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Endocrinology Vol. 145, No. 9 4204-4212
Copyright © 2004 by The Endocrine Society

Regulation of Luteal Cell Big Stanniocalcin Production and Secretion

Mark Paciga, Gabriel E. DiMattia and Graham F. Wagner

Departments of Physiology and Pharmacology (M.P., G.F.W.), Biochemistry (G.E.D.), and Obstetrics and Gynecology (G.E.D.) and Faculty of Medicine and Dentistry, University of Western Ontario, and London Regional Cancer Center (G.E.D.), London, Ontario, Canada N6A 5C1

Address all correspondence and requests for reprints to: Dr. Graham F. Wagner, Department of Physiology and Pharmacology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1. E-mail: graham.wagner{at}fmd.uwo.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In mammals, the ovaries have the highest levels of stanniocalcin (STC) gene expression, most or all of which is confined to androgen-producing thecal-interstitial cells (TICs). Ovarian TICs also synthesize a different STC that consists of three high molecular weight species collectively known as big STC. Upon release in response to LH stimulation, TIC-derived big STC is sequestered locally by target cells, particularly steroidogenic cells of the corpus luteum, via a receptor-mediated process. Although there is little or no STC gene expression in luteal cells in the in vivo setting, this report describes how the gene is turned on, STC mRNA becomes readily detectable, and big STC is secreted when bovine luteal cells are cultured in vitro. STC gene expression and secretion were both positively regulated by activation of the adenylate cyclase/protein kinase A signaling pathway (forskolin and 8-bromo-cAMP). However, prostaglandin E2 was the only natural luteal cell ligand capable of replicating the effects of forskolin and 8-bromo-cAMP (LH had no consistent effect). Sex steroids such as 17ß-estradiol, androstenedione, and progesterone significantly decreased luteal cell STC expression and secretion. However, only androstenedione was capable of reducing STC production and secretion to undetectable levels. This report is the first to show that once removed from their normal context within the ovary, luteal cells are capable of synthesizing and secreting big STC. It is also the first to delineate the regulatory mechanisms involved in STC production and secretion by luteal cells. These results therefore suggest that under certain physiological conditions, the corpus luteum could very well serve as a source of STC production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A LACK OF information on cell signaling pathways and the receptor have seriously hampered our understanding of stanniocalcin (STC) function. Initially, it was assumed that STC in mammals would simply mimic the actions of its fish counterpart in the regulation of mineral homeostasis. And indeed, there is good evidence to support this view, because STC has been shown to control the movements of calcium and/or phosphate across transporting epithelia in mammalian kidney and gut (1, 2). Recently, however, it has become clear that STC has more widespread effects in mammals based on its tissue pattern of expression (3), transgenic studies in mice (4, 5), new information pertaining to the subcellular targeting of both ligand and receptor (6, 7), and the realization that there are several different ligands emanating from the STC gene (8). The structure of the STC receptor and the signal transduction pathways activated by ligand binding have not been elucidated. What is known, however, is that saturable, high affinity binding sites are present on the plasma membranes, outer and inner mitochondrial membranes, and cholesterol lipid droplets of the various cell types targeted by the ligand. These binding sites are specific for STC and presumably are responsible for the high degree of ligand sequestration observed in cells targeted by the ligand (6, 7). Studies of gain of function STC transgenic mice have shown that STC overexpression causes 1) 30–50% reductions in postnatal body size independently of GH or IGF-I, 2) embryonic growth restriction, 3) compromised female reproduction, and 4) deleterious effects on maternal lactation/nursing behavior (4). These findings have been independently confirmed in a second study, which revealed that STC transgenic mice consumed significantly more food and oxygen than their wild-type counterparts despite their smaller size (5). Collectively, the data suggest that STC has effects on metabolism, reproduction, and development in addition to affecting mineral homeostasis. Microarray studies have revealed how the expression patterns of the STC genes (STC and STCrp/STC2) can be correlated with pathologies such as cancer, hypoxia, inflammation, and viral infections (9). Collectively, these findings suggest that the STCs probably influence many different aspects of mammalian biology.

In pursuit of its more reproductive-related functions, we have been examining the role of STC in the ovary. The ovaries have by far the highest tissue levels of STC gene expression, virtually all of which is confined to the androgen-producing, secondary interstitial cells between developing follicles and theca interna cells surrounding growing and mature follicles, collectively known as thecal-interstitial cells (TICs) (3, 10). The ovaries are also unique in that they produce a number of high molecular weight STC variants that we refer to collectively as big STC. Unlike the 50-kDa form of the hormone found in most tissues (STC50), big STC comprises at least three proteins of 84, 112, and 135 kDa (8). Big STC also has a different subcellular targeting pathway. Unlike STC50, which is targeted to the inner mitochondrial matrix, TIC-derived big STC is targeted to the cholesterol lipid droplets of nearby luteal cells to suppress progesterone synthesis (7).

Intriguingly, the pattern of STC protein distribution within the ovary is markedly different from that of the transcript. As expected, TICs, which are the source of STC production, also contain high levels of STC protein. However, oocytes and cells of the corpus luteum that are essentially devoid of gene expression contain equally high levels of STC immunoreactivity (10). There are two possible explanations to account for this. The first is that luteal cells and oocytes do not express the gene and are simply targets of TIC-derived big STC, which they sequester in large amounts. This would be reminiscent of STC50 signaling in kidney nephrons, where collecting duct cells produce the hormone for targeting to upstream segments such as the distal convoluted tubules (6, 11). Indeed, there is good evidence to support this scenario, because big STC and its receptors have both been identified on the lipid storage droplets of small and large luteal cells (7). The second possibility is that luteal cells are, in fact, capable of STC production, but perhaps only under specific circumstances. It is conceivable that the isotopic in situ hybridization procedure we employed in the past to ascertain whether STC gene expression was confined solely to TICs may have been insufficiently sensitive to reveal lower levels of expression in luteal cells (3, 10). Moreover, because only approximately 50% of the corpus luteum is made up of small and large luteal cells (12, 13), isotopic in situ hybridization may have been similarly incapable of discriminating between signal emanating from steroidogenic cells and that from the equally pervasive endothelial cells.

Having characterized the targeting pathway and actions of big STC on luteal cell progesterone release, the purpose of this study was to address the possibility that under some circumstances, the cells might also be capable of STC production. In terms of possible approaches to this question, we reasoned that an in vitro setting employing highly purified luteal cells afforded the best opportunity for determining whether the cells were capable of STC synthesis. Remarkably, our findings revealed that the cells began to synthesize and secrete large amounts of STC upon removal from their context within the ovary, and that there probably are physiological conditions during which luteal cells serve as the source of the hormone in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Prostaglandin E2 (PGE2), PGF2{alpha}, H-89, forskolin, 1,9-dideoxyforskolin, 8-bromo-cAMP (8Br-cAMP), and all the steroid hormones used in this study were obtained from Sigma-Aldrich Corp. (St. Louis, MO). UltraCulture medium was purchased from BioWhittaker (Walkersville, MD; catalog no. 12-725F).

Bovine luteal cell (BLC) culture
Bovine ovaries were obtained from a local abattoir. The corpora lutea were dissected out of the ovary and washed several times in Hanks’ Balanced Salt Solution containing 0.1% BSA (Sigma-Aldrich Corp.). A highly pure population of dispersed large and small luteal cells was obtained by mincing the corpora lutea and subsequently digesting the tissue in an enzymatic mixture of collagenase type II (Invitrogen Life Technologies, Burlington, Canada) and deoxyribonuclease I (Roche, Laval, Canada) as previously described (14, 15). Cell viability was assessed by trypan blue exclusion. Cells were seeded at a density of 500,000 cells/ml in 24-well tissue culture plates and maintained at 37 C in UltraCulture medium supplemented with an antibiotic-antimycotic solution (Invitrogen Life Technologies). The proportion of steroidogenic cells was determined by 3ß-hydroxysteroid dehydrogenase (3ßHSD) enzyme biochemistry as previously described (16). The ability of cells to respond to hormone stimulation was assessed by measuring progesterone output in response to LH (obtained from Dr. A. F. Parlow, National Hormone and Pituitary Program, University of California-Los Angeles Medical Center, Torrance, CA). Experiments testing the effects of H-89, forskolin, 1,9-dideoxyforskolin, 8Br-cAMP, and steroid hormones on STC gene expression and secretion were always conducted on triplicate wells of cells at the concentrations and times indicated. The agents being tested were added to the cells from 100-fold concentrates, and controls always received equivalents volumes of solvent in lieu of hormone or test reagent.

Western blot analysis
Conditioned cell culture media were obtained as previously described (8). Protein concentrations were standardized using the Bio-Rad protein assay kit (Hercules, CA). After SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes (Roche) and incubated with polyclonal human STC (hSTC) antiserum (1:40,000), followed by horseradish-peroxidase-conjugated donkey antirabbit antibody (1:50,000; Amersham Pharmacia Biotech, Arlington Heights, IL), and subsequently developed with an enhanced chemiluminescence Western blotting detection kit (Amersham Pharmacia Biotech).

RIA
The RIA employed for measurements of medium STC levels employs a polyclonal antibody to recombinant hSTC and purified hSTC for tracer and standards. The assay has been extensively characterized for specificity, and there is no significant displacement of the tracer by a variety of polypeptide hormones at concentrations as high as 10 µg/ml (these included LH, FSH, human chorionic gonadotropin, placental lactogen, ACTH, PTH, TSH, prolactin, GH, follistatin, and STC2) (17). The sensitivity of the STC RIA was 0.2 ng/ml. For measurement of medium progesterone levels, we used a charcoal separation RIA employing a progesterone antiserum raised in rabbits (provided by Dr. Tom Kennedy, University of Western Ontario). The sensitivity of this assay was 0.1 ng/ml, and the antibody specificity for progesterone was previously determined (18). Both assays had coefficients of variation for intra- and interassay variability that were less than 10% and 14%, respectively. Conditioned medium was added directly to both assays for measurement of hormone content as previously described (8).

Northern blot analysis
Total RNA from corpora lutea as well as from purified luteal cells was isolated using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. The RNA was then resolved on 1% agarose/3% paraformaldehyde gels, and Northern blots were hybridized against a random-primed, 32P-labeled, 742-bp bovine STC-coding region-specific cDNA using Ultrahyb (Ambion, Inc., Austin, TX) as previously described (8).

Immunocytochemistry
Immunocytochemistry was carried out on paraformaldehyde-fixed, paraffin-embedded sections of bovine corpus luteum using a specific polyclonal antibody to hSTC as previously described (8).

Experimental design and statistical analysis
All experiments performed on cultured cells were replicated two to five times, in each case on different lots of cells. Within individual experiments, each treatment was performed on a minimum of three replicate wells of cells. The data from individual experiments were analyzed by ANOVA, and Dunnett’s test was employed for post hoc analysis. Statistical significance was assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
STC gene expression and STC protein distribution in intact bovine corpus luteum
To assess the levels of STC gene expression in the intact corpus luteum, Northern blot analysis was performed on freshly isolated bovine corpora lutea at various stages of the estrous cycle. The results showed that STC gene expression was undetectable in corpora lutea from early (<10 d), mid (10–15 d), and late (>15 d) stages of the estrous cycle (Fig. 1Go). In contrast, immunocytochemical analysis of tissue sections from bovine corpus luteum revealed the presence of immunoreactive STC in both large and small luteal cells (Fig. 2Go), similar to that observed in rodents (3).



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  		FIG. 1. Northern blot analysis of STC mRNA in bovine corpus luteum. Each lane contains 25 µg total RNA isolated from individual corpora lutea obtained from different animals harvested on four separate occasions at various stages of the estrous cycle. No STC gene expression was observed at any stage of the cycle (Early, <10 d; Mid, 10–15 d; Late, >15 d). The control lane contains approximately 5 µg total bovine thecal cell RNA, where a signal was detected after a 48-h exposure. No signal was detected in corpus luteum even after prolonged exposures. The Northern blot shown was repeated three times, with the same results obtained on each occasion.

 


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  		FIG. 2. Immunocytochemical localization of STC protein in bovine corpus luteum. A, Low magnification field of bovine corpus luteum after immunocytochemical staining for STC-immunoreactive cells. The same staining pattern was observed at all stages of the estrous cycle. B, Higher magnification of the box in A, showing STC-positive small (arrows) and large luteal cells (arrows). C, No staining was observed in control sections when the antiserum was preincubated with excess hSTC before use. This pattern of STC immunostaining was observed in tissue sections from corpora lutea harvested on at least three separate occasions.

 
Steroidogenic capacity and LH responsiveness of cultured BLCs
To determine whether BLCs were steroidogenically active, cells were monitored by 3ßHSD histochemistry, and the majority (>85%) were found to be 3ßHSD positive. To ensure that BLCs were still hormone responsive in vitro, cells were challenged with LH. As shown in Fig. 3Go, LH had concentration-dependent effects on progesterone (P4) output to a maximum of 4-fold in response to 100 ng/ml LH. Higher doses of LH had no greater effect. Hence, the BLC cultures were steroidogenically active and hormonally responsive.



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  		FIG. 3. Progesterone secretion by cultured BLCs. Cells were plated as described in Materials and Methods. To ensure that they were still functional and hormonally responsive, all BLC preparations were challenged with increasing concentrations of LH for 24 h. The P4 content of the medium was then assessed by RIA. Shown here is a typical response to LH by BLC cultures. This experiment was repeated at least three times. Each bar represents the mean ± SEM from three wells. *, P < 0.05; **, P < 0.01 (compared with controls, by ANOVA/Dunnett’s test).

 
STC production and secretion by cultured BLCs
Having established functional cultures of BLCs, we proceeded to examine STC gene expression in vitro. As shown in Fig. 4Go, whereas STC gene expression was barely detectable in BLC cultures at 6 and 12 h postplating, a dramatic increase in STC mRNA abundance occurred between 12 and 24 h (>2-fold) and continued to rise over the next 12 h (~0.3-fold increase). Thereafter, STC gene expression remained constant. This pattern of STC gene expression was observed in BLCs obtained from corpora lutea in all stages of the bovine estrous cycle (early, mid, and late; data not shown).



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  		FIG. 4. The induction of STC gene expression and secretion in cultured BLCs. Total RNA was isolated from cultured BLCs at various times postplating, and equal amounts were subjected to Northern blot analysis (~10 µg/lane). A, Representative Northern blot of STC and 18S RNA levels. Although STC gene expression was barely detectable at 6 and 12 h postplating, there was a significant induction in message levels by 24 and 36 h. B, Graphical display of the data in A. C, The pattern of STC secretion mirrored that of gene expression. Each bar in the data shown in C is the mean ± SEM of three replicate determinations. **, P < 0.01 compared with 6 h (by ANOVA/Dunnett’s test). These time-course experiments were conducted on three separate occasions. D, Molecular mass of secreted STC assessed by Western blotting. Luteal cells secreted big STC, consisting of the high molecular mass species of 84, 112, and 135 kDa.

 
Having discovered that STC gene expression was induced in vitro in BLCs, the possibility that synthesized STC was secreted was also explored. This was accomplished by measuring the accumulation of STC in culture medium by RIA (Fig. 4CGo). Under basal conditions, barely detectable levels of STC were present in the culture medium at 6 and 12 h postplating (0.25 and 0.32 ng/ml, respectively). By 24–36 h, however, the accumulation of STC in the culture medium had increased significantly (~1 ng/ml; P < 0.01). Thereafter, no further significant increase in the amount of STC secreted by luteal cells was observed. The molecular weight of secreted STC was also assessed by Western blotting to determine whether luteal cells synthesized STC50 or big STC. As shown in Fig. 4DGo, secreted STC consisted of three high molecular weight species of 84, 112, and 135 kDa. In a separate study, basal levels of STC and P4 secretion were monitored over time. In agreement with an earlier study showing that big STC functions as an inhibitor of luteal cell P4 release (7), the accumulation of P4 in the culture medium decreased in proportion to the rise in STC output (Fig. 5Go, A and B). Regression analysis revealed that there was a strong negative correlation between the two parameters (Fig. 5CGo; r2 = 0.83; P < 0.0001).



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  		FIG. 5. Basal P4 and big STC secretion by BLCs in culture. Cells were plated as described in Materials and Methods. P4 and big STC secretion were quantified by RIA at the times indicated. B, Stepwise increase in big STC secretion between 12 and 72 h; B, stepwise decrease in P4 secretion over the same time frame. Each bar represents the mean ± SEM of three replicate determinations. C, Strong negative correlation between the two parameters after linear regression analysis (r2 = 0.83; P < 0.0001).

 
Regulation of big STC secretion by cultured BLCs
We wanted to determine whether luteal cell big STC secretion was regulated by protein kinase A, as in the case of bovine TICs (8). Forskolin, an activator of adenylate cyclase, significantly enhanced secretion. As shown in Fig. 6AGo, low concentrations of forskolin (0.1 and 1 µM) had no effect on secretion compared with controls. This was in contrast to bovine thecal cells, which are sensitive to low concentrations of forskolin (8). However, luteal cell big STC secretion was significantly increased, approximately 2- and 3-fold by 10 and 100 µM forskolin, respectively. In time-course studies, stimulation by forskolin was evident by 24 h postplating and became more pronounced by 48–72 h postplating (Fig. 6BGo). The forskolin analog, 1,9-dideoxyforskolin, did not affect STC secretion (not shown). A similar effect on secretion was observed when cultures were treated with the cAMP analog, 8Br-cAMP. As shown in Fig. 6CGo, there was a dose-dependent increase in STC secretion in response to increasing concentrations of 8Br-cAMP (~1 ng/ml in control cultures compared with ~3 ng/ml in cells treated with 1 mM 8Br-cAMP).



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  		FIG. 6. Effects of forskolin and 8Br-cAMP on big STC secretion by cultured BLCs. Cells were plated as described in Materials and Methods. Big STC secretion in response to forskolin and 8Br-cAMP was assessed by RIA of the culture medium. A, Concentration-dependent effect of forskolin on big STC secretion after 24 h. B, The stimulatory effect of forskolin on luteal cell big STC secretion was evident by 24 h and persisted up to 72 h. C, The cAMP analog, 8Br-cAMP, had a similar effect on luteal cell STC secretion after a 24-h exposure. Shown here are representative data from one experiment, where each bar represents the mean ± SEM of three wells. *, P < 0.05; **, P < 0.001 (compared with controls). The forskolin and 8Br-cAMP dose-response experiments were both conducted on five separate occasions, whereas the forskolin time-course experiment was conducted twice.

 
STC expression and secretion in response to PGE2
In view of the consistently positive effects of adenylate cyclase activation on big STC secretion by cultured BLCs, we proceeded to test known regulators of luteal cell function that act via the adenylate cyclase/protein kinase A signal transduction pathway, including LH (0.1–1000 ng/ml), human chorionic gonadotropin, FSH, isoproterenol (all at 0.1–100 ng/ml), PGF2{alpha}, and PGE2 (both at 0.2–2000 ng/ml). However, the only ligand capable of consistently replicating the effects of forskolin and 8Br-cAMP proved to be PGE2, which had dose-dependent stimulatory effects on secretion between 2 and 200 ng/ml (Fig. 7AGo). The stimulatory effects of PGE2 were maximal by 24 h and persisted up to 36 h postplating (Fig. 7BGo). Interestingly, when P4 secretion was compared with that of big STC in PGE2-treated cells, a different pattern was evident (Fig. 7CGo). As observed above, P4 secretion by untreated cells decreased progressively over time. However, in response to PGE2 treatment, this decline in P4 secretion was significantly attenuated.



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  		FIG. 7. The effects of PGE2 on big STC and P4 secretion by BLCs. STC content in the medium was assessed by RIA after PGE2 treatment. A, PGE2 had dose-dependent stimulatory effects on STC secretion after a 24-h exposure. B, The stimulatory effects of PGE2 were evident by 24 h and persisted up to 36 h. C, PGE2 treatment attenuated the temporal decline in P4 secretion. D, The stimulatory effects of PGE2 on luteal cell STC secretion were abolished by H-89, a specific inhibitor of protein kinase A (10 µM for 24 h). Although H-89 by itself did not affect basal luteal cell STC secretion, it significantly reduced PGE2-stimulated STC secretion compared with that in untreated control cells. Each bar represents the mean and SEM of three wells. **, P < 0.01 (by ANOVA/Dunnett’s test). The PGE2 dose-response studies were carried out five times, whereas the time-course study was conducted twice. The study in C was also performed twice. Shown here are representative data from one experiment.

 
To further confirm the role of protein kinase A in regulating big STC secretion, BLC cultures were treated with PGE2 plus H-89 (10 µM), a selective inhibitor of protein kinase A. As shown in Fig. 7DGo, H-89 by itself did not have significant effects on STC secretion. However, STC secretion was significantly decreased in cultures treated with PGE2 plus H-89. In accordance with the secretion data, STC mRNA levels were elevated 3- to 4-fold in BLC cultures treated for 24 h with forskolin, 8Br-cAMP, and PGE2, (Fig. 8Go).



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  		FIG. 8. Effects of forskolin and 8Br-cAMP and PGE2 on luteal cell STC gene expression. A, Effects of forskolin (10 µM) and 8Br-cAMP (1 mM) on bovine luteal cell STC gene expression after a 24-h exposure. STC mRNA expression was significantly increased in response to both agents. Forskolin increased STC mRNA expression about 2-fold, whereas an approximately 3-fold increase was observed in response to 8Br-cAMP. B, Graphical presentation of the STC and 18S RNA levels shown in A. C, Northern blot showing the up-regulation of STC gene expression in response to PGE2 (200 ng/ml). The low levels of STC mRNA levels in untreated BLCs increased about 5-fold in response to PGE2 treatment. D, Graphical representation of the data in C. These experiments were conducted on two separate occasions. Shown here are representative data from one experiment.

 
Effects of steroids on luteal cell big STC secretion, and gene expression
The effects of locally produced ovarian factors (such as progesterone, androstenedione, and 17ß-estradiol; all tested at concentrations of up to 2 µM) and the glucocorticoid, dexamethasone (2 µM), were also tested for possible effects on luteal cell STC secretion. Whereas dexamethasone had no significant effect on secretion (not shown), P4, 17ß-estradiol, and androstenedione all significantly decreased big STC secretion by the cells (Fig. 9Go).



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  		FIG. 9. Sex steroids reduce luteal cell big STC secretion. Cells were plated as described in Materials and Methods. Cultures were treated with either UltraCulture medium containing ethanol (vehicle control) or UltraCulture medium containing P4, 17ß-estradiol (E2), or androstenedione (A). The medium STC content was assessed after 24 h by RIA. All steroids reduced big STC output, with progesterone having the least effect, and androstenedione the greatest effect. Each bar represents the mean and SEM of four wells. *, P < 0.05; **, P < 0.001 (vs. control). Shown here are representative data from one experiment that was repeated twice more.

 
Having identified steroids with inhibitory effects on secretion, we proceeded to examine whether STC gene expression was similarly affected. Remarkably, STC gene expression was significantly suppressed in BLCs treated with 2 µM androstenedione between 6 and 24 h postplating (Fig. 10AGo), whereas 17ß-estradiol and P4 had no significant effect (not shown). Big STC secretion was also markedly suppressed in response to androstenedione treatment (Fig. 10BGo; P < 0.01). Coincident with the suppression of STC production by androstenedione, there was a corresponding induction in progesterone output, as much as 7-fold by 24 h (Fig. 10CGo; P < 0.01).



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  		FIG. 10. Androgens suppress luteal cell big STC gene expression and secretion. Cells were plated and maintained in UltraCulture medium alone (control) or with 2 x 10–6 M androstenedione. RNA was extracted for Northern analysis at 6, 12, and 24 h postplating. Twenty-five micrograms of total RNA were loaded in each lane. The experiment was repeated four times. A, Representative Northern blot from one experiment. Androstenedione inhibited luteal cell STC gene expression at all times examined. B, STC secretion was also suppressed in cultures treated with androstenedione. Although basal STC secretion increased in control cultures over time, very little immunoassayable STC was detected in the medium of androstenedione-treated cells. C, P4 secretion was significantly increased by androstenedione treatment. Each bar represents the mean and SEM of three wells. **, P < 0.01 vs. control cells. D, STC secretion, compared with that by untreated cells (A), was inhibited in ascending order by 5{alpha}-androstan-3ß,17ß-diol (B), dihydroxytestosterone (C), 1-dehydrotestosterone (D), androsterone (E), testosterone (F), and androstenedione (G). The concentration of all steroids used was 2 µM.

 
Big STC secretion was also suppressed, albeit to lesser degrees, by 5{alpha}-androstan-3ß,17ß-diol, dihydroxytestosterone, 1-dehydrotestosterone, and testosterone (see Fig. 10DGo). Androstenedione caused the greatest suppression of basal STC secretion (~90% suppression compared with control cultures), whereas 5{alpha}-androstan-3ß,17ß-diol caused the least suppression (~33% suppression compared with control cultures).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study is the first to show that STC gene expression is induced when luteal cells are maintained in culture. Furthermore, the form of STC produced as a result proved to be big STC, comprising the same three high molecular weight species that have been previously identified in ovarian TICs (8). The production and release of newly synthesized hormone were also enhanced through activation of the adenylate cyclase/protein kinase A pathway. Finally, we found that sex steroids, in particular androgens, had highly suppressive effects on STC gene expression and secretion, suggesting that the close physical proximity of luteal cells to the androgen-producing TICs might be responsible for their lack of STC production in the in vivo setting (3, 10).

BLCs have proven useful in delineating the regulatory effects of big STC on P4 production and release. The addition of big STC to cultured BLCs produces concentration-dependent inhibitory effects on both basal and hormone-stimulated P4 output through a mechanism that involves the targeting of both ligand and receptor to the cholesterol/lipid storage droplets (7). However, it now appears that once removed from the ovary, luteal cells begin to synthesize and secrete big STC. The isolation of the cells appeared to be essential for gene induction, as the intact corpus luteum contained no measurable STC mRNA at any stage of the estrous cycle, and induction occurred as early as 6 h postplating.

Having discovered that luteal cells were capable of big STC synthesis, we explored the regulatory mechanisms involved. Interestingly, we discovered that LH was not the same regulator of STC production it proved to be in TICs (8). Whereas LH is a potent stimulator of TIC STC output, it was without effect on luteal cells even at concentrations above the physiological range (0.1–100 ng/ml). Instead, luteal cell STC release proved to be positively regulated by PGE2 through the protein kinase A pathway and negatively regulated by sex steroids. P4 and estradiol both suppressed hormone release, and interestingly, both are autocrine regulators of luteal cell function in vivo (19, 20, 21, 22, 23, 24). Hence, it is possible that these steroids tonically suppress luteal cell big STC synthesis in the intact ovary. Even more pronounced inhibitory effects were obtained with androgens. Of those tested, androstenedione, most likely acting via the androgen receptor (25), exerted the greatest inhibitory effect on big STC secretion and was the only steroid capable of reducing STC expression and secretion to undetectable levels. In vivo, the close proximity of luteal cells to the source of androgen production (TICs) might be responsible for the suppression of STC production, at least when androgen production is high. If true, this opens up the intriguing possibility that corpus luteal cells may, in fact, synthesize big STC in vivo under conditions where androgen production is low or naturally suppressed, such as the luteal phase of the estrous cycle or after parturition (26, 27). However, we must also consider the possibility that the androgens might be blood-borne and either ovarian or adrenal in origin. Finally, it is interesting to note that none of the sex steroids, including the androgens, has been shown to have any regulatory effect on big STC production and release by ovarian TICs (8). Thus, there is a clear difference in how the STC gene and its products are regulated in the different cell types.

The corpus luteum is formed from the mass of granulosa and thecal cells that undergo luteinization after ovulation, where granulosa cells are believed to give rise to large luteal cells, and thecal cells become small luteal cells (28, 29, 30, 31). Hence, in future studies it would be of interest to determine whether big STC synthesis occurs primarily in the smaller, thecal-lutein cells or the larger, granulosa-lutein cells. Because granulosa cells lack the capacity for big STC synthesis (3, 10), it is possible that big STC is only made in theca-derived luteal cells for subsequent signaling on small and large cells, both of which possess big STC receptors after luteinization.

In summary, we have demonstrated that after removal from their normal context within the ovary, luteal cells begin to synthesize big STC at rates comparable to those in TICs. A large part of the in vivo suppression may be due to thecal cell-derived androgens, whose main purpose until now has been to serve as an estrogen precursor. In view of these findings it may well be worth revisiting the luteal cell in the in vivo setting and exploring the possibility of there being physiological states in which the corpus luteum serves as a source of big STC production.


   Footnotes
 
This work was supported by a grant from the Canadian Institutes of Health Research and the Kidney Foundation of Canada.

Abbreviations: BLC, Bovine luteal cell; 8Br-cAMP, 8-bromo-cAMP; h, human; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; P4, progesterone; PGE2, prostaglandin E2; STC, stanniocalcin; STC50, 50-kDa form of stanniocalcin; TIC, thecal-interstitial cell.

Received December 1, 2003.

Accepted for publication June 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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