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Departments of Physiology (M.P., A.J.W., G.F.W.), Oncology (G.E.D.), and Obstetrics and Gynecology (A.J.W., G.E.D.), 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, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1. E-mail: graham.wagner{at}fmd.uwo.ca.
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Abstract |
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Introduction |
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In a previous study we examined ovarian STC expression during estrus, pregnancy, and lactation in the mouse to determine whether production changed in accordance with specific reproductive events (8). While there were no changes over estrus, steady-state levels of STC mRNA rose dramatically over the course of gestation (15-fold). Expression peaked between d 10–14 of pregnancy and then dropped sharply at parturition (8). The levels of expression increased once more postpartum, but interestingly this second rise was entirely dependent on the presence of a suckling litter. Serum STC levels in the maternal circulation rose and fell in concert with ovarian STC gene expression (8). As STC is normally undetectable in mammalian serum, this suggested that ovarian STC was capable of signaling systemically during pregnancy and lactation. Collectively, these findings suggested that in addition to its role in mineral metabolism, mammalian STC had evolved into an endocrine modulator of female reproduction.
The induction of ovarian STC gene expression during gestation and lactation likely involves other hormones. Identifying these hormones and their signaling pathways would enhance our understanding of how ovarian STC production is regulated and provide clues about which aspects of female reproduction are regulated in turn by STC. Therefore, the goal of this study was to establish viable cultures of thecal-interstitial cells (TICs) to address these questions. To our knowledge, this study is the first to show that TICs are capable of maintaining a high level of STC gene expression in vitro. More importantly, it shows that human (h) chorionic gonadotropin (CG) signaling via the protein kinase A (PKA) pathway is the principal regulator of ovarian STC production and secretion. Finally, this study reveals that ovarian STC is physically different from the 50-kDa protein found in most other tissues.
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Materials and Methods |
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Rat and bovine TIC cultures
Immature (25-d-old) female Sprague Dawley rats were obtained from Charles River Laboratories, Inc. (St. Constant, Canada). The animals were injected sc with 17ß-estradiol (1 mg/0.3 ml) in sesame oil for 3 consecutive days starting on d 28 of age. On the day after the last injection, the animals were killed by CO2 asphyxiation. All treatments and procedures were performed in accordance with the guidelines set by the Canadian Council on Animal Care. The ovaries were harvested and placed in ice-cold medium 199 (supplemented with 25 mM HEPES, 2 mM L-glutamine, and 1 mg/ml BSA). After removing the surrounding fat pad, follicles were punctured with a hypodermic needle to eliminate granulosa cells. Subsequently, the ovaries were minced and digested in an enzymatic mixture of collagenase type II and deoxyribonuclease I as previously described (10, 11). An enriched population of TICs was obtained by subjecting the resulting suspension of dispersed ovarian cells to Percoll density gradient centrifugation (12). Rat TIC viability and purity were assessed by trypan blue exclusion and immunocytochemistry, respectively. Cells were seeded at a density of 0.4 x 106 cells/ml and maintained at 37 C in McCoy’s 5a medium supplemented with L-glutamine (2 mM), BSA (1 mg/ml), and antibiotic-antimycotic solution (Life Technologies, Inc.) or UltraCulture medium supplemented with antibiotic-antimycotic solution.
Bovine ovaries were obtained from a local abattoir. Follicles (2–8 mm) were aspirated with an 18-gauge needle as described previously (13). After removal of the oocyte-cumulus complex, the remaining cell suspension was washed several times in 0.1% BSA in Hanks’ balanced salts. A highly enriched population of TICs was obtained by Percoll density gradient centrifugation as previously described (14). Bovine TIC purity was also assessed by immunocytochemistry as described below. Bovine cells were seeded at a density of 0.4 x 106 cells/ml and were maintained at 37 C in UltraCulture medium. The addition of hormones and reagents to both types of cultures were made from 100-fold concentrated stocks.
Histochemical characterization of isolated TICs
Immunocytochemistry was used to determine the purity of rat and bovine TICs after Percoll-gradient purification. Immunocytochemistry was performed using the following antisera: mouse antivimentin (Novocastra Laboratories, Newcastle Upon Tyne, UK), mouse anticytokeratin (DAKO Corp., Mississauga, Canada), rabbit antifactor VIII (Novocastra Laboratories), rabbit anti-P450 17
-hydroxylase-C17,20-lyase (CYP17; provided by Dr. Anita Payne, Stanford University School of Medicine, Stanford, CA), and rabbit antihuman stanniocalcin (hSTC) (7, 9, 15). Freshly isolated TICs were centrifuged in a Cytospin apparatus (Shandon, Inc., Pittsburgh, PA) and fixed in 4% paraformaldehyde (in PBS, pH 7.5) for 10 min at room temperature. After blocking endogenous peroxidase activity (0.3% hydrogen peroxide in methanol) and nonspecific binding (10% normal goat serum in PBS), cells were incubated overnight at 4 C with primary antiserum. The following day, cells were washed and incubated with either biotinylated goat antimouse IgG or biotinylated goat antirabbit IgG secondary antiserum. The biotinylated secondary antibodies were visualized with the Vectastain ABC peroxidase detection system (Vector Laboratories, Inc., Burlingame, CA). Cells were then counterstained with hematoxylin, dehydrated, and mounted.
In addition to immunocytochemical characterization, the proportion of steroidogenic TICs obtained was monitored by 3ß-hydroxysteroid dehydrogenase enzyme biochemistry as previously described (16, 17).
Northern analysis
Total TIC RNA was extracted using TRIzol reagent (Life Technologies, Inc.) and was resolved on 1% agarose/3% paraformaldehyde gels. Northern blots were hybridized against a random primed 32P-labeled mouse STC cDNA (7) or a 742-bp bovine STC-coding region-specific cDNA using ULTRAhyb hybridization buffer according to manufacturer’s instructions (Ambion, Inc., Austin, TX). Autoradiography was performed using Kodak BioMax MS film with a BioMax TranScreen-HE intensifying screen (Eastman Kodak Co., Rochester, NY), followed by densitometric quantification of the STC mRNA signal. To standardize the STC mRNA signal, blots were subsequently probed with a 32P-labeled cDNA encoding 18S ribosomal RNA (7). The results were expressed as STC/18S RNA ratios.
RIA
Conditioned media were collected from TIC cultures at various times after treatment, centrifuged at 12,000 x g for 15 min, transferred to clean microcentrifuge tubes, and stored at -20 C until assay for STC and P4 content by RIA. The sensitivities of the STC and P4 RIAs were 0.2 and 0.1 ng/ml, respectively, and both assays were highly specific for their respective ligands (15, 18).
Western blot analysis
Conditioned TIC culture medium (48 h) was filtered through an 0.8-µm pore size syringe filter and concentrated using a Centricon YM-30 centrifugal filter device (Millipore Corp., Bedford, MA). Intracellular TIC protein was obtained by homogenizing cells in Hanks’ balanced salt solution containing 0.5 mM EDTA and 1 mM phenylmethylsulfonylfluoride, followed by centrifugation at 500 x g, and filtering the resulting supernatant through an 0.8-µm pore size syringe filter. Protein concentrations of media and cell lysates were standardized using the Bio-Rad Laboratories, Inc., protein assay. After SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes (Roche, Montréal, Canada) and incubated with polyclonal hSTC antisera (1:40,000), followed by horseradish peroxidase-conjugated donkey antirabbit antibody (1:50,000; Amersham Pharmacia Biotech, Baie d’Urfe, Canada), and subsequently developed with an ECL Western blotting detection kit (Roche and/or Amersham Pharmacia Biotech). Recombinant hSTC (Human Genome Science, Rockville, MD) was used as a positive control for Western blot analysis and for preabsorbing the primary antiserum.
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Results |
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95% for both bovine and rat). On the other hand, contamination by granulosa cells and endothelial cells was minimal (<5%, as detected by cytokeratin and factor VIII immunostaining). The pattern and proportion of vimentin-positive immunostaining also corresponded well to that of STC and CYP17, as both rat and bovine TICs were 90–95% immunoreactive for STC and CYP17. In addition, 66–75% of isolated TICs were routinely 3ß-hydroxysteroid dehydrogenase positive, indicating that the majority of cells were steroidogenically active.
Both rat and bovine TIC cultures responded to LH stimulation with increased P4 secretion, as shown in Fig. 1
. Under basal conditions, bovine TICs secreted 2.2 ± 0.5 ng/ml P4 over 24 h (mean ± SEM), and secretion was increased 3-fold to 6 ± 1 ng/ml in the presence of LH (10 ng/ml). Treatment of rat TICs with LH produced a similar increase in P4 secretion (Fig. 1). These observations are in agreement with previous data (20, 21, 22) and confirmed that the TIC cultures were functional and hormone responsive.
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shows how 10 µM forskolin caused a 4-fold increase in STC secretion after 24 h, whereas the biologically inactive forskolin analog 1,9-dideoxyforskolin (10 µM) had no effect. Interestingly, the use of UltraCulture, an enriched serum-free medium, also significantly enhanced STC secretion from rat TIC cultures (1.6 ± 0.08 ng/ml; P < 0.01) compared with that from cells maintained in McCoy’s 5a medium without serum (0.6 ± 0.08 ng/ml). Figure 2B shows that P4 secretion by rat TICs was also augmented by forskolin and UltraCulture medium compared with that by nontreated cultures in McCoy’s 5a medium. Similar effects were obtained for STC gene expression. Rat TICs treated with forskolin in McCoy’s 5a and those maintained in UltraCulture medium had 7-fold higher steady state levels of STC mRNA compared with TICs in McCoy’s 5a medium alone (Fig. 2C).
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, A and B). Steady-state levels of STC mRNA increased 2-fold between 12–36 h and 2-fold again between 36–60 h post plating. In contrast, the rate of STC output was relatively constant over the same time frame, suggesting that under basal conditions STC production and secretion rates were not necessarily correlated (Fig. 3C).
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shows that over a period of 24 h, forskolin significantly increased STC secretion by bovine TICs at all doses tested (1, 10, and 100 µM). Stimulation was evident by 6 h and persisted for up to 24 h (Fig. 4B). In contrast, the biologically inactive analog 1,9-dideoxyforskolin had no effect at similar doses (1, 10, and 100 µM; results not shown).
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, C and D). This was also true in the case of LH. In the end, the only ligand capable of replicating the effects of forskolin on STC secretion proved to be hCG. Figure 5A shows that after a 24-h exposure to 1 ng/ml hCG, STC secretion increased from 1.4 ± 0.03 ng/ml in nontreated cultures to 2.2 ± 0.9 ng/ml. Exposure to 5 and 10 ng/ml hCG further increased the release of STC to 2.5 ± 0.09 and 3.7 ± 0.1 ng/ml, respectively. Higher hCG doses (100 ng/ml) had no greater effect, indicating that 10 ng/ml hCG was a near-maximal dose for inducing STC release.
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, IMBX alone had no effect on basal STC secretion. However, in the presence of IBMX, hCG-stimulated STC secretion was enhanced ever more than by hCG alone (P < 0.05).
Steroidogenesis, as measured by P4 accumulation in the culture medium, increased in a manner similar to that of STC. Figure 5B shows that nontreated bovine TIC cultures released 28 ± 2 ng/ml P4 over 24 h. As expected, treatment with increasing concentrations of hCG significantly enhanced P4 release, reaching a peak of 75 ± 1.7 ng/ml in the presence of 10 ng/ml hCG. However, in contrast to STC, IBMX alone increased P4 secretion almost 2-fold to 44 ± 2.5 ng/ml. IBMX also augmented hCG-induced P4 output.
When the STC and P4 data from Fig. 5 were regressed, there was a strong and statistically significant, positive correlation between the two variables. The correlation was weakest at the lowest does of LH and IBMX (Fig. 6A; r2 = 0.71) and became increasingly strongly at the medium (Fig. 6B; r2 = 0.81) and high (Fig. 6C; r2 = 0.84) doses. In all cases, these correlations were highly significant (P < 0.001). Interestingly, the same two variables were poorly correlated in nonstimulated cells (Fig. 6D; r2 = 0.046; P = 0.12).
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). However, the addition of H-89 not only suppressed hCG-stimulated STC secretion, but also significantly decreased basal STC release by approximately 50% compared with the controls. Figure 7B contains the corresponding P4 secretion data by the cultures. At all times examined (6, 12, and 24 h), the addition of hCG significantly increased the rate of P4 secretion by TICs compared with that in control cultures. However, in contrast to STC, H-89 had little or no effect on basal or hCG-stimulated P4 secretion.
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shows, however, that under nonreducing conditions, TIC-derived STC consisted of three higher molecular mass bands of 84, 112, and 135 kDa. The same banding pattern was obtained for both stored and secreted forms of the hormone. Furthermore, staining of all three bands was abolished when the primary antiserum was preabsorbed with hSTC (1 µg/ml; results not shown). Interestingly, STC derived from the human fibrosarcoma cell line HT1080 and the mouse corticotrope cell line AtT-20 produced the same banding pattern. Under reducing conditions, TIC-derived STC migrated as a 45-kDa band compared with the 27-kDa band for recombinant STC (Fig. 8B). However, unlike recombinant STC, treatment of TIC-derived STC with PNGase F to remove N-linked carbohydrate moieties did not produce a downward shift in molecular mass (Fig. 8C). Nonetheless, both TIC- and HT1080-derived STC exhibited parallelism in the RIA, indicating that the material displacing the [125I]hSTC tracer was immunologically indistinguishable from recombinant 50-kDa hSTC (Fig. 8D).
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Discussion |
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Identifying the functions of ovarian STC will enhance our overall understanding of female reproduction, and in this report we sought to determine how its production and release are regulated. This first required the development of a primary culture system for TICs in which STC production was sustained, a task that was aided in large part by the extensive literature on rat and bovine TIC primary cultures (10, 11, 12, 14, 22, 23, 24, 35, 36). Immunocytochemical analysis indicated that the majority of our rat and bovine TICs were vimentin positive and that contamination by granulosa and endothelial cells was minimal, in agreement with previous studies (19, 37). Furthermore, the proportion of vimentin-positive cells corresponded well to those that were STC and CYP17 positive. As CYP17 is localized to TICs in both rat and bovine ovaries (36, 38, 39), these results clearly indicated that the majority of cells were thecal in origin. To ensure that the cells were functional, we assessed their responsiveness to LH stimulation. In this case both rat and bovine TICs showed increased P4 production in response to LH stimulation, also in agreement with previous studies (10, 11, 20, 21, 22, 35). Although the LH steroidogenic response was similar in rat and bovine cells (3-fold), the rat response may have been muted by estrogen priming, as estrogens are potent down-regulators of CYP17 (40). However, estradiol had no effects on STC expression and secretion by rat or bovine cells.
Typically rat TICs maintained in McCoy’s 5a medium appeared healthy, continued to express the STC gene, and responded well to LH stimulation. However, cells maintained in UltraCulture medium had greater STC output and higher basal levels of STC expression. UltraCulture also proved to be a superior cell attachment and growth medium for bovine TICs. To our knowledge UltraCulture has not been used previously on TICs, so it remains to be seen whether other pathways are affected. Our results also indicated that not only was STC gene expression in both rat and bovine TICs cell autonomous, but STC production by bovine TICs actually increased over time in vitro. The consistency in STC/18S RNA ratios indicated that this was not due to an increase in cell number, suggesting that endogenous factors may exert inhibitory effects on TIC STC gene expression in vivo. Although STC gene expression increased over time, the rate of STC secretion was constant after 12 h post plating, suggesting that under basal conditions STC production and release are differentially regulated.
Previous studies have shown that ovarian STC gene expression is subject to regulation by hormone manipulations in vivo. Using a mouse model of superovulation, we have seen transient inductions in expression after hCG administration (2.5-fold) suggestive of STC regulation by LH (8). To determine whether these effects might be direct, we treated TIC cultures with LH and hCG, both of which bind to the LH receptor and ultimately lead to enhanced cAMP production and PKA activation (41). Initially we tested the effects of LH and forskolin on STC secretion, and whereas we always observed increased steroid (P4) output, LH treatment did not consistently increase STC secretion. Forskolin, on the other hand, significantly increased STC output by both rat and bovine cells. Similarly, isoproterenol and prostaglandin E2, both of which operate through the PKA pathway, had only slight stimulatory effects on STC release. In the end, the only hormone capable of consistently mimicking the forskolin effects on STC secretion proved to be hCG. The inability of LH to induce STC secretion is surprising in view of its consistent effects on P4 output in both our own and previous studies. However, compared with hCG, LH does have a lower receptor binding affinity and steroidogenic potency due to its faster rates of dissociation and internalization and sulfated terminal carbohydrate moieties that shorten its half-life (42), all of which could imply that P4 and STC secretion are subject to differing levels of PKA activation.
In addition to demonstrating an effect of hCG on STC secretion, we demonstrated its dependency on PKA activation. STC secretion was augmented in TICs treated with hCG in combination with IBMX, a phosphodiesterase inhibitor, whereas the selective PKA inhibitor, H-89, completely abolished hCG-stimulated release. On its own, H-89 significantly reduced basal STC output without affecting that of P4, again suggesting differential regulation of STC and P4 secretion by the PKA pathway. In the case of P4, both its production and secretion were enhanced in cultures treated with hCG plus IBMX, and compared with STC, P4 secretion was much more responsive to the selective addition of IBMX. There were also strong, positive correlations between STC and P4 output under stimulatory conditions.
There are numerous instances in which gonadotropin-regulated TIC products interact with other steroidogenic cells in the ovary (granulosa and luteal cells), including FSH-modulated IGF-binding protein-3 (43) and IGF-binding protein-6 release (44), hCG-modulated TGFß2 release (29), and relaxin-like factor release in response to LH (23). In the case of STC, the evidence suggests that it is targeted to oocytes and luteal cells (7, 8). Oocytes do not produce STC, yet they contain extraordinarily high levels of both STC (8) and its receptor (our unpublished observations), suggesting that they sequester thecal cell-derived STC in the normal course of STC signaling. Luteal cells heavily sequester STC (7, 8) in addition to other hormones, such as PRL, a process that is also receptor mediated, as PRL receptors colocalize to the same cells that sequester the hormone (45).
The underlying purpose of STC sequestering by luteal cells and oocytes has yet to be determined, but could be related to their differentiation and/or development. The bovine reproductive cycle ranges from 21–24 d (46, 47). During estrus the mature/dominant follicle ruptures, releasing an oocyte in response to the LH surge. Those thecal and granulosa cells comprising the ruptured follicle then undergo luteinization, a process also mediated in large part by LH. Because STC secretion by TICs is also LH regulated, it is conceivable that STC has roles in both processes. In the case of follicular development and/or ovulation, STC may act as a trophic factor on the dominant follicle(s). For instance, bovine follicular cells isolated before the LH surge are susceptible to Fas ligand-induced apoptosis, whereas those isolated afterward are not (48). Hence, a role in apoptosis should not be ruled out. Indeed, antiapoptotic effects of STC have been noted in neuronal cells after ischemic insult (49). In the case of luteal cells, STC could have a role in their development and maintenance during pregnancy as well as in P4 synthesis.
The mammalian STC cDNA sequence predicts a protein of 247 residues with a single glycosylation consensus site. There is also an unpaired cysteine residue in the molecule, which allows for the formation of homodimers (4, 50). Lectin binding studies and Western blotting have confirmed this to be the case, as in all tissues analyzed mammalian STC is a glycosylated, 50-kDa homodimer, as predicted (28). It was surprising, therefore, that when the same analysis was carried out on TIC extracts and conditioned media, both cellular and secreted STC proved to be substantially larger. In the native state, TIC-derived STC comprised three molecular mass species of 84, 112, and 135 kDa. Indeed, there was no evidence for the smaller, 50-kDa form of the hormone (STC50). The increased molecular mass was not due to differential glycosylation, as PNGase F treatment produced no downward shift in molecular mass, nor do these higher molecular mass forms bind to the plant lectin, concanavalin A (our unpublished observations), suggesting that they are differentially glycosylated compared with STC50 or are not glycosylated at all. The fact that chemical reduction collapsed all three bands into a 45-kDa species indicates that they are polymers of two or more subunits, stabilized by disulfide linkages. The greater size of ovarian STC could be due to posttranslational modifications to the STC core sequence, perhaps entailing the covalent attachment of other proteins. The existence of additional, uncharacterized exons also cannot be ruled out. The latter seems unlikely, however, as HT1080 cells produce the same higher molecular mass forms of the hormone despite the fact that HT1080 STC mRNA encodes the typical 247-residue STC monomer (50).
The structural uniqueness of ovarian STC may also extend to differences in its clearance kinetics. One characteristic of STC50 is the extreme rapidity with which it is rendered nonimmunoreactive after injection into the bloodstream (15). Until now, this feature has served to explain the general absence of STC immunoreactivity in mammalian serum (except during pregnancy and lactation). In view of the magnitude in structural differences there now appear to be between ovarian STC and STC50, it would not be surprising to find differences in their clearance kinetics. This in itself might be sufficient to explain the persistence of STC in the serum of pregnant and lactating mice (8).
This study is the first to characterize ovarian TIC-derived STC and to demonstrate that in vitro STC gene expression and secretion are regulated by LH/hCG via the PKA pathway. Our findings also suggest that in the ovary, mammalian STC has evolved into a local and systemic mediator of female reproduction, a transition that has also been accompanied by marked changes in the physical character of the hormone. It is intriguing that in fish, the ovarian form of STC is structurally unique from that produced by the corpuscles of Stannius, in this case in the size of the carbohydrate moiety (3). Future studies will no doubt reveal that the physical differences in ovarian STC are integral to its uniquely reproductive functions. Finally, in keeping with the established practice of naming high molecular mass hormone variants (e.g. big GH and big PRL), we suggest that the STC variants described here be collectively referred to in future as big STC.
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Acknowledgments |
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Footnotes |
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Abbreviations: CG, Chorionic gonadotropin; h, human; IBMX, 3-isobutyl-1-methylxanthine; P4, progesterone; PKA, protein kinase A; PNGase F, peptide N-glycosidase F; STC, stanniocalcin; STC50, 50-kDa STC; TIC, thecal-interstitial cell.
Received March 25, 2002.
Accepted for publication June 21, 2002.
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