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BOR - Papers in Press, published online ahead of print January 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.020461
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BIOLOGY OF REPRODUCTION 70, 1333–1339 (2004)
DOI: 10.1095/biolreprod.103.020461
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Acquisition of Luteolytic Capacity: Changes in Prostaglandin F2{alpha} Regulation of Steroid Hormone Receptors and Estradiol Biosynthesis in Pig Corpora Lutea1

Francisco J. Diaz, and Milo C. Wiltbank2

Endocrinology-Reproductive Physiology Program and Department of Dairy Science, University of Wisconsin-Madison, Madison, Wisconsin 53706


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pig corpora lutea (CL) acquires luteolytic capacity at about Day 13 of the estrous cycle, after which luteolysis occurs in response to prostaglandin F2{alpha} (PGF2{alpha}) treatment. We postulated that differences in transcription factors such as the steroid hormone receptors may be responsible for the differences in PGF2{alpha}-induced gene expression after acquisition of luteolytic capacity. In these studies, we evaluated the effect of PGF2{alpha} on luteal expression of receptors for progesterone (nuclear and membrane progesterone receptor [PR]), estradiol (ER{alpha} and ERß), glucocorticoid, androgens, and two enzymes in estradiol synthesis (P450-17{alpha} and aromatase). Two experiments were conducted to examine the early (0.5 h, experiment I) and late (10 h, experiment II) effects of PGF2{alpha} on the expression of these receptors in CL with (Day 17) or without (Day 9) luteolytic capacity. PGF2{alpha} decreased ER{alpha} mRNA (35%) and increased ERß mRNA (558%) and protein (376%) only in Day 17 CL and not Day 9. The estradiol biosynthetic pathway was upregulated by PGF2{alpha} in Day 17 CL but not Day 9 CL, with a dramatic increase in aromatase mRNA and luteal estradiol content. Nuclear PR was not affected by PGF2{alpha}, but was greater (176%) in Day 9 CL, while a putative membrane PR was greater (156%) in Day 17 than Day 9 CL. There were no detectable changes in glucocorticoid or androgen receptor mRNA. Thus, luteolytic capacity is associated with upregulation of estradiol biosynthesis, which in conjunction with increased ERß expression and altered PR expression may promote luteolysis in the pig CL.

corpus luteum, estradiol, steroid hormone receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Following the LH surge and ovulation, granulosa and theca cells begin to differentiate into luteal cells. Hormone production shifts from estradiol to progesterone and there is dramatic remodeling and hypertrophy of the new corpus luteum (CL). Near the end of the estrous cycle, the development of the CL concludes with regression or luteolysis. During luteolysis, prostaglandin F2{alpha} (PGF2{alpha}) causes a dramatic decrease in progesterone production and structural regression of the CL [1, 2]. One quality of a fully differentiated CL is the ability to undergo luteolysis in response to exogenous PGF2{alpha}, a property that has been termed luteolytic capacity. For example, in the pig, a single exogenous treatment with PGF2{alpha} before Day 13 of the estrous cycle does not cause luteolysis [3], indicating that the porcine CL acquires luteolytic capacity at a relatively late time in the estrous cycle as compared with other species. The mechanisms and factors responsible for acquisition of luteolytic capacity have not yet been fully defined.

Receptors for PGF2{alpha} (FP receptors) are highly abundant in the porcine CL by Day 5 [4] and thus the absence of FP receptors is not the reason for lack of luteolytic capacity before Day 13. In addition, treatment with PGF2{alpha} induces a number of changes in gene expression that are similar regardless of whether the CL has acquired luteolytic capacity; however, the full luteolytic program is not activated [5–8]. Differential gene expression in response to PGF2{alpha} is consistent with alterations in transcription factors being a key aspect of acquisition of luteolytic capacity. One key family of transcription factors that is known to be expressed in the pig CL [9–11] is the receptors for the steroid hormones. Steroids binding to these receptors are known to regulate many physiological processes, particularly reproductive processes.

The CL is a primary source of progesterone and the role of progesterone in regulating luteal function is becoming better understood. Recent evidence points to a role of progesterone and/or the nuclear progesterone receptor (nPR) in protecting the CL from apoptosis [12–14]. In addition, luteal membranes are known to specifically bind progesterone with high affinity [15, 16], possibly due to a putative membrane progesterone receptor (mPR) recently identified in several species including pigs [17].

Estradiol is also produced by the CL and thus may be a target for estrogen action. There is evidence supporting both luteotrophic [18, 19] and luteolytic [20–23] roles for estrogen and estrogen receptors (ER{alpha} and ERß) in the CL. The mechanisms underlying luteal estradiol production and the physiological function of luteal estradiol have not been defined. The roles of glucocorticoid and testosterone are not well understood in the CL. Testosterone is clearly produced by the pig CL [20]; however, it is not clear whether testosterone plays a functional role in the CL or is simply serving as substrate for estradiol synthesis. Glucocorticoid is not produced in the pig ovary, and the synthetic glucocorticoid, dexamethasome, does not have any effect on progesterone production from mature CL [24].

The present study investigated the PGF2{alpha}-induced and developmental changes in luteal concentrations of nuclear steroid hormone receptors, the estradiol biosynthetic pathway, and levels of a putative membrane progesterone receptor in pig CL before and after acquisition of luteolytic capacity. We hypothesized that PGF2{alpha} would differentially regulate expression of these steroid hormone receptors before versus after acquisition of luteolytic capacity. In addition, we hypothesized that PGF2{alpha} would induce the estradiol biosynthetic pathway only in CL with luteolytic capacity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents

Cloprostenol was purchased from Bayer Corporation (Shawnee Mission, KS), ketamine was from Fort Dodge Animal Health (Fort Dodge, IO), and xylazine was from Phoenix Pharmaceuticals (St. Joseph, MO). 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). 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. The first day of estrus was designated as Day 0. Because luteolysis in the pig begins very near the time of acquisition of luteolytic capacity (Day 13), we found it difficult in preliminary experiments to reliably find animals in the late estrous cycle that had luteolytic capacity but had not yet been exposed to a luteolytic signal. Therefore, we chose to use pseudopregnant gilts based on previous results that pseudopregnant gilts readily undergo luteolysis in response to PGF2{alpha} treatment [25]. Pseudopregnancy was induced 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 was maintained with halothane. Ovaries were collected via midventral laparotomy and CL were dissected away from ovarian stroma and frozen in liquid nitrogen. The Research Animal Resource Center Committee at University of Wisconsin- Madison approved all procedures performed on animals.

Experiment I

This experiment examined the acute in vivo (0.5 h) regulation of mRNA for ER{alpha}, ERß, nPR, putative mPR, glucocorticoid receptor (GR), aromatase, and P450-17{alpha} by PGF2{alpha}. A paired experimental design was used to ensure the timing of CL collection because it would have been difficult to anesthetize and surgically collect CL within 30 min after PGF2{alpha} treatment. On Day 9 after estrus (n = 4) or Day 17 of pseudopregnancy (n = 5), gilts were anesthetized and one ovary collected (control CL). Following removal of the control ovary, PGF2{alpha} (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 in liquid nitrogen.

Experiment II

This experiment examined the late in vivo (10 h) regulation of mRNA for ER{alpha}, ERß, nPR, putative mPR, GR, androgen receptor (AR), aromatase, and P450-17{alpha} by PGF2{alpha}. Gilts 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 PGF2{alpha} (500 µg cloprostenol; n = 4), Day 17 saline (n = 5) and Day 17 PGF2{alpha} (500 µg cloprostenol; n = 5). On Day 9 of the estrous cycle or Day 17 of pseudopregnancy, gilts received either saline or PGF2{alpha} 10 h before ovary removal. Corpora lutea were collected and frozen in liquid nitrogen.

Isolation of Total RNA

Total RNA was isolated using the RNAgents total RNA isolation system (Promega). Briefly, CL were ground in a mortar and pestle cooled with liquid nitrogen. Approximately 30 mg of tissue was transferred to a fresh tube containing 900 µl of denaturing solution and homogenized for 20 sec using a polytron tissue grinder. Ninety microliters of 2 M sodium acetate and 900 µl of phenol/chloroform/isoamyl alcohol were added to the lysate and incubated on ice for 15 min. Samples were centrifuged for 20 min at 16 000 x g in a refrigerated microcentrifuge. Supernate was transferred to a fresh tube and RNA was precipitated with an equal volume of isopropanol and incubated at –20°C for 1 h. Samples were then centrifuged at 16 000 x g for 10 min to pellet RNA and washed with 1 ml 70% ethanol. The RNA pellet was dried and resuspended in 30 µl diethyl pyrocarbonate-treated water, and RNA purity and quantity were measured by absorbance at 260/280 nm in a spectrophotometer.

Reverse Transcription-Polymerase Chain Reaction

Quantitation of ER{alpha}, ERß, nPR, mPR, GR, AR, aromatase, and P450- 17{alpha} hydroxylase (P450-17{alpha}) mRNAs was done 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-base pair (bp) product. ER{alpha} (Z37167), ERß (AF164957), nPR (S49016), mPR (AF313616), AR (AF202775), GR (AF141371), aromatase (U92245), and P450-17{alpha} (M63507) primers were synthesized from published genebank sequences to produce the expected 562-, 214-, 197-, 435-, 435-, 482-, 358-, and 440- bp products (see Table 1 for primer sequences). Reverse transcription was carried out with 19 µl of 1x master mix (1x reverse transcription [RT] buffer, 0.2 mM dNTPs, 100 pmol random primer, and 40 U reverse transcriptase) and 2 µl of total RNA for 1.5 h at 37°C. For polymerase chain reaction [PCR], 4 µl of RT reaction were added to 1x 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 25–30 cycles of PCR (95°C: 30 sec, 54–57°C: 30 sec, and 72°C: 30 sec) followed by a final extension at 72°C for 5 min. Evaluation of mPR and AR mRNA was accomplished by amplifying AR and mPR products for 6 cycles before addition of G3PDH primers for an additional 24 cycles. This was to ensure both PCR products were in the linear amplification phase. Reaction products were separated on 5% PAGE gel and stained with ethidium bromide. For each sample, two products were quantified using Collage imaging system (Fotodyne, Hartland, WI). Values were calculated as the ratio of gene-specific band intensity/G3PDH band intensity.


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  		TABLE 1. Primer sequences used for PCR.a

Western Blotting

ERß protein was analyzed by immunoblot. 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. Protein concentration in supernate was measured by BCA assay (Pierce Biochemical, Rockford, IL). Sixty micrograms of protein extract was combined with 10 µl of 6x loading buffer and 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 fluoride membrane using the mini-protean 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-ERß (HC-150, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:2000 dilution for 2 h at 25°C, followed by 3x washing (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20). Anti-rabbit horseradish peroxidase (HRP) (Santa Cruz Biotechnology) was added at 1:5000 dilution for 1 h at 25°C, followed by 3x washing. Specific proteins were detected with enhanced chemiluminescent reagent (NEN Life Science Products, Boston, MA). Blots were exposed to x-ray film for 5 min and quantified using a collage photoimaging system.

Estradiol Assay

The procedure used for determination of estradiol has been described previously [26]. Luteal tissue (~100 mg) was homogenized in 1 ml of 5% trichloroacetic acid (TCA) using a polytron tissue grinder. The lysate was centrifuged at 16 000 x g for 5 min to clarify and the supernatant stored at –20°C. TCA extracts were diluted 1:500 in assay buffer (40 mM MOPS, 0.12 M NaCl, 10 mM EDTA, 0.1% gelatin, 0.5% Tween 20, and 0.005% chlorohexidine digluconate, pH 7.4). Goat anti-sheep IgG (2 µg/ ml, Calbiochem, San Diego, CA) were coated onto 96-well plates and they were washed with wash buffer (0.02 M MOPS, 0.025% Tween-20, pH 7.2) three times. Antiestradiol polyclonal antiserum (CSU-224, donated by G.D. Niswender, Colorado State University, Fort Collins, CO) was incubated on the plate at 1:10 000 dilution for 1 h to allow binding to anti- sheep IgG. Samples and standards were then added (100 µl) and allowed to bind for 1 h. Fifty microliters of estradiol-HRP made by the method of Munro and Stabenfeldt [27] using estradiol-3-hemisuccinate (Steraloids, Newport, RI) were added and incubated on the plates for 1 h. The plates were washed three times and 125 µl of substrate solution added (0.05 M sodium acetate, 0.3 mM H2O2, and 0.1 mg/ml 3,3',5,5' tetramethyl benzidine). The reaction was stopped with 50 µl of 0.5 M sulfuric acid. Optical density was measured with an optical plate reader (Bio-Tek Instruments, Winooski, VT) using 450 and 600 nm wavelength filters. The sensitivity of the assay was 11.07 pg and the intra- and interassay coefficients of variation were 3% and 7%, respectively.

Statistical Analyses

Results for experiment II (10 h) were analyzed by two-way analysis of variance (ANOVA) using the general linear model (GLM) procedure of the Statistical Analysis System (SAS) [28]. Results from experiment I (0.5 h) were analyzed by paired t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Estrogen Receptor {alpha}

Treatment with PGF2{alpha} had no acute (0.5 h) effect on ER{alpha} mRNA in either Day 9 or Day 17 CL (Table 2). However, 10 h after PGF2{alpha} treatment, ER{alpha} was decreased in Day 17 CL but unchanged in Day 9 CL (Fig. 1A, P < 0.05). In CL from untreated animals, ER{alpha} was lower in Day 9 CL than Day 17 CL in both experiment I (Table 2, P < 0.05) and experiment II (Fig. 1A, P < 0.05).


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  		TABLE 2. Acute (0.5 h) effects of PGF2{alpha} on steady-state concentrations of specific mRNA transcripts in pig CL.a



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  		FIG. 1. Steady-state concentrations of ER{alpha} (A) and ERß (B) mRNA (ratio of specific ER mRNA:G3PDH mRNA) at 10 h after PGF2{alpha} treatment in Day 9 (n = 4–5 per group) and Day 17 (n = 5 per group). Representative gel photos of PCR products for ER{alpha} or ERß and G3PDH mRNA from two Day 9 and two Day 17 animals untreated or treated (+) with PGF2{alpha}. a,b,c Denote significant differences, P < 0.05

Estrogen Receptor ß

The mRNA for ERß was differentially regulated by PGF2{alpha}. Acute (0.5 h) treatment with PGF2{alpha} increased ERß mRNA 2.6-fold in Day 17 CL but not in Day 9 CL (Table 2, P < 0.05). Likewise, ERß mRNA was increased 3.1- fold at 10 h after PGF2{alpha} treatment in Day 17 CL (Fig. 1B, P < 0.05) but not Day 9 CL (Fig. 1B). Immunoblot revealed an increase in ERß protein after PGF2{alpha}. Treatment with PGF2{alpha} increased ERß protein (Fig. 2) in Day 17 CL (control 2655 ± 213; PGF2{alpha} 9985 ± 1149 relative density units, P < 0.05) but not in Day 9 CL (saline 4506 ± 1234; PGF2{alpha} 3086 ± 575 relative density units) 10 h after treatment.



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  		FIG. 2. Quantification of ERß protein showing (A) graphical summary of results (mean ± SEM pixel density) and (B) representative immunoblot of CL from untreated and treated Day 9 (n = 4–5 per group) and Day 17 (n = 5 per group) animals 10 h after treatment. Recombinant human ERß (rhERß) was used as a positive control. a,b Denote significant differences, P < 0.05

Nuclear and Membrane Progesterone Receptors

Both the nuclear and putative membrane forms of progesterone receptors were unaffected by acute (0.5 h) treatment with PGF2{alpha} in either Day 9 or Day 17 CL (Table 2). Likewise, 10 h after treatment, PGF2{alpha} had no effect on nPR (Fig. 3A) or mPR (Fig. 3B) in either Day 9 or Day 17 CL. In CL from untreated animals, there were differences in the concentrations of nPR and mPR mRNA between CL collected on Day 9 or Day 17. In CL from untreated Day 9 animals, nPR was greater compared with CL from untreated Day 17 animals (Table 2 and Fig. 3A, P < 0.05). Conversely, mPR was greater in Day 17 CL compared with Day 9 CL (Table 2 and Fig. 3B, P < 0.05).



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  		FIG. 3. Steady-state concentrations of nPR (A) and putative mPR (B) mRNA (ratio of specific PR mRNA:G3PDH mRNA) 10 h after PGF2{alpha} treatment of Day 9 (n = 4–5 per group) and Day 17 (n = 5 per group) gilts. Representative gel photos of PCR products for nPR or mPR and G3PDH mRNA are also shown

Glucocorticoid and Androgen Receptor

The mRNA for androgen receptor was not significantly different at 10 h after PGF2{alpha} treatment in either Day 9 or Day 17 CL (Table 3). Glucocorticoid receptor was also not different after PGF2{alpha} treatment at 0.5 h (Table 2) or 10 h (Table 3) after treatment in Day 9 or Day 17 CL. The concentrations of AR and GR were similar in CL from untreated animals collected on Day 9 and Day 17 (Tables 2 and 3).


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  		TABLE 3. Late (10 h) effects of PGF2{alpha} on steady-state concentrations of specific mRNA transcripts in pig CL

Estradiol Biosynthetic Pathway

Luteal estradiol-17ß concentrations were increased after PGF2{alpha} treatment in Day 17 CL but not in Day 9 CL (Fig. 4A, P < 0.05). Acute (0.5 h) treatment with PGF2{alpha} had no effect on P450-17{alpha} mRNA in either Day 9 or Day 17 CL (Table 2). However, at 10 h after treatment, PGF2{alpha} decreased P450-17-{alpha} mRNA in Day 17 CL but not in Day 9 CL (Table 3, P < 0.05). In CL from untreated animals, P450-17-{alpha} mRNA was greater in Day 9 compared with Day 17 CL (Tables 2 and 3, P < 0.05). The PGF2{alpha}-induced increase in luteal estrogen production was closely associated with a large increase in aromatase mRNA. Acutely (0.5 h), PGF2{alpha} had no effect on aromatase mRNA in either Day 9 or Day 17 CL (Table 2). However, at 10 h after treatment, PGF2{alpha} induced a large increase in aromatase mRNA only in Day 17 and not in Day 9 CL (Fig. 4, B and C, P < 0.05). In CL from untreated animals, aromatase mRNA was virtually undetectable (Table 2, Fig. 4, B and C).



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  		FIG. 4. A) Concentrations of luteal estradiol content, (B) gel photo of G3PDH and aromatase PCR products, and (C) steady-state concentrations of aromatase mRNA (ratio of aromatase mRNA:G3PDH mRNA) in CL 10 h after PGF2{alpha} treatment of Day 9 (n = 4–5 per group) and Day 17 (n = 5 per group) gilts. a,b Denote significant differences, P < 0.05


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Luteolysis involves major cellular and secretory changes that appear to occur only in CL that have acquired luteolytic capacity or the ability to respond to PGF2{alpha} with the full complement of responses required for luteolysis. In this study, we examined the hypothesis that luteal estradiol production and luteal expression of steroid receptors would be regulated by PGF2{alpha} only in CL with and not CL without luteolytic capacity. One of the most striking changes that we found in this study was a dramatic induction of aromatase expression by PGF2{alpha} that led to a specific increase in estradiol production within CL with luteolytic capacity. In addition, we report that PGF2{alpha} increased ERß mRNA and protein and decreased ER{alpha} mRNA only in CL with luteolytic capacity. These results are consistent with our hypothesis and suggest a role for luteal estradiol production and action in the process of luteolysis. In contrast, PGF2{alpha} treatment did not alter expression of nuclear steroid hormone receptors for androgen, glucocorticoid, or progesterone (nPR), or of a putative membrane receptor for progesterone. However, nPR mRNA was decreased and mPR mRNA increased in CL from untreated animals with luteolytic capacity. These results, combined with previous research, findings provide insights into the potential physiology underlying the luteolytic process.

The preovulatory follicle secretes estradiol in increasing amounts until a GnRH and LH surge is triggered. The surge in LH, in turn, downregulates estradiol production and initiates a shift from follicular estradiol production to luteal progesterone production due to downregulation of aromatase activity in granulosa cells. Intriguingly, during luteolysis, there is a shift back to lower progesterone and greater estradiol production [20] apparently within the large luteal cell [29]. From our results, the shift to luteal estradiol production appears to involve regulation of aromatase expression. In the pig CL with luteolytic capacity, PGF2{alpha} dramatically increased aromatase mRNA from almost nondetectable levels to high concentrations by 10 h after treatment (Fig. 4). In general, PGF2{alpha} appears to activate intracellular effector systems other than protein kinase A (PKA), such as protein kinase C (PKC) and mitogen-activated protein kinase [30, 31]. Thus, the regulation of the aromatase gene apparently shifts from PKA regulation in granulosa cells to regulation by PGF2{alpha} acting through other second messenger pathways in large luteal cells. Similarly, previous studies have shown that the cycloxygenase-2 promoter shifts from PKA to PKC regulation as bovine granulosa cells differentiate into large luteal cells with luteolytic capacity [32]. However, in Day 9 CL, aromatase mRNA was not induced by PGF2{alpha}, indicating that the pathways needed for induction of this gene were incomplete or blocked in CL without luteolytic capacity.

The physiological role of this increase in luteal estradiol production is unclear. Estradiol has been proposed to have both luteotrophic and luteolytic effects in pigs and other mammals. In rabbits, estradiol is critical for maintenance of progesterone production [33]. In pigs, estradiol implants promote greater CL development than nonimplanted CL in the same animal [18, 34]. Also, porcine CL implanted with a microdialysis system and infused with estradiol respond with an increase in progesterone, an effect blocked by tamoxifen [19]. In both these studies, the CL were treated at a time when the CL would lack luteolytic capacity. The finding of elevated estradiol production (Fig. 4) and increased ERß expression in regressing CL (Figs. 1 and 2) suggests a luteolytic role for estradiol in the pig. The failure to upregulate these pathways in Day 9 CL may be responsible, in part, for the inability of PGF2{alpha} to induce luteolysis at this time. However, direct manipulative studies will be needed to clearly demonstrate and provide mechanistic information about the effect of estradiol during luteolysis. In addition to a potential autocrine/paracrine function within the CL, it seems likely that the increase in luteal estradiol production would increase circulating estradiol at the time circulating progesterone was decreasing and this could have important systemic actions during the porcine estrous cycle such as regulating circulating FSH and/or LH.

Seemingly contradictory roles of estrogen in the CL could be explained by changes in estrogen receptors. In the present study, PGF2{alpha} induced ERß mRNA and protein and decreased ER{alpha} mRNA only in Day 17 CL and not Day 9 CL. In the primate CL, ERß protein expression changes throughout the luteal phase with an increase during the time of luteolysis [21]. Growing evidence suggests that ER{alpha} and ERß have different, even opposing, effects on cell function and that ERs interact with other transcription factors to produce specific physiological actions. In addition, the ERß subtype has been associated with induction of cell death in an epithelial cell line [35] and through the Fas/FasL system in neuronal cells [36]. Recently, Fas-mediated cell death has been demonstrated in rat and bovine CL [37–39]. In these studies, FasL-mediated cell death was blocked by progesterone [40] or by an anti-Fas antibody [38]. Thus, there seems to be an association between ERß expression, FasL expression, and cell death in the CL.

The inhibition of FasL-induced cell death by progesterone [40] is consistent with an extensively discussed protective effect of progesterone on the CL [12–14, 41, 42]. In our study, we detected substantial luteal expression of the nuclear form of the progesterone receptor, consistent with previous reports in pigs [9] but not in rats [12, 42]. In bovine luteal cells, a PR antagonist causes cell death of luteal cells [13]. In rat CL, progesterone inhibits the luteolytic effects of prolactin [40] and the antiprogesterone RU468 increases luteal cell death [12]. In the primate CL, blocking progesterone production causes premature luteal regression [14]. These observations implicate progesterone and PR in protecting the CL from premature luteal regression. In our study, PR mRNA was greater in Day 9 CL than in Day 17 CL, suggesting a potential role of changes in PR expression in acquisition of luteolytic capacity. Nevertheless, the dramatic decrease in luteal progesterone production that only occurs in CL with luteolytic capacity, combined with the observed reduction in luteal expression of nPR, could reduce the protective effect of progesterone in the CL with luteolytic capacity.

In addition to the nuclear PR, a putative membrane PR has recently been identified and cloned in several species [17]. This membrane protein could be responsible for the previously reported ability of luteal membranes to bind progesterone [15, 16]. In the present study, we observed that pig CL express mRNA for the putative mPR (Fig. 3). In contrast with nPR, the steady-state concentrations of putative mPR mRNA were greater in Day 17 than in Day 9 CL. Thus, nPR and mPR may have differing or even opposing actions on CL function.

One potentially confusing aspect of integrating the role of ER and PR into luteolysis is that the steroids that are activating these receptors are produced locally in extremely high amounts. Based on dose-response studies in other tissues, it would be anticipated that estradiol and progesterone binding would saturate their cognate receptors in the CL even during times of lowered steroid production. The affinities of estrogen and progesterone for their receptors are 0.1 nM for ER{alpha}, 0.4 nM for ERß [43], and 4.8 nM for nPR [44]. In the pig CL, basal concentrations of estradiol and progesterone in luteal tissue are 8.4 nM (Fig. 4, 2.3 ng/g tissue) and 194 µM [5], respectively, exceeding the Kd for their receptors by 10 to 10 000 fold. Thus, in the presence of high ligand concentrations, a mechanism must exist for regulating the signaling of estradiol and progesterone in the CL. Recently, progesterone has been shown to induce a negative regulator of PR, FKBP51, in human cancer cell lines, thus decreasing sensitivity to progesterone action [45]. Similar desensitization mechanisms could occur inthe CL.

It is still difficult to integrate our results on steroid receptors into a comprehensive model of luteolysis and acquisition of luteolytic capacity. In the pig and a number of other species, PGF2{alpha} is the primary luteolysin and binds to luteal FP receptors in CL with or without luteolytic capacity. In the CL without luteolytic capacity, PGF2{alpha} treatment either does not decrease or produces only a transient decrease in luteal progesterone production. In contrast, in CL with luteolytic capacity, there is a shift in luteal steroid production after PGF2{alpha} with decreased production of progesterone and increased luteal estradiol production due to a surprisingly dramatic increase in luteal aromatase expression (Fig. 4). In addition, CL without luteolytic capacity have elevated nPR that could also increase the protective PR-mediated effect of progesterone. Luteal estradiol could also have altered functional effects in CL with luteolytic capacity due to a PGF2{alpha}-induced decrease in ER{alpha} (removal of trophic/mitogenic support) and increase in ERß (increase in FasL-associated cell death). Obviously, other key cytokines and regulatory factors such as endothelin-1 and monocyte chemoattractant protein-1 would also need to be integrated into a complete model for luteolysis. The mechanism(s) preventing induction of luteolytic pathways in CL without luteolytic capacity remains to be determined. Nonetheless, the present results are consistent with dramatic shifts in steroid production and luteal steroid hormone receptors being a key part of the complete program for regression of the CL.


   FOOTNOTES
 
1 Supported by USDA-00-35203-9134, HD-32623, and an Advanced Opportunity Fellowship to F.J.D. Back

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 Back

Received: 18 June 2003.

First decision: 9 July 2003.

Accepted: 16 December 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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