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Prostaglandin F₂ Induces Distinct Physiological Responses in Porcine Corpora Lutea after Acquisition of Luteolytic Capacity¹

^a Endocrinology-Reproductive Physiology Program, ^b Department of Dairy Science, and ^c Department of Animal Science, University of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT

This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F₂ analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 µg cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF₂ secretion. In experiment I, progesterone remained high and PGF₂ low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF₂ production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12,13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF₂ production only from Day 17 luteal cells. Thus, PGF₂ production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF₂ production is not explained by a difference in induction of PGHS-2 mRNA or protein.

corpus luteum, corpus luteum function

INTRODUCTION

Prostaglandin F₂ (PGF₂) is considered the natural luteolysin in many mammalian species including the pig [1–3]. In pigs, PGF₂ acts by a systemic and a local mechanism. Unilateral hysterectomy leads to regression of corpora lutea (CL) on both sides, indicating a systemic effect of uterine PGF₂ [4]. However, if three fourths of the remaining horn is removed, only CL on the same side as the remaining uterine tissue regress, indicating a local effect of PGF₂ [5]. In either case uterine PGF₂ initiates luteolysis in pigs [3]. In the CL, PGF₂ binds to a G-protein-coupled receptor with seven transmembrane domains, termed the FP receptor. The FP receptor is predominantly present on the plasma membrane of the steroidogenic large luteal cells [6]. Binding of PGF₂ to the FP receptor leads to elevation of free intracellular calcium [Ca²⁺]_i and activation of protein kinase C (PKC) [7, 8]. The events occurring after PKC activation and elevation in [Ca²⁺]_i and before complete luteolysis have not been fully described. Some observed physiological changes are decreased concentrations of mRNA for steroidogenic enzymes [9, 10] including: 3ß-hydroxysteroid dehydrogenase (3ßHSD), P450-cholesterol side-chain cleavage, and steroidogenic acute regulatory protein (StAR). Prostaglandin F₂ also initiates programmed cell death of the CL [11, 12]. This process is thought to involve alterations in oxygen free radical formation [13]. The luteolytic effect of PGF₂ can be overcome with the addition of the antioxidant vitamin E [14]. In the CL, another antioxidant, vitamin C, is present at high levels [15] and may serve as a luteoprotective agent. Rapid depletion of vitamin C occurs following PGF₂ treatment [10, 15] and may be one of the luteolytic mechanisms activated by PGF₂.

In vivo cloprostenol treatment can induce PGF₂ production from CL tissue cultured in vitro [16, 17] and recently has been shown to increase the key enzyme in PG biosynthesis, prostaglandin G/H synthase (PGHS)-2, in ovine [18] and bovine CL [10]. Thus, the small amount of uterine PGF₂ that reaches the CL can initiate an autoamplification pathway that causes intraluteal production of PGF₂. In some species, such as primates and rabbits, hysterectomy does not prolong luteal life span, and intraluteal production of PGF₂ may be the primary agent of luteolysis [19, 20]. In the pig, an elegant series of experiments showed that in vivo treatment with cloprostenol dramatically increased PGF₂ production from luteal tissue subsequently cultured in vitro [17, 21]. We have used this model system to examine the intracellular mechanisms involved in this response to PGF₂ by the pig CL.

In many species including the cow, marmoset, rabbit, rat, mare, and pig there is a period during the early part of the luteal phase in which the CL will not regress after a single treatment with PGF₂ or cloprostenol [22–27]. In most of these species, the CL acquires the capacity to undergo luteolysis (luteolytic capacity) between Days 6 to 9 of the estrous cycle. The pig CL acquires luteolytic capacity relatively late in the cycle, near Day 13. Thus, the pig CL has reached mature size and maximal progesterone production for a number of days before luteolytic capacity is established.

The mechanisms involved in acquisition of luteolytic capacity have been examined. In cattle and rats, there is no difference in FP receptor concentration in CL with or without luteolytic capacity [27, 28]. In pigs, FP receptors increase near the time of acquisition of luteolytic capacity [29, 30]; however, there were still substantial numbers of FP receptors (1 million FP receptors/large luteal cell) in CL prior to acquisition of luteolytic capacity. Thus, absence of FP receptors does not appear to explain lack of luteolytic capacity. Recent findings in the cow indicate that PGF₂ administration induces similar decreases in luteal vitamin C and mRNA for FP receptor and 3ßHSD in either Day 4 or Day 11 CL, in spite of the fact that luteolysis was induced only in the Day 11 CL [10]. As mentioned above, PGF₂ or cloprostenol induce PGF₂ production from ovine and porcine CL. In bovine CL, PGF₂ induced PGHS-2 mRNA in Day 11 but not Day 4 CL, suggesting that autoamplification of luteal PGF₂ may be a key component of luteolytic capacity in bovine CL [10]. Interestingly, Guthrie and Rexroad [21] mentioned in their discussion that, "luteal PGF release in vitro was not increased by cloprostenol treatment of pigs on Day 8 of the cycle"; although, they showed no supporting data. They speculated that this lack of response could be due to lack of PGF₂ receptors. In the present study we hypothesized that cloprostenol would induce PGF₂ secretion only from CL with luteolytic capacity and that this induction would be related to an increase in PGHS-2 mRNA and protein in CL with luteolytic capacity.

MATERIALS AND METHODS

Chemicals and Reagents

Cloprostenol was purchased from Bayer Corporation (Shawnee Mission, KS), ketamine was from Fort Dodge Animal Health (Fort Dodge, IA), and xylazine was from Phoenix Pharmaceuticals (St. Joseph, MO). The T7 RNA polymerase, Taq polymerase, reverse transcriptase, dNTPs, RNAsin, and DNAase I were purchased from Promega (Madison, WI). Molecular weight markers were from Gibco/BRL (Gaithersburg, MD). Magnetight oligo(dT) beads were from Novagen (Madison, WI). Protein A/G plus agarose, anti-PGHS-2 polyclonal antibody, anti-goat horseradish peroxidase (HRP) antibody and PGHS-2 blocking peptide were from Santa Cruz Biotechnology (Santa Cruz, CA). Unless otherwise specified other chemicals and reagents used in these studies were purchased from Sigma (St. Louis, MO).

Animals

Crossbred gilts (Cambrough x Line 19) 6–8 mo of age were obtained from the university herd or purchased from Pig Improvement Company (PIC, Franklin, KY). Animals were kept in individual pens with free access to water and were fed a maintenance diet of corn and soybean meal. For all studies, animals were checked daily for standing estrus with a mature boar. First day of estrus was designated as Day 0. Pseudopregnancy was induced in some gilts with daily injections of estradiol benzoate (2 mg i.m.) on Days 11–15. On the day ovaries were collected, anesthesia was induced with i.m. injection of ketamine (15 mg/kg) and xylazine (0.3 mg/kg). Gilts were intubated and surgical plane of anesthesia maintained with halothane. Ovaries were collected via midventral laparotomy, and CL were dissected away from ovarian stroma and either frozen in liquid nitrogen or transported to the laboratory in cold media (m199, 100 IU/ml penicillin, 10 mg/ml streptomycin, 0.1% BSA) for further processing. The Research Animal Resource Center Committee at University of Wisconsin-Madison approved all procedures performed on animals.

Preparation of Native and Competitor RNA Standards

Bovine primers for PGHS-2 designed in our laboratory [31, 32] were used to amplify a cDNA fragment of 484 base pairs (bp) (PGHS-2) from porcine uterine mRNA. The PGHS-2 fragment was cloned into pCR II vector (Invitrogen) and sequence analysis was performed using the ABI prism dye-terminator sequencing kit (University of Wisconsin-Madison Biotechnology Center). Porcine-specific primers were designed to amplify a 422-bp product of PGHS-2. This region of porcine PGHS-2 mRNA shows 100% identity with the recently sequenced porcine PGHS-2 mRNA (AF207824). Bovine primers for FP receptor [31, 32] generated a 288-bp product that was cloned into pGEM-T (Promega) vector and sequenced as above. The FP receptor sequence overlapped with a previously published partial porcine cDNA sequence (U91520) and together these two sequences yield a composite sequence of 437 bp. This composite FP receptor sequence was used to generate primers to amplify a 322-bp product from uterine mRNA and this was cloned into pGEM-T vector. This region of porcine FP receptor mRNA sequence showed 88% identity to bovine (D17395), 90% to ovine (U73798), 85% to rat (U26663), and 87% to human (L24470) FP receptor sequence. Competitor fragments were produced by deletion of 80 bp from the native FP receptor and 105 bp from the native PGHS-2 sequence using a PCR-based internal deletion method [33]. The competitor fragments of 242 bp (FP receptor) and 317 bp (PGHS-2) were cloned into pGEM-T vector. Native and competitor RNA standards were produced by in vitro transcription of the appropriate plasmids using T7 RNA polymerase as previously described [31]. For cytosolic phospholipase A₂ (PLA₂), bovine primers previously developed in our laboratory were used to generate native and competitor standards using methodology similar to the method described above for PGHS-2 and FP receptor. Bovine cytosolic (c)PLA₂ primers amplified the expected product of approximately 510 bp from pig mRNA.

Isolation of mRNA

Messenger RNA was isolated from CL tissue and cultures of mixed luteal cells using Magnetight oligo(dT) magnetic beads. Frozen CL tissue was ground to a fine powder using a mortar and pestle filled with liquid nitrogen. Approximately 20 mg of this luteal powder was homogenized in 400 ml lysis buffer (4 M guanidium isothiocyanate, 0.5% sarcosyl, 10 mM Tris-HCl, pH 8.0 and 1% dithiothreitol) using a glass homogenizer. Chromosomal DNA was sheared by passing the homogenate through a 25-gauge needle 10 times. Two volumes of binding buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA and 400 mM NaCl) were added to homogenate and mixed well. Samples were centrifuged at 16 000 x g for 5 min at 4°C to pellet cellular debris. Supernatant was transferred to a tube containing 800 µg of oligo(dT) beads and allowed to hybridize for 10 min. Beads were captured on a magnetic stand, and supernatant was saved for DNA determination. Beads were washed four times with 500 µl wash buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) and mRNA was eluted with 30 µl elution buffer (2 mM EDTA) after heating to 65°C for 5 min. The mRNA was stored at -70°C until used. The cell culture samples were processed as above, except cells were lysed directly in 100 µl lysis buffer/200 µl binding buffer, washed three times with 300 µl wash buffer, and eluted in 10 µl of elution buffer.

Quantification of mRNA using Quantitative Competitive Reverse Transcription PCR

Messenger RNA from tissue or luteal cells were analyzed as described previously [31] by a quantitative-competitive (QC) reverse transcription (RT)-PCR assay with standard curve methodology. Standard curves for FP receptor, PGHS-2, and cPLA₂ were created by adding increasing amounts of native standards to a constant amount of competitor in a 20-µl final volume of RT master mix (1x RT buffer, 0.2 mM dNTPs, 100 pmol random primer, and 40 U reverse transcriptase). For all standard curves eight 2x serial dilutions were prepared. For FP receptor and cPLA₂ standard curves, native amounts ranged from 0.312 to 40 amol per reaction with a constant 4 amol of competitor. The standard curve for PGHS-2 used 0.16 to 20 amol per reaction of native with a constant 1.5 amol of competitor. Master mix (19 µl) was dispensed into 0.2-ml thin-wall PCR tubes and 1 µl of either native standards or samples was added to individual tubes. Reverse transcription was carried out at 37°C for 1.5 h followed by heating to 95°C for 10 min in a programmable thermocycler (MJ Research, Watertown, MA). Four 4 µl of RT reaction were added to PCR master mix (1x thermophilic buffer supplied with enzyme, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.4 µM each of forward and reverse primers and 0.5 U Taq DNA polymerase) in a 20-µl final volume and amplified for 30 cycles of PCR (95°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec followed by a final extension at 72°C for 5 min). Reactions were separated on a 5% PAGE gel and stained with ethidium bromide. Products were quantified using a Collage imaging system (Fotodyne, Heartland, WI). Standard curves were plotted as log ratio of native:competitor density readings on the y-axis versus log of native added in attomoles on the x-axis. Unknown samples were compared to the standard curve to calculate the copy number. Cell number was calculated from the DNA content in the supernatant collected during mRNA isolation as described previously [34].

Semiquantitative RT-PCR

Evaluation of 15-hydroxyprostaglandin dehydrogenase (PGDH) mRNA was accomplished using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA as an internal control. Primers for G3PDH were designed from the published partial porcine sequence (X94251) to produce the expected 285-bp product. The PGDH primers (water buffalo) were obtained from Colorado State University, Animal Reproduction Laboratory (Laboratory of Dr. G.D. Niswender). The PGDH primers amplified a product from pig mRNA of the expected size (394 bp). Reverse transcription was carried out with 19 µl of 1x master mix (same as above, but without competitor) and 1 µl of sample for 1.5 h at 37°C. For PCR, 4 µl of RT reaction was added to 1x PCR master mix (same as above, but with both G3PDH and PGDH primer sets at a concentration of 0.4 µM) for 30 cycles (95°C for 30 sec, 54°C for 30 sec, and 72°C for 30 sec, followed by a final extension at 72°C for 5 min). Reactions were separated on a 5% PAGE gel and stained with ethidium bromide. For each sample two products were quantified using the Collage imaging system. Values were calculated as the ratio of PGDH band intensity:G3PDH band intensity.

Immunoprecipitation and Western Blotting

Frozen luteal tissue (60 mg) was homogenized in 900 µl of cold homogenization buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1% Triton X-100, and 0.25% deoxycholate) using a polytron tissue grinder. Lysate was centrifuged twice at 16 000 x g for 10 min to obtain a clear lysate. To each sample 20 µl of protein A/G conjugated to agarose beads (Santa Cruz Biotechnology) were added and incubated for 4 h at 4°C. The samples were centrifuged at 16 000 x g for 5 min to pellet the agarose beads, and the supernatant was transferred to a fresh tube. Two micrograms of specific anti-PGHS-2 antibody was added followed by incubation overnight at 4°C. Immune complex was precipitated with 20 µl of protein A/G conjugated to agarose beads for 4 h at 4°C, followed by centrifugation at 16 000 x g for 5 min. Samples were washed four times with cold homogenization buffer. After the final wash, 40 µl of 2x SDS-loading dye were added, and samples were steamed for 5 min. The entire sample was loaded on a 10% SDS-PAGE gel, and proteins were separated at 120 mA for 1.5 h. Proteins were transferred to polyvinylidene difluoride membrane using the miniprotean II gel transfer system (Bio-Rad, Hercules, CA). Following transfer, blots were incubated in blocking buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk) overnight at 4°C. Immunoblotting proceeded by incubating blots with anti-PGHS-2 antibody (Santa Cruz Biotechnology) at 1:2000 dilution for 2 h at 37°C, followed by three washes (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20). Anti-goat HRP (Santa Cruz Biotechnology) was added at 1:20 000 dilution for 1 h at 37°C, followed by three washes. Specific proteins were detected with enhanced chemiluminescent reagent (NEN Life Science Products, Boston, MA). Blots were exposed to x-ray film for 10 min and quantified using the Collage photo imaging system (Heartland, WI).

Hormonal and Ascorbic Acid Assays

The procedure used for the vitamin C assay has been described previously [10, 35]. Luteal tissue (60 mg) was homogenized in 1 ml of 5% trichloroacetic acid (TCA) using a polytron tissue grinder. The lysate was centrifuged at 10 000 x g for 5 min to clarify and the supernatant stored at -20°C. To 50 µl of sample, 50 µl ethanol, 25 µl 0.4% H₃PO₄-EtOH, 50 µl 0.5% bathophenanthroline, and 0.03% FeCl₃-EtOH were added in the order shown. The plate was incubated for 1 h at 37°C with shaking to allow for color development. Absorbance was measured at 525 nm using an enzyme immunoassay (EIA) plate reader (Bio-Tek Instruments, model EL310; Fisher, Pittsburgh, PA).

For progesterone determination, media from luteal incubations and luteal TCA extracts were diluted 1:100 and 1:1000, respectively, in assay buffer (40 mM morpholinepropanesulfonic acid, 0.12 M NaCl, 10 mM EDTA, 0.1% gelatin, 0.5% Tween 20, and 0.005% chlorohexidine digluconate, pH 7.4). The progesterone assay is a competitive ELISA assay and has been described previously [36]. An ELISA for PGF₂ was developed in our laboratory. Media from luteal incubations or cell culture experiments were diluted 20- to 100-fold in assay buffer. Rabbit anti-sheep IgG (2 µg/ml, Calbiochem, San Diego, CA) was coated onto 96-well plates and they were washed with PBS buffer three times. Sheep anti-PGF₂ (1:50 000, Assay Design, Inc., Ann Arbor, MI) was incubated on the plate for 1 h to allow binding to anti-sheep IgG. Samples and standards were then added and allowed to bind for 15 min. A PGF₂-HRP conjugate made by the mixed anhydride method of Hayashi and Yamamoto [37] was incubated on the plate for 1 h. The plates were washed three times and developed as above. Prostaglandin E₂ was measured in pooled samples of luteal tissue incubations (two samples/animal) using a PGE₂ EIA assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.

Experiment I

Gilts 8 mo of age were checked for estrus daily with a mature boar. Animals were randomly assigned to one of four groups: Day 9 saline (n = 5), Day 9 cloprostenol (500 µg; n = 4), Day 17 saline (n = 5), and Day 17 cloprostenol (500 µg; n = 5). Cloprostenol was used in an attempt to avoid cross-reactivity in the PGF₂ assay; however, cloprostenol does bind to the antibody used in the ELISA. Nevertheless, by 10 h after treatment, all cloprostenol has been cleared from the circulation. On Day 9 of the estrous cycle or 17 of pseudopregnancy, gilts received either saline or cloprostenol 10 h prior to ovary removal. Corpora lutea were collected via midventral laparotomy and were transported to the laboratory within 5 min. For each animal, 400 mg of finely minced CL tissue was cultured in a six-well tissue culture dish (six wells/animal) for 2 h at 37°C with shaking. Following incubation, media were collected for PGF₂ and progesterone determination. Tissue in each well was weighed, allowing for expression of results per unit mass tissue. The remainder of the luteal tissue collected during surgery was frozen in liquid nitrogen for determination of total ascorbic acid, luteal progesterone, and several mRNAs (PGHS-2, cPLA₂, PGDH, and FP receptor). Frozen luteal tissue was also analyzed for PGHS-2 protein concentrations using the immunoprecipitation/Western blotting protocol.

Experiment IIa

To examine the temporal regulation of PGHS-2 mRNA in vitro, CL from saline-treated gilts in experiment 1 (Day 9, n = 5; Day 17, n = 5) were dissociated. Sliced luteal tissue was incubated for two 45-min periods with dissociation media (M199, 100 IU/ml penicillin, 10 mg/ml streptomycin, 0.1% BSA, 2.5 mg/ml collagenase type IV [Worthington Biochemical Corp., Freehold, NJ], and 0.5 mg/ml DNAase I [Boehringer Mannheim, Indianapolis, IN]) at 37°C with vigorous shaking. Cells were filtered through a 70-µm screen, washed four times, and counted using a hematocytometer. Viability was assessed with trypan blue dye exclusion and was greater than 80% for small and large luteal cells. Mixed luteal cells containing 20 000 large luteal cells were plated on 24-well plates (M199, 100 IU/ml penicillin, 10 mg/ml streptomycin, 0.1% BSA, and 1% fetal calf serum) overnight. The next day, cells were washed three times with media and treated with media only, 10 nM PDD (a PKC activator), or 100 nM cloprostenol. At 0, 1, 2, 4, 8, and 12 h after treatment, cells were lysed with 100 µl lysis buffer per well (see Messenger RNA Isolation) and 200 µl binding buffer and stored at -80°C until mRNA isolation. Samples were quantified for PGHS-2 mRNA. Media samples collected 24 h after treatment were analyzed for PGF₂ and progesterone.

Experiment IIb

In a separate experiment, luteal cells from Day 9 after estrus or Day 17 of pseudopregnancy, prepared as above, were treated with 30 nM cloprostenol for 10 min to mimic acute exposure to PGF₂, as would occur in vivo. After exposure, cells were washed four times and media replaced. Samples were collected for PGHS-2 mRNA determination 1 h after treatment. Media samples were also collected at 24 h after treatments for analysis of progesterone and PGF₂ secretion.

Experiment III

This experiment examined the acute in vivo (30 min) regulation of mRNA for PGHS-2, FP receptor, PGDH, and cPLA₂ by cloprostenol. On Day 9 after estrus (n = 4) or Day 17 of pseudopregnancy (n = 4), gilts were anesthetized and one ovary collected (control CL). Following removal of control ovary, 500 µg of cloprostenol was given i.m. and the other ovary was collected 0.5 h later (treated CL). Corpora lutea were collected and frozen for quantitation of mRNAs and luteal vitamin C.

Statistical Analyses

Results from experiment I, including luteal ascorbic acid, luteal progesterone, tissue secretion of PGE₂, PGF₂ and progesterone, and mRNAs for PGHS-2, FP receptor, cPLA₂, and PGDH, were analyzed by one-way ANOVA using the general linear method (GLM) procedure of the Statistical Analysis System (SAS) [38]. Mean separation was performed by Fisher's least significant different (LSD). Results for PGHS-2 protein were evaluated by t-tests. In experiment IIa, PGHS-2 mRNA levels after in vitro cloprostenol or PDD were analyzed separately for Day 9 and Day 17 luteal cells using the GLM procedure of SAS. Pig was used as block, and time was included in the model. Results from experiment IIb, luteal cell secretion of progesterone and PGF₂ and PGHS-2 mRNA levels were analyzed within day using ANOVA followed by Fisher's LSD. Results from experiment III, including acute induction of mRNAs for PGHS-2, cPLA₂, PGDH, and FPr and vitamin C, were analyzed by paired t-test.

RESULTS

Experiment I

Cloprostenol treatment in vivo increased (P < 0.05) PGF₂ (Fig. 1) and PGE₂ (Table 1) accumulation in media cultured with luteal tissue from Day 17 CL but not Day 9 CL. Progesterone secretion decreased (P < 0.05) only in Day 17 cloprostenol-treated CL compared to all other groups (Fig. 1). Luteal progesterone concentrations mirrored the results from luteal tissue incubations with cloprostenol decreasing progesterone only in Day 17 CL (Table 1). Total luteal vitamin C decreased to 46% (P < 0.05) after cloprostenol treatment in Day 17 but not Day 9 CL (Table 1).

View larger version (16K): [in this window] [in a new window]   FIG 1. Secretion of progesterone (A) and PGF2 (B) from luteal cells after 2 h incubation in vitro. Animals (n = 4 or 5/group) were treated in vivo with saline or 500 µg cloprostenol 10 h prior to surgical collection of CL. Six replicate wells were analyzed for each animal and tissue was weighed to allow expression of results per unit mass of tissue. Different superscripts indicate differences. P < 0.05

The PGHS-2 mRNA was not different (P > 0.05) in either Day 9 or Day 17 CL 10 h after cloprostenol treatment. The FP receptor mRNA was decreased (P < 0.05) following cloprostenol treatment in both Day 9 and Day 17 CL (Table 1). The mRNA for PGDH was not different between any treatment groups. The mRNA for cPLA₂ was greater in Day 9 than Day 17 CL regardless of cloprostenol treatment (P < 0.05). Cloprostenol decreased cPLA₂ mRNA (P < 0.05) in Day 17 but not Day 9 CL.

Figure 2 shows Western blots for PGHS-2 protein. The luteal PGHS-2 protein demonstrated two bands of approximately 78 and 74 kDa. Preincubation of the PGHS-2 antibody with a PGHS-2 peptide (Fig. 2B) completely eliminated these two bands indicating the specificity of the observed bands. As shown in Figure 2, A and C, the PGHS-2 protein was increased at 10 h after cloprostenol treatment in Day 9 (P < 0.05) or Day 17 (P = 0.097) CL.

View larger version (40K): [in this window] [in a new window]   FIG 2. A) Western blot of immunoprecipitated PGHS-2 protein 10 h after saline or 500 µg cloprostenol treatment in vivo (PC, purified ovine PGHS-2 standard; [-] saline; [+] cloprostenol). B) Western blot of same samples as in panel A using anti-PGHS-2 antibody preincubated with PGHS-2 blocking peptide. C) Graphical representation of densitometric readings from PGHS-2 Western blots (n = 4 or 5/group). *P = 0.097, **P < 0.05

Experiment IIa

Cloprostenol increased (P < 0.05) PGHS-2 mRNA in a time-dependent fashion in luteal cells from both Day 9 and Day 17 CL (Fig. 3A). Maximum induction occurred by 4 h. The PKC activator PDD also increased (P < 0.05) PGHS-2 mRNA in luteal cells from both Day 9 and Day 17 CL (Fig. 3B). Progesterone production was inhibited by cloprostenol only in luteal cells from Day 17 CL; however, PDD decreased (P < 0.05) progesterone production in both groups (Fig. 4A). Production of PGF₂ after continuous cloprostenol treatment could not be accurately measured because of cross-reactivity of cloprostenol in the PGF₂ assay. Production of PGF₂ was increased (P < 0.05) by PDD treatment of luteal cells from both Day 9 and Day 17 CL (Fig. 4B).

View larger version (26K): [in this window] [in a new window]   FIG 3. Induction of PGHS-2 mRNA by cloprostenol (A) or the PKC activator PDD (B) in pig luteal cells taken from CL on Day 9 of the estrous cycle or Day 17 of pseudopregnancy (n = 5/group). There was a time effect (P < 0.05) within day for both cloprostenol and PDD

View larger version (21K): [in this window] [in a new window]   FIG 4. A) Media progesterone from Day 9 or Day 17 luteal cells (n = 5/group) 24 h after treatment with media (control), 100 nM cloprostenol, or 10 nM PDD. B) Media PGF2 from Day 9 or Day 17 luteal cells (n = 5/group) 24 h after treatment with media only (control) or 10 nM PDD. Different superscripts within day indicate differences (P < 0.05)

Experiment IIb

Acute cloprostenol treatment (30 nM), for 10 min increased PGHS-2 mRNA (P < 0.05) in luteal cells from both Day 9 and Day 17 CL (Fig. 5A). In contrast, treatment with cloprostenol for 10 min increased PGF₂ secretion and decreased progesterone secretion only in luteal cells from Day 17 CL (Fig. 5, B and C).

View larger version (19K): [in this window] [in a new window]   FIG 5. Effect of acute (10 min) treatment with 30 nM cloprostenol on A) PGHS-2 mRNA (1 h after treatment), B) media progesterone, and C) media PGF2 (24 h after treatment) from Day 9 or Day 17 luteal cells. Different superscripts within day indicate differences (P < 0.05)

Experiment III

Cloprostenol treatment in vivo increased (P < 0.05) PGHS-2 mRNA in both Day 9 and Day 17 CL (Table 2) 0.5 h after treatment. Luteal vitamin C tended to decrease (P = 0.07) after cloprostenol in Day 17 but not Day 9 CL. The mRNAs for FP receptor, PGDH, and cPLA₂ were not changed by cloprostenol on either Day 9 or Day 17 at 0.5 h after cloprostenol treatment (Table 2).

DISCUSSION

This study was designed to investigate the intracellular mechanisms involved in acquisition of luteolytic capacity. Luteolytic capacity is the ability of the CL to undergo complete luteolysis in response to a single treatment with exogenous PGF₂. Herein, we present evidence linking acquisition of luteolytic capacity in the pig CL with changes in physiological responses induced by cloprostenol.

Luteolytic capacity is not achieved in the porcine CL until after Day 12 of the estrous cycle [23]. This is very near the time of natural luteolysis in the pig. We therefore used a pseudopregnant pig model in this study. Pseudopregnancy can be induced with exogenous estrogen given on Days 11–15 [39]. The CL of pseudopregnancy are similar to CL of early pregnancy, both are able to undergo luteolysis in response to PGF₂ [40, 41]. The estradiol benzoate protocol used in this study maintained luteal function in all Day 17 saline-treated CL, as indicated by the high luteal progesterone. In addition, progesterone secretion from luteal tissue was similar between saline-treated Day 17 and Day 9 CL.

In contrast, there were dramatic differences between Day 9 and 17 CL after in vivo treatment with cloprostenol. Figure 6 shows an intracellular model of the distinct physiological responses induced following FP receptor activation before and after acquisition of luteolytic capacity. Progesterone production from luteal tissue in vitro was identical in Day 9 CL whether or not they had received cloprostenol in vivo; whereas, cloprostenol treatment decreased in vitro progesterone production in Day 17 CL to 13% of saline-treated group. The intracellular mechanism that produced this inhibition only in Day 17 cloprostenol-treated CL has not yet been investigated but could involve changes in StAR mRNA or protein [9]. The inhibition of progesterone production was not due to a global decrease in metabolic activity after cloprostenol treatment because there was a dramatic increase in PGF₂ and PGE₂ production in Day 17 but not Day 9 CL. Thus, as shown in Figure 6, the cloprostenol-induced increase in PGF₂ production and decrease in progesterone production are clearly associated with acquisition of luteolytic capacity in the pig CL. As indicated previously in ovine, bovine, and porcine CL [10, 18, 21], there appears to be an autoamplification loop that causes intraluteal production of PGF₂ and this may be critical for completion of luteolysis. Several pulses of uterine PGF₂ may be necessary to induce the biosynthetic machinery within some luteal cells for intraluteal PGF₂ production [42, 43]. Intraluteal autoamplification of PGF₂ would maintain high PGF₂ concentration in the CL to stimulate luteolysis maximally.

View larger version (32K): [in this window] [in a new window]   FIG 6. Conceptual model depicting the physiological responses induced by PGF2 in pig CL before and after acquisition of luteolytic capacity. AA, Arachidonic acid; FPr, prostaglandin F receptor; cPLA2, cytosolic phospholipase A2; PGHS-2, prostaglandin G/H synthase

The first committed step in PG synthesis is catalyzed by PGHS-2 (also called COX-2), converting arachidonic acid to PGH₂. In the cow, PGF₂ induces PGHS-2 mRNA only in CL with luteolytic capacity (Day 11) [10]. However, in the pig we observed that PGHS-2 mRNA was induced by cloprostenol in both Day 9 and Day 17 CL. Even more surprising, cloprostenol induced PGHS-2 protein in Day 9 and tended to increase PGHS-2 protein in Day 17 CL (P = 0.097). The induction of PGHS-2 mRNA and protein in Day 9 CL is puzzling because there clearly was no increase in luteal PGF₂ accumulation in media of tissue slices. In Figure 6, possible regulatory points of PGF₂ synthesis are indicated by question marks in the luteal cell without luteolytic capacity. There may be an inhibitor of PGHS-2 activity present in Day 9 CL that is lost by Day 17. This would explain the induction of PGHS-2 protein without an increase in PGF₂ secretion. Alternatively, either substrate (arachidonic acid) for PG synthesis is limiting or downstream events such as conversion of PGH₂ to PGF₂ or secretion of PGF₂ are limiting. If substrate availability is the key regulatory event, the most likely target is cPLA₂. The finding that cPLA₂ mRNA is lower in Day 17 as compared to Day 9 CL is not consistent with changes in cPLA₂ mRNA explaining differences in PGF₂ production. Future studies could determine the role of cPLA₂ by focusing on changes in luteal cPLA₂ activity and protein. Thus, although acquisition of luteolytic capacity is associated with a PGF₂ autoamplification pathway in the CL of both cattle and pigs, it appears that the mechanisms that prevent or induce this pathway differ between the cow and gilt. Nevertheless, a direct comparison between the CL in the bovine study [10] and the pig CL in this study may not be appropriate because porcine CL acquire luteolytic capacity at a much later stage of the luteal phase than do bovine CL.

Another possibility is that PGF₂ is being metabolized in the CL by the enzyme PGDH, as previously suggested [44]. We found that PGDH mRNA is highly abundant in the pig CL. It is present in about a 1:1 ratio with the housekeeping gene G3PDH (Tables 1 and 2). However, it does not appear to be regulated at the mRNA level by cloprostenol, but this does not exclude possible changes in protein activity. In the sheep CL, PGDH activity increases during maternal recognition of pregnancy, indicating a possible luteoprotective role in the ovine CL [44]. Prostaglandin F₂ may also be converted to another PG. For example, PGE-9-keto-reductase converts PGF₂ to PGE₂. We measured PGE₂ in media from luteal tissue incubations and found a similar pattern to PGF₂ with induction of PGE₂ production by cloprostenol in Day 17 but not Day 9 CL (Table 1). Thus, it appears that conversion to PGE₂ does not explain differences in PGF₂ production by Day 9 CL.

Prostaglandin F₂ arrives at the early CL (without luteolytic capacity) and activates some intracellular systems. This is evidenced by cloprostenol increasing PGHS-2 protein and mRNA and decreasing FP receptor mRNA in both Day 9 CL and Day 17 CL. These results are in agreement with previous findings showing a decrease in PGF₂ binding sites after PGF₂ treatment in the pig [45]. However, some physiological responses to cloprostenol differ between Day 9 and Day 17 CL. The most striking is the difference in PGF₂ production discussed above. The results with luteal vitamin C also indicate differential regulation in Day 9 versus Day 17 CL. There was no acute (0.5 h) or delayed (10 h) effect of cloprostenol on luteal vitamin C in Day 9 CL; but a significant decrease in vitamin C occurred after cloprostenol treatment in Day 17 CL (Table 1).

Our original intent in doing experiments with the pig CL was to extend our previous results on luteolytic capacity to a nonruminant species and to find an animal model in which luteal cells with or without luteolytic capacity could be obtained for in vitro studies. The cell culture experiments confirm that cultured porcine luteal cells continue to respond to PGF₂ in ways that are similar to in vivo responses. Thus, it may be possible to determine the intracellular mechanisms involved in acquisition of luteolytic capacity using this in vitro system. In this regard, continuous treatment with PDD, a PKC activator, increased PGHS-2 mRNA, decreased progesterone, and increased PGF₂ secretion by both Day 9 and Day 17 luteal cells (Figs. 3 and 4). This suggests that continuous, potent activation of the PKC system can overcome the mechanisms that prevent luteolysis in the CL without luteolytic capacity. It is possible that PGF₂ does not potently activate the PKC intracellular effector system in the CL without luteolytic capacity. Alternatively, inhibitors of PKC may be present in early CL and may prevent full expression of the luteolytic process as has been suggested in the sheep CL [46]. Possible changes in activation of intracellular signaling during acquisition of luteolytic capacity may be part of a differentiation program occurring in luteal cells that begins with the LH surge. Luteinization is mainly a PKA-mediated event, while luteolysis is a PKC/[Ca²⁺]_i-mediated event. The CL without luteolytic capacity may represent an intermediate stage of differentiation between PKA- and PKC-mediated function.

In conclusion, the pathways leading to luteolytic capacity remain to be defined fully. Data from multiple species indicate that autoamplification of luteal PGF₂ is a critical component of luteolytic capacity; although, the mechanisms leading to this autoamplification appear to differ between species. Other endpoints, such as luteal vitamin C and progesterone production are also differentially regulated by PGF₂ in CL with or without luteolytic capacity. One hypothesis for acquisition of luteolytic capacity implicates a difference in activation of intracellular signaling pathways, such as the PKC pathway, upon binding of PGF₂ to the FP receptor. The present results are consistent with this hypothesis but should be considered highly speculative until more direct experiments are performed. The data presented clearly support part of our original hypothesis that PGF₂ production increases after cloprostenol treatment only in CL with luteolytic capacity. However, our proposed mechanism, induction of PGHS-2, does not explain the difference in cloprostenol-induced PGF₂ production between CL with and without luteolytic capacity. Understanding the control of intraluteal PGF₂ production may provide a greater understanding of the cellular and molecular basis for luteolytic capacity and may be useful in developing methods to promote or prevent luteal regression.

ACKNOWLEDGMENTS

We thank Drs. G.D. Niswender and P.J. Silva for developing and providing the PGDH primers; R. Sartori, C. Cordoba, Y.-L. Wu, L.E. Anderson, S. Sangsritavong, A. Gumen, and J. Haughian for surgical assistance; and Ms. Josie Lewandowski for technical assistance.

FOOTNOTES

First decision: 18 April 2000.

¹ This work was supported by NIH grant HD-32623.

² Correspondence: Milo C. Wiltbank, Department of Dairy Science, University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI 53706. FAX: 608 263 9412; wiltbank{at}calshp.cals.wisc.edu

Accepted: June 21, 2000.

Received: March 13, 2000.

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