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Obstetrics and Gynaecology, Department of Reproductive and Developmental Sciences, University of Edinburgh (M.M., M.C.L., S.v.d.D., S.G.H., W.C.D.), and Royal Infirmary of Edinburgh (N.M., K.J.T.), Centre for Reproductive Biology, Queen’s Institute of Medical Research, Edinburgh EH16 4TJ, United Kingdom
Address all correspondence and requests for reprints to: Michelle Myers, Obstetrics and Gynaecology, The Queen’s Medical Research Institute Centre for Reproductive Biology, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: m.myers{at}sms.ed.ac.uk.
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Abstract |
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Introduction |
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Within the ovary, ovulation and the resulting folliculo-luteal transition has been likened to an inflammatory response, as a result of the acute hemodynamic, cellular, and biochemical changes that occur at the site of follicle rupture (5, 6). Key studies have clearly shown that the repetitive damage from consecutive ovulations must be quickly repaired in anticipation for the next ovulatory cycle, and locally produced glucocorticoids are involved (7, 8, 9). Cortisol, the most important glucocorticoid, is well known to minimize inflammatory damage to tissues by encouraging wound healing and subsequent repair. Clear evidence of this phenomenon is apparent from the switch of 11ß-hydroxysteroid dehydrogenase (11ßHSD) isoforms over the ovarian cycle, complemented by the increased concentrations of cortisol in follicular fluid after the LH surge (10). At present, both 11ßHSD type 1 (that tends to generate cortisol) and 11ßHSD type 2 (that tends to inactivate cortisol) isoforms, and their activities, have been reported in the ovaries of several species (11).
Throughout the human body, glucocorticoids are well known to exhibit a plethora of physiological roles. Although most of these actions have been best characterized in body systems such as kidney, liver, adrenal, and local inflammatory responses, influences on human fertility, oocyte maturation, and the establishment of a functional corpus luteum have been suggested (12, 13, 14, 15). It has also been reported that because glucocorticoids can inhibit endothelial cell proliferation (16), they may have a role in the local regulation of angiogenesis.
With the knowledge that glucocorticoids are involved in inflammatory-associated events in the ovary and may adversely affect angiogenesis, we hypothesized that local cortisol action may have a role in the luteolytic process. Although governed by different luteolytic mechanisms than in women, elegant studies in the rat corpus luteum have identified a potential role for 11ßHSD type 2 in the regressing corpus luteum (17). Therefore, based upon the findings in the rat study, coupled with much evidence for glucocorticoids in tissue and scar regeneration, we hypothesized that glucocorticoids were involved across the luteal life span. Therefore, the aims of the current study were to 1) investigate luteal glucocorticoid metabolism, reception, and subsequent regulation using carefully dated human corpora lutea and primary cell cultures of luteinized granulosa cells and 2) establish whether changes in cortisol synthesis and metabolism might be a key event in the regulation of the tissue remodeling associated with luteolysis.
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Materials and Methods |
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Isolation of human luteinized granulosa cells and fibroblast-like cells
The medical ethics committee separately approved the collection of cells from patients undergoing assisted conception. With patient consent, follicular fluid was collected from women undergoing transvaginal oocyte retrieval for in vitro fertilization after ovarian stimulation using a standard procedure (22). Isolation of luteinized granulosa cells using Percoll density gradient centrifugation was carried out as described previously (20, 23). Fibroblast-like cells were obtained from prolonged cultures of follicular aspirates as described previously (20).
Primary cell culture treatments
Pooled luteinized granulosa cells (100,000 per well of three to five patients) were cultured in 24-well plates precoated with Matrigel (BD Biosciences, Bedford, MA) in serum-free medium (supplemented DMEM/Ham’s F12 mixture), as described previously (20). Each pooled experiment for the following treatments was carried out at least three times to avoid biological bias.
Assessment of the acute effects of hCG and progesterone
Luteinized granulosa cells plated as above had fresh medium changed every 2–3 d over the course of culture, and treatment was carried out on d 6 or 7 of culture. The treatments groups were 1) controls with low-density lipoprotein (LDL, 50 mg/liter; Sigma-Aldrich Corp., Dorset, UK), 2) hCG (10 ng/ml; Serono, Welwyn Garden City, UK) with LDL (50 mg/liter), and 3) hCG (10 ng/ml) and LDL (50 mg/liter) in conjunction with 100 µM aminoglutethimide (Sigma-Aldrich). After 24 h, medium and cells were stored for steroid analysis and mRNA extractions, respectively. Progesterone concentrations in the culture media were determined by an in-house RIA as described previously (20).
Manipulation of hCG in prolonged cultures of luteinized granulosa cells
To mimic the luteal phase in primary cell culture, luteinized granulosa cells were plated as described above and grown for 12 d as described previously (22). Briefly, cells were stimulated with low-dose hCG (1 ng/ml) with LDL (50 mg/liter) beginning on d 2 and this was repeated every second day until d 7 when treatments were replaced with maximal doses of 100 ng/ml hCG/LDL or LDL alone. Cells were analyzed after 7 d with hCG and on d 12 in the presence or absence of hCG to mimic the progesterone secretion profile of late-luteal and luteal rescue stages, respectively (22).
Relative cell counts for various steroid and steroid inhibitor treatments
Pooled luteinized granulosa cells were cultured as above for 7 d in the presence of the same carrier ethanol concentrations in each well. The experiments were piloted to determine the appropriate concentrations of reagents, and final experiments were repeated three times in triplicate. The treatments were 1) control, 2) aminoglutethimide (100 µM), 3) RU486 (100 µM; Sigma), 4) RU486 (100 µM) with progesterone (500 µM; Sigma), 5) RU486 (100 µM) with cortisol (500 µM; Sigma), and 6) RU486 (100 µM) with hCG (100 ng/ml). After 7 d, the cells were removed by trypsinization, resuspended, and counted using a hemocytometer. Values were taken as the mean of at least four separate counts by an observer blinded to the treatments and related to controls.
Treatment of cultured fibroblast-like cells with cortisol
Cultures of fibroblast-like cells were derived as described above and transferred to 24-well plates at a concentration of 60,000–80,000 cells per well. After 6 h in serum-free culture, the medium was removed and replaced with medium containing either cortisol (100 nM) or an equivalent amount of the ethanol carrier as a control. After 24 h, the culture medium was collected for subsequent zymography, and the cells were used for mRNA extraction.
Preparation of cDNA from human corpora lutea and cultured cells
Messenger RNA was batch extracted from frozen human corpora lutea and reverse transcribed into cDNA using random hexamers as described previously (20). Luteinized granulosa cell mRNA was extracted using RNeasy mini-spin columns after lysis by the addition of RNeasy lysis buffer (QIAGEN, Crawley, Sussex, UK). Lysates were frozen until processed as per manufacturers’ protocols and then DNase treated with on-column DNase I (QIAGEN) and quantified using the NanoDrop ND-1000 Spectrophometer (NanoDrop Technologies, Wilmington, DE). mRNA was then reverse transcribed into cDNA using random hexamers (Applied Biosystems, Foster City, CA).
Quantitative analysis of gene expression by real-time PCR
Quantitative real-time PCR was carried out on the ABI PRISM 7700 sequence detection system (Applied Biosystems) using specific primers and probes (Eurogentec, Southampton, UK) for each gene of interest (Table 1
), and levels were related to a ribosomal 18S internal control (Applied Biosystems). Quantitative real-time PCR was performed with an extension temperature of 72 C and 30 cycles of amplification. All samples were performed in duplicate, and a relative comparison was made to an appropriate tissue control, either human placental or liver cDNA.
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Immunohistochemistry
Immunolocalization of glucocorticoid receptor (GR) was carried out using a rabbit polyclonal antibody (ABR; Cambridge BioScience, Cambridge, UK) in 5-µm paraffin tissue sections of human corpora lutea prepared on poly-L-lysine-coated microscope slides. These sections were dewaxed, rehydrated, washed in PBS, subjected to microwave antigen retrieval in 0.01 M citric acid (pH 6.0) for 10 min, and left to cool to room temperature. All sections were washed and placed in 3% H2O2/methanol for 30 min, followed by an avidin and biotin block and another block using normal goat serum (NGS; Diagnostics Scotland, Edinburgh, UK) diluted 1:4 in PBS containing 5% BSA [NGS/Tris-buffered saline (TBS)/BSA] for 1 h at room temperature.
GR antibody was diluted 1:2000 in blocking solution and incubated on sections overnight at 4 C. After rinsing, sections were incubated with the biotinylated goat antirabbit IgG (diluted 1:500 in NGS/TBS/BSA) secondary antibody (Dako Corp., Cambridge, UK) for 1 h. After washing, the sections were incubated in avidin-biotin complex-horseradish peroxidase (Dako), and binding was visualized by incubation with liquid 3,3'-diaminobenzidine tetrahydrochloride (Dako). Sections were counterstained lightly with hematoxylin to enable cell identification. Negative controls were performed identically to the above protocol with primary antibody incubations substituted with blocking serum containing nonspecific Igs at the same concentration. Images were captured using an Olympus Provis microscope (Olympus Corp. Optical Co., London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak Co., Rochester, NY), stored on an HP computer and assembled using Photoshop 7.0.1 (Adobe, Mountain View, CA).
Fluorescent immunohistochemistry
Sections were washed, subjected to antigen retrieval, and blocked as described above, and negative controls were performed as above. Rabbit anti-GR and rabbit anti-11ßHSD type 1 (Cayman/IDS Ltd., Bolton UK) diluted 1:100 in NGS/TBS/BSA were incubated on sections overnight at 4 C. Sections were washed, and slides were incubated with goat antirabbit IgG 488 (Dako) diluted 1:200 in PBS for 1 h.
Sections that were labeled with anti-GR were subjected to further colocalization experiments. Sections were reblocked with NGS/PBS/BSA for 1 h and then incubated with mouse monoclonal antibodies anti-CD31 (Dako; 1:20 in block), anti-CD68 (Dako; 1:20 in block), or anti-
-smooth muscle actin (-SMA, Dako; 1:500 in block) overnight at 4 C. Sections were washed and incubated with the fluorochrome streptavidin 546 Alexafluor (Molecular Probes, Eugene, OR) diluted 1:200 in PBS for 1 h.
Sections that were labeled with anti-11ßHSD type 1 were reblocked with normal donkey serum/PBS/BSA for 1 h and then incubated with sheep anti-11ßHSD type 2 (kind gift from Prof. Ian Mason, The University of Edinburgh, Edinburgh, UK) diluted 1:50 in donkey serum. Sections were washed and incubated with donkey antisheep peroxidase (Dako) 1:200 in normal donkey serum/PBS/BSA for 30 min before washing and incubating for 10 min with tyramide Cy3 (TSA plus cyanine 3 system; PerkinElmer Life Sciences, Boston, MA) diluted 1:50 in the supplied buffer to amplify the 11ßHSD type 2 immunostaining with red fluorescence.
Nucleic acids were labeled with To-Pro 3 and washed and mounted in Permafluor (Beckman Coulter, High Wycombe, UK). Fluorescent images were captured using an LSM 510 Axiovert 100M confocal microscope (Carl Zeiss Ltd., Welwyn Garden City, UK). Images of 11ßHSD type 1 and 11ßHSD type 2 were analyzed comparatively by standardizing the computer settings for each isoform. Therefore, the relative intensity of staining for each isoform corresponds to abundance of protein levels. All images were compiled using Photoshop 7.0.1 (Adobe Systems Inc., San Jose, CA).
Measurement of net 11ßHSD oxidoreductase activity
Interconversion of cortisone to cortisol via 11-oxoreductase activity was assessed in the presence and absence of hCG. Pooled luteinized granulosa cells were stimulated with either 100 ng/ml hCG in serum-free medium or serum-free medium alone for 24 h. Controls included incubations containing no cells with only Matrigel. After hCG stimulation, culture medium was discarded from wells and replaced with culture medium containing 100 nM cortisone substrate and 0.1 µCi [3H]cortisone to give a final volume of 500 µl/well. All incubations were in triplicate for 4 h at 37 C with 95% air-5% CO2. After incubation, medium was added to glass tubes containing 5-ml aliquots of dichloromethane and vortexed thoroughly. To separate the aqueous and organic phases, tubes were centrifuged at 12,000 rpm for 10 min. After the aqueous phase was removed, samples were evaporated to dryness under nitrogen gas at 45 C. Steroid residues were resuspended in 100 µl dichloromethane and samples along with one [3H]cortisol and one [3H]cortisone blot were transferred to silica gel-precoated plastic sheets for thin-layer chromatographic separation of precursor and product in the solvent system of chloroform-ethanol (92:8 by volume) (Merck, Haddeson, Hertfordshire, UK). Thin-layer chromatography plates were then scanned using a Bioscan System 200 detector (Lablogic Systems, Sheffield, UK), and corresponding peaks were analyzed for enzymatic activity in each sample and consequently each treatment group. Results are expressed as amount of cortisone converted to cortisol (picomoles) per hour.
Gelatin zymography
Cell culture medium was collected from serum-free cultures and subsequently frozen at –20 C. Aliquots of 200 µl were subjected to freeze drying for 2–3 h until they resembled a powder and then reconstituted in 20 µl sterile dH2O. One microliter of the reconstituted sample was added to sample buffer [10% (vol/vol) glycerol, 1% (wt/vol) SDS, and 0.04% (vol/vol) bromophenol blue] and applied to an 11% (wt/vol) polyacrylamide gel containing 1 mg/ml gelatin and 0.1% (wt/vol) SDS. Gels were incubated in 2.5% Triton X-100 for 45 min after electrophoretic separation of proteins and then incubated at 37 C overnight in digestion buffer [50 mmol/liter Tris-HCl (pH 7.6) containing 0.2 mol/liter NaCl, 5 mmol/liter CaCl2, and 0.02% (wt/vol) Brij 35] as described previously (2). Gels were stained in staining solution [30% (vol/vol) methanol, 10% glacial acetic acid, and 0.5% (wt/vol) Coomassie brilliant blue G250] and then destained in the same solution minus the Coomassie staining dye. The bands on the zymography gels reflect the activity of matrix metalloproteinase 2 (MMP-2) and were analyzed by transmission densitometry (G-700 densitometer; Bio-Rad, Hemel Hempstead, Hertfordshire, UK), and integrated software (Quantity One, Bio-Rad). All densitometry measurements were made between samples on the same gel or between gels run under identical conditions with a common control sample on each gel to ensure comparability.
Statistical analysis
Parametric statistics were used when the data were distributed normally, with appropriate tests highlighted in the figure legends. Differences were considered significant at P < 0.05 level.
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). Both isoforms of the cortisol-metabolizing enzyme 11ßHSD (type 1 and type 2) could be immunolocalized to the cytoplasm of granulosa-lutein cells in human corpora lutea (Fig. 1, B and C), although the staining intensity of 11ßHSD type 1 was much greater than that of 11ßHSD type 2. Specific immunostaining for each isoform could be detected in each corpus luteum analyzed from each stage of the functional luteal phase. No staining could be detected in negative control sections (Fig. 1B, inset). The corpus luteum of women therefore has the capacity for the local production and metabolism of cortisol.
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), but unlike the 11ßHSD isoforms, GR could be localized to nonsteroidogenic cell types. Dual-staining immunohistochemistry with -SMA demonstrated GR expression in stromal myofibroblasts and pericytes (Fig. 1, E and F). Dual immunostaining with CD68 and CD31 demonstrated specific nuclear GR expression in macrophages (Fig. 1G) and endothelial cells (Fig. 1H) in each corpus luteum examined. This pattern of GR immunostaining was detected in corpora lutea from each stage of the luteal phase. Various different cells in the human corpus luteum therefore have the capacity to respond to both systemic and locally produced cortisol in an autocrine, paracrine, or endocrine manner.
The effect of hCG on 11ßHSD and GR expression in primary cultures of luteinized granulosa cells
To determine whether the expression of GR and cortisol-metabolizing enzymes in steroidogenic cells could be acutely regulated, the effect of hCG was examined in primary cell cultures of human luteinized granulosa cells. The addition of hCG for 24 h resulted in a 30-fold up-regulation of 11ßHSD type 1 expression (P < 0.05, Kruskal-Wallis) (Fig. 2A). At the same time, 11ßHSD type 2 was down-regulated (P < 0.05, Kruskal-Wallis) (Fig. 2B), whereas GR expression was also up-regulated (P < 0.05, ANOVA) (Fig. 2C). To determine the functionality of the enzyme and the direction of 11ßHSD activity, 11-oxoreductase activity was assessed in the presence and absence of hCG. This confirmed that hCG stimulated reductase activity and acted to generate cortisol (P < 0.05, t test) (Fig. 2E).
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). Aminoglutethimide reduced progesterone (range 160–400 ng/ml) secretion to control levels (l70–200 ng/ml) in the presence of hCG (Fig. 2D). Although aminoglutethimide had no effect on hCG-stimulated up-regulation of 11ßHSD type 1 and GR, it inhibited the hCG-induced down-regulation of 11ßHSD type 2 (Fig. 2B). This suggests that progesterone may be involved in the local regulation of 11ßHSD isoform expression. Overall, these data suggest that cortisol metabolism and reception may be hormonally regulated in luteal steroidogenic cells.
The effect of chronic manipulation of hCG in cultures of human luteinized granulosa cells
To determine whether hCG could regulate the expression of 11ßHSDs and GR in more physiologically relevant prolonged culture conditions, 12-d cultures of luteinized granulosa cells, designed to mimic the luteal phase (20), were examined. Withdrawal of hCG in culture down-regulated 11ßHSD type 1 (P < 0.05, Kruskal-Wallis), whereas its expression was maintained in the presence of hCG (Fig. 3A). Conversely, hCG withdrawal had no effect on 11ßHSD type 2 expression, whereas maintenance of hCG did not alter its expression because the trend toward reduction did not reach significance (P > 0.05, Kruskal-Wallis) (Fig. 3B). The expression of GR showed a similar pattern to that of 11ßHSD type 1, but there were no significant changes detected (Fig. 3C) (P > 0.05, ANOVA). These data suggest that cortisol-metabolizing enzymes may continue to be differentially regulated by hCG under chronic conditions.
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). Exogenous hCG tended to up-regulate 11ßHSD type 1 (Fig. 4A) but tended to down-regulate 11ßHSD type 2 (Fig. 4B). There were no specific effects of hCG on the expression of GR in the corpus luteum of women (Fig. 4C). These data suggest that the differential regulation of cortisol metabolism in luteinized granulosa cells in vitro may also occur in the human corpus luteum in vivo.
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). This effect was not seen when progesterone synthesis was inhibited by aminoglutethimide (Fig. 5). This decrease in cell number in the presence of RU486 could be prevented by the addition of cortisol (P > 0.05, ANOVA) so that cell numbers were no different from controls (Fig. 5). Conversely, the addition of saturating concentrations of progesterone or hCG in the presence of RU486 did not increase the cell number beyond that of RU486 alone (P > 0.05, ANOVA) (Fig. 5). These data crudely suggest that cortisol may have direct actions on luteinized granulosa cells in vitro.
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). In vitro, our fibroblast-like cells express MMP-2 mRNA and proteolytic activity. The addition of exogenous cortisol inhibited both the expression (P < 0.001, t test) and activity (P < 0.0001, t test) of MMP-2 (Fig. 6, B and C). This suggests that cortisol may have the potential to have relevant effects on neighboring nonsteroidogenic cells.
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Discussion |
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It is uncertain what role cortisol has in the human corpus luteum. Roles for active glucocorticoids in the ovary have certainly been proposed and described during folliculogenesis and ovulation (11). Previous experiments in our laboratory demonstrated that hCG has the ability to regulate many different cell types and their molecular functions within the corpus luteum and highlighted the important role for locally produced intermediate regulatory molecules (18, 20, 21, 23). Herein, we propose that glucocorticoids may be regulatory molecules with signaling roles within the corpus luteum. We believe that hCG promotes the generation of active glucocorticoids that have a luteotropic role in the human corpus luteum such that the structural remodeling associated with luteolysis is inhibited and maintenance of early pregnancy is facilitated. Similarly, removal of local cortisol may facilitate luteal regression in the absence of hCG.
The 11ßHSDs catalyze the interconversion of active (cortisol) and inactive (cortisone) glucocorticoids by isoforms type 1 and 2, respectively. The temporal and spatial expressions of both isoforms have been documented in the ovary of other species (24, 25, 26, 27). It is well established that 11ßHSD type 2 is the predominant isoform during the follicular phase of the ovarian cycle, localized to the nonluteinized granulosa cells before the LH surge (26). This is an important concept because too much cortisol during the follicular phase is reported to disrupt FSH-stimulated granulosa cell development/function (and presumably estradiol production) (28), which would consequently inhibit successful folliculogenesis. In addition, during folliculogenesis, the predominant steroid is estradiol, and unlike progesterone (as discussed below), estradiol has a very low affinity for cortisol-binding protein (CBP) (29) and thus will not displace cortisol. Because it has also been reported that estradiol can increase the hepatic production of CBP (30), another mechanism may exist during the follicular phase to lower free cortisol. It seems that in the follicular phase, local cortisol generation and action tends to be inhibited.
This is not the case, however, in the periovulatory period. Once the dominant follicle is exposed to the midcycle preovulatory gonadotropin surge, the predominant isoform switches from type 2 to type 1 11ßHSD (26, 27). This phenomenon can also be seen by a rise of free cortisol that is 50 times higher in follicular fluid after the LH surge (10, 31), indicative that expression levels of the 11ßHSD enzymes in the ovary are an accurate measure for the direction of glucocorticoid biosynthesis. Indeed, it has been suggested that this process is involved in facilitating fertility. Some studies report that IVF patients with a higher cortisol to cortisone ratio in their follicular fluid have greater pregnancy success rates (32, 33), although others do not agree (31, 34). It is likely, however, that the switch in isoforms around ovulation has effects on the oocyte as well as regulating the inflammatory reaction associated with follicular rupture and its resolution (6). Such findings further support the hypothesis that 11ßHSD enzyme activities in the human ovary are developmentally and hormonally regulated (27).
The expression of 11ßHSD type 1 in luteinizing granulosa cells is maintained in the granulosa lutein cells of the corpus luteum. We also detected 11ßHSD type 2 in the corpus luteum. Type 2 11ßHSD mRNA expression and protein has previously been reported to be very low or undetectable in freshly isolated luteinized granulosa cells of both rats and humans (26, 35, 36, 37). This suggests that as the corpus luteum is formed there may be a recovery of 11ßHSD type 2 expression. Indeed, all our cultures of luteinized granulosa cells expressed 11ßHSD type 2, and they had been cultured for at least 7 d before analysis. Although 11ßHSD type 1 remains the most abundant isoform in luteinized granulosa cells in culture and in the corpus luteum, both isoforms are expressed. hCG inhibited the expression of 11ßHSD type 2 in vitro and tended to do the same in vivo. In contrast, hCG stimulated 11ßHSD type 1 expression acutely and in prolonged cultures and tended to do the same in vivo. The regulation of 11ßHSD type 1, like in the periovulatory period, seems to a direct effect of hCG signaling. However, as the inhibition of 11ßHSD type 2 by hCG was blocked when progesterone synthesis was inhibited, progesterone rather than hCG may be involved in the inhibition of 11ßHSD type 2 expression. This finding supports a similar experiment in granulosa cells by Thurston et al. (37) and demonstrates the same principles of progesterone actions observed in the kidney and placenta (38, 39, 40) whereby progesterone inhibits 11ßHSD type 2. Indeed, because progesterone receptor expression is down-regulated as the corpus luteum matures, this may be a mechanism for the reemergence of steroidogenic cell 11ßHSD type 2 expression in the corpus luteum (22).
The effect of hCG on 11ßHSD type 1 expression was mirrored by 11-oxoreductase activity levels and the generation of cortisol from cortisone. We are unable, however, to assess cortisol metabolism in the late luteal and rescued corpus luteum to confirm this in vivo. Although we believe that changes in the expression of different 11ßHSD isoforms in tissues informs the direction of cortisol/cortisone metabolism (27), it is clear that 11ßHSD type 1 has the potential to act as a bidirectional enzyme (given the appropriate coenzyme environment) (41). However, previous detailed studies on the direction of metabolism in luteinized granulosa cells (27, 37), ovarian surface epithelial cells (8, 9, 42), and other tissues (43, 44, 45) as well as the phenotype of 11ßHSD knockout mice (46) have suggested that in vivo 11ßHSD type 1 preferentially generates cortisol. However, as pointed out by Jonas et al. (47), recent studies have established that the net direction of 11ßHSD type 1 is dependant upon the cell availability of hexose-6-phosphase-dependent NADPH, which may be different in highly steroidogenically active tissues and the direction of cortisol metabolism in vivo remains to be studied.
The human corpus luteum has the potential to respond to locally generated cortisol because it expresses nuclear GR. Indeed, GR has been localized previously to many cell compartments in the ovary (36). Although the primary receptor for cortisol is GR, cortisol also has a high affinity for the mineralocorticoid receptor. We did not analyze the expression of mineralocorticoid receptor, but it has been reported to be expressed in the ovary (36). We have localized the expression of GR to the nuclei of steroidogenic cells. These cells of the human corpus luteum are also reported to express other important nuclear steroid receptors such as estrogen and progesterone receptors (48, 49, 50). It is unclear whether other steroids can influence glucocorticoid signaling by receptor-dependent mechanisms, but it is likely that cortisol has direct effects on the cells expressing the 11ßHSD enzymes involved in its synthesis and metabolism.
It is not clear whether the expression of GR in the corpus luteum is regulated. We have shown that steroidogenic cell progesterone receptors are differentially regulated in the corpus luteum across the luteal phase (22), although their role has not yet been elucidated (51). In contrast, we could see no obvious changes in steroidogenic cell GR immunostaining across the luteal phase. Indeed, in the endometrium, where glandular progesterone receptor expression in the secretory phase shows changes similar to that in luteal steroidogenic cells (22, 52), there was no change in GR expression across the functional menstrual cycle (53). However, in vitro hCG tended to up-regulate GR expression similar to its effects on 11ßHSD type 1. Whether GR expression in corpora lutea is hormonally regulated but masked by detection methods is not entirely clear. What is clear is that multiple cell types in each corpus luteum express nuclear GR.
Luteal endothelial cells and macrophages express nuclear GR. Protein colocalization of GR with CD31 demonstrates specific nuclear staining of endothelial cells. The effect of cortisol on luteal endothelial cells is not clear, but glucocorticoids have been shown to suppress angiogenesis (54), and this is most notably due to the suppression of vascular endothelial growth factor action (54, 55). In the human corpus luteum, however, there continues to be some angiogenesis stimulated by hCG during luteal rescue (56) in response to the marked up-regulation of vascular endothelial growth factor after exogenous hCG (57). Immune cells, most notably macrophages (CD68-positive cells), which also express GR, accumulate in the corpus luteum during luteolysis and show a marked reduction in number during hCG-induced luteal rescue (1). The role of cortisol in luteal macrophage accumulation and action is not clear, but because glucocorticoids are known to regulate cytokine signaling and macrophages in both health and disease (58), it may affect the immune cell-mediated processes during luteolysis. Luteal myofibroblasts are known to express macrophage chemoattractant protein-1 (59), and these cells also express nuclear GR.
When considering a role for glucocorticoids in the corpus luteum, it is very important to establish the relationship between cortisol and the marked excess of structurally related progesterone. Cortisol exists in one of two forms, bound and free, that regulate its bioavailability. In most systems, the majority of the steroid is bound to plasma proteins (notably CBP) with only a fraction free and unbound (12). Although CBP has the highest affinity for cortisol of all the binding globulins, other steroids such as progesterone and particularly 17OH-progesterone have high binding affinities to CBP (12, 29). Therefore, very high concentrations of progesterone and progesterone metabolites (such as in the corpus luteum) will displace cortisol from CBP, that acts as a buffer reservoir, resulting in the environment becoming enriched with free cortisol. This scenario is known as the free hormone hypothesis (12, 29) and predicts that the bioactivity of cortisol is proportional to free hormone concentrations and not total concentration, which includes the protein-bound fraction. This is an important paradigm to consider because the concentration of free biologically active cortisol in preovulatory follicular fluid is 10 times greater than that of serum (31). It is likely that, because of high local progesterone concentrations, rather than being mainly bound to CBP in the corpus luteum, locally generated cortisol is more likely to be free and functional at lower concentrations.
It is still not clear what effects cortisol has on luteal cells. We hypothesized that it may affect the survival of luteinized granulosa cells. Our observations and those of others (60) suggested that RU486 reduced survival of these cells in culture. We used simple cell counting to document the effect on RU486 on cell survival. Because cells treated with the progesterone synthesis disruptor aminoglutethimide, which blocks P450 side-chain cleavage, showed no change in their morphology or viability, we tested whether the effect of RU486 could be reversed by hCG or exogenous progesterone. Unlike a previous study using human luteinized granulosa cells, under slightly different conditions (60), we could not fully reverse the RU486 effects using progesterone. However, we were able to do so using cortisol. It is not clear whether this effect is at the level of the receptor because the concentration of cortisol would not be expected to fully displace the RU486. It may be that cortisol affects the cell susceptibility to RU486 in different ways. There may be specific effects of RU486 not mediated by hormone antagonism. Direct actions of RU486 have been reported in ovarian epithelial cancer cells and human endometrium, whereby it down-regulated molecules involved in signal transduction pathways by cytokines, growth factors, and other physiological stimuli that control cell functions (61). Indeed, in cultured luteinized granulosa cells, cortisol and dexamethasone offer protection against serum deprivation and induced apoptosis by bcl-2 and TNF- (62, 63) through mechanisms that include stabilization of the actin cytoskeleton (64). Whatever the mechanism of action, our results crudely suggest that cortisol may have direct effects on luteal steroidogenic cells. Indeed, if cortisol has any direct genomic effects in vivo, they are more likely to promote, rather than inhibit, steroidogenic cell survival and function.
If cortisol does have luteotropic actions, it may also have specific actions on the nonsteroidogenic cells of the corpus luteum that are key regulators of tissue remodeling during luteolysis. Luteal fibroblasts are the main source of MMP-2, a key proteolytic enzyme involved in tissue remodeling associated with luteolysis in women (2) and in many other species (65, 66, 67). Both primates (67) and women (2) show maximal MMP-2 expression in the late luteal phase. However, during maternal recognition of pregnancy, MMP-2 production is considerably reduced (2), suggestive that hCG is regulating the enzymatic expression through intermediate molecules (18). Furthermore, it is tempting to speculate that active glucocorticoids may also prevent luteolysis by inhibition of intraluteal prostaglandin synthesis (68). Although this is an attractive suggestion, the actual role of prostaglandins in the human corpus luteum remains elusive.
In the present study, our novel findings suggest that cortisol may be involved in paracrine interactions that control tissue remodeling. Recently, an in vitro study from our laboratory modeling the human corpus luteum has shown that activin A is a paracrine factor secreted from luteinized granulosa cells that may up-regulate fibroblast MMP-2 secretion (23) in the absence of hCG. In contrast to activin A (23), cortisol treatment of luteal fibroblast-like cells in culture resulted in a reduction in the production of MMP-2, a pattern reflecting MMP-2 expression in exogenous hCG-rescued luteal tissue (2). Indeed, glucocorticoids decreased MMP-2 activity in rat aortic smooth muscle cells (69) and in a fibrosarcoma cell line (70). It seems that the nonsteroidogenic cells forming the corpus luteum have the ability to directly respond to cortisol. If these effects do occur in vivo, it is likely that cortisol tends to inhibit rather than stimulate the remodeling associated with luteolysis and may therefore be considered to be luteotropic in nature.
We hypothesized that during luteolysis, more cortisol is generated in the local environment, consequently preventing aberrant scarring to the tissue. It is clear, however, from the present study that luteolysis is not associated with an increase in cortisol, and the opposite is true. We have shown that hCG tends to generate cortisol by up-regulating 11ßHSD type 1 and down-regulating 11ßHSD type 2. We have shown that the corpus luteum has the potential to react to this cortisol and that the effect on steroidogenic and neighboring cells tends to be luteotropic rather than luteolytic. In summary, our observational and interventional in vivo and in vitro models have generated results that suggest that cortisol tends to be withdrawn during luteolysis and maintained during luteal rescue. Glucocorticoids may have a role in the local luteal regulation of maternal recognition of pregnancy in women.
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Acknowledgments |
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Disclosure Statement: The authors of this manuscript have nothing to declare.
First Published Online September 13, 2007
Abbreviations: CBP, Cortisol-binding protein; GR, glucocorticoid receptor; hCG, human chorionic gonadotropin; 11ßHSD, 11ß-hydroxysteroid dehydrogenase; LDL, low-density lipoprotein; MMP-2, matrix metalloproteinase 2; NGS, normal goat serum; TBS, Tris-buffered saline.
Received June 5, 2007.
Accepted for publication September 4, 2007.
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determined by microarray analysis. J Endocrinol 183:19–28 and ß mRNA in corpus luteum of human subjects. Mol Hum Reprod 5:17–21 in the human corpus luteum. Mol Hum Reprod 7:137–145This article has been cited by other articles:
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