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Biology of Reproduction 61, 1527-1534 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Insulin-Like Growth Factor (IGF)-I, IGF-I Receptor, and IGF Binding Protein-3 Messenger Ribonucleic Acids and Protein in Corpora Lutea from ProstaglandinF2{alpha}-Treated Gilts1

Whitney C.E. Nicholsona, Zhaoping Gea, David M. Plotnera, Charlotte E. Farinb, and John E. Gadsby2,a

a Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine and b Department of Animal Science, North Carolina State University, Raleigh, North Carolina 27695


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F2{alpha} (PGF2{alpha}) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF2{alpha} (or saline) on Days 5–9 of the cycle to induce premature luteolysis. CL were collected on Days 6–9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF2{alpha}-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8–9, while IGFBP-2 was higher on Days 6–7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF2{alpha}-treated animals vs. control animals (P < 0.05). In conclusion, PGF2{alpha}-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6–7), it is possible that they may mediate the luteolytic actions of PGF2{alpha} by sequestering IGF-I and preventing its interaction with the IGF-IR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The corpus luteum (CL), by producing progesterone, is responsible for maintaining pregnancy or, in the absence of pregnancy, regulating the length of the estrous cycle [1]. In the nonpregnant pig, the life span of the CL is 16–18 days [2], and at the end of this period the CL undergoes luteolysis as a result of secretion of prostaglandin F2{alpha} (PGF2{alpha}) by the uterus [3]. The pig is rather unique among the livestock species in that its CL display an extended period of refractoriness to the luteolytic effects of exogenous PGF2{alpha}, which lasts approximately 12 days [4]. However, this insensitivity can be overcome by repeated administration of PGF2{alpha} on Days 5–10 following the onset of estrus, successfully reducing the mean inter-estrus period from 19.8 ± 0.6 to 13.3 ± 0.5 days and inducing premature CL regression [5, 6]. In addition, the porcine CL is capable of functioning autonomously of pituitary gonadotropins for approximately 12–14 days [2], although little is known of the control mechanisms underlying this process.

Recent studies have investigated the insulin-like growth factor (IGF) system (IGF-I, IGF-II, IGF binding proteins, and IGF receptors) as an important autocrine/paracrine system regulating ovarian function (reviewed in [7–11]). IGF-I is stimulatory to progesterone, oxytocin, and relaxin release by luteal tissue in vitro, supporting a luteotropic role for IGF-I in the CL [12–18]. IGF-I mRNA has been shown to be expressed in CL of several species, including the sheep [19], rat [16], cow [14], and pig [20]. We have shown that steady-state levels of IGF-I mRNA were expressed in small luteal cells to a greater extent in the early (Days 4–10) vs. the late (Days 12–16) luteal phase, suggesting that IGF-I may play an important role as an autocrine/paracrine growth factor during the period of luteal autonomy [21].

Previous studies have reported inhibitory actions of the IGF binding proteins (IGFBPs) in the ovary [22], whereas in other tissues, both stimulatory and inhibitory actions have been reported [23]. Several of the IGFBPs are present within the ovary, and there is evidence for local expression within the ovarian follicle [10]. In addition, studies performed in our laboratory have shown mRNA transcripts for IGFBP-2, -3, -4, and -5 by Northern blot analysis in the porcine CL [21]. Treatment with PGF2{alpha} resulted in a 2.6-fold increase in IGFBP-3 production in luteinized porcine granulosa cells in culture, while prostaglandin E2, a luteotropic compound, reduced IGFBP-3 production to half of control levels [24]. From these studies it was proposed that IGFBP-3 may play an inhibitory role on granulosa cell function, as had been suggested previously [25]. Furthermore, since IGFBP-3 mRNA expression was elevated in CL undergoing luteolysis in the rat [26], it has been suggested that IGFBP-3 may play a role in luteolysis. In the current study we focused on IGFBP-3 since it appears to be the most significantly regulated IGFBP in the porcine CL [27], as a potential mediator of the luteolytic actions of PGF2{alpha} [24].

The action of IGF-I is dependent on its interaction with the type I IGF receptor (IGF-IR), which is also expressed in the porcine CL [28, 29]. Little is known about the regulation and expression of IGF-IR in the porcine CL, although this information is important because changes in receptor availability could greatly modify the actions of IGF-I in this tissue.

The purpose of this study was to examine the effects of treatment with PGF2{alpha} during the early luteal phase, on the intraluteal IGF system. Our first hypothesis was that treatment with PGF2{alpha} would inhibit IGF-I production in the CL. We also hypothesized that IGFBP-3 expression would increase in response to PGF2{alpha} treatment and thus act as an inhibitor of the luteotropic actions of IGF-I in the porcine CL. Finally, we hypothesized that expression of the IGF-I receptor would be reduced in response to PGF2{alpha} treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Collection

The experimental protocol involving animal care and use was performed in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction, and the approval of the North Carolina State University Institutional Animal Care and Use committee. Forty crossbred gilts (Landrace/Yorkshire x Hampshire/Duroc) were checked daily for signs of estrus (standing heat). Day 0 was defined as the first day of standing heat. On Days 5–9 gilts were given 12.5 mg PGF2{alpha} (Lutalyse; Upjohn Co., Kalamazoo, MI) or saline (control) at 12-h intervals [5, 6]. On Days 6, 7, 8, and 9, animals (n = 5 per day/treatment) were anesthetized, and ovaries were surgically removed [30]. CL were bisected, excised from the ovaries, snap-frozen in liquid nitrogen, and stored at -80°C until extraction of RNA or protein could be performed.

CL Weight

Weights of frozen CL were determined for all CL before RNA or protein extraction for slot blots, RIA for IGF-I and progesterone, and ligand and Western blots.

Progesterone RIA

To confirm that luteolysis had occurred in PGF2{alpha}-treated animals as previously reported [5, 6], progesterone was extracted from 1 CL per animal using the method validated by Whaley and colleagues [31]. Briefly, luteal tissue (~100–200 mg) was weighed, pulverized, and homogenized in 4.5 ml ice-cold 0.01 M PBS using three 30-sec bursts with a hand-held homogenizer. Homogenates were incubated on ice for 1 h and centrifuged at 1700 x g for 10 min at 4°C, and the supernatant was recovered. The extraction efficiency of progesterone from luteal tissues was determined for every fifth sample by addition of 20 000 cpm [3H]progesterone to homogenates. After recovery of the supernatant, an aliquot was counted by liquid scintillation spectrophotometry (LKB, Turku, Finland). Progesterone extraction efficiency was 92.9 ± 1.8% for eight CL examined. Determination of progesterone concentration in CL extracts was performed using the Coat-A-Count RIA kit (Diagnostic Products Corp., Los Angeles, CA) after dilution (1:100) in PBS-gel (0.1 M phosphate-buffered saline containing 0.1% gelatin pH 7.2). All samples were assayed within a single assay with a standard curve of 0.01–4 ng/tube. The intraassay coefficient of variation was 5.9%.

RNA Analysis

Whole-cell RNA (wcRNA) was extracted from 2 CL for each animal with Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH), according the manufacturer's instructions [32, 33]. Briefly, the tissue was pulverized under liquid nitrogen, weighed, and homogenized in Tri-Reagent. Whole-cell RNA was then extracted with chloroform and precipitated with isopropanol. RNA pellets were resuspended in 50 mM Tris/EDTA and stored at -80°C.

Northern blots were prepared by subjecting 30 µg wcRNA per lane to 1% formaldehyde agarose gel electrophoresis. After the ethidium bromide-stained gels were viewed to confirm the integrity of 28 and 18S ribosomal RNA, the wcRNA was subsequently blotted to Zeta Probe+ membrane (Bio-Rad, Richmond, CA) overnight, and the membranes were baked for 1 h at 80°C. Slot blots were prepared using 2.5 and 5 µg wcRNA that had been treated with ribonuclease (RNase)-free deoxyribonuclease (DNase; Boehringer Mannheim, Indianapolis, IN). RNA was applied to Zeta Probe+ membranes using a minifold slot blot apparatus (Schleicher & Schuell, Keene, NH). After application of wcRNA, membranes were baked for 1 h at 80°C. Whole-cell RNA from a CL tissue pool was run on all blots for assessment of blot-to-blot variation.

The same hybridization conditions were used for both Northern and slot blots. Prehybridization (65°C for at least 10 min) was carried out in 0.25 M Na2HPO4 (pH = 7.2) and 7% SDS. Blots were then hybridized with 32P-labeled porcine cDNA probes for IGF-I [34] (obtained from Dr. F. Simmen, University of Florida, Gainesville, FL), IGFBP-3 [35] (from Drs. Shimasaki and Ling, Whittier Institute, La Jolla, CA), or IGF-IR [29]. Hybridizations were conducted for either 40 h (IGF-I) or overnight (all other probes) in fresh prehybridization buffer. After incubation, blots were washed twice at room temperature for 30 min in 0.1-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) and 0.1% SDS, washed twice at 65°C in 20 mM Na2HPO4 (pH = 7.2) with 1% SDS for 15 min, placed in a Phosphorimager cassette (Molecular Dynamics, Sunnyvale, CA) for 20 h, and then subjected to Phosphorimager analysis. After analysis, all blots were stripped by washing twice in 0.1-strength SSC/0.5% SDS at 95°C for 20 min and re-probed with [32P]-3-phosphoglyceraldehyde dehydrogenase (pGAD; used as a housekeeping gene) [21]. After incubating and washing, blots were placed in a Phosphorimager cassette and analyzed for pGAD hybridization signals. The steady-state levels of pGAD mRNA did not change significantly in response to day or treatment, and thus all RNA data were normalized for pGAD levels and were expressed as the following ratios: IGF-I:pGAD, IGFBP-3:pGAD, or IGF-IR:pGAD.

Protein Analysis

IGF-I RIA IGF-I protein was extracted from luteal tissue as reported previously [36–38]. Briefly, frozen tissue was pulverized under liquid nitrogen, weighed, homogenized in 1 M acetic acid (5 ml per gram tissue), incubated on ice for 2 h, and centrifuged at 1000 x g for 10 min. Samples were then neutralized with 5 M NaOH, re-acidified with 1% trifluoroacetic acid, and passed through Sep-Pak Plus C18 cartridges (Waters Corp., Milford, MA) to separate IGF-I from its binding proteins [19, 39]. Samples were dried under nitrogen at 37°C and resuspended in IGF-I assay buffer (30 mM sodium phosphate, 10 mM EDTA, 0.2 g/L protamine sulfate, 0.05% Tween 20). Samples were briefly centrifuged, and the supernatant was stored at -20°C until the time of the assay. IGF-I in luteal extracts was assayed using a double-antibody RIA previously validated for porcine serum [40, 41]. The RIA was performed using 50 µl of unknown or pool sample in each tube, UB2–495 (National Hormone and Pituitary Program) as the primary antibody, and a goat anti-rabbit serum (Linco Research Inc., St. Charles, MO) as the second precipitating antibody. IGF-I standards (recombinant human) were obtained from Monsanto (St. Louis, MO) and ranged from 0.016 to 1 ng/tube. IGF-I peptide was iodinated using a chloramine T iodination procedure, and 20 000 cpm of 125I-IGF-I was used in each tube. Further validation of this assay for luteal extracts was performed. Extraction efficiency for IGF-I was calculated as 72.9 ± 2.1%. Dose responsivity of the assay, and parallelism between the standard curve and increasing doses of CL extract and IGF-I standard were also confirmed. All samples were analyzed in a single assay, and intraassay CV was 4.44%. Total IGF content (nanograms per CL) for each CL (1 per animal) was calculated as the total IGF extracted from an individual CL. IGF concentration (ng/g CL tissue) was calculated as the total IGF extracted from an individual CL divided by the weight of that CL.

IGFBP ligand blots IGFBPs were extracted from CL tissue by pulverization under liquid nitrogen followed by homogenization of tissue in cold buffer containing 1% Triton-X 100, 2 mM EGTA, 2 mM EDTA, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 4 µg/ml PMSF, in 20 mM Hepes. After homogenization, samples were centrifuged at 14 000 x g at 4°C for 10 min, and then at 100 000 x g at 4°C for 1 h. Supernatants were aliquoted and stored at -20°C until determination of protein concentration using the BCA (bicinchoninic acid—detection reagent) protein assay (Pierce, Rockford, IL) and ligand blot analysis. For ligand blots, protein samples were separated on 10% nondenaturing SDS-PAGE gels using 350 µg of protein from individual CL extracts per lane. After electrophoresis, the gels were transferred to nitrocellulose using a wet transfer system (Bio-Rad). Membranes were blocked sequentially in buffer (10 mM Tris, 150 mM NaCl, 0.5 mg/ml sodium azide) supplemented with 1) 3% Nonidet P-40, 2) 1% BSA, and 3) 0.1% Tween 20. Blots were then incubated with 100 000 cpm/ml 125I-IGF-I in buffer with 1% BSA and 0.1% Tween 20 overnight at 4°C. Blots were washed 3 times in 0.1% Tween 20, 10 mM Tris, 150 mM NaCl, and 0.5 mg/ml sodium azide, and 3 times in this buffer without 0.1% Tween 20. Blots were allowed to air-dry, placed in a Phosphorimager cassette for 48 h, and subjected to Phosphorimager analysis.

IGF-IR Western blots IGF-IR was extracted from CL tissue by pulverization and subsequent homogenization under the same conditions as for the IGFBPs (above). After homogenization, samples were centrifuged at 14 000 x g at 4°C for 10 min; the supernatant was then transferred to new tubes and centrifuged at 100 000 x g at 4°C for 1 h. Supernatants were aliquoted and stored at -20°C until determination of protein concentration using the BCA protein assay (Pierce) and Western blot analysis. The Western blot protocol was modified from Oemar et al. [42]; briefly, proteins were separated on 3–10% gradient SDS-PAGE gels under reducing conditions, using 300 µg of protein from individual CL extracts per lane. After electrophoresis, the gels were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using a wet transfer system (Bio-Rad). After nonspecific binding sites were blocked with 5% nonfat dry milk in 5 mM Tris-HCl pH 7.4, 136 mM NaCl, and 0.1% Tween 20 (TBST buffer), blots were incubated overnight at 4°C with a 1:1000 dilution of IGF-IRß polyclonal antibody (sc-713; Santa Cruz Biotechnology Inc., Santa Cruz, CA). The blots were then washed with water twice for 5 min each. The blots were incubated for 1 h at room temperature with a 1:10 000 dilution of horseradish peroxidase-labeled anti-rabbit IgG (Amersham, Buckinghamshire, England) and washed 2 x 5 min in water, then 2 x 5 min in TBST buffer, rinsed 3x in water, and then soaked in water for 30 min. The IGF-IR antibody binding was detected using enhanced chemiluminescence (ECL) reagents (Amersham). Blots were exposed to Kodak X-OMAT-AR film (Eastman Kodak, Rochester, NY), and binding was quantified using densitometric analysis.

Statistical Analysis

All data were analyzed by ANOVA using the General Linear Model procedures of Statistical Analysis Systems [43]. The final model included the main effect of day and treatment and their interactions. When a significant effect was detected, differences between means were analyzed by least significant difference, using {alpha} (or P) < 0.05 to indicate significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Luteal Weights

The weights of CL (Table 1) of control animals increased significantly (P < 0.05) from Days 6 to 7 and then remained unchanged through Day 9. On Day 6, mean CL weight was 0.21 ± 0.02 g, and on Days 7 through 9 CL weights ranged from 0.34–0.36 g. In PGF2{alpha}-treated animals, luteal weights were maximal on Day 6 and declined steadily throughout the treatment period to reach lowest values on Day 9 (P < 0.05 compared to Day 6). On Day 6, CL weights were higher (P < 0.05) in PGF2{alpha}-treated animals than in controls and on Days 7–9 were lower (P < 0.05) in PGF2{alpha}-treated vs. control animals. Gross observation of CL from PGF2{alpha}-treated animals on Day 9 indicated that they were much paler and less vascular than those from all other treatment groups.


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  		TABLE 1. CL weight and progesterone concentrations for control and PGF2{alpha}-treated animals

Luteal Progesterone Concentration

Progesterone concentrations in luteal extracts (Table 1) remained high in controls, ranging from 72.33 to 84.55 µg/g tissue and did not change significantly (NS) throughout the treatment period. In PGF2{alpha}-treated animals, luteal progesterone concentrations were not statistically different from those of controls on Days 6–8 and ranged from 62.61 to 65.31 µg/g of CL tissue. However, luteal progesterone concentrations in PGF2{alpha}-treated animals declined significantly (P < 0.05 vs. those of control animals and of Day 6–8 treated animals) on Day 9, falling to 15.15 µg/g of tissue.

Northern Blots

Northern blot analysis (data not shown) of wcRNA hybridized with [32P]IGF-I cDNA revealed two major transcripts at 6.7 and 0.9 kilobases (kb) for IGF-I as had been previously reported [21]. The predominant transcript for IGF-I was 6.7 kb, while the 0.9-kb transcript was of lesser intensity. Northern blot analysis of pGAD (data not shown) demonstrated a single transcript of 1.6 kb. Analysis of IGFBP-3 mRNA revealed a single transcript of 2.8 kb as previously reported [21, 27] (data not shown). Northern blot analysis for IGF-IR (data not shown) revealed a predominant transcript of 11 kb [29].

Slot Blots

On the basis of slot blot analysis, no significant changes in the steady-state level of IGF-I mRNA occurred in response to PGF2{alpha} treatment on any day studied (Fig. 1A). The ratio of IGF-I to pGAD in controls increased significantly from Days 6 to 8 (P < 0.05) and remained higher on Day 9 (range of 0.9 to 1.2). In PGF2{alpha}-treated animals, the IGF-I:pGAD ratio did not change significantly during the study period and remained between 1.06 and 1.15.



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  		FIG. 1. Steady-state levels of A) IGF-I, B) IGFBP-3, and C) IGF-IR mRNA in CL from control and PGF2{alpha}-treated gilts. Whole-cell RNA preparations were subjected to slot blot analysis. Data from each treatment/day were averaged and expressed as mean ± SEM (Day 6 control n = 4; n = 5 per day per treatment for all other groups). Different superscript letters represent differences (P < 0.05) between days within treatment. No significant differences were seen by day for control (IGF-IR mRNA) or PGF2{alpha}-treated (IGF-I, IGFBP-3) animals. *Significant (P < 0.05) difference between treatments, within day; i.e., IGF-IR mRNA on Day 9

Similarly, little change was observed in steady-state levels of IGFBP-3 mRNA (Fig. 1B) throughout the treatment period, and no significant change was observed with PGF2{alpha} treatment. The IGFBP-3:pGAD ratio in controls increased slightly during the period of the study and was significantly elevated on Day 9 compared with Day 6 (P < 0.05). No significant trends were seen in IGFBP-3 mRNA expression in PGF2{alpha}-treated animals.

Steady-state levels of IGF-IR mRNA (Fig. 1C) did not change significantly in controls during the treatment period and ranged from 15.1 on Day 6 to 11.2 on Day 9. In contrast, in PGF2{alpha}-treated animals, steady-state levels of IGF-IR mRNA increased significantly (P < 0.05) on Day 9 (vs. Day 7). Additionally, IGF-IR levels in PGF2{alpha}-treated animals were numerically higher than in controls on all days of the study and were significantly elevated on Day 9, when there was a 2-fold increase in IGF-IR (23.9 ± 1.2 vs. 11.2 ± 1.0).

Luteal IGF-I Concentration

Luteal concentrations of IGF-I in controls ranged between 17.4 and 23.4 ng/g tissue and did not significantly change (Fig. 2A) during the period studied. In PGF2{alpha}-treated animals, luteal IGF-I concentrations did not differ on Days 6 and 7 but were increased significantly on Days 8 and 9 (P < 0.05). When compared to CL from controls, luteal IGF-I concentrations in PGF2{alpha}-treated animals were numerically higher on Day 8 (not significant) but were significantly elevated (P < 0.05) on Day 9 (approximately 2-fold). However, when data were corrected for changes in luteal weight, no significant day or treatment effects were seen (Fig. 2B, total IGF-I content, ng/CL).



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  		FIG. 2. A) IGF-I concentration (ng/g of tissue) and B) content (ng/CL) in CL from control and PGF2{alpha}-treated gilts. Data from each treatment/day were averaged and expressed as mean ± SEM (Day 6 control n = 4; n = 5 per day per treatment for all other groups). Different superscript letters represent differences (P < 0.05) between days within treatment. No significant differences were seen by day for control (IGF-I concentration or content) or PGF2{alpha}-treated (IGF-I content) animals. *Significant (P < 0.05) difference between treatments, within day; i.e., IGF-I concentration on Day 9 (A)

IGFBP Ligand Blots

A representative ligand blot is shown in Figure 3, demonstrating 5 distinct protein bands with molecular masses of 44, 40, 34, ~30, and 24 kDa. The 44- and 40-kDa binding protein bands were confirmed to be the IGFBP-3 doublet by Western blot using a specific IGFBP-3 antibody in our laboratory (data not shown). The 34-kDa protein corresponds to the reported size for IGFBP-2, the ~30-kDa protein band to that of IGFBP-5 and glycosylated IGFBP-4, and the 24-kDa protein to that reported for nonglycosylated IGFBP-4 in the pig [44]. Accordingly, with the exception of IGFBP-3, we designated the IGF binding proteins detected on ligand blots as IGFBP-2, -4, and -5 according to their apparent similarity in size to those reported previously [44].



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  		FIG. 3. Representative ligand blot analysis of CL protein. Lanes indicate individual CL samples (e.g., 9c, Day 9 control; 6t, Day 6 PGF2{alpha}-treated)

In controls, luteal concentration of IGFBP-2 increased from Day 6 to Day 7 (P < 0.05) and remained elevated for the duration of the treatment period (Fig. 4A). There was a significant increase in IGFBP-2 in CL from PGF2{alpha}-treated animals from Day 6 to Day 7, followed by a decrease on Days 8 and 9 (P < 0.05 vs. Days 6–8). IGFBP-2 was significantly higher (P < 0.05) in the CL of treated vs. control animals on Days 6 and 7. Although there was no difference observed between PGF2{alpha}-treated vs. control animals on Day 8, luteal IGFBP-2 levels were significantly decreased (P < 0.05) to approximately half that of controls on Day 9.



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  		FIG. 4. A) IGFBP-2 and B) IGFBP-3 protein in CL from control and PGF2{alpha}-treated gilts. Data are presented as mean pixel intensity ± SEM per gram of CL tissue for each treatment group (Day 6 control n = 4; n = 5 per day per treatment for all other groups). Different superscript letters represent differences (P < 0.05) between days within treatment. *Significant (P < 0.05) difference between treatments, within day; i.e., IGFBP-2 on Days 6, 7, and 9, and IGFBP-3 on Days 6, 8, and 9

IGFBP-3 concentrations in CL from control animals increased steadily from Day 6 to Day 8 (P < 0.05 vs. Day 6), but by Day 9, they had returned to approximately the levels seen on Day 6 (Fig. 4B). In PGF2{alpha}-treated animals, luteal IGFBP-3 concentrations declined steadily and significantly throughout the study. Luteal levels of IGFBP-3 were significantly (P < 0.05) increased in PGF2{alpha}-treated animals compared with controls (P < 0.05) on Day 6 but were significantly reduced compared with controls (P < 0.05) on Days 8 and 9, reaching lowest values on Day 9.

Evaluation of the ~30-kDa (putative IGFBP-5/glycosylated IGFBP-4) and the 24-kDa (putative nonglycosylated IGFBP-4) protein bands showed few if any changes in the CL from control animals throughout the study period (data not shown). Similarly, the levels of these binding proteins did not differ in CL from PGF2{alpha}-treated animals except on Day 9, when there was a marked (and significant; P < 0.05) decrease compared with control levels (data not shown).

IGF-IR Western Blots

A representative Western blot is shown in Figure 5A, demonstrating the single ~95-kDa band of IGF-IR. IGF-IR concentrations in controls (Fig. 5B) remained steady on Days 6–8 but decreased significantly on Day 9 (P < 0.05 vs. Days 6–8). In PGF2{alpha}-treated animals, IGF-IR levels increased from Day 6 to Day 7 (P < 0.05 vs. Day 6) and fell steadily throughout the rest of the treatment period. IGF-IR levels in PGF2{alpha}-treated animals were not different from control levels on Days 6 or 8, but were higher on Day 7 (P < 0.05) and lower on Day 9 (P < 0.05) compared with those of controls.



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  		FIG. 5. A) Representative Western blot of IGF-IR in CL protein extracts. Lanes indicate individual CL samples (e.g., 9c, Day 9 control; 6t, Day 6 PGF2{alpha}-treated). B) IGF-IR concentration in CL from control and PGF2{alpha}-treated gilts. Data from each treatment/day were averaged and expressed as mean ± SEM (Day 6 control n = 4; n = 5 per day per treatment for all other groups). Different superscript letters represent differences (P < 0.05) between days within treatment. *Significant (P < 0.05) difference between treatments, within day; i.e., Days 7 and 9


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was designed to investigate possible interactions between the luteolytic effects of PGF2{alpha} and the intraluteal IGF system. We hypothesized that PGF2{alpha} would inhibit IGF-I and IGF-IR but stimulate the production of IGFBP-3 in the CL, any of which actions could provide a pathway by which luteolysis might occur. The experimental model used in this study, involving twice-daily injections of PGF2{alpha} on Days 5–9, has been shown to induce premature luteolysis in the pig in previous studies from our laboratory [5, 6]. In the present study, we confirmed these findings and showed that luteal progesterone concentration and CL weights were decreased in Day 9 PGF2{alpha}-treated animals, indicating functional and structural premature luteolysis.

By Northern blot analysis, mRNA transcripts for IGF-I, IGFBP-3, and IGF-IR were readily demonstrated. The sizes of these transcripts—6.7 and 0.9 kb for IGF-I, 2.8 kb for IGFBP-3, 11 kb for IGF-IR, and 1.6 kb for pGAD—matched those previously reported from our laboratory and by others [21, 27, 29].

Using slot blot analysis to quantify the steady-state levels of these mRNAs, we detected no changes in IGF-I in response to PGF2{alpha} administration. The lack of change in IGF-I mRNA is in agreement with data in the sheep, in which administration of PGF2{alpha} had no effect on mRNAs for IGF-I or growth hormone receptor [45]. At the protein level, luteal IGF-I concentrations (ng/g tissue) were significantly higher on Day 9 and showed a tendency to increase on Day 8 in PGF2{alpha}-treated animals, although when expressed as total luteal IGF-I content (ng/CL) this did not change in PGF2{alpha}-treated animals, a finding consistent with the unchanging mRNA levels. An increase in IGF-I (protein) concentration has been observed in the corpora albicantia of sheep on Day 3 of the subsequent luteal phase, although the authors did not report their data as corrected for CL weights [19]. On the basis of these data, it appears that PGF2{alpha} administration does not affect overall intraluteal expression of IGF-I (mRNA or protein), and thus, IGF-I availability to luteal cells does not appear to be compromised by treatment with PGF2{alpha} in the pig, contrary to our original hypothesis.

It should be noted that our determinations of luteal IGF-I concentrations by RIA (or even IGFBPs 2 and 3 by ligand blot analysis—see below) cannot completely rule out a contribution from the blood. However, in some preliminary studies in which we attempted to correct CL tissue values for blood contamination, by monitoring hemoglobin levels [37], we determined that blood contamination was less than 5% (v:v). Consequently, we believe that the levels of IGF-I (and IGFBPs 2 and 3) measured in this study predominantly reflect local synthesis.

Our observations that IGFBP-3 mRNA steady-state levels in CL of PGF2{alpha}-treated animals were unchanged is in agreement with Grimes and colleagues [24], who also reported no change in IGFBP-3 mRNA in luteinized porcine granulosa cells treated with PGF2{alpha}. However, these authors observed a 2.6-fold increase in IGFBP-3 protein by ligand blots [24], which is consistent with our data on Day 6 showing that IGFBP-3 protein levels were increased in PGF2{alpha}-treated animals. IGFBP-3 has been shown to be inhibitory to granulosa cell function in the pig [46] and in other species [25], and it has also been shown to inhibit IGF-I-stimulated production of progesterone by bovine thecal cells [47]. Additionally, Erickson and colleagues [26] showed that IGFBP-3 mRNA expression was limited to involuting CL in the rat and suggested a role for IGFBP-3 in normal luteolysis. Furthermore we observed that luteal IGFBP-2 protein concentrations increased on Days 6 and 7 in our study, in response to PGF2{alpha} treatment. IGFBP-2 has been linked to atresia in porcine follicles [48, 49], presumably via its capacity to bind available IGF-I, thus inhibiting IGF-I's actions. Thus, overall, when considered with our data, these observations may suggest a role for both IGFBP-2 and -3 in PGF2{alpha}-induced luteolysis in the pig.

Little is known about IGFBPs 4 and 5 in the ovary of the pig, compared with other species such as the rat, in which increased mRNA expression for these binding proteins is associated with atresia in graafian follicles [50, 51]. In the pig, IGFBP-4 mRNA has been linked to the emergence of LH receptors in granulosa cells of preovulatory follicles, is expressed in the theca of medium to large growing follicles [28], and is quite abundant in the CL [21], but it is not detected in atretic follicles [28]. IGFBP-5 is also expressed in the porcine CL [21], but it appears to be expressed in capillary endothelial cells [28]. In cultured porcine granulosa cells, IGFBP-5 production was reduced by FSH, while IGFBP-5 mRNA and protein was stimulated by IGF-I [44, 52]. In our study, we saw no significant changes in the levels of IGFBPs 4 or 5 before Day 9, although we were not able to identify these IGFBPs definitively on the basis of their location on ligand blots.

The decreased levels of all binding proteins observed on Day 9 in CL taken from PGF2{alpha}-treated gilts undergoing luteolysis could result from decreased production of these proteins or increased degradation by specific proteases [53] as the luteal tissue regressed. However, as this decrease in binding protein levels did not occur before Day 9, it is likely that this decrease is a result, rather than a cause, of the luteolytic effects of PGF2{alpha}.

The biological significance of the increase in steady-state levels of IGF-IR mRNA in response to PGF2{alpha} treatment is unclear, especially in light of the marked decrease seen in the levels of IGF-IR (protein). An increase in IGF-IR mRNA has been observed in the CL of the rat on Day 21 of pregnancy, at which time the CL undergoes luteolysis [16]. In addition, IGF-IR mRNA was shown to be elevated in the corpora albicantia of sheep, being highest in expression on Day 3 of the following cycle [19]. From preliminary studies in our own laboratory, we have shown that expression of mRNA for IGF-IR increased from early to late luteal phase, reaching a maximum on Days 13–16 of the estrous cycle [29]. Thus the increase in IGF-IR mRNA seen on Day 9 observed in PGF2{alpha}-treated animals in the present study may reflect the changes seen in expression during normal luteolysis. Furthermore, it is difficult to explain the increase in IGF-IR protein levels in PGF2{alpha}-treated animals on Day 7. It is possible that there is an up-regulation in IGF-IR in response to a decrease in available IGF-I, brought about by the increase in binding protein concentrations. In several past studies in other cell types, a decrease in available IGF-I up-regulated IGF-IR mRNA and protein expression [54–56]. The decrease in luteal IGF-IR protein seen in PGF2{alpha}-treated vs. control animals on Day 9 may have been due to the degradation of the tissue as luteolysis progressed, and therefore is likely to have been a result of luteolysis rather than a possible cause. Further studies are needed to provide a greater understanding of the regulation of IGF-IR and its role in promoting luteal function throughout the estrous cycle, in order for us to understand more clearly the significance of these data.

In summary, we have shown that repeated administration of PGF2{alpha} induced premature luteolysis in the early luteal phase, indicated by decreased luteal weights and progesterone concentrations in the CL of treated animals. PGF2{alpha}-induced luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. Protein levels of IGFBP-2, IGFBP-3, and IGF-IR increased early in the treatment period (Days 6–7). Elevation in the levels of binding proteins may mediate the luteolytic actions of PGF2{alpha} by sequestering IGF-I and preventing its interaction with the IGF-IR.


   ACKNOWLEDGMENTS
 
We would like to thank Dr. Frank Simmen, Drs. Shunichi Shimasaki and Nicholas Ling, and James Hammond for their generous donations of cDNAs for IGF-I, IGFBP-3, and pGAD, respectively. We also gratefully acknowledge Dr. Karen Swanchara and Dr. Robert Harrell for their assistance with Northern and ligand blots, respectively, and Bennett Crowell and Dr. William Flowers for their assistance with the statistical analysis.


   FOOTNOTES
 
1 This work was supported by funds from the North Carolina Pork Producer's Association and the State of North Carolina. Back

2 Correspondence: John Gadsby, College of Veterinary Medicine, 4700 Hillsborough St., Raleigh, NC 27606. FAX: 919 515 4237; john_gadsby{at}ncsu.edu Back

Accepted: August 2, 1999.

Received: August 3, 1998.


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 MATERIALS AND METHODS
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