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Articles |
a Departments of Internal Medicine,
b Obstetrics and Gynecology,
c Anatomy and Cell Biology,
d Pharmacology,
e Physiology, and
f Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p 
0.01) in dex-treated than in control rats (44.5 ± 2.3 vs. 23.0 ± 5.6 ng/ml). Dexamethasone treatment increased lipid droplets and lipid in the corpora lutea as revealed by electron microscopy and oil red O staining. Cholesterol esters were higher in corpora lutea of dex-treated rats compared to controls (14.8 ± 1.1 vs. 2.2 ± 0.5 µg/mg corpora lutea wet tissue, respectively; p 0.05). Another group of hypophysectomized rats was treated with either a high or a lower dosage of corticosterone, both of which caused an elevation to > 2-fold of plasma 20-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxysteroid dehydrogenase in a luteal cell line and in primary cultures of rat corpora lutea. Adashi et al. [4] reported a similar effect of glucocorticoids on the activity of 20-hydroxysteroid dehydrogenase in rat granulosa cells. Telleria et al. [11] found that dexamethasone inhibited the expression of interleukin-6 in rat luteal tissue. These in vitro effects of dexamethasone [2, 11] were mimicked by the addition of progesterone, which was proposed to act through the glucocorticoid receptor, since progesterone receptor is apparently not expressed in rat luteal tissue [12]. In a recent investigation of the potential anti-inflammatory actions of dexamethasone in the corpora lutea of hypophysectomized rats [13], we observed that the administration of dexamethasone stimulated the production of progestins by the corpora lutea and changed the appearance of the corpora lutea in a manner suggestive of the accumulation of lipid. In the present work, we have pursued this initial finding to determine whether the enhancement of steroidogenesis in corpora lutea of hypophysectomized rats treated with glucocorticoids is associated with the accumulation of lipid in the corpora lutea.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female Sprague-Dawley rats were purchased from Charles River (Portage, MI) after gonadotropin-induced ovulation followed by hypophysectomy. The rats received an injection of eCG (5 IU, s.c.) at 29 days of age followed by an injection of hCG (5 IU, s.c.) 56 h later to induce ovulation and the development of a single set of corpora lutea. This protocol was adapted from Taya and Greenwald [14] and has been successfully used by our laboratory [15]. The animals were hypophysectomized by the vendor at 32 days of age and then shipped to our laboratory. The rats (90–95 g) were housed in a room with a 12L:12D cycle and provided with free access to rat chow, 5% glucose water, and sliced oranges.
Experiment 1: Effects of Dexamethasone Treatment
The rats were divided into dexamethasone-treated or vehicle-treated (control) groups. Beginning at 40 days of age, rats were injected s.c. with either dexamethasone (Gensia Pharmaceuticals, Irvine, CA; lot P7HO13) twice daily (200 µg/rat/day) or vehicle (control). This is an anti-inflammatory dose of dexamethasone that effectively blocks inflammatory cytokine responses to an injection of lipopolysaccharide in mice [16]. The treatments were instituted for 4 days, and the rats were killed by decapitation at 44 days of age. In this and all other experiments involving hypophysectomized rats, the removal of the pituitary was verified by examination of the sella turcica using a stereomicroscope. The few animals in which pituitary fragments were observed were excluded from the experiment. Trunk blood was collected for the assay of plasma progestins. Ovaries from some rats were frozen and prepared for microscopy. From other rats, ovaries were removed and placed on ice, and the corpora lutea were dissected. These corpora lutea and the remainder (nonluteal tissues) of the ovary were frozen in liquid nitrogen for subsequent measurement of cholesterol concentrations.
Experiment 2: Effect of Corticosterone Treatments on Plasma 20-Dihydroprogesterone Concentration
After hormonally induced ovulation, the rats were hypophysectomized at 32 days of age as described above. At 38 days of age, the rats were anesthetized by Metofane inhalation (Malinckrodt Veterinary Inc., Mudelein, IL). An incision was then made through the dorsal skin, and either placebo or corticosterone pellets (Innovative Research of America, Sarasota, FL) were placed subcutaneously. The dosages were either four 50-mg corticosterone pellets (200 mg/rat) for the high corticosterone dosage, or as two 50-mg corticosterone pellets (100 mg/rat) plus two placebo pellets for the lower dosage. Four placebo pellets were used for the control animals. The 200-mg dosage of corticosterone was selected because it has been shown to produce high corticosterone levels in plasma of adrenalectomized rats [17]. The lower dosage was selected because it is within a dosage range reported to replace normal glucocorticoid function in adrenalectomized rats [18, 19]. The rats were maintained with the implants for a total period of seven days, at which time they were killed by decapitation and trunk blood was collected for RIA of 20-dihydroprogesterone.
Experiment 3: Identification of Glucocorticoid Receptor in Ovarian Tissues of Hypophysectomized Rats
Ovaries from 23 ovulated, hypophysectomized rats (as described above) were removed and placed on ice. The corpora lutea were dissected and pooled, as were the nonluteal portions of the ovaries. Samples of liver, a tissue that is known to express glucocorticoid receptor, were also taken from several rats. The pooled tissues were weighed quickly and placed in ice-cold homogenization buffer (10 mM HEPES, 1 mM EDTA, 20 mM molybdate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 250 µM PMSF, pH 7.4) at a w:v ratio of 1:1.5. The pooled corpora lutea or nonluteal ovarian tissues were homogenized in an ice-cold glass homogenizer fitted with a glass pestle (2 x 10 strokes) and centrifuged at 100 000 x g for 20 min, at 4°C. The supernatant containing the cytosolic fraction was subsequently immunoadsorbed, and immunoblotting followed, according to the procedures of Bresnick et al. [20], as described below. The glucocorticoid receptor was immunoadsorbed from 160 µl of cytosol by rotation for 2 h at 4°C with 30 µl of BuGR2 monoclonal anti-glucocorticoid receptor antibody (Affinity Bioreagents, Golden, CO) prebound to 12 µl of protein A-sepharose. Immunopellets were washed three times by suspension in 1 ml of TEGM buffer, pH 7.6 (10 mM TES [2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid], 50 mM NaCl, 4 mM EDTA, 10% [w:v] glycerol, 20 mM sodium molybdate), and proteins were resolved by SDS-PAGE. Immunopellets were boiled in SDS sample buffer, and the proteins were resolved on a 10% SDS-polyacrylamide gel. Proteins were transferred to Immobilon-P membranes and probed with 1 µg/ml BuGR2 antibody for the glucocorticoid receptor. The immunoblots were then incubated with 125I-conjugated goat anti-mouse counterantibody (Dupont NEN, Boston, MA) to visualize the immunoreactive bands.
Electron Microscopy
Isolated, dissected corpora lutea from control and dexamethasone-treated rats were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. Samples were buffer-rinsed, then postfixed in 1% OsO4. The samples then received additional buffer rinses, which were followed by dehydration and embedding in epon araldite. Sections were poststained with 2% uranyl acetate in 50% ethanol and lead citrate as described by Reynolds [21], and then photographed in a Philips CM100 transmission electron microscope (Philips Electron Optics Inc., Mahwah, NJ).
Light Microscopy
Whole ovaries from control and dexamethasone-treated rats were embedded in OCT compound (Sakura Finetek USA, Inc., Torrance, CA) and frozen in slush 2-methylbutane cooled in liquid nitrogen. Sections (7 µm) were picked up on Superfrost/Plus microslides (Fisher Scientific, Pittsburgh, PA), air-dried, and then fixed in 4% formaldehyde, 0.5% glutaraldehyde, and 1% CaCl2 in 0.1 M cacodylate buffer pH 7.3 for 40 min. Slides were rinsed in buffer and dH2O, and then in 60% isopropanol before staining with oil red O, according to procedures of Lillie [22] and Humason [23]. After rinsing with 60% isopropanol, the tissues were counterstained in Gill's #3 hematoxylin (Fisher Scientific). All solutions were kept at 4°C. Slides were coverslipped in Aqua-Poly Mount (Polysciences Inc., Warrington, PA) and stored at 4°C. Photographs were made with a Sony (Glen Mills, PA) DKC-5000 digital photo camera.
Lipid Extraction and Cholesterol Determination
Dissected corpora lutea and nonluteal ovarian tissues were homogenized in 0.5 ml ice-cold PBS. Lipids were extracted from the homogenates by the addition of 1.5 ml of ice-cold CHCl3:MeOH (1:2) followed by vortexing. The homogenate mix was then stored under nitrogen overnight at 4°C. On the following morning, 0.5 ml of ice-cold dH2O was added followed by 1.5 ml CHCl3 at 4°C; this final mix was vortexed and centrifuged at 2000 rpm for 10 min. The solvent layer was collected and dried under nitrogen, after which 0.5 ml isopropanol were added and the mixture was vortexed. The samples were then stored at 4°C under nitrogen for 1–2 h until the performance of the cholesterol assay. The measurement of total cholesterol, free cholesterol, and cholesterol esters was conducted according to the method of Deacon and Dawson [24], which involves the enzymatic hydrolysis of cholesterol by cholesterol oxidase and the formation of hydrogen peroxide in the absence and presence of cholesterol ester hydrolase.
RIAs
Plasma was obtained by collection of trunk blood into heparinized tubes followed by centrifugation at 1740 x g for 20 min. Aliquots of plasma were extracted, and progesterone and 20-dihydroprogesterone were measured following the procedures reported by Elbaum et al. [25] and Bender et al. [26], respectively.
Statistical Analysis
Means were compared using Bonferroni's Multiple Comparisons Test for the effect of corticosterone implants, or Student's t-test for all other experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovulation by the stated regimen of eCG and hCG, followed by hypophysectomy the following day, resulted in an average of 12 ± 2 corpora lutea, which appeared at autopsy 12 days later in vehicle-treated animals as well-vascularized, solid structures of uniform appearance. In dexamethasone-treated animals, the corpora lutea were also vascular but appeared more opaque.
Experiment 1: The Effects of Dexamethasone Treatment
Plasma progestin
The effect of four days of dexamethasone treatment on plasma concentration of progestins is shown in Figure 1. Plasma progesterone concentration was not different between dexamethasone- and vehicle-treated rats (4.7 ± 0.7 vs. 3.4 ± 0.8; mean ± SEM), respectively. However, the metabolite, 20-dihydroprogesterone, which is a major steroid produced by corpora lutea of hypophysectomized rats [14, 27], was significantly elevated in plasma in dexamethasone-treated animals (44.5 ± 2.3 vs. 23.0 ± 5.6; p < 0.05).
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Microscopy
Figure 2 shows electron micrographs of representative areas of corpora lutea of vehicle- and dexamethasone-treated rats. Whereas most luteal cells of vehicle-treated rats have few lipid droplets, the luteal cells of dexamethasone-treated rats have abundant lipid droplets, which fill the cytoplasm.
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Figure 3 shows representative areas of ovarian tissues stained with oil red O. In both ovaries, the stromal-interstitial tissue stained brightly and intensely for lipid; no difference in staining intensity for stromal-interstitial tissue for the two treatments could be detected. The granulosa cells of follicles for both treatments were not stained. Consistent with the results of electron microscopy, the corpora lutea of dexamethasone-treated animals displayed lipid staining that was markedly more intense than that in the vehicle-treated animals. Thus, dexamethasone enhanced staining for lipid in the corpora lutea but not in the other ovarian compartments.
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Tissue Cholesterol and Cholesterol Ester Content
The effect of dexamethasone treatment on sterol content of luteal tissue is shown in Figure 4. Dexamethasone treatment was associated with a significant increase in free cholesterol content: 2.3 ± 0.05 (vehicle-treated animals) vs. 4.8 ± 0.1 µg/mg luteal tissue (dexamethasone-treated), p < 0.05. Dexamethasone treatment also caused an increase to 7-fold in luteal tissue cholesterol esters: 2.2 ± 0.5 vs. 14.8 ± 1.1 µg/mg tissue, p < 0.05.
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Figure 5 shows the effect of dexamethasone treatment on sterol content of the nonluteal ovarian tissues. A small but significant increase in cholesterol content was observed in dexamethasone-treated animals: 2.8 ± 0.25 µg/mg tissue (dexamethasone-treated) vs. 2.0 ± 0.15 (vehicle-treated animals), p < 0.05. However, dexamethasone treatment failed to stimulate a significant accumulation of cholesterol esters: 12.4 ± 1.5 µg/mg tissue (dexamethasone-treated) vs. 9.3 ± 1.0 (vehicle-treated), p > 0.05.
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Experiment 2: Corticosterone Treatments; the Effect on Plasma 20-Dihydroprogesterone Concentration
The effect of corticosterone or placebo implants on plasma 20-dihydroprogesterone concentration is illustrated in Figure 6. The 100-mg replacement "low" dosage resulted in plasma concentrations of 20-dihydroprogesterone of 29.8 ± 3.8 ng/ml, compared to a mean concentration of 12.6 ± 3.4 ng/ml in the placebo-implanted rats, p < 0.05. The rats with the 200 mg "high"-level corticosterone implants also showed a significant increase in concentration of plasma 20-dihydroprogesterone, with a mean value of 33.0 ± 3.6 ng/ml, p < 0.05.
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Experiment 3: Detection of Glucocorticoid Receptor
The results of the experiment to identify the glucocorticoid receptor in rat ovarian tissues are illustrated in Figure 7. The radiograph shows a band of about 92 kDa, immunologically identified as the glucocorticoid receptor in both luteal and nonluteal ovarian tissues. The receptor band had mobility identical to that seen in liver.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydroprogesterone, which is a major steroid produced by corpora lutea of hypophysectomized rats [14, 27]. Accompanying the increased plasma progestin was an increase in luteal lipids detected microscopically with oil red O staining, and an increase in lipid droplets detected by electron microscopy. Measurement of cholesterol and cholesterol esters in isolated corpora lutea revealed marked enhancement of luteal sterols in the rats treated with dexamethasone. Therefore, from these observations we have concluded that one striking effect of dexamethasone treatment is a marked enhancement of lipid accumulation in the corpora lutea, associated with increased plasma concentration of 20-dihydroprogesterone, suggesting increased rates of steroidogenesis. This dosage of dexamethasone is in the range of dosages that would be classified as anti-inflammatory [16]. Therefore, we wanted to know whether the naturally occurring glucocorticoid, corticosterone, is capable of producing a similar effect on plasma 20-dihydroprogesterone concentration. Two dosages were used, one high [17] and the other approximating a replacement dosage of corticosterone in rats [18, 19]. Plasma 20-dihydroprogesterone concentration was elevated in rats to a similar extent with both corticosterone treatments, indicating the efficacy of corticosterone to stimulate luteal steroid production even at a lower dosage. The identification of glucocorticoid receptor protein in corpora lutea of hypophysectomized rats is consistent with a recent report of mRNA for glucocorticoid receptor in the rat corpus luteum [2] and with an earlier report of glucocorticoid receptor in rat ovary, on the basis of specific binding characteristics [1]. The receptor is an ~92-kDa protein, which is found in other tissues of the rat [28]. The identification of glucocorticoid receptor in the corpus luteum and the observed responses to glucocorticoids naturally lead to questions concerning the cellular mechanisms involved and the physiological significance of such observations. In previous reports [6–8, 10], the observed effects of glucocorticoids on luteal function were complicated by the presence of the pituitary and the consequent potential indirect effects mediated by pituitary hormones. In the present investigation, indirect effects of glucocorticoid administration on the ovary cannot be dismissed, yet the pituitary hormones were not involved. It is of interest to note that the nonluteal compartment of the ovary also expressed glucocorticoid receptor. The presence of glucocorticoid binding [1] and direct actions of glucocorticoid in rat granulosa cells [4, 5] suggest the potential for a role of glucocorticoids in the rat follicle. We entertain the possibility that glucocorticoid receptor might also be expressed in the stromal-interstitial tissues, although we have no direct data to support this. The nonluteal tissues did not respond robustly to dexamethasone treatment by increasing cholesterol ester accumulation as in the corpora lutea. However, the stromal-interstitial tissues of control (vehicle-treated) rats had relatively high lipid content, revealed by intensive oil red O staining.
The physiological significance of glucocorticoid action in the corpus luteum is not clear. In the present study, as well as in other reports [14, 15, 27], it is clear that the corpora lutea can survive and produce steroids in the absence of pituitary hormones, and presumably with greatly reduced corticosterone in the circulation [29]. The glucocorticoid receptor in the corpus luteum may be activated under normal circumstances by progesterone, which is known to bind the receptor [30, 31]. Although a progesterone receptor has not been found in the rat corpus luteum [12], either progesterone or dexamethasone has been shown to affect the expression of 20-hydroxysteroid dehydrogenase in rat luteal cells and in a rat luteal cell line [2]. The authors interpret these observations to suggest that the glucocorticoid receptor mediates these actions [2], consistent with the thesis by Rothchild [32] that progesterone has an autocrine action in the corpus luteum. Thus, our results presented here, the evidence that glucocorticoid can act directly in rat luteal cells, and the knowledge that the rat corpus luteum expresses 11ß-hydroxysteroid dehydrogenase type 2 [3] lead us to propose that the potential exists for a normal physiological action of glucocorticoid in this tissue. Such an action might be evident in regressing corpora lutea in which 20-dihydroprogesterone is a major secreted progesterone metabolite, as is the case in hypophysectomized rats [14, 27]. The increases in luteal cholesterol esters may be related to the increased production of progestins observed previously [13]. Recent studies also have provided a conceptual framework for potential molecular steps involved in the trophic effect of glucocorticoids. Russell et al. [33] have shown in rat luteal tissues that the trophic actions of prolactin involve the activation of signal transducers and activators of transcription (Stat), specifically Stat3 and Stat5. Also, Stoecklin et al. [34] reported that dexamethasone and prolactin can induce the activation of Stat5 in COS cells transfected with the prolactin receptor, the glucocorticoid receptor, and Stat5. These reports suggest that there are steps of signal transduction that are common to prolactin and to glucocorticoids. Further studies are warranted to explore the potential physiological actions and the mechanism of action of glucocorticoid in the corpus luteum.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: R. Towns, Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Michigan Medical School, 1331 E. Ann St., Room 5111, Ann Arbor, MI 48109-0580. FAX: 734 647 2307; rtowns{at}umich.edu
Accepted: March 17, 1999.
Received: January 27, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor. Endocrinology 1997; 138:4497–4500.-reductase activity in determining the responsiveness of hypophysectomized rats to ACTH. Endocrinology 1975; 96:1153–1157.[Abstract]2-macroglobulin (2M) promoter: relation to the expression of a 2M in the rat ovary. Biol Reprod 1996; 55:1029–1038.[Abstract]This article has been cited by other articles:
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  Z. Cai and C. Stocco Expression and Regulation of Progestin Membrane Receptors in the Rat Corpus Luteum Endocrinology, December 1, 2005; 146(12): 5522 - 5532. [Abstract] [Full Text] [PDF]
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 R. Sasson and A. Amsterdam Stimulation of Apoptosis in Human Granulosa Cells from in Vitro Fertilization Patients and Its Prevention by Dexamethasone: Involvement of Cell Contact and Bcl-2 Expression J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3441 - 3451. [Abstract] [Full Text] [PDF]
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 F. Gaytan, C. Morales, C. Bellido, and J. E. Sanchez-Criado Selective Apoptosis of Luteal Endothelial Cells in Dexamethasone-Treated Rats Leads to Ischemic Necrosis of Luteal Tissue Biol Reprod, January 1, 2002; 66(1): 232 - 240. [Abstract] [Full Text]
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