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Biology of Reproduction 63, 21-29 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Increased Expression of Insulin-Like Growth Factor Binding Protein-1 During Induced Regression of Bovine Corpora Lutea1

B.L. Sayrea, R. Tafta, E.K. Inskeep2,a, and J. Killefera

a Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, West Virginia 26506-6108

ABSTRACT

Three experiments were conducted to examine gene expression during induced luteal regression in the cow; the initial purpose was the identification of potential embryotoxins. In experiment 1, changes in gene expression in the corpus luteum (CL) were identified by differential display reverse transcription–polymerase chain reaction (DD-PCR) during the first 72 h of luteal regression in cows treated with prostaglandin F2{alpha} (PGF2{alpha}) on Days 4–7 after estrus. Expression of insulin-like growth factor–binding protein-1 (IGFBP-1) was up-regulated, with greatest expression at 24 h (P < 0.05) after treatment with PGF2{alpha} began. In experiment. 2, IGFBP-1 and its mRNA were quantified in CL collected 24 or 48 h after treatment with PGF2{alpha} on Day 4 or 10 after estrus. Because local mechanisms for exchange of hormones between the ovary and uterus are known in ruminants, uterine flushings were assayed for IGFBP-1 to seek evidence of local transfer of luteal IGFBP-1 to the uterus. IGFBP-1 mRNA was increased (P < 0.05) in CL 24 h after treatment when PGF2{alpha} that began on Day 10, and by 48 h after treatment that began on Day 4. Concentrations of IGFBP-1 increased (P < 0.05) in a pattern similar to mRNA, by 24 h on Day 10, and by 48 h on Day 4. Concentrations of IGFBP-1 in uterine flushings did not change on either day. Concentrations of progesterone decreased (P < 0.05) by 8 h after treatment with PGF2{alpha} that began on Day 10, but not until 24 h after treatment that began on Day 4. In experiment 3, cows received either saline or PGF2{alpha} and CL were collected 2 or 10 h after a single treatment, or 2 h after a second treatment that was given 8 h after the first. Expression of IGFBP-1 was increased by 2 h after treatment with PGF2{alpha} on both Days 4 and 10 after estrus. In conclusion, secretion of IGFBP-1 is increased during luteolysis, and may inhibit the steroidogenic effects of insulin-like growth factor-I (IGF-I), but no evidence was found to implicate IGFBP-1 in the embryotoxic effect of regressing CL.

corpus luteum, corpus luteum function

INTRODUCTION

In a series of studies conducted during the last decade at the West Virginia Agricultural and Forestry Experiment Station [1], early embryonic death occurred in postpartum cows with short luteal phases despite replacement therapy with progestogen. Loss of embryos was shown to be due to a combination of premature secretion of prostaglandin F2{alpha} (PGF2{alpha}) and a product of the regressing corpus luteum (CL) [2, 3]. Buford et al. [3] developed a model for the embryotoxic effect by treating mated, nonlactating cows with PGF2{alpha} every 8 h on Days 4 through 7 or 5 through 8 after estrus, coincident with replacement therapy with progestogen [2]. An embryotoxic effect was seen when luteal regression was induced between Days 4 and 8 of pregnancy, but not after Day 10 of pregnancy [3, 4].

In the studies reported here, changes in gene expression (mRNA) were investigated in CL during induced regression; the initial purpose was to identify potential luteal embryotoxins. As expected, some messages were down-regulated, including steroidogenic acute regulatory protein (StAR), as previously shown in the ewe treated with PGF2{alpha} [5]. Several messages were up-regulated, including prostaglandin H synthase (PGHS-2, also known as Cox-2) and PGF2{alpha} synthase (PGFS) [6]. Of particular interest, insulin-like growth factor-binding protein-1 (IGFBP-1) was up-regulated. The association of IGFBP-1 with luteolysis in ruminants is apparently a novel finding and is the focal point of this report.

First isolated from human endometrium during pregnancy, IGFBP-1 was called either placental protein-12 or {alpha}1-progesterone-dependent endometrial globulin ({alpha}1-PEG) [7]. IGFBP-1 is present in bovine endometrium by Day 13 of pregnancy [8] and could be involved in regulating the effects of IGF-I and IGF-II [7] on embryonic development [9–11]. It was proposed that IGFBP-1 secreted during induced luteolysis might be transported to the uterus through the oviductal vein, as has been shown for progesterone [12–14], or through local vascular diffusion pathways (recently reviewed by Bonnin et al. [15]), which transfer prostaglandin from the uterus to the ovary. Fogwell et al. [16] demonstrated that PGF2{alpha} injected intrafollicularly left the ovary and returned via the ovarian artery to cause luteolysis. Therefore, based upon the anatomical relationships of utero-ovarian vessels elucidated by Ginther [17], it follows that transfer from the ovarian vein to the uterine artery could occur. The early presence of IGFBP-1 could cause untimely changes in availability or action of IGFs, and thus contribute to the luteal embryotoxic effect previously identified during early pregnancy in cows [1, 3].

Increased concentrations of IGFBP-1 may inhibit the effects of IGF-I during luteolysis. IGFBP-1 is stimulated by phorbol esters [18, 19]; thus, increased protein kinase C activity in response to PGF2{alpha} [20] may increase expression of IGFBP-1 in luteal tissue. IGF-I and its mRNA increased in CL during the midluteal phase of the estrous cycle [21, 22], and treatment of bovine luteal cells with IGF-I stimulated production of progesterone [23, 24]. Stimulation of IGFBP-1 production by PGF2{alpha} may antagonize the effects of IGF-1, hastening the demise of the CL. The experiments had three objectives: 1) to identify differences in gene expression in nonregressing and regressing CL during the first 72 h after treatment with saline or PGF2{alpha} on Days 4–7 after estrus, 2) to determine if changes in expression of IGFBP-1 differed when luteal regression was induced on Day 4 or 10 after estrus, and 3) to determine if concentrations of IGFBP-1 were increased in uterine luminal fluid during luteal regression.

MATERIALS AND METHODS

Experiment 1

Treatments and tissue collection Estrus was synchronized in 15 nonlactating beef cows with a single injection of PGF2{alpha} (25 mg; Lutalyse; Pharmacia and Upjohn, Inc., Kalamazoo, MI) followed by estradiol benzoate (400 µg) 40 h later [25]. On Day 4 postestrus, all cows received either PGF2{alpha} (15 mg, n = 11) or saline (3 ml, n = 4) i.m. every 8 h until ovariectomy. Corpora lutea were collected by blunt dissection after ovariectomy via supravaginal incision under epidural anesthesia (6–9 ml of 2% lidocaine for cows 450–700 kg) [3, 26]. Ovaries were removed 24 h (n = 2) and 48 h (n = 2) after the initiation of treatment with saline (data were pooled to form a single control group because no effect of time was expected in saline-treated controls) and 24 h (n = 4), 48 h (n = 4), or 72 h (n = 3) after the initiation of treatment with PGF2{alpha}. After removal, CL were washed in diethylpyrocarbonate (DEPC)-treated H2O and bisected. Each half was homogenized in 10 ml of denaturing buffer (4 M guanidinium thiocyanate, 25 mM Na-citrate, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol) and stored at -80°C.

Differential display RT-PCR Differences in patterns of gene expression were determined using a modified differential display reverse transcription-polymerase chain reaction (DD-PCR) protocol described by Liang and Pardee [27]. Briefly, total RNA was extracted from each CL by the method described by Chomoczynski and Sacchi [28]. The RNA pellet was resuspended in 100 µl of DEPC-treated H2O and cDNA was synthesized from total RNA (300 ng) using a dT(11)VV anchor primer, where V equals A, C, or G. A total reaction volume of 30 µl contained 1x RT buffer (50 mM Tris-HCl, 40 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol, pH 8.3), 30 units of Maloney murine leukemia virus (MMLV; Promega, Madison, WI) reverse transcriptase, 20 µM dNTPs, and 2.5 µM of dT(11)VV anchor primer. The reaction was incubated at 70°C for 10 min, 40°C for 1 h, and 95°C for 5 min. The cDNA was stored at -80°C.

The resulting cDNAs (1 µl) were amplified by PCR with a decamer primer (a 10-nucleotide oligomer of random sequence, with 50% GC content) and a dT(11)VV anchor primer. A total reaction of 25 µl contained 1x PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), 3 mM MgCl2, 200 µM dNTPs, and 1 unit of Taq DNA polymerase (Display Systems Biotech, Vista, CA). Amplification was performed using incubation at 94°C for 3 min for initial denaturation, then 40 cycles were carried out as follows: 94°C for 30 sec for denaturing, 40°C for 1 min for annealing, and 72°C for 1 min for extension, followed by a final 5 min at 72°C for extension (MJ Research, Incline Village, NV). Amplified products were resolved on a 4% nondenaturing polyacrylamide gel and visualized with SYBR Green I fluorescent DNA stain (FMC BioProducts, Rockland, ME) and with silver staining procedures [29]. Independent DD-PCR were conducted on each CL RNA sample to compare variation among samples. In addition, PCR reactions containing either RNA, cDNA without Taq DNA polymerase, or water to replace template cDNA were conducted to control for contamination.

Sequencing Products of DD-PCR that increased or decreased during regression compared with control CL were reamplified by removing a portion of the gel containing the product, and incubating it in 50 µl of Tris-EDTA (10 mM Tris-HCl [pH 7.5], 1 mM EDTA) buffer for 1 h at 25°C to elute the product. A 1-µl aliquot of eluted product was reamplified with the same PCR protocol used for the initial amplification. Reamplified products were visualized on a 6% nondenaturing polyacrylamide gel by SYBR Green I staining procedures.

Products amplified by PCR and eluted from the gel were cloned into a pCR Script(+) plasmid and transfected into pBluescript-competent cells using the pCR Script(+) Cloning kit (Stratagene, La Jolla, CA). Plasmid DNA was extracted using QIAprep miniprep purification columns (QIAGEN, Valencia, CA).

Cloned DD-PCR fragments were sequenced using the standard protocols of Sanger et al. [30] as modified for Silver Sequence DNA sequencing (Promega). The mass of each of the cloned PCR products was estimated by comparison with a DNA mass ladder after agarose gel electrophoresis was performed. For each product, 1 µg of plasmid DNA was sequenced using the M13 forward primer according to the instructions of the manufacturer. Sequencing reactions were subjected to electrophoresis on a 6% denaturing polyacrylamide gel. After electrophoresis, the gel was stained using a standard silver staining technique described by Bassam et al. [29]. Sequences were read from the gels and compared with genetic databases for identification, and for assignment of potential functions. The databases (GenBank and EMBL) were searched using the Blast server at the National Center for Biotechnology Information Web site (http://www.ncbi.gov/).

Expression of IGFBP-1 Specific primers were developed for the amplification of one of the identified differentially expressed genes. Primers specific for bovine IGFBP-1 were 5'-CTGTTGGCATTTGGGGTC-3' and 5'-GGAGCCCTGCCAGCGAGAAC-3' and amplified a 280-base pair (bp) fragment as previously reported [31]. Amplification was performed using incubation at 94°C for 3 min for initial denaturation, then 40 cycles were carried out as follows: 94°C for 30 sec for denaturing, 55°C for 1 min for annealing, and 80°C for 1 min for extension, followed by a final 5 min at 80°C for extension. Using an oligo-dT primer, cDNA was made from total RNA and the cDNA was amplified by PCR. The amplified product was visualized on a 1.5 %-agarose gel stained with SYBR Green I DNA stain. A product was amplified from each CL and quantified by densitometric analysis of the stained gel. Primers specific for ß-actin (5'-TCATGAAGTGTGACGTTGACATCCGT-3' and 5'-CTAGAAGCATTTGCGGTGCAGGATG-3') were used to amplify a 285-bp product as an internal standard, to verify the level of amplification. Densitometry data were examined by least squares analysis of variance using the Statistical Analysis System (SAS) general linear model (GLM) procedure [32]. The effects of treatment for 24, 48, or 72 h were compared with the effects in the pooled control group (24 and 48 h) by a one-way analysis of variance followed by a test of least significant difference.

Experiment 2

Treatments and tissue collection Cyclic cows (n = 30) were treated on Day 4 or Day 10 with either saline or PGF2{alpha} every 8 h until the CL were removed at 24 or 48 h. Jugular blood samples were taken at 8 h intervals from 24 h before treatments began and until CL removal. Progesterone was measured in serum as described previously [33]. Inter- and intraassay coefficients of variation were 6.9 and 2.9, respectively. Data for progesterone were examined by least squares analysis of variance with the SAS GLM procedure [32] as described for repeated measures [34]. Treatment and day of initiation of treatment were in the main plot and time after treatment and interaction of treatment and time after treatment were in the subplot.

Uteri were flushed nonsurgically with 200 ml of sterile saline, and the flushings were lyophilized and stored at -20°C. Corpora lutea were collected by blunt dissection from the ovary via supravaginal incision under epidural anesthesia [3, 26]. After collection, the CL were washed immediately with sterile saline to remove any blood, and 500 mg of tissue was added to 5 ml of TriReagent (Sigma Chemical Co., St. Louis, MO) and homogenized with a glass Tenbroeck tissue grinder. Homogenized CL were stored at -80°C.

Expression of luteal products Total RNA and proteins were isolated from the tissue samples with TriReagent according to the manufacturer's instructions. Complementary DNA was synthesized from RNA (2 µg) with an oligo dT(15) primer. The PCR amplification was conducted using the ß-actin primer and other specific primers: IGFBP-1 and PGF2{alpha} receptor (PGFR): 5'- GTAAAAAGGGTTTCACAGG-3' and 5'- CAAAGACTGGGAAGATAGGTT-3', PGHS-2: 5' AGGTGTATGTATGAGTGTAGGA-3' and 5'-GTGCTGGGCAAAGAATGCAA-3', PGFS: 5'-TGGAACCTATGCACCTGAG-3' and 5'-ACGTGTGACAGAGATCCACC-3', StAR: 5'-CCAGCCAGCACACACATGGA-3' and 5'-CTCTACAGCGACCAAGAGCT-3', cholesterol side chain cleavage enzyme (P450scc): 5'-CCAGAAGTATGGCCCCGATT-3' and 5'-GGAGC CCTGCTGCTTGATGC-3', 3ß-hydroxysteroid dehydrogenase (3ß-HSD): 5'-TGTTGGTGGAGGAGAAGG-3' and 5'-GGCCGTCTTGGATGATCT-3'. Amplification was performed using incubation at 94°C for 3 min for initial denaturation, then 35 cycles were carried out as follows: 94°C for 30 sec for denaturing, 55°C for 1 min for annealing, and 80°C for 1 min for extension, followed by a final 5 min at 80°C for extension. After PCR, specific products were separated on a 1.5% agarose gel by electrophoresis and visualized with ethidium bromide stain. Products were quantified by densitometric analysis of the stained gel. Primers specific for ß-actin were used as an internal standard to verify the level of amplification. Densitometry data were examined by least squares analysis of variance with the SAS GLM procedure [32]. The effects of three treatments (control, treated on Day 4, and treated on Day 10), time after treatment (24 or 48 h), and the treatment x time interaction were included in the model. When interaction effects were detected, differences among individual treatments were determined by the test of least significant difference, as calculated by SAS [32].

Analysis of IGFBP-1 by Western ligand blots Relative amounts of IGFBPs were determined by Western ligand blotting assay as described by Hossenlopp et al. [35] with some minor modifications. Proteins from uterine flushings and homogenates of luteal tissue were quantified (Bio-Rad protein assay; Bio-Rad Laboratories, Hercules, CA). Equal quantities of protein from uterine flushings and CL were separated on 12.5% nonreducing SDS polyacrylamide gels with 3% stacking gels. Although data from individual animals were analyzed, samples were pooled to generate the representative autoradiographs presented. Proteins were transferred to nitrocellulose membranes (0.2 mm; Schleicher and Schuell, Inc., Keene, NH) overnight at 200 mA at 4°C in Towbin buffer (25 mM Tris, pH 8.3, 192 mM glycine, 0.03% SDS, and 20% methanol) using a BioRad Transblot unit (Bio-Rad). After transfer, the membranes were dried, incubated with 3% Nonidet P-40 and Tris-buffered saline (TBS; 10 mM Tris, pH 7.4, 150 mM NaCl, and 0.05% sodium azide) for 30 min, then incubated with 1% BSA in TBS with 0.1% Tween 20 (TBST) for 2 h. The membranes were washed with TBST, then incubated overnight with [125I]IGF-I (100 000 dpm/ml). The membranes were washed twice with TBST and twice with TBS, dried, and exposed to X-Omat AR film (Eastman Kodak, Rochester, NY) for 36 h at -80°C.

The location of IGFBP-1 on the gel was determined with human IGFBP-1 standard and prestained molecular weight markers. Human IGFBP-1 was used because its mass is similar to bovine IGFBP-1 [36, 37] and because an antibody specific for bovine IGFBP-1 could not be located. Other IGFBPs are of similar mass and, therefore, this approach alone is not definitive. Because changes in the 29- to 31-kDa protein paralleled changes in mRNA it is quite likely that the changes observed on the ligand blot represent IGFBP-1. Densitometric data were examined as described for the data on mRNAs.

Experiment 3

Cyclic cows (n = 35) were treated on Day 4 (n = 18) or Day 10 (n = 17) with either saline or PGF2{alpha} and the CL were removed at 2 h (n =11) or 10 h (n = 12) after a single treatment or 2 h after a second treatment (n = 12) given 8 h after the first treatment. Corpora lutea were collected and processed as described for experiment 2. Expression of IGFBP-1, StAR, cytochrome P450 side chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were determined by PCR. Data were examined as a 2 x 3 factorial by least squares analysis of variance by GLM-SAS [32] for effects of day, treatment group and treatment group by day. When interaction effects were detected, differences among individual treatments were determined by the test of least significant difference, as calculated by SAS [32].

RESULTS

Experiment 1

Differences in the patterns of some differential display products were detected. Amplicons present in three of four or two of three animals in the treated groups, but not present in time-matched controls were classified as differentially expressed. A representative differential display gel is shown in Figure 1. PCR products of each CL incubated with one anchor primer and one random primer are displayed on this gel. A total of 9 anchor primers and 6 random primers were tested. Results were similar among cows at the same stage of regression (Fig. 1). Amplification of reactions that contained RNA, cDNA without Taq DNA polymerase, or water did not result in visible banding patterns.



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  		FIG. 1. Representative gel displaying DD-PCR results from bovine CL collected at 24 (lanes 2 and 3) and 48 h (lanes 4 and 5) after saline or 24 (lanes 6–9), 48 (lanes 10–13), and 72 h (lanes 14–16) after PGF2{alpha} treatment. Each lane represents a CL collected from a different cow at each time period. Differences in PCR product patterns indicate a change in expression of a gene between nonregressing (lanes 2–5) and regressing (lanes 6–16) CL. White arrowheads indicate up-regulated genes and black arrowheads indicate down-regulated genes. Lanes 1 and 17 show the positions of 100-bp DNA markers

Six fragments that were expressed differentially in treated and control CL were isolated. Five of the isolated fragments were increased after treatment with PGF2{alpha} and one was decreased. The six fragments were sequenced and compared with GenBank and EMBL databases, allowing determination of probable identities of three. The fragment that decreased had a sequence similar to a tRNA ribosyltransferase, which likely would not have effects outside of the luteal cell. One of the fragments for which expression increased was identified as ribosomal protein L7. Ribosomal protein L7 functions primarily in the presentation of mRNA to the ribosomal complex; it is thought to have some control over which mRNAs are translated [38, 39]. Thus, ribosomal protein L7 is not likely to have direct effects outside of the luteal cell, but may affect luteal cell function and translation of luteal products. Another fragment for which expression was increased was identified as IGFBP-1. The IGF-binding proteins are secreted into the circulation, so it seemed possible that luteal IGFBP-1 may affect IGF activity in either the CL, the uterus, or both. Thus, this gene product may have functional significance in either luteal regression or interruption of pregnancy in animals on replacement therapy with progestogen.

The intensity of the PCR product representing IGFBP-1 on differential display increased by 48 h and remained greater than control intensity (9, 11, 67, and 105 arbitrary densitometry units for the fragment representing IGFBP-1 for control and treatment with PGF2{alpha} for 24, 48, and 72 h, respectively). Expression of IGFBP-1 after treatment with PGF2{alpha} (Fig. 2A) was measured by specific RT-PCR to compare to the results of differential display. In response to treatment, the amount of IGFBP-1 mRNA was increased at 24 h (P < 0.05), and 48 h (P < 0.01), but did not differ from control amounts at 72 h (Fig. 2A). The amount of ß-actin mRNA was not different among treatment groups or time periods. The contrast in timing of changes in IGFBP-1 mRNA by the two methods may be due to the use of nonspecific primers and (or) competition among the many PCR products generated during differential display.



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  		FIG. 2. Expression of IGFBP-1 was increased during PGF2{alpha}-induced luteal regression. A) Luteal expression of IGFBP-1 either after treatment with saline or after PGF2{alpha} for 24, 48, or 72 h every 8 h beginning on Day 4 of experiment 1. Graph represents the means of arbitrary densitometry units for each treatment. SE = 45. B) Expression of IGFBP-1 in bovine CL after treatment with PGF2{alpha} every 8 h for 24 or 48 h beginning on Day 4 (solid bars) or 10 (striped bars), experiment 2. Data expressed as means of arbitrary densitometric units of the PCR fragment amplified from IGFBP-1 cDNA. SE = 68. *Indicates the mean differed (P < 0.05) from control

Experiment 2

Luteal concentrations of IGFBP-1 mRNA were affected by treatment (P < 0.01) and the day x treatment interaction (P < 0.001; Fig. 2A). IGFBP-1 mRNA was increased at 48 h after treatment that began on Day 4 (P < 0.05; Fig. 2B) and at 24 h but not at 48 h when treatment began on Day 10 (P < 0.05; Fig. 2B). Concentration of ß-actin mRNA did not change with treatment or time.

Analyses of data from Western ligand blots indicated that concentrations of an IGFBP with an approximate mass of 29–31 kDa in bovine CL were increased (P < 0.05) by 48 h when cows began treatment on Day 4, whereas increased (P < 0.05) concentrations were observed by 24 h and remained increased at 48 h when treatment began on Day 10 of the estrous cycle (Fig. 3, A and B). Concentrations of this IGFBP in uterine flushings were not increased by treatment with PGF2{alpha} on either Day 4 or Day 10 (296, 201, and 240 arbitrary densitometric units for control, and treatment with PGF2{alpha} for 24 h and 48 h, respectively). This IGFBP was similar in mass to human IGFBP-1 (29–31 kDa) and probably is bovine IGFBP-1, because changes in the quantity of this IGFBP paralleled changes in mRNA for IGFBP-1. However, the presence of other IGFBPs cannot be excluded at this time.



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  		FIG. 3. Concentrations of IGFBP-1 in bovine CL after treatment with PGF2{alpha} every 8 h for 24 or 48 h beginning on Day 4 or 10 (experiment 2). A) Western ligand blot of pooled luteal protein samples for each treatment group and human IGFBP-1 standard. B) Means of arbitrary densitometric units of IGFBP-1 in CL collected on Day 4 (solid bars) or 10 (striped bars). SE = 195. *Indicates the mean differed (P < 0.05) from control

Expression of mRNA for PGFR was decreased (P < 0.05) by treatment with PGF2{alpha} by 24 h when treatment began on Day 10, but not until 48 h when treatment began on Day 4. Expression of PGHS-2 mRNA was increased at 24 h after treatment with PGF2{alpha} on Day 4 (P < 0.05; Fig. 4); no increase was seen after treatment on Day 10, but PGHS-2 mRNA had decreased to undetectable values by 48 h (Fig. 4). When treatment with PGF2{alpha} began on Day 10, mRNA for PGFS was increased (P < 0.05) by 24 h, while increased expression was not detected until 48 h in the group that began treatment on Day 4 (Fig. 4).



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  		FIG. 4. Expression of PGF receptor (PGFR), prostaglandin G/H synthase-2 (PGHS-2), and PGF synthase (PGFS) mRNA by bovine CL after treatment with PGF2{alpha} every 8 h for control (solid bars), 24 h (striped bars), or 48 h (hatched bars) beginning on Day 4 (A) or 10 (B) in experiment 2. Data expressed as means of arbitrary densitometric units of the PCR fragment amplified from cDNA. *Indicates the mean differed (P < 0.05) from control

Decreased (P < 0.05) concentrations of progesterone from cows that began treatment with PGF2{alpha} on Day 4 were not evident until 24 h (Fig. 5A). However, concentrations of progesterone in serum were decreased (P < 0.05) by 8 h in cows treated with PGF2{alpha} on Day 10 after estrus, and continued to decrease over 24 h (Fig. 5B).



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  		FIG. 5. Serum concentrations of progesterone after treatment (time = 0) with either saline or PGF2{alpha} beginning on Day 4 (A) or 10 (B) after estrus in experiment 2. Treatments continued every 8 h until tissue collection at 24 or 48 h. Concentrations of progesterone did not differ with time of collection within treatment or between treatments before Time 0, so the values were combined. SEM = 0.25

Expression of StAR and 3ß-HSD were decreased (P < 0.05) by 24 h, and further reduced by 48 h when treatment with PGF2{alpha} began on Day 10, however expression was not decreased until 48 h when treatment began on Day 4 (Fig. 6). Expression of P450scc was unaffected by treatment with PGF2{alpha} beginning on Day 4, but was decreased by 48 h when treatment began on Day 10 (P < 0.05; Fig. 6).



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  		FIG. 6. Expression of cholesterol side chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and steroidogenic acute regulatory protein (StAR) mRNA by bovine CL after treatment with PGF2{alpha} every 8 h for control (solid bars), 24 h (striped bars), or 48 h (hatched bars) beginning on Day 4 (A) or 10 (B). Data expressed as means of arbitrary densitometric units of the PCR fragment amplified from cDNA. *Indicates the mean differed (P < 0.05) from control

Experiment 3

Expression of IGFBP-1 was increased (P < 0.05) at 2 h after either the first or second (8 h after the first) treatments with PGF2{alpha} on both Day 4 and 10. Expression of IGFBP-1 returned to values similar to control values by 10 h after a single treatment with PGF2{alpha} on Day 4, but remained increased on Day 10 (Fig. 7). Expression of StAR did not differ with treatment on Day 4 (Fig. 8A), and was not changed at 2 h after the first treatment with PGF2{alpha}, but was decreased by 10 h after the first treatment or 2 h after the second treatment with PGF2{alpha} on Day 10 (P < 0.05; Fig. 8B). Expression of 3ß-HSD did not differ significantly at the times studied for treatment on either Day 4 or 10 (Day 4: 331, 383, 345, and 242; Day 10: 379, 382, 273, and 284 arbitrary densitometric units for control and treatment with PGF2{alpha} at 2 or 10 h after a single treatment, or 2 h after a second treatment that was given 8 h after the first treatment, respectively).



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  		FIG. 7. Expression of IGFBP-1 mRNA by bovine CL after treatment on Day 4 (A) or 10 (B) with either saline (solid bars) or PGF2{alpha} (striped bars) and the CL were removed at 2 h (1x 2 h) or 10 h (1x 10 h) after a single treatment or 2 h (2x 2 h) after a second treatment, 8 h after the first. Data expressed as means of arbitrary densitometric units of the PCR fragment amplified from cDNA. SEM = 69. *Indicates the mean differed (P < 0.05) from saline treatment



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  		FIG. 8. Expression of StAR mRNA by bovine CL after treatment on Day 4 (A) or 10 (B) with either saline (solid bars) or PGF2{alpha} (striped bars) and the CL were removed at 2 h (1x 2 h) or 10 h (1x 10 h) after a single treatment or 2 h (2x 2 h) after a second treatment, 8 h after the first. Data expressed as means of arbitrary densitometric units of the PCR fragment amplified from cDNA. SEM = 43. *Indicates the mean differed (P < 0.05) from saline treatment

DISCUSSION

Differential display RT-PCR was used to identify substances produced by the CL during regression that might be embryotoxic. One up-regulated gene product identified during luteal regression was identified as IGFBP-1 mRNA. It was reasoned that IGFBP-1 could be secreted, travel to the uterus via the oviductal vein [12–14] or local vascular diffusion pathway [15], and interfere with embryonic development [11]. Bovine embryos produce both IGF-I and IGF-II, and contain receptors for IGF-I and IGF-II at all stages of development from oocyte to blastocyst [9, 31]. Culture of bovine embryos with IGF-I and estrous cow serum increased the number of embryos developing into blastocysts and hatching from the zona pellucida [10]. IGFs also stimulated the secretion of ovine trophoblastic protein-1 in vitro [40]. IGFBP-1 was expressed by bovine endometrium by Day 13 of pregnancy and only on Day 15 of pregnancy in ovine endometrium [8, 41]. In transgenic mice that overexpressed IGFBP-1, fewer blastocysts were harvested and fewer embryos implanted or were carried to term [42]. Thus, increased luteal production of IGFBP-1 might increase concentrations of IGFBP-1 within the uterine lumen and prevent the embryotrophic effects of IGF-I and/or IGF-II. Because the reduction in pregnancy rate in cows with transferred embryos did not differ in uterine horns adjacent to or opposite from the regressing corpus luteum [43], the embryotoxin may travel through the uterine lumen after local transfer, but systemic distribution has not been ruled out.

Two findings in experiment 2 provided evidence against an embryotoxic role for IGFBP-1. First, concentrations of IGFBP-1 were not increased in uterine flushings. Second, IGFBP-1 and its mRNA increased in CL of cows treated with PGF2{alpha} on Day 10, when luteal regression did not cause loss of pregnancy in cows receiving replacement therapy with progestogen [4]. Thus, luteal IGFBP-1 does not appear to be a component of the embryotoxic effect of luteal regression found in previous experiments [1].

Other evidence obtained in these studies supports the concept that luteal oxytocin and luteal and uterine PGF2{alpha} are key components of the embryotoxic effect as proposed by Lemaster et al. [44]. The expression of genes necessary for prostaglandin synthesis increased following multiple injections of PGF2{alpha} beginning on Day 4 in the experiments reported here and increased luteal production of prostaglandins has been observed following treatment with PGF2{alpha} [45] or of luteal cells with oxytocin in vitro [46]. Uterine PGF2{alpha} could be increased, in part, as a result of local transfer mechanisms between the ovary and uterus. PGF2{alpha} interferes with early embryonic development in the rabbit [47], rat [48], and cow [49] when added to culture medium and could be the embryotoxin in cows undergoing early luteal regression. Indeed, Schrick et al. [50] found higher concentrations of PGF2{alpha} in uterine flushings of postpartum beef cows on Day 6 of a short luteal phase.

In postpartum cows that were mated at the estrus before a short estrous cycle was expected, treatment with a PGHS inhibitor, flunixin meglamine, tended to improve pregnancy rates, but did not return them to values expected in cows with a normal estrous cycle [3]. However, the combination of removal of the CL and treatment with the prostaglandin inhibitor restored pregnancy rates when progesterone supplementation was provided [3]. Treatment of cows with oxytocin on Days 4–8 after estrus reduced pregnancy rates to a degree similar to rates seen in postpartum cows or cyclic cows treated with PGF2{alpha} and given supplemental progestogen [44]. Concurrent treatment with a PGHS inhibitor overcame the effects of oxytocin on pregnancy rates. Therefore, increased concentrations of uterine PGF2{alpha} may be the factor involved in the embryotoxic effect found previously [1]. Transfer of oxytocin and PGF2{alpha} from the regressing CL to the uterus through either local vascular diffusion [15] or via the oviductal vein [12–14] could increase both uterine secretion of PGF2{alpha} and concentrations of PGF2{alpha} in the uterine lumen, thus causing the embryotoxic effect.

While a role for IGFBP-1 as an embryotoxic factor was excluded by the results of the experiments reported here, IGFBP-1 may promote luteal regression by antagonizing the effects of IGF-1 on luteal function. Treatment with PGF2{alpha} increased production of IGFBP-1 and its mRNA in bovine CL in the present study. The effect was seen as early as 2 h after initial treatment with PGF2{alpha}; therefore, expression of IGFBP-1 mRNA was up-regulated before detectable changes in concentrations of progesterone or in the expression of enzymes controlling rate-limiting steps in progesterone synthesis. In cows treated on Day 4, IGFBP-1 mRNA had returned to control values by 10 h unless a second injection of PGF2{alpha} was given 8 h after the first. In cows treated on Day 10 (at which time a single injection of PGF2{alpha} can cause luteolysis [51, 52]), the increase in IGFBP-1 mRNA was maintained at 10 h after a single dose of PGF2{alpha}.

Luteal concentrations of IGF-I increase from Day 1 to 5 to Day 12 to 17 of the estrous cycle, and decrease rapidly at luteolysis [20]. Type I and II receptors for IGF are present in luteal membrane preparations [23] and have been detected using immunocytochemistry [11]. Binding of IGF-I to receptors on luteal cell membranes increases luteal secretion of progesterone, supporting the hypothesis that IGF-1 can act in a paracrine fashion to affect luteal function [53]. Conversely, treatment with IGFBP-3 inhibited IGF-I-induced progesterone production by human granulosa-luteal cells [54]. Together, these studies and the present experiments provide evidence that IGF-I is present in the CL, that the CL can respond to IGF-I, and that PGF2{alpha} increases the production of IGFBPs that can antagonize IGF-I stimulated luteal steroidogenesis. Therefore, IGFBP-1 may antagonize the effects of IGF-I, resulting in decreased luteal production of progesterone.

Peripheral concentrations of progesterone decreased within 8 h of treatment with PGF2{alpha} on Day 10, as expected from the literature [51], but did not decrease until 24 h after treatment with PGF2{alpha} on Day 4. Inhibition of progesterone secretion appears to be due to effects on the transport of cholesterol to the mitochondrial site of cytochrome P450scc, a process mediated by StAR. Treatment with PGF2{alpha} inhibited the expression of mRNA for StAR protein in corpora lutea of ewes and cows [5, 55]. Others have shown that treatment with PGF2{alpha} does not affect the activity, concentration, or mRNA level of P450scc [56–58], or the concentrations of 3ß-HSD, but decreases expression of 3ß-HSD mRNA [59]. In the present study, expression of mRNAs for StAR and 3ß-HSD were decreased at 10, 24, and 48 h when luteal regression was initiated on Day 10, but only at 48 h when treatment was initiated on Day 4.

On Day 4, CL are resistant to a single injection of PGF2{alpha} and do not regress, despite the presence of functional high-affinity receptors for PGF2{alpha} [52] in concentrations similar to those in midcycle CL. Skarzynski and Okuda [60] have suggested that the reduced sensitivity of the early CL results from down-regulation of receptors for PGF2{alpha} or postreceptor changes that depend on locally produced hormones. Treatment with repeated dosages of PGF2{alpha} decreases expression of PGFR by 24 h in midcycle CL and by 48 h in early CL. Repeated treatment with PGF2{alpha} beginning on Day 10 increased expression of PGHS and PGFS, as found [61] after a single treatment with PGF2{alpha}. In contrast, continued treatment beginning on Day 4 increased mRNA for PGHS by 24 h and mRNA for PGFS by 48 h in the present study, whereas Tsai and Wiltbank [52] found decreased expression of PGHS at 4 h after a single treatment on Day 4. In the pig, the response of PGHS to treatment with PGF2{alpha} did not differ between Days 9 and 17 [62]. Thus, the mechanism for increased luteal production of PGF2{alpha} was active or could be activated by continued treatment, both early in the cycle, when the CL did not regress after treatment with PGF2{alpha}, and in older CL that did respond to PGF2{alpha}.

Increased luteal production of PGF2{alpha} may amplify the effect of exogenous PGF2{alpha} on secretion of progesterone by increasing luteal production of mediators, such as endothelin-1 (ET-1) [63] and IGFBP-1. Binding of ET-1 to the ETA receptor decreased progesterone secretion by luteal tissue, and an inhibitor of ETA can block the effects of PGF2{alpha} on secretion of progesterone [63]. Mamluk et al. [64] found that concentrations of ETA receptor in luteal tissue were decreased by increased concentrations of IGF-1. Similarly, concentrations of ETA are low during the estrous cycle until luteal regression [62], whereas concentrations of IGF-1 decreased at luteal regression [20]. Increased IGFBP-1, found in the present study by 2 h after PGF2{alpha} treatment on both Days 4 and 10 may reduce the availability of IGF-1, thus increasing concentrations of ETA and the response to PGF2{alpha}.

Recently, Silva [65] observed that concentrations of mRNA for prostaglandin dehydrogenase (PGDH), which metabolizes PGF2{alpha} to 15-keto-PGF2{alpha}, were greater in early than in midcycle CL of the ewe. The increased amount of this enzyme may protect the early CL from effective concentrations of PGF2{alpha}, thus explaining why early CL are less responsive to exogenous PGF2{alpha}.

In conclusion, the use of DD-PCR allowed identification of novel genes, such as IGFBP-1, that exhibited increased or decreased expression in the regressing CL. Expression of IGFBP-1 mRNA was increased after treatment with PGF2{alpha}, and the luteal response, in both expression of IGFBP-1 and decreased secretion of progesterone, appeared to be more rapid on Day 10 than on Day 4. Increased IGFBP-1 may antagonize the effects of IGF-1 and contribute to the altered luteal function that impairs luteal steroidogenesis and results in regression of the CL.

ACKNOWLEDGMENTS

The authors thank Diana Kirkpatrick-Keller and Larry Whetsell for careful assistance in laboratory analyses, and Pharmacia and Upjohn, Inc. for generous supplies of Lutalyse.

FOOTNOTES

First decision: 19 January 2000.

1 This work was supported by WV Hatch Project 321 (NE-161) and is published with the approval of the Director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Paper No. 2699. Back

2 Correspondence: E.K. Inskeep, Div. Animal and Veterinary Sciences, West Virginia University, P.O. Box 6108, Morgantown, WV 26506-6108. FAX: 304 293 2232; einskeep{at}wvu.edu Back

Accepted: February 24, 2000.

Received: December 15, 1998.

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