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BOR - Papers in Press, published online ahead of print March 14, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.055889
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BIOLOGY OF REPRODUCTION 77, 156–164 (2007)
DOI: 10.1095/biolreprod.106.055889
© 2007 by the Society for the Study of Reproduction, Inc.

Vasoactive Peptides in the Luteolytic Process Activated by PGF2alpha in Pseudopregnant Rabbits at Different Luteal Stages1

Cristiano Boiti 2 3, Margherita Maranesi 3, Cecilia Dall'Aglio 4, Luisa Pascucci 4, Gabriele Brecchia 3, Anna Gobbetti 5, and Massimo Zerani 5

Dipartimento di Scienze Biopatologiche ed Igiene delle Produzioni Animali e Alimentari, Sezione di Fisiologia veterinaria, Laboratorio di Biotecnologie Fisiologiche,3 and Sezione di Anatomia Veterinaria,4 Università degli Studi di Perugia, I-06100 Perugia, Italy Dipartimento di Biologia Molecolare, Cellulare e Animale,5 Università degli Studi di Camerino I-62032 Camerino, Italy

ABSTRACT

To study the role of endothelial factors in luteal function, the dynamic profiles of genes for endothelin 1 (EDN1), its receptor subtypes, EDNRA and EDNRB, and angiotensin converting enzyme (ACE) were examined in corpora lutea (CL) obtained from rabbits on Days 4 and 9 of pseudopregnancy after prostaglandin (PG) F2{alpha} analogue (alfaprostol) treatment. The cell type distribution of EDN1 in the ovaries and its mechanisms of actions in vitro and in vivo were also studied. Positive immunostaining for EDN1 was localized in the luteal and endothelial cells, in granulosa cells of the follicles, and in the ovarian epithelium. The basal mRNA levels for EDNRA, EDNRB, and ACE were lower (P ≤ 0.01) in Day-4 CL than in Day-9 CL, whereas those for EDN1 did not differ between these two time-points. On Day 4, the luteal EDN1, EDNRA, EDNRB, and ACE mRNA levels were similarly increased two-fold (P ≤ 0.01) 1.5 h after alfaprostol injection, and did not show further changes in the subsequent 24 h. On Day 9, alfaprostol challenge transiently up-regulated (P ≤ 0.01) the luteal ACE transcripts at 1.5 h, and those of EDN1 at 1.5 h and 3 h, whereas the EDNRA and EDNRB transcript levels remained unchanged during the course of luteal regression. EDN1 decreased (P ≤ 0.01) progesterone release and increased (P ≤ 0.01) PGF2{alpha} secretion and NOS activity via the PLC/PKC pathway in Day-9 CL, but not in Day-4 CL, cultured in vitro. EDN1-induced, but not alfaprostol-induced luteolysis, was blocked by cotreatment in vivo with the ACE antagonist captopril. These findings support the hypothesis that PGF2{alpha} regulates luteolysis through intraluteal activation of the renin-angiotensin/EDN1 systems in CL that have acquired luteolytic competence.

ACE, corpus luteum function, EDN1, EDNRA, EDNRB, luteolysis, ovary

INTRODUCTION

Luteolysis is a streamlined process that involves functional and structural changes that end with the complete demise of corpora lutea (CL) through apoptotic pathways [1, 2]. In the rabbit, which is a reflex ovulator, the luteolytic mechanism comes into play either at the end of gestation or after an infertile mating around Day 14 of pseudopregnancy [3]. In this species, CL regression is driven by prostaglandin (PG) F2{alpha} (PGF2{alpha}) of uterine origin [4], given that hysterectomy [5], endometritis [6], and indomethacin treatment [7] prolong luteal function.

Besides the key role of PGF2{alpha}, several distinct intraluteal pathways have emerged as potential candidate mediators of its luteolytic action [8]. Prominent among these mediators is endothelin–1 (EDN1), a potent vasoconstrictor that is synthesized by the vascular endothelium [9–11], which inhibits progesterone release both in vivo and in vitro in a number of species [12–15]. Recently, using the pseudopregnant rabbit model in the mid-luteal stage, we have shown that the luteolytic action of EDN1 is mediated by complex interactions with the prostanoid cascade via its receptor subtype EDNRA; for reasons that remain unclear, PGF2{alpha} does not require EDN1 to elicit its luteolytic effect [16].

An increasing body of evidence indicates that angiotensin II (ANGII), which is the main effector of the renin-angiotensin system (RAS) that regulates systemic blood pressure and electrolyte balance, may act together with EDN1 in the control of CL life-span [17, 18]. In fact, ANGII is released by midcycle bovine CL in vitro and suppresses progesterone release [19]. Moreover, all the components of the RAS have been identified in the ovaries of rats and cows, including renin and angiotensin I converting enzyme (ACE), the enzymes that catalyze ANGI and ANGII production, respectively, as well as the ANGII peptide and its receptor [17, 20–26]. In addition, between PGF2{alpha} and both EDN1 and ANGII, several modulated feedback and auto-amplifying mechanisms have been described in the CL of ruminants [27–29].

However, despite the extensive studies on luteal regression, the physiological relevance of these vasoactive peptides in the control of luteal function is still uncertain. Similarly, little is known about which mechanisms protect the growing CL from the functional luteolysis that occurs in the early luteal stage up to Day 9 of pseudopregnancy, when the CL of rabbits shift from refractoriness to complete responsiveness to exogenous PGF2{alpha} [30, 31, 32] and EDN1 [16].

Therefore, the main objective of the present study was to elucidate the basal and PGF2{alpha} analogue (alfaprostol)-induced temporal expression changes for early luteal (Day-4) and midluteal stage (Day-9) CL of pseudopregnancy, in order to characterize the role of the EDN1/RAS system in the acquisition of luteal sensitivity to PGF2{alpha}. In addition, using in vivo and in vitro studies, we further investigated the luteolytic mechanisms of action of EDN1.

MATERIALS AND METHODS

Reagents

Primers for the mRNAs of EDN1, EDNRA, EDNRB, and ACE, random hexamer primers, DNAase I, RNAse H-reverse transcriptase (Superscript II), E. coli RNase H, DNA molecular mass ladders, TRIzol, Taq DNA polymerase (Platinum), 10x PCR Buffer, 50 mM MgCl2, RNAse-free tubes, RNAse-free water, and deoxy-NTPs were obtained from Invitrogen (S. Giuliano Milanese, Milan, Italy). Primers for 18S rRNA and the corresponding competimers (QuantumRNA 18S Internal Standards) were acquired from Ambion (Austin, TX). Tritiated hormones and [2,3–3H]L-arginine, with a specific activity of 30–40 Ci/mmol, were purchased from Amersham Biosciences (Amersham Biosciences Ltd., Little Chalfont, Bucks, UK), while progesterone and PGF2{alpha} antisera and nonradioactive hormones were from Sigma (St Louis, MO). The NOS Detect Assay Kit was purchased from Alexis Corp. (Läufelfingen, Switzerland). The primary mouse monoclonal anti-EDN1 antibody, which was used for immunohistochemistry, was supplied by ABR Affinity Bioreagents (Golden, CO). The biotinylated secondary antibody, goat anti-mouse IgG, was purchased from Vector Laboratories (Burlingame, CA). The avidin-biotin complex (ABC, Vector Elite Kit) and the chromogen 3,3'-diaminobenzidine tetrachloride (DAB) were also from Vector Laboratories. The kit for protein assays was purchased from Bio-Rad Laboratories (Hercules, CA).

Incubation wells were obtained from Becton Dickinson (Clifton, NJ), medium 199 and Earles Balanced Salt Solution (EBSS) were from GIBCO (Grand Island, NY). Phospholipase A2 (PLA2, 4-bromophenacyl bromide), PLC (compound 4880), PKC (staurosporine), ACE (captopril) inhibitors, Hepes, and BSA were purchased from Sigma, whereas all other pure grade chemical and reagents were obtained locally. EDN1 was purchased from Calbiochem (San Diego, CA).

The following hormonal preparations were administered via i.m. injection: GnRH analogue (Receptal; Hoechst-Roussel Vet, Milan, Italy), eCG (Folligon; Intervet, Milan, Italy), and the PGF2{alpha} analogue alfaprostol (Gabbrostim; Centralvet, Milan, Italy).

Animals, Hormonal Regimen, and Tissue Collection

The protocols involving the use of the animals in these experiments were approved by the Bioethics Committee of the University of Perugia. Unmated New Zealand White rabbits of 5 mo of age and weighing 3.5–3.8 kg were caged individually in quarters at the University of Perugia Central Animal Facility and maintained under controlled conditions of 14L:10D and 18°C. The animals were provided with commercial rabbit chow and tap water ad libitum. All rabbits were treated with 20 IU eCG, followed 3 days later by i.m. injection of 0.8 µg GnRH (Day 0) to induce pseudopregnancy [33]. The rabbits were maintained in accordance with the standards of animal care.

On Day 4 or Day 9 of pseudopregnancy, rabbits (n = 18/group) were administered i.m. 200 µg alfaprostol. At each luteal stage, three rabbits were killed by cervical dislocation just before (time 0) and then 1.5, 3, 6, 12, and 24 h after PGF2{alpha} analogue administration. Reproductive tracts, which were promptly removed from each animal, were thoroughly washed with saline. For the analyses of luteal gene expression by RT-PCR, the CL were excised from ovaries and after careful dissection of nonluteal tissue using fine forceps under stereoscopic magnification, the specimens were rinsed with RNAse-free PBS and immediately frozen at –80°C. For the in vitro study, CL were harvested from three does that were killed on Days 4 and 9 of pseudopregnancy, as previously reported [31].

For the purpose of the present study, functional luteolysis was defined as a 50% drop in plasma progesterone relative to the pretreatment values, while complete luteolysis was judged to be the failure of CL to secrete progesterone, such that the levels of progesterone in the blood fell below 1.0 ng/ml, which is the concentration detected in estrus rabbits [34].

Immunohistochemistry of EDN1

For the immunohistochemical detection of EDN1, two additional animals for each time-point were killed just prior (time 0) to alfaprostol injection and 1.5 h and 3 h after alfaprostol injection administered on either Day 4 or Day 9 of pseudopregnancy. The ovaries were immediately excised, fixed by immersion in 4% (v/v) formaldehyde in PBS (pH 7.4) for 24 h at room temperature, and subsequently processed for embedding in paraffin following routine tissue preparation procedures. Serial 7-µm-thick sections mounted on poly-L-lysine-coated glass slides were dewaxed in xylene and hydrated through a graded ethanol series. The sections were microwave irradiated for 5 min at 750 W in 10 mM citric acid buffer (pH 6.0) for antigen retrieval. All subsequent steps were carried out in a moist chamber at room temperature. The sections were first treated with a 0.5% solution of hydrogen peroxide in methanol for 10 min, in order to inactivate the endogenous pseudoperoxidase activity, then rinsed with PBS and incubated for 30 min with normal goat serum, to minimize the nonspecific binding of reagent in the subsequent steps. To reduce variability in staining, within each luteal stage, all the tissue sections were incubated together. The monoclonal mouse anti-EDN1 antibody was raised against a peptide that maps within an internal region of human EDN1, which shows 100% amino acid sequence homology with the same region in the rabbit EDN1. The primary antiserum was diluted 1:100 in PBS and added to the sections overnight. The next day, the sections were washed in PBS and incubated with a biotinylated secondary antibody (1:200 in PBS) for 30 min. After PBS washes, the sections were exposed to avidin-biotin-peroxidase complex (1:2500 in PBS) for 30 min, followed by the chromogen DAB for 5 min, to visualize the site of reaction. The slides were then washed in PBS, counterstained with Gill's hematoxylin, and dehydrated before mounting in Canada balsam natural. Positive reactions were recognized as reddish brown precipitates. Sections for which the primary antibody was omitted or substituted by preimmune goat {gamma}-globulin were used as negative controls for nonspecific staining.

RNA Extraction and Reverse-Transcription Multiplex RT-PCR Amplification

For each rabbit, total RNA was extracted from a pool of eight CL (approximately 90 mg of tissue) and reverse-transcribed into cDNA, while multiplex PCR amplification was carried out with EDN1, EDNRA, EDNRB, and 18S sense and antisense primers, as reported elsewhere [16]. The primer sequences for ACE (168-bp product) were: forward, 5'-CCTCTACCTGAACCTGC-3' and reverse, 5'-TGGGCTTGTCTGGAAAGG-3'. Preliminary experiments were carried out to establish the optimal ratio of 18S primers to their competimers, i.e., primers that were modified only at their 3'-ends to block extension by DNA polymerase, to be used in the reaction mix. The ratios were 2:8 for EDN1 and ACE and 3:7 for EDNRA and EDNRB RT-PCRs, respectively. The 18S rRNA is an ideal internal control for quantitative RNA analysis because its expression remains invariant across tissues and treatments, whereas its relatively high abundance makes it difficult to use in RT-PCR experiments, in which target mRNA genes are far less abundant. However, by mixing the appropriate amounts of 18S primers with their competimers, the PCR amplification efficiency of 18S cDNA can be reduced to a level that is roughly similar to that of the gene under study. Between 30 and 40 cycles, both the target and 18S products were in a linear exponential phase of amplification (data not shown). All the PCR reactions consisted of an initial denaturation step at 94°C for 75 sec, followed by amplification for 35 cycles of 94°C for 15 sec, 60°C for 30 sec, and 72°C for 45 sec, with a final extension step of 72°C for 10 min. Within each experiment and for each gene analyzed, the complete set of samples was processed in parallel in a single PCR, using aliquots of the same PCR master mix. The amplified PCR-generated products were analyzed by electrophoresis on 2% agarose gels using ethidium bromide staining for visualization. The analysis of amplification products was carried out as reported elsewhere [16].

In Vivo Induction of Luteolysis

EDN1 was dissolved in saline at a concentration of 10 µg/ml and administered i.v. via a catheter inserted in the central ear vein, as previously reported [16]. The ACE inhibitor captopril was dissolved in saline at a concentration of 5 mg/ml and injected s.c. (1 mg/kg) 1 h before EDN1 or PGF2{alpha} analogue administration and again 8 h later. Both the dosage and treatment protocols for captopril were based on the recently published experimental data for cows [18].

On Day 9 of pseudopregnancy, rabbits (n = 5 per group) were randomly assigned to receive one of the following treatments: saline (control), EDN1 (10 µg), EDN1 plus ACE inhibitor, alfaprostol (100 µg), and alfaprostol plus ACE inhibitor.

From each rabbit, blood samples were collected by venopuncture of the marginal ear vein just before (basal) and 2, 4, 8, 24, and 48 h after treatment. The samples, which were collected in EDTA-containing tubes, were immediately centrifuged at 3000 x g for 15 min, and the plasma was stored frozen until assayed for progesterone concentration, to assess CL functional status.

In Vitro Incubations

For the in vitro study, Day-4 or Day-9 CL were randomly distributed (one CL/well) into incubation wells in 1 ml of culture medium 199 with EBSS that contained 2.2 mg/ml sodium bicarbonate, 2.3 mg Hepes, and 3% BSA, referred to here as M199. Before treatment, CL were placed inside each well using fine forceps. Each incubation set of wells was divided into five experimental groups of five wells as follows: (I) medium alone as control; (II) EDN1 (100 µM); (III) EDN1 plus PLA2 inhibitor (100 nM); (IV) EDN1 plus PLC inhibitor (100 nM); and (V) EDN1 plus PKC inhibitor (100 nM). The culture plates were incubated at 37°C in air with 5% CO2 as reported elsewhere [16]. The medium in each well was collected after 4 h of incubation and stored immediately at –20°C for later determinations of progesterone and PGF2{alpha}. Each CL was weighed and stored immediately at –20°C for later determinations of NOS activity and protein concentration. Preliminary evidence, together with the previous results [16], guided our choices of incubation conditions and the minimum effective dosages of the substances used in the present in vivo study (data not shown).

Progesterone and PGF2{alpha} Assays

Progesterone and PGF2{alpha} concentrations were determined by RIA with specific antibodies, according to the procedures reported elsewhere [32]. The assay sensitivity was 0.08 ng/ml for progesterone and 19 pg/ml for PGF2{alpha}. The intraassay and interassay coefficients of variation were 5.3% and 10.2% for progesterone and 8% and 12% for PGF2{alpha}, respectively.

NO Synthase Activity Determination

In rabbit CL, NOS activity was determined by monitoring the conversion of [3H]L-arginine into [3H]L-citrulline using a commercial NOS assay kit according to the procedure described previously [32].

Statistical Analysis

At each stage of pseudopregnancy, the ratios of each PCR product for the target genes (EDN1, EDNRA, EDNRB, and ACE) normalized against the coamplified 18S product were analyzed by two-way ANOVA (time after alfaprostol treatment and gel being the two sources of variability) and the Newman-Keuls multi-comparison post-test. Data relative to overall in vivo treatment effects on plasma progesterone during the 48-h time-course of EDN1- or PGF2{alpha} analogue-induced luteolysis were analyzed by ANOVA for repeated measurements according to a model that included treatment group, doe within group, time period, and interaction between group and time period; doe within treatment was used as the error term. Progesterone, PGF2{alpha}, and enzyme activity from the in vitro data were evaluated by ANOVA. All statistical analyses were performed using Prism (GraphPad Software Inc., San Diego, CA). Comparisons among all the data for days and time-points were evaluated using the Student t-test. A value of P < 0.01 was considered significant.

RESULTS

EDN1 Immunolocalization in Rabbit CL

Using a monoclonal antibody, positive staining for EDN1 was localized in the endothelial cells of the luteal blood vessels of ovaries collected before treatment on either Day 4 (Fig. 1d) or Day 9 of pseudopregnancy (Fig. 2d). In the same unstimulated ovaries, cytoplasmic positive signals were also identified in the luteal (Figs. 1 and 2d) and granulosa cells of the follicles (Figs. 1 and 2f), as well as in the ovarian epithelial cells (Figs. 1 and 2e). In each positive cell type, staining was evident 3 h after PGF2{alpha} analogue injection independently of luteal stage (Figs. 1 and 2, g–i) and thereafter (data not shown). Staining was completely abolished when the primary antibody was substituted with nonimmune serum (Figs. 1 and 2, a–c).


Figure 01
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  		FIG. 1. Cellular immunolocalization of EDN1 in rabbit ovaries collected on Day 4 of pseudopregnancy before (d–f) and 3 h (g–i) after PGF2{alpha} analogue (alfaprostol) injection, showing positive staining in luteal cells (lc), endothelial cells (en), ovarian epithelium (ep), and granulosa cells of follicles (gc). Control sections (a–c) incubated in the absence of primary antibody show no reactivities in the lc, en, ep, and gc. Bar = 20 µm.


Figure 02
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  		FIG. 2. Cellular immunolocalization of EDN1 in rabbit ovaries collected on Day 9 of pseudopregnancy before (d–f) and 3 h (g–i) after PGF2{alpha} analogue (alfaprostol) injection, showing positive staining in luteal cells (lc), endothelial cells (en), ovarian epithelium (ep), and granulosa cells of follicles (gc). Control sections (a–c) incubated in the absence of primary antibody show no reactivities in the lc, en, ep, and gc. Bar = 20 µm.

Luteal Gene Expression

The corresponding amplification products obtained using primers designed for each gene matched the expected sizes (Figs. 3–6, panels B and C).


Figure 03
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  		FIG. 3. Gene expression patterns of EDN1 mRNAs in CL of rabbits collected 0, 1.5, 3, 6, 12, and 24 h after PGF2{alpha} analogue (alfaprostol) injection on Day 4 or Day 9 of pseudopregnancy. A) Summarized data (means ± SEM) derived from densitometric analyses of EDN1 in CL, reported in arbitrary units relative to 18S expression. For each day of pseudopregnancy and for each time-point, the values shown represent the combined results for three different rabbits. Different letters above the bars indicate significantly different values (P < 0.01) among treatment days and time-points. B, C) Representative photographs (Day 4, B; Day 9, C) of typical 2% agarose, ethidium bromide-stained gels, showing the presence of the expected products yielded after RT-PCR using primers for the target EDN1 and 18S. Lane LD shows the kilobase DNA marker, lane PCR represents a negative control of RNA that was not reverse-transcribed but was subjected to PCR amplification, while the other lanes represent samples taken at the corresponding hours after alfaprostol injection.


Figure 06
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  		FIG. 6. Gene expression patterns of ACE mRNAs in the CL of rabbits collected 0, 1.5, 3, 6, 12, and 24 h after PGF2{alpha} analogue (alfaprostol) injection on Day 4 or Day 9 of pseudopregnancy. A) Summarized data (means ± SEM) derived from densitometric analyses of ACE in CL, reported in arbitrary units relative to 18S expression. For each day of pseudopregnancy and for each time-point, he values shown represent the combined results for three different rabbits. Different letters above the bars indicate significantly different values (P ≤ 0.01) among days and time-points. B, C) Representative photographs of typical 2% agarose, ethidium-bromide stained gels, showing the presence of the expected products yielded after RT-PCR using primers for the target ACE (168 bp) and 18S (489 bp). Lane LD shows the kilobase DNA marker, lane PCR represents a negative control of RNA that was not reverse-transcribed but was subjected to PCR amplification, while the other lanes represent samples taken at the corresponding hours after alfaprostol injection.

EDN1 mRNA. Prior to treatment, the relative abundances of EDN1 mRNA did not differ between Day-4 and Day-9 CL (Fig. 3A). At Day 4 of pseudopregnancy, the luteal EDN1 mRNA levels rose two-fold (P ≤ 0.01) 1.5 h after alfaprostol treatment, and remained at approximately the same high levels (P ≤ 0.01) for the following 24 h (Fig. 3A). At Day 9 of pseudopregnancy, the numbers of EDN1 mRNA luteal transcript were increased (P ≤ 0.01) 1.5 h and 3 h after PGF2{alpha} analogue challenge (Fig. 3A). Thereafter, the numbers of EDN1 transcript dropped (P ≤ 0.01) to the pretreatment values (Fig. 3A).

EDNRA and EDNRB mRNAs. Before treatment, both the EDNRA (Fig. 4A) and EDNRB (Fig. 5A) mRNA transcripts were poorly expressed in Day-4 CL compared to Day-9 CL (P ≤ 0.01). Upon alfaprostol administration at Day 4 of pseudopregnancy, both the EDNRA and EDNRB mRNA levels were increased (P ≤ 0.01) two- to three-fold, and remained high (P ≤ 0.01) for the following 24 h (Figs. 4 and 5A). Conversely, at Day 9 of pseudopregnancy, the luteal EDNRA and EDNRB genes did not show any clear patterns, remaining at approximately the same levels for up to 24 h after PGF2{alpha} analogue challenge (Figs. 4 and 5A).


Figure 04
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  		FIG. 4. Gene expression patterns of EDNRA mRNAs in CL of rabbits collected 0, 1.5, 3, 6, 12, and 24 h after PGF2{alpha} analogue (alfaprostol) injection on Day 4 or Day 9 of pseudopregnancy. A) Summarized data (means ± SEM) derived from densitometric analyses of EDNRA in CL, reported in arbitrary units relative to 18S expression. For each day of pseudopregnancy and for each time-point, the values shown represent the combined results for three different rabbits. Different letters above the bars indicate significantly different values (P < 0.01) among days and time-points. B, C) Representative photographs (Day 4, B; Day 9, C) of typical 2% agarose, ethidium bromide-stained gels, showing the presence of the expected products yielded after RT-PCR using primers for the target EDNRA and 18S. Lane LD shows the kilobase DNA marker, lane PCR represents a negative control of RNA that was not reverse-transcribed but was subjected to PCR amplification, while the other lanes represent samples taken at the corresponding hours after alfaprostol injection.


Figure 05
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  		FIG. 5. Gene expression patterns of EDNRB mRNAs in the CL of rabbits collected 0, 1.5, 3, 6, 12, and 24 h after PGF2{alpha} analogue (alfaprostol) injection on Day 4 or Day 9 of pseudopregnancy. A) Summarized data (means ± SEM) derived from densitometric analyses of EDNRB in CL, reported in arbitrary units relative to 18S expression. For each day of pseudopregnancy and for each time-point, the values shown represent the combined results for three different rabbits. Different letters above the bars indicate significantly different values (P < 0.01) among days and time-points. B, C) Representative photographs (Day 4, B; Day 9, C) of typical 2% agarose, ethidium bromide-stained gels, showing the presence of the expected products yielded after RT-PCR using primers for the target EDNRB and 18S. Lane LD shows the kilobase DNA marker, lane PCR represents a negative control of RNA that was not reverse-transcribed but was subjected to PCR amplification, while the other lanes represent samples taken at the corresponding hours after alfaprostol injection.

ACE mRNA. Before treatment, the steady-state level of ACE mRNA was lower (P ≤ 0.01) at Day 4 than at Day 9 of pseudopregnancy (Fig. 6A). At Day 4, the ACE mRNA levels rose almost two-fold (P ≤ 0.01) within the first 1.5 h after alfaprostol administration and remained at approximately the same relatively high levels (P ≤ 0.01) for the following 24 h (Fig. 6A). Conversely, at Day 9 of pseudopregnancy, the relative abundance of ACE mRNA increased (P ≤ 0.01) 1.5 h after alfaprostol challenge, but then declined to basal values during the course of luteal regression (Fig. 6A).

In Vitro Culturing of CL

For Day-9 CL cultured in vitro, EDN1 decreased (P ≤ 0.01) progesterone release (Fig. 7A) but increased (P ≤ 0.01) PGF2{alpha} secretion (Fig. 7B) and NOS activity (Fig. 7C). Coincubation of EDN1 with the PLA2-inhibitor did not modify the EDN1-dependent effects either on hormone or NOS activity (Fig. 7). Conversely, the addition of either PLC or PKC inhibitors blocked the luteolytic action of EDN1 on both progesterone and PGF2{alpha} release, as well as on NOS activity, all of which remained at control values (Fig. 7). In Day-4 CL, EDN1 did not cause any changes among the experimental groups in terms of hormone release and enzyme activity (data not shown).


Figure 07
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  		FIG. 7. In vitro effects of medium alone, endothelin 1 (EDN1), EDN1 plus PLA2 inhibitor, EDN1 plus PLC inhibitor, and EDN1 plus PKC inhibitor on both progesterone (A) and PGF2{alpha} (B) release, and total NOS activity (C) by rabbit CL harvested on Day 9 of pseudopregnancy. The results shown are the means ± SD of five replicates. Different letters above the bars indicate significantly different values (P ≤ 0.01).

In Vivo Induction of Luteolysis

The plasma P4 concentrations, which are used as a marker of luteal functional activity, decreased similarly in rabbits 8 h after EDN1 or alfaprostol injection on Day 9 of pseudopregnancy, and complete functional luteolysis was achieved 24 h later when the plasma P4 levels dropped to 0.3 ng/ml in both groups (Fig. 8). EDN1-induced functional luteolysis was blocked by pretreatment with the ACE inhibitor captopril (Fig. 8). Conversely, alfaprostol-induced luteolysis was not affected by cotreatment with captopril (Fig. 8).


Figure 08
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  		FIG. 8. In vivo effects of EDN1 (10 µg) and EDN1 plus ACE inhibitor (captopril, 1 mg/kg s.c. given twice) (left panel), and PGF2{alpha} analogue (alfaprostol, 100 µg) and PGF2{alpha} plus captopril (right panel) on progesterone plasma levels at Day 9 of pseudopregnancy. Values are mean ± SEM for 5 animals/group. *, P ≤ 0.01 compared to the control (saline).

DISCUSSION

The present study provides evidence that exogenous PGF2{alpha} differentially modulates the expression of EDN1, its receptors, and ACE in both the early luteal- and midluteal-phase CL of pseudopregnant rabbits. However, only in the latter, which have acquired luteolytic competence, does a positive and reciprocal feed-back mechanism exist between PGF2{alpha} and EDN1, likely mediated by ACE, come into play to induce functional and structural luteolysis.

The cell-type distribution of EDN1 within the rabbit ovary was precisely identified by the IHC technique. Independently of the luteal stage examined, either before or after PGF2{alpha} treatment, EDN1 staining was clearly evidenced in the vascular endothelial and luteal cells of the CL. In contrast, Schams et al. [18], using the IHC technique on bovine midcycle CL, revealed no or only very weak staining for EDN1 in both endothelial and steroidogenic cells, before and up to 4 h after PGF2{alpha} injection. This discrepancy is likely due to interspecies differences and/or to methodological causes rather than to the specificity of the primary antibody used. In fact, the mature forms of EDN1 are highly conserved among species, and the rabbit EDN1 nucleotide sequence is identical to that of human, porcine, rat, bovine, and canine origin [35]. In the present study, positive immunoreactivity for EDN1 was evidenced in the cytoplasm of follicular granulosa cells, as previously found for other species [36–40], and in the ovarian surface epithelial cells, which are the sites of an inflammatory-like response during ovulation [41, 42].

Before treatment, the relative abundance of EDN1 mRNA was comparable between young and mature CL, whereas those of the EDNRA, EDNRB, and ACE mRNAs were much lower in Day 4 than in Day 9 CL. These results confirm that all the EDN1 system members and ACE are constitutively expressed, albeit at different levels, in rabbit CL, as reported in other species [43]. These differences may reflect developmental changes in the ratio between steroidogenic and endothelial cell compartments over the total cell population of rabbit CL [44] or in their gene expression levels. Subtle differences in the intraovarian hormonal milieu during the luteal growing phase [11] could also be responsible for the discrepancy with the previous report [16] in terms of the relative abundances of EDN1 and EDNRB transcripts in Day-4 CL.

In the present study, exogenous PGF2{alpha} analogue challenge clearly up-regulated the transcription of both the EDN1 and ACE genes, independently of luteal stage and well in advance of progesterone decline [45]. Interestingly, whereas the expression levels of these mRNAs remained high and quite stable in young CL for the 24 h following PGF2{alpha} treatment, in mature CL they were up-regulated only for the first 1.5–3 h. PGF2{alpha} up-regulated both the EDNRA and EDNRB genes in the early-stage CL, whereas no modulation was evident in the midluteal-stage CL, probably because they were already being expressed at their maximal levels. On the other hand, the basal EDNRA and EDNRB mRNA levels in the midluteal CL were similar to those induced by PGF2{alpha} in the early luteal CL. The EDN1, EDNRA, EDNRB, and ACE expression differences suggest that the luteal cell components of growing and mature CL react differently to the same dosage of exogenous PGF2{alpha}, which supports the idea of a functional role for the RAS/EDN1 system in the luteolytic cascade evoked by PGF2{alpha}. However, it remains to be established whether the stimulatory action of PGF2{alpha} on the rabbit CL EDN1 gene is exerted directly or indirectly, given that in bovine carotid artery endothelial cells, ANGII immediately and dose-dependently up-regulates the expression of preproendothelin 1 mRNA [46]. However, the insensitivity of CL to the luteolytic action of PGF2{alpha} in the early luteal stage cannot be ascribed to a faulty response of the local EDN1/RAS system activation, but rather to some other mechanism, including the lack of endogenous PGF2{alpha} production which modulates CL function, as previously indicated for other species [47, 48] and for rabbits [49].

PGF2{alpha}-induced EDN1 gene expression increases have also been observed in midluteal-phase CL of cycling ewes [15] and cows [10, 11, 18, 50, 51], but not in early luteal-phase bovine CL [11, 52]. In cattle, PGF2{alpha} injection rapidly increases EDN1 release within the regressing CL, as well as into the ovarian venous blood [53]. Furthermore, the regulatory mechanisms that control the expression of ACE have been demonstrated in bovine CL during the estrus cycle and after the induction of luteal regression, when luteal ACE mRNA expression increases continuously, peaking 12 h and 24 h after PGF2{alpha} injection on Days 8 and 12 of the estrus cycle [17, 18, 54]. Interestingly, in cows, the overall response of CL EDN1 receptors to the luteolytic challenge dose of PGF2{alpha} showed the opposite pattern, since on Day 4 it failed to up-regulate EDNRA [11] but was effective at up-regulating both subtypes for up to 48 h when administered on Days 8–10 of the cycle. Thus, in contrast to what has been found in cows [11, 52], the EDN1 system in the CL of rabbits during the early luteal phase are not completely refractory to PGF2{alpha}, even if it is not able to decrease progesterone production.

Our in vitro results show that EDN1, after engagement of its cognate binding site, likely EDNRA [16], activates the G protein-dependent PLC/PKC pathway, which inhibits progesterone release and enhances PGF2{alpha} synthesis and NOS activity in Day-9 CL. These data are in agreement with previous reports on EDN1 intracellular signaling systems in other tissues [55, 56], as well as in ovine, bovine, and human luteal cells [15, 52, 57, 58]. In contrast, EDN1 did not affect the basal production levels of progesterone and PGF2{alpha} or NOS activity in Day-4 CL, in agreement with our previous results obtained in vivo [16]. These findings can be justified by the relatively low levels of receptors for EDN1, as compared to those present in Day-9 CL; alternatively, they can be explained by changes in the EDN1 intracellular signaling pathways, rather than to reduced or impaired synthesis of EDN1, since PGF2{alpha} up-regulates its expression. In bovine CL, Choudhary et al. [59] found that the luteolytic efficacy of EDN1 was the same on Days 4 and 10, suggesting that this vasoactive agent is a tonic inhibitor of progesterone production rather than a mediator of PGF2{alpha} action. These contrasting findings suggest that the mechanism of acquisition of luteolytic competence is different in rabbit and bovine CL.

In the present study, we have shown that EDN1-induced luteolysis at Day 9 of pseudopregnancy is inhibited by antagonizing ACE with captopril. Previously, we found that the luteolytic action of EDN1 was also blocked by pretreatment with indomethacin, a cyclooxygenase inhibitor [16]. Taken together, these findings further support the putative role of the RAS/EDN1 system in the luteolytic process, which is probably also mediated by the prostanoid pathway in the rabbit, similar to what has been described in cows [18, 23, 60]. However, our present data show that PGF2{alpha}-induced luteolysis is not affected by the blockade of ACE, which confirms that, at least in the pseudopregnant rabbit model, the cascade mechanism triggered by the prostaglandin analogue alfaprostol does not require the RAS system for the stimulation of luteolysis, in accordance with previous results obtained for the cow [18].

The findings of the present study, together with the results of previous studies conducted by us [16] and others [48, 61, 62], suggest that in midluteal-phase CL of pseudopregnant rabbits, positive reciprocal feedback mechanisms exist between RAS/EDN1 and PGF2{alpha}, which are capable of inducing functional and structural luteolysis. With a short time-lag, exogenous PGF2{alpha} stimulates endothelial and luteal cells to express increasing levels of EDN1 transcripts. EDN1, via autocrine and paracrine mechanisms, engages luteal EDNRA receptors and stimulates, through ACE mediation, the intraluteal synthesis of new PGF2{alpha}, which in turn binds to its luteal FP receptors and up-regulates EDN1 expression in both endothelial and luteal cells in both paracrine and autocrine fashions. Although the present data add new pieces to the biological puzzle of luteolysis in the rabbit model, further studies are needed to improve our understanding of the system of fine tuning that controls the CL life span in this species and the differential, age-dependent resistance to the luteolytic action of exogenous PGF2{alpha}.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. James Burge for revision of the English text. The authors thank Mrs. G. Mancini for excellent technical assistance in the preparation of the immunohistochemistry experiments.

FOOTNOTES

1Supported by a grant from Ministero dell'Istruzione, dell'Università e della Ricerca (Contract no. 2005078028). Back

Correspondence: 2Cristiano Boiti, Dipartimento di Biopatologiche ed Igiene delle Produzioni Animali e Alimentari, Università of Perugia, S. Costanzo 4, 06126 Perugia, Italy. FAX: 39 075 5857654; e-mail: cristiano.boiti{at}unipg.it

Received: 29 August 2006.

First decision: 12 October 2006.

Accepted: 9 March 2007.

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