards run in parallel. Their average was plotted as a percentage relative to controls ± SEM

Expression of rIAP3 in In Vitro CL Organ Culture

The expression of rIAP3 mRNA in the CL under conditions of spontaneous apoptosis was assessed in whole CL organ culture. CL were incubated for 2 h in minimal medium in the absence of trophic support with and without the antioxidant SOD. The control was snap-frozen CL. Figure 5 shows the relative expression of rIAP3 mRNA by real-time RT-PCR. Unexpectedly, quantitative analysis revealed a change in rIAP3 expression at 2 h of incubation under these conditions; however, the increase was not statistically significant. This difference was seen in both the absence and the presence of SOD. Localization of rIAP3 in CL by in situ hybridization in the control (Fig. 6A) and 2 h in the absence of trophic support (Fig. 6B) showed no difference in the cytoplasmic signal. Figure 6C presents a negative control hybridized with the sense probe. We also performed in situ labeling of fragmented DNA to identify nuclei of dying cells. Figure 6, D and E, shows results of the TUNEL reaction, which was performed to document the onset of DNA fragmentation that is absent at time 0 h (Fig. 6D) but clearly present in some 30–40% of the luteal cells at 2 h of trophic-free incubation (Fig. 6E).



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  		FIG. 5. Quantitative analysis of rIAP3 mRNA expression in CL after trophic-free organ culture using real-time RT-PCR. Corpora lutea were incubated for 2 h in trophic-free media with and without SOD. The 0-h control was snap-frozen without incubation. No statistical significance was seen between treatment groups. Results were expressed as the mean of three individual samples per time point ± SEM. Test samples were quantitated by serially diluted, external standards run in parallel and plotted as percentage values relative to the 0-h control



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  		FIG. 6. Localization of rIAP3 mRNA expression in the rat CL after in vitro organ culture in trophic-free medium was investigated by in situ hybridization. A and B) Expression of rIAP3 in CL incubated for 0 h (without incubation) and 2 h, respectively. C) CL hybridized with sense probe as a negative control showed no signal. D and E) A TUNEL reaction was performed on CL incubated for 0 and 2 h demonstrating nuclei of dying cells stained brown in (E). Abbreviations: de, dying endothelial cell; dl, dying luteal cell; ec, endothelial cell; lc, luteal cell


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The IAPs are a family of proteins capable of suppressing apoptosis occurring in response to a variety of stimuli. The role of IAPs in the female reproductive system has received little attention. The present data confirm the presence of IAPs in the ovary and suggest their involvement in follicle survival by mechanisms that have already been established for other tissues [25, 33–35, 38, 47]. The cyclical nature of ovarian function, particularly the formation of the CL, qualifies the ovary as an especially interesting model to study apoptosis [9, 48]. To date, there is little information available about the role of IAPs in the rat CL. Therefore, the aims of this study were to establish the presence of IAPs and examine their regulation in the rat CL.

In the present study, we report the presence of IAP transcripts in the rat ovary. We used an ovarian lambda cDNA library prepared from Day 15 of pregnancy and isolated several clones. We isolated two identical full-length clones with close homology to the XIAP (BIRC 4) group, termed rIAP3, and partial clones with closest homology to the cellular IAP of the BIRC 3 group (sequences not shown). The putative protein encoded by the rIAP3 cDNAs had a unique carboxyl terminus when compared to the two other published rIAP3 sequences. This difference may have been due to tissue specificity. The large regulated transcript was almost identical in size to that previously reported for rat [24] and its counterpart in the mouse [29].

The rat pregnancy model of CL regression enabled us to demonstrate regulation of rIAP3 at the mRNA level. A reduction in expression was seen in the CL at the time of structural regression (i.e., at the postpartum stage). Our previous studies have indicated that while progesterone production in the rat declines by Day 22 of pregnancy, the wet weight of the CL does not fall until after parturition [4]. In fact, just after parturition is the time when the most significant increase in cell death is apparent [49]. Furthermore, our previous work has shown that at Day 22 of pregnancy, the CL demonstrated an increase in Fas/Fas ligand expression, a proapoptotic pathway [12]. Therefore, our results suggest that rIAP3 may be relevant for the survival of the CL, and the reduced expression may make luteal cells susceptible to preexisting, default proapoptotic pathways. In fact, this reduction in rIAP3 and not its absence suggests that a controlled reduction in rIAP3 expression may be a mechanism for limiting the rate of cell death since, in fact, the demise of the CL is gradual over the following estrus cycles [4].

We used the well-characterized model of whole CL organ culture [9, 10, 12, 50] to induce spontaneous apoptosis, which can be inhibited by the addition of SOD. We found that in this model, the expression of rIAP3 mRNA showed variation but was not statistical. At 2 h of trophic-free culture, at a time when there already was significant apoptotic death shown by TUNEL and quantitatively by 3'end-labeling of DNA [42], rIAP3 expression had not changed significantly. The SOD treatment, which prevented apoptosis [42], did not elicit a significant change in rIAP3 mRNA expression. A recent study has shown that when rat granulosa cells were cultured in the presence of the proapoptotic cytokine TNF{alpha}, XIAP was up-regulated [39]. Apoptotic death did not occur in the presence of TNF{alpha} until XIAP expression was inhibited by either antisense cDNA or inhibition of nuclear factor kappa B, a transcriptional activator of IAPs [51, 52]. This response has also been found in other cell culture systems [19, 51, 53]. However, in the absence of total trophic support, the possible inhibition by IAP of the caspase-dependent apoptotic pathways would be limited and probably short-lived as other cell regulatory pathways would soon collapse. Most probably, mitochondrial induction of apoptosis via the Apaf-1/cytochrome C/caspase 9 and Smac/DIABLO pathway would be responsible for the cell death under these trophic-free conditions [54]. Our previous studies demonstrated that at 2 h of trophic-free incubation of rabbit CL, the Bcl-2 family proteins demonstrated a shift to proapoptotic expression. In the presence of SOD the shift in expression was reversed [10]. The up-regulation of the antiapoptotic Bcl-2 may be a necessary step for the inhibition of apoptosis through rIAP3, as Bcl-2 has been shown to inhibit Smac, which is a specific inhibitor of IAPs [55–57]. Therefore, in the absence of Smac inhibition, direct blocking of rIAP3 enabled death to proceed. Under the same culture conditions, with the addition of SOD, the inhibition of Smac through the Bcl-2 enabled rIAP3 to exert its antiapoptotic effect.

In situ hybridization of the whole ovary confirmed a previous study that demonstrated that XIAP (rIAP3) and Hiap-2 (rIAP2) expression appeared to be linked to follicular development in the rat [38]. They demonstrated strong protein IAP expression in granulosa cells of medium to large antral follicles, follicular structures that precede the formation of the CL. We found antral follicles and follicles that exhibited signs of structural regression often stained less or not at all for rIAP3 mRNA. However, all evidently healthy antral follicles expressed rIAP3. This positive association of rIAP3 expression with follicular survival appears to also correlate with CL survival. This is not surprising, as the ruptured follicle gives rise to the CL and the granulosa cells differentiate into luteal cells, that, if not structurally, then biochemically appear to be the same, albeit producing larger quantities of progesterone in consequence of up-regulation of steroid-producing enzymes [2].

We previously reported that administration of GnRH-Ag induces apoptosis during early pregnancy [14]. As early as 8 h after the commencement of treatment, luteal cell apoptosis occur with the evidence provided by a fall in progesterone levels and an increased low-molecular-weight DNA fragmentation. Our data also demonstrated an increase in Bax expression following GnRH-Ag treatment. Interestingly, in this study, the GnRH-Ag treatment had a marked effect on rIAP3 mRNA expression. The significant reduction in the expression of this IAP at 24 h after treatment provides support to the hypothesis that GnRH-Ag treatment induces apoptosis by decreasing the expression of IAPs. This observation is further substantiated by our earlier finding that the translated product of this mRNA was significantly decreased at 24 h after the GnRH-Ag treatment [58].

Staining of endothelial cells in the CL was not apparent; however, this was difficult to determine accurately due to the lack of counterstaining. Endothelial cells are relatively resistant to the initial regression of the CL [4]. We assessed the expression of rIAP3, but there may be other IAP(s) present in endothelial cells possibly responsible for repression of apoptotic pathways. In fact, two recent studies have shown an initial up-regulation of IAP in endothelial cells when treated with TNF{alpha}, apparently to counteract the proapoptotic effect [19, 59]. In these studies, only the rIAP1 (BIRC 2) family member was cloned from endothelial cells of porcine and human, suggesting tissue specificity.

In conclusion, the results of this study indicate, for the first time, the presence and regulation of an IAP in the rat CL. Furthermore, the data suggest a role for rIAP3 in the development and death of the rat CL in vivo, demonstrating an inverse relationship with the structural regression of the CL. We also suggest that the gradual demise of the CL after pregnancy may be controlled by the lowering of rIAP3 expression levels, just as the lower expression of IAPs in preantral follicles may make them more susceptible to atresia. Further studies are required to determine the exact role of IAPs in the suppression of apoptosis within the CL.


   ACKNOWLEDGMENTS
 
The authors would like to gratefully acknowledge Professor Alan Bittles from the Center for Human Genetics, Edith Cowan University, for his generous contribution of materials and the gift of GnRH-Ag from Dr. A. Corbin, Wyeth-Ayerst Laboratories, Philadelphia.


   FOOTNOTES
 
1 This study was supported by the following grants: the Australian Research Council, National Health and Medical Research Council, Raine Medical Foundation (all to A.M.D.), and the Dora Lush Biomedical Postgraduate Scholarship (to R.R.L.); the National Institutes of Health (GM08248) and by the National Aeronautics and Space Administration (NAG9-963), Washington, DC (to R.S.); and by grants from the Bernische Krebsliga, the Stiftung fur-klinisch-experimentelle Tumorforschung and the Swiss National Science Foundation (31-63700.00) (to R.R.F.). Back

2 Correspondence: Arun M. Dharmarajan, School of Anatomy and Human Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. FAX: 61 8 9380 1051; dharma{at}anhb.uwa.edu.au Back

Received: 14 November 2002.

First decision: 27 November 2002.

Accepted: 23 January 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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