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Endocrinology Vol. 144, No. 6 2437-2445
Copyright © 2003 by The Endocrine Society

Lipoprotein Enhancement of Ovarian Theca-Interstitial Cell Steroidogenesis: Relative Contribution of Scavenger Receptor Class B (Type I) and Adenosine 5'-Triphosphate- Binding Cassette (Type A1) Transporter in High-Density Lipoprotein-Cholesterol Transport and Androgen Synthesis

Qian Wu, Susan Sucheta, Salman Azhar and K. M. J. Menon

Departments of Obstetrics/Gynecology and Biological Chemistry (Q.W., K.M.J.M.), University of Michigan Medical School, Ann Arbor, Michigan 48109; and Geriatric Research, Education and Clinical Center (S.S., S.A.), VA Palo Alto Health Care System, Palo Alto, California 94304

Address all correspondence and requests for reprints to: K. M. J. Menon, University of Michigan Medical School, 6428 Medical Sciences Building I, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0617. E-mail: kmjmenon{at}med.umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The theca-interstitial cells take up plasma high-density lipoprotein (HDL)- and low-density-lipoprotein-derived cholesterol to convert into steroid hormones. The uptake of HDL-derived cholesterol is mediated by the scavenger receptor, class B, type I (SR-BI). In nonsteroidogenic cells, HDL-stimulated efflux of cholesterol has been shown to be mediated by the ATP-binding cassette A1 (ABCA1) transporter. Its expression has not been documented in steroidogenic cells. The goal of the present study was to determine: 1) the role of SR-BI in theca-interstitial cell androgen production; 2) whether theca-interstitial cells express ABCA1 transporter mRNA; and 3) the relative roles of SR-BI and ABCA1 transporter in androgen production. The ABCA1 transporter mRNA expression in rat theca-interstitial cells was shown using RT-PCR and Northern blot analyses. The role of SR-BI and ABCA1 in androstenedione production was also examined by treating cells with anti-SR-BI and 2-hydroxypropyl-ß-cyclodextrin in the presence and absence of human chorionic gonadotropin and/or human HDL3. The treatment of theca-interstitial cells with anti-SR-BI antibody blocked more than 90% of HDL plus human chorionic gonadotropin-stimulated androstenedione production, and selective HDL-CE uptake. On the other hand, the use of inhibitors of ABCA1 transporter function had no discernible effect on HDL-supported androgen production. These data demonstrate that, although theca-interstitial cells express both SR-BI and ABCA1 transporter mRNA, the SR-BI pathway supplies the majority of the cholesterol required for androgen production. Furthermore, the present study presents evidence for a crucial role for SR-BI in HDL-mediated androgen production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THECA-INTERSTITIAL CELLS PLAY a crucial role in controlling follicular growth and atresia, regulating ovarian steroidogenesis and providing a supporting structural framework for ovarian follicles (1, 2, 3, 4, 5). These cells are also the site of androgen production, and the thecal androgens are transferred to the granulosa cells, where they serve as substrate for estrogen biosynthesis (6, 7, 8, 9, 10). Moreover, steroids and locally produced growth factors play an important role in regulating the proliferation and steroidogenic function of granulosa cells (1, 2, 3, 4, 5). In humans, function of the theca-interstitial cell compartment and the accompanying hyperandrogenism have been linked to pathophysiological conditions such as polycystic ovary syndrome, hyperthecosis, and anovulation (11, 12, 13). In experimental animals, a hyperandrogenic state, created by androgen administration, has also been shown to cause anovulation (14).

LH is the principal hormone responsible for regulating androgen synthesis in theca-interstitial cells (15, 16, 17). However, there is growing evidence that locally produced growth factors and cytokines, as well as insulin/IGF-I, play an important role in steroid production in theca-interstitial cells, either alone or in combination with trophic hormone (15, 16, 17, 18, 19, 20, 21, 22). Theca-interstitial cells, like other steroid-producing cells, require cholesterol for the initial step in the steroidogenic process, and it seems that these cells receive cholesterol exogenously from circulating lipoproteins (23, 24). Indeed, both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) have been shown to effectively support androgen biosynthesis in these cells (23, 24). The primary pathway for cellular uptake of LDL-derived CE involves the LDL receptor and other members of the LDL receptor family (25). These receptors function via the endocytic uptake and lysosomal degradation of intact lipoprotein particles, to release cholesterol and other lipids within the cell. An alternate pathway, which occurs primarily with HDL, is the so-called selective pathway, in which HDL-CE is preferentially taken up into the cell without the parallel uptake and degradation of the intact HDL particle (26, 27). Previous studies have shown that adrenal and ovarian granulosa cells and luteal cells acquire cholesterol from HDL through the selective pathway (28, 29). A recently identified HDL receptor (the scavenger receptor, class B, type I) (SR-BI), functions as an authentic HDL receptor and initiates the extracellular phase of the selective CE-uptake process (30, 31). The direct involvement of SR-BI in steroidogenesis has not been established in the ovarian tissue.

In addition to CE uptake, SR-BI can promote cholesterol efflux by reorganizing membrane cholesterol domains and initiating the aqueous diffusion of cholesterol to exogenous acceptors (32, 33). Recently, the ATP-binding cassette A1 (ABCA1) transporter has also been shown to play a role in cholesterol efflux in nonsteroidogenic cells, where it serves as a cholesterol efflux regulatory protein, possibly acting as an antagonist of SR-BI (34, 35, 36, 37, 38). The identity and expression of ABCA1 have not yet been demonstrated in any of the steroid synthesizing tissues or cell systems. This issue is of special interest because steroid-producing cells internalize large quantities of cholesterol to satisfy their steroidogenic needs, and also because of the fact that cholesterol efflux is not an issue in these systems.

Previous studies from our laboratory have shown the presence of SR-BI and gonadotropin and insulin-mediated regulation of SR-BI expression in the theca-interstitial cells, under in vivo and in vitro conditions (39, 40). The present study was undertaken to examine the role of SR-BI in androgen synthesis in theca-interstitial cells. A second goal was to determine whether ABCA1 is expressed in theca-interstitial cells and to evaluate the relative contribution of SR-BI and ABCA1 in HDL-supported androgen production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Iodine-125I radionucleotide (carrier free, ~17 Ci/mg) was purchased from NEN Life Science Products (Boston, MA). [1{alpha}, 2{alpha} (N)-3H] cholesteryl oleolyl ether (COE) (48.0 Ci/mmol) was a product of Amersham Pharmacia Biotech (Arlington Heights, IL). [{alpha}-32P] deoxy-CTP (3000 Ci/mmol) was obtained from ICN Biomedicals, Inc. (Irvine, CA). Peroxidase and cholesterol oxidase were obtained from Calbiochem (La Jolla, CA). Cholesterol esterase, bovine insulin, human chorionic gonadotropin (hCG), and 2-hydroxypropyl-ß-cyclodextrin (HCD) were purchased from Sigma (St. Louis, MO). The RTS RadPrime DNA labeling kit, One Step RT-PCR kit, medium 199, L-glutamine, McCoy’s 5A medium, and TRIzol reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). X-Omat AR film was purchased from Eastman Kodak Co. (Rochester, NY). Collagenase (CLS I; 260 U/mg) was purchased from Worthington Biochemical Corp. (Freehold, NJ). Reagents for androstenedione RIA (DSL-4200) were obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). The antirat/mouse SR-BI polyclonal antibodies that were used have been previously described and characterized (41). All other chemicals and reagents used were of analytical grade.

Animals
Sprague Dawley female rats (~25 d old) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and, for the most part, were used without any further treatment. The animals were killed by CO2 asphyxiation, and ovaries were excised under sterile conditions and processed immediately for the isolation of theca-interstitial cells or stored frozen in liquid nitrogen. For some studies, groups of six rats were treated with 2 U insulin, 12.5 IU hCG, or 2 U insulin, followed in 1 h by 12.5 IU hCG; and all animals were killed 12 h later. The excised ovaries were processed immediately for the isolation of theca-interstitial cells.

Isolation and culture of theca-interstitial cells
The theca-interstitial cells were isolated and cultured using a previously described procedure (24, 40, 42). In brief, freshly excised ovaries were collected in medium 199 supplemented with 25 mM HEPES (pH 7.4), 2 mM L-glutamine, 1 mg/ml BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin. The ovaries were dissected of adhering fat, punctured with a needle under a dissecting microscope to release granulosa cells, and thoroughly flushed with medium to remove any remaining adhering granulosa cells. The remaining tissue was then minced and incubated for 90 min at 37 C in a medium containing 2.5 mg/ml collagenase plus 10 µg/ml deoxyribonuclease. After mechanical dispersion, the released theca-interstitial cells were centrifuged at 250 x g for 5 min and washed two times in medium. Subsequently, the dispersed cells were subjected to a single 5-min unit gravity purification. Cell viability was checked by trypan blue exclusion and ranged from 70–80%. The dispersed cells were either seeded in 60-mm dishes to determine steroid production (3 x 106 viable cells/dish) or to isolate total cellular RNA (4 x 106 viable cells per dish), or in 35-mm dishes (2 x 106 viable cells/dish) to measure lipoprotein-derived cholesteryl ester (CE) transport. All dishes were maintained overnight in McCoy’s 5A medium containing 2 mM L-glutamine, 1 mg/ml BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin, in a humidified atmosphere of 95% air-5% CO2 at 37 C. The purity of the cells in the culture plates was assessed by histochemical staining (43) and averaged around 95%.

Measurement of androstenedione production
To examine the functional role of SR-BI and ABCA1 in androgen production, the cultured theca-interstitial cells were pretreated with either anti-SR-BI antibody (1:60) for 24 h or with HCD (2 mM) for 2 h. Short-term exposure of cells to HCD induces rapid depletion of plasma membrane-associated cholesterol and leads to inhibition of ABCA1 function, i.e. cholesterol efflux (44). For studies requiring coincubation with these two reagents, HCD was added during the final 2 h of incubation with anti-SR-BI. After preincubation, the dishes were maintained for an additional 24 h in the presence or absence of hCG (100 ng/ml), human HDL3 (hHDL3) (50 µg/ml cholesterol), or hHDL3 plus hCG. At the end of the final incubation, the medium was collected from each dish and stored at -20 C until assayed for androstenedione by RIA.

RNA isolation and demonstration of ABCA1 transporter expression in theca-interstitial cells by RT-PCR
RT-PCR was used to demonstrate the presence of ABCA1 in theca-interstitial cells and to prepare a probe for ABCA1 mRNA analyses by Northern blotting. Total RNA was isolated from cultured dishes (~4 x 106 cells/dish) using the TRIzol reagent (Life Technologies, Inc.), following the manufacturer’s protocol. Twenty micrograms of total RNA were reverse-transcribed using the one-step RT-PCR System (Life Technologies, Inc.) at 48 C for 45 min. After reverse transcription, the PCR reaction was carried out. The primers for murine ABCA1 were derived from mRNA sequence available in the GenBank (accession no. X75926) (45). The upstream primer contained the following sequence: 5'-GGG TGG GGG CCT GAA GAT C-3'. The downstream sequence was 5'-AGA GCC ATT TGG GGA CTG AAC ATC-3'. PCR amplification conditions included 32 cycles at 94 C for 30 sec, 56 C for 1 min, and 68 C for 2 min, with a final extension at 72 C for 7 min. The PCR-amplified products were separated by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining and UV illumination. The PCR products of the expected size (~500 bp) were purified and ligated into PCR TOPO vector and transformed into competent Escherichia coli (DH 5{alpha}) cells. Plasmids containing inserts of expected size were sequenced to confirm the identity of the product.

RNA extraction and Northern blot analysis
Total RNA was isolated from theca-interstitial cells using the procedure of Chomczynski and Sacchi (46). Briefly, cells were suspended in a solution of guanidine isothiocyanate and extracted with water-saturated phenol and chloroform-isoamyl alcohol (49:1). The RNA in the aqueous phase was precipitated with 3 vol of ethanol at -20 C overnight. The RNA was quantified, and the purity was determined by the A260/A280 ratio. Aliquots of total RNA (30 µg) were separated by electrophoresis in 1.2% agarose formaldehyde gels and transferred to nitrocellulose membranes using 10x saline sodium citrate. Blots were UV cross-linked and prehybridized at 42 C for 2 h in a solution containing salmon sperm DNA (0.5 mg/ml) and 2x hybridization buffer diluted 1:1 with deionized formamide. Probes for ABCA1 (502 bp) and 18S ribosomal RNA were labeled by random primer extension with [{alpha}32P] deoxy-CTP. Membranes were hybridized overnight at 42 C in fresh hybridization buffer using 2 x 107 cpm labeled probe. After hybridization, the membranes were washed four times with 2x saline sodium citrate, containing 0.1% sodium dodecyl sulfate at room temperature (10 min each), and once at 60 C (30 min). The washed membranes were subjected to autoradiography using XAR film (Eastman Kodak Co.) with intensifying screens at -70 C for an appropriate length of time. The intensity of ABCA1 bands was quantified using a densitometric scanner (Arcus II). After stripping, the membranes were reprobed with 32P-labeled cDNA probe for 18S ribosomal RNA, exposed to x-ray film, and scanned. ABCA1 data were normalized relative to expression of 18S ribosomal RNA.

Uptake and internalization of lipoprotein-derived CEs
For this set of experiments, the theca-interstitial cells were cultured and maintained in 35-mm dishes. After overnight attachment, cells were incubated at 37 C for 24 h without or with hCG (100 ng/ml) and SR-BI anti-serum (1:60 dilution) or preimmune serum (PIS). Subsequently, the medium in each dish was replaced with fresh medium containing these substances and hHDL3 equipped with radiolabeled, nonreleasable apolipoprotein and CE tags that accumulate within the cells even when degraded (47, 48). The incubations were carried out with the [125I]-labeled dilactitol tyramine (DLT)-[3H]COE-hHDL3 (50 µg protein/ml) ± hCG and/or ± anti-SR-BI for 4 or 12 h at 37 C. At the end of incubation, the dishes were washed and cells solubilized with sodium hydroxide. Aliquots were taken, to which equal volumes of 20% trichloroacetic acid (TCA) were added to determine soluble and insoluble 125I-radioactivity, or they were extracted with organic solvents to determine 3H-radioactivity (47, 48).

Endocytic uptake of lipoprotein particles is calculated from TCA-soluble 125I label only. The difference between total and TCA-soluble radioactivity is taken as the cell surface associated 125I-radioactivity. Because both 125I and 3H labels are on the same particle, the surface-bound 3H is also equal to the surface-bound 125I. Thus, total 3H minus surface-bound 3H equals the total amount of 3H internalized. To calculate the selective uptake of CE, soluble 125I radioactivity is subtracted from soluble 3H radioactivity.

Miscellaneous techniques
Human LDL and human apolipoprotein E-free HDL were isolated and characterized as previously described (49). For uptake and internalization studies, hHDL3 preparations were conjugated with residualizing labels, i.e. [125I]-labeled DLT and [3H]COE (47, 48). The DNA content of the cells was quantified colorimetrically (50). The procedure of Markwell et al. (51) was used to quantify the protein content of lipoproteins. Cellular lysate protein concentration was determined using the bicinchoninic acid procedure of Smith et al. (52). Cholesterol content of the lipoproteins was determined according to the method of Deacon and Duncan (53).

Statistical analysis
The statistical analysis was initially carried out using ANOVA. If ANOVA indicated significant differences within the data sets, paired comparisons were made using unpaired Student’s t test. Each experiment was repeated at least three times, with comparable results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and hormonal regulation of ABCA1 transporter mRNA in theca-interstitial cells
Our initial objective was to identify and characterize hormonal regulation of ABCA1 mRNA expression. To identify ABCA1 expression in rat theca-interstitial cells, total RNA, isolated from the cells, was reverse-transcribed to cDNA and amplified by PCR using primers described in Materials and Methods. A 502-bp cDNA was amplified (Fig. 1Go). The identity of the PCR product was confirmed by DNA sequencing and comparison with the published sequence for murine ABCA1; the sequence of the amplified rat ABCA1 product was 98% homologous to its murine counterpart. Thus, these studies demonstrate that ABCA1 transporter is expressed in rat ovarian theca-interstitial cells. The presence of ABCA1 mRNA in rat theca-interstitial cells was further confirmed by Northern blot analysis. A single transcript (7.8 kb) was identified using the PCR product as the probe (data not shown).



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  		Figure 1. Identification of mRNA transcript for ABCA1 in rat theca-interstitial cells. Total RNA, from theca-interstitial cells cultured overnight, was subjected to RT-PCR, with mouse-specific primers for ABCA1, and analyzed by agarose gel (1%) electrophoresis, followed by staining with ethidium bromide. Lanes 1 and 2, 3 and 4, and 5 and 6, Results of duplicate RT-PCR of three separate experiments; lane 7, DNA size markers.

 
The hormonal regulation of ABCA1 mRNA expression in theca-interstitial cells was then examined under in vivo conditions. Groups of six rats were treated either with vehicle, 12.5 IU hCG, 2 U insulin or 2 U insulin for 1 h followed by 12.5 IU hCG. All animals were killed 12 h after hCG or insulin treatment, ovaries were excised, and the theca-interstitial cells were used for RNA isolation. Aliquots of total RNA from various groups were subjected to Northern blot analysis as described in Materials and Methods. The RNA blots and densitometric scanning data of the blots are shown in Fig. 2Go. Treatment with hCG resulted in a 100% increase in ABCA1 transcript, as compared with the vehicle-treated control group. Similarly, treatment with insulin significantly increased the steady-state levels of ABCA1 transcript, compared with the vehicle-treated controls. Sequential treatment with insulin and hCG resulted in further and synergistic stimulation of ABCA1 transcript, relative to vehicle-treated controls. These results demonstrate that theca-interstitial cells express ABCA1 mRNA and that agents known to stimulate steroidogenic function also regulate ABCA1 expression. Furthermore, given that both hCG and insulin enhanced ABCA1 mRNA expression individually and in concert, the present observation suggests that the two hormones may use independent pathways to up-regulate the expression of ABCA1 transcripts and that, at some point, their respective pathways may converge, resulting in the synergistic stimulation of ABCA1 mRNA levels.



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  		Figure 2. Hormonal regulation of ABCA1 mRNA expression in theca-interstitial cells in vivo. A and B, Northern blot analysis of ABCA1 mRNA and 18S rRNA expression in theca-interstitial cells isolated from rats treated for 12 h with saline (control), 12.5 IU hCG, 2 U insulin, or 2 U insulin followed by 12.5 IU hCG. C, Quantification of ABCA1 mRNA expression under basal conditions and in response to hCG and/or insulin. The intensities of ABCA1 bands were quantified and normalized to 18S rRNA and are shown in histogram form. Results are representative of three separate experiments and are the mean ± SEM. The bars with identical letters (i.e. a or b) represent statistical significance (P < 0.005) between the groups that bear the letters. The bars with asterisks (***) denote statistical significance (P < 0.005), compared with the control.

 
Experiments were then performed to study the separate and combined actions of hCG and insulin on ABCA1 mRNA levels in cultured theca-interstitial cells in vitro. Triplicate dishes of cultured theca-interstitial cells were treated with vehicle (control), insulin, hCG, or insulin plus hCG, and RNA was isolated and subjected to Northern blot analysis for ABCA1. As shown in Fig. 3Go (A and B), insulin treatment increased steady-state level of ABCA1 transcript by approximately 100%. Unlike insulin, in vitro addition of hCG had no discernible effect on the expression of ABCA1 mRNA. This lack of effect of hCG is most likely attributable to the loss of functional LH receptors in cultured cells, a phenomenon which, along with the subsequent restitution of the receptor by insulin, has been well documented by our laboratory (40). In contrast, when insulin and hCG were present together, hCG further potentiated the stimulatory action of insulin on ABCA1 mRNA levels by approximately 400%, when compared with control.



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  		Figure 3. Hormonal regulation of ABCA1 mRNA expression in theca-interstitial cells in vitro. Theca-interstitial cells were isolated and cultured in 60-mm tissue culture plates at a density of 4 x 106 cells/dish. After overnight culture, triplicate dishes were treated without or with hCG (100 ng/ml), insulin (10 µg/ml), or insulin (10 µg/ml) plus hCG (100 ng/ml) for 24 h. After incubation, total RNA was isolated from each dish using TRIzol reagent (Life Technologies, Inc.), and ABCA1 transcripts were determined by Northern blot analysis and normalized to the values for 18S mRNA. A and B, ABCA1 mRNA and 18S rRNA signals, respectively, from one representative experiment. C, Densitometric scan of ABCA1 normalized for 18S. Results are representative of three separate experiments. Values are mean ± SEM. The bars with identical letters represent statistical significance (P < 0.005) between the groups bearing the same letters. The bars with asterisks denote statistical significance (P < 0.005), compared with the control.

 
Anti-SR-BI antibody inhibits HDL-supported androstenedione production in cultured theca-interstitial cells
Previously, we have shown that theca-interstitial cells express significant amounts of SR-BI (a receptor that mediates selective lipoprotein-CE transport) and that insulin and hCG both independently and synergistically up-regulate its expression (39, 40). The results presented above demonstrate that theca-interstitial cells also express ABCA1 transporter mRNA, a protein that participates in the efflux of cellular free cholesterol (34, 35, 36, 37, 38), and that its expression is also regulated by insulin and hCG. Thus, a series of follow-up studies were carried out to examine the relative contribution of these two cholesterol-processing pathways in the utilization of HDL-derived cholesterol precursor for androgen production. To achieve this goal, and based on preliminary observations, we made use of anti-SR-BI and HCD to inhibit SR-BI-mediated selective CE delivery into, and ABCA1-dependent efflux of free cholesterol from, theca-interstitial cells, respectively. HCD is known to indirectly inhibit ABCA1 function by depleting the ABCA1-sensitive pool of free cholesterol (44).

Cultures of theca-interstitial cells were treated without or with PIS, anti-SR-BI, or HCD ± anti-SR-BI in the presence or absence of hCG or hCG plus hHDL3 for 24 h. The incubation media were collected and quantified for androstenedione production, by RIA. As shown in Fig. 4AGo (left panel), treatment of theca-interstitial cells with PIS, anti-SR-BI antibody, or HCD had no demonstrable effect on basal androstenedione production. On the other hand, as expected, treatment with hCG produced approximately a 3-fold increase in androstenedione production in control cells (Fig. 4BGo, middle panel). Simultaneous addition of hCG and anti-SR-BI resulted in an inhibition of steroid synthesis, as compared with hCG alone. Similar inhibition was also observed when anti-SR-BI was replaced with PIS, and this is consistent with a nonspecific action of serum proteins. Likewise, incubation of cells with HCD caused an inhibition in androstenedione production that was further potentiated when HCD- and anti-SR-BI were added together. Because no exogenous cholesterol source was used during incubation, the observed reduction in androstenedione production after HCD treatment may also be nonspecific. The results in Fig. 4CGo (right panel) show the separate and combined actions of anti-SR-BI and HCD on hCG plus hHDL3-stimulated androstenedione production. Coincubation with hCG plus HDL produced, as expected, a robust stimulation of androstenedione production in control cells (13-fold), as compared with hCG alone. These data are consistent with published studies reporting that both trophic hormone and HDL-cholesterol are required for maximal androgen production (24). To evaluate the contribution of SR-BI in HDL-supported and gonadotropin-stimulated steroid production, the cells were exposed to HDL and hCG in the presence of anti-SR-BI or PIS. As shown in Fig. 4CGo, pretreatment of theca-interstitial cells with anti-SR-BI almost completely inhibited (~95%) androstenedione production. On the other hand, addition of PIS reduced steroid production only slightly. Likewise, incubation of cells with the cholesterol acceptor, HCD, also reduced androstenedione production (Fig. 4CGo), whereas the combined addition of anti-SR-BI and HCD completely inhibited androstenedione production. These results demonstrate that in theca-interstitial cells, the SR-BI selective pathway provides the bulk of the HDL-derived cholesterol necessary for maximal androstenedione production. Additionally, because some inhibition of steroidogenesis was also observed with HCD, it seems likely that the plasma membrane-associated free-cholesterol pool may also function as a minor contributor to androgen synthesis in these cells.



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  		Figure 4. Effects of anti-SR-BI antibody and cyclodextrin on androstenedione production. Theca-interstitial cells were isolated and cultured as described in Materials and Methods. After overnight attachment, cells were preincubated with medium alone, PIS, SR-BI antibody (1:60 final dilution), HCD (20 mM), or a combination of SR-BI antibody and HCD in the presence or absence of hCG or hCG + hHDL3 for 24 h, as described under Materials and Methods. A, Basal; B, hCG (100 ng/ml); and C, hCG plus hHDL3 (50 µg total cholesterol/ml). Results are representative of three separate experiments. The bars bearing the same letters represent statistical significance (P < 0.005) between bars bearing the letters. In B and C, all treatment groups were significantly (P < 0.005) different from the corresponding control group.

 
We next examined the time course of inhibitory action of anti-SR-BI on HDL-supported androstenedione production. As can be seen from the data presented in Fig. 5Go, the HDL-supported and hCG-induced steroidogenesis decreased precipitously between 12 and 24 h after anti-SR-BI treatment (i.e. between 60–95% decrease). To test the specificity of the inhibitory action of SR-BI antibody, similar studies were carried out using human LDL as a cholesterol donor, and the data are summarized in Fig. 6Go. As expected, simultaneous exposure of theca-interstitial cells to LDL and hCG produced, on average, an increase of approximately 18-fold in androstenedione production. Inclusion of PIS, however, only slightly inhibited the steroidogenic response. Likewise, addition of anti-SR-BI had no appreciable effect on androstenedione production in response to LDL and hCG. Collectively, these results demonstrate that anti-SR-BI selectively blocks HDL- but not LDL-supported androgen production.



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  		Figure 5. Time-dependent inhibitory effect of anti-SR-BI antibody on androstenedione production by theca-interstitial cells in response to hCG and HDL. Cultured theca-interstitial cells were pretreated with SR-BI antibody (1:60) for various times; subsequently, hCG (100 ng/ml) and HDL (50 µg total cholesterol/ml) were added, and the dishes were incubated for an additional 24 h. After incubation, aliquots of the media were assayed for androstenedione, by RIA. Values are the mean ± SEM (n = 4).

 


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  		Figure 6. Lack of an inhibitory effect of anti-SR-BI antibody on LDL-supported androstenedione production in theca-interstitial cells. Theca-interstitial cells were cultured in medium containing anti-SR-BI antibody (1:60) or PIS for 24 h, or HCD (20 mM) for 2 h, as described in Materials and Methods, followed by the addition of hCG (100 ng/ml) and/or LDL (50 µg total cholesterol/ml), and all dishes were incubated for an additional 24 h. After incubation, aliquots of the media were quantified for androstenedione by RIA. Values are mean ± SEM. Results are representative of three separate experiments.

 
Effect of anti-SR-BI on selective CE uptake
To determine whether SR-BI mediates the selective uptake of HDL-CE, cultured theca-interstitial cells were incubated for 4 or 12 h with 125I/3H-hHDL3 in the presence and absence of hCG and/or anti-SR-BI, after which hHDL3 cell surface association and hHDL3-CE-selective uptake were measured. As shown in Fig. 7AGo (left panel), theca-interstitial cells showed an hCG-dependent increase in the cell surface association of hHDL3 that was approximately 60–70% greater than control (medium alone) cells. Measurement of hHDL3-CE-selective uptake showed even more dramatic increase, especially at 12 h (~600%), in hCG-treated cells, as compared with control cells (Fig. 8AGo, left panel). These data indicate that cell-surface binding of hHDL3 to theca-interstitial cells leads to efficient CE uptake by the selective pathway and that hormone treatment results in a more robust uptake of CE by this pathway.



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  		Figure 7. Anti-SR-BI inhibition of 125I-hHDL3-cell surface association in control and hCG-treated theca-interstitial cells. Theca-interstitial cells were cultured in the absence or presence of hCG (100 ng/ml) and anti-SR-BI antibody (1:60) or PIS for 24 h. Subsequently, 125I/3H-hHDL3 was added, and the dishes were further incubated for 4 or 12 h at 37 C. The cells were then processed for the determination of hHDL3 cell surface association. Values represent the mean ± SE (n = 3).

 


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  		Figure 8. Effect of anti-SR-BI on hHDL3-CE-selective uptake in control and hCG-treated cells. Incubation conditions were identical to those described under Fig. 7Go except that the cell homogenates were also processed for the determination of hHDL3-CE-selective uptake. Values represent the mean ± SE (n = 3).

 
To examine the relationship between SR-BI and hHDL3-CE-selective uptake, inhibition experiments were performed by preincubating theca-interstitial cells with or without anti-SR-BI and/or hCG before the addition of double-labeled hHDL3. Figure 7BGo (right panel) shows that anti-SR-BI effectively blocked the cell association of 125I-hHDL3 to both control and hCG-treated cells. This pattern of anti-SR-BI inhibition is even more pronounced for the selective uptake of hHDL3-CE. In this case, hHDL3-[3H] CE-selective uptake was drastically reduced in both control and hCG-treated cells, and maximum inhibition (>80%) was observed, at 12 h, in the presence of hCG (Fig. 8BGo, right panel). Thus, from these data, we conclude that hHDL3-CE-selective uptake is SR-BI dependent and that the inhibitory effect of anti-SR-BI antibody on hCG-stimulated androstenedione production is attributable to inhibition of SR-BI (HDL receptor) function and subsequent prevention of selective CE uptake.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to examine the role of SR-BI in androstene production and to evaluate the relative roles of SR-BI and the ABCA1 transporter on the uptake of exogenous HDL-CEs and HDL-supported androgen production in theca-interstitial cells. The role of SR-BI function in steroidogenesis was examined with the help of anti-SR-BI antibody. The theca-interstitial cells, like other ovarian cell types (54, 55, 56), require large quantities of exogenous cholesterol for steroidogenesis, and they actively use cholesterol from both LDL and HDL (23, 24). Moreover, the ovarian cells, in general, acquire HDL-derived cholesterol for steroid biosynthesis and CE storage via the selective pathway (29). In this pathway, in which SR-BI initiates the extracellular phase of the selective CE uptake, the lipoprotein-derived CE is preferentially taken up into the cell without the parallel uptake and degradation of intact lipoprotein particles (29, 57).

Before examining HDL-supported steroidogenesis and the selective-CE uptake pathway, we first established that theca-interstitial cells express ABCA1. Indeed, both RT-PCR and Northern blot analysis showed that significant levels of ABCA1 transcripts are expressed in theca-interstitial cells. In addition, the expression of ABCA1 was further up-regulated by hCG and insulin, two hormones known to stimulate androgen production. These results are similar to those of previous studies from our laboratory in which theca-interstitial cells were shown to express significant levels of SR-BI mRNA, and that SR-BI expression was also enhanced by treatment with hCG and insulin (39, 40). These data suggest that both insulin and gonadotropins not only regulate androgen production but also exert a positive influence on the expression of SR-BI and ABCA1.

HDL-supported androstenedione production was significantly reduced when the theca-interstitial cells were pretreated with anti-SR-BI antibody. Although HCD, an inhibitor of membrane channel protein function, somewhat inhibited androstenedione production, this seemed to be nonspecific, presumably arising from the loss of the plasma-membrane-associated pool of free cholesterol. ABCA1 is known to function as a facilitator of cholesterol efflux in nonsteroidogenic cells. However, because the steroidogenic cells internalize large quantities of lipoprotein (HDL)-derived cholesterol to meet their high cholesterol needs, the efflux of cholesterol is not a significant factor. In the current studies, we have considered an alternative possibility, where the ABCA1-mediated efflux system could serve as a default mechanism to obtain cholesterol from HDL under conditions in which the SR-BI-mediated cholesterol transport pathway is blocked, thereby depriving the cells of cholesterol supply through this pathway. Our results suggest that this seems not to be the case. If ABCA1 were to function, like SR-BI, to facilitate cholesterol import instead of its well-established efflux function, then anti-SR-BI would not have blocked androgen synthesis to the extent that was observed (Fig. 4CGo). From these data, we conclude that the SR-BI-mediated selective pathway is the major delivery system of the HDL-derived cholesterol required for steroidogenesis in the theca-interstitial cells. However, at present, we cannot completely rule out the possibility that ABCA1 may play some other regulatory roles in these cells.

To provide further evidence that anti-SR-BI inhibits androstenedione production by interfering with selective CE transport, we made use of double-labeled HDL particles in which the apoproteins were labeled with residualizing 125I-DLT, and the CE with [3H]COE. This approach allows the precise quantification of selective HDL-CE uptake under a variety of experimental conditions (41, 55, 56, 57). As shown in Fig. 8Go, use of anti-SR-BI antibody efficiently and completely blocked the selective uptake of CE from HDL donor particles. Furthermore, the fact that anti-SR-BI also decreased 125I-DLT binding to cell surface (Fig. 7Go) suggests that anti-SR-BI interferes with the ability of HDL to bind to SR-BI.

The mechanism by which anti-SR-BI inhibits HDL-supported steroidogenesis and HDL-CE uptake is presently unclear. The anti-SR-BI antibody used in the current studies was raised against the carboxyl terminus of mouse/rat SR-BI (41). Because of this reason, use of the antibody was not expected to exert any inhibitory action when tested in intact cultured cells. However, the evidence presented here clearly indicates that anti-SR-BI effectively blocked both selective HDL-CE uptake and HDL-supported steroidogenesis. One likely explanation is that anti-SR-BI antibody molecules might diffuse slowly into the theca-interstitial cells. A second possible explanation is that the proteolytic enzymes used for theca-interstitial cell isolation may damage the cell surface, which, in turn, may lead to nonspecific translocation of anti-SR-BI into the interior of the cell. However, the cells were cultured for approximately 24 h before the addition of antibody, which is likely sufficient time for the cells to completely recover from this presumptive damage. Moreover, the fact that the cells retained fully functional insulin and hCG signaling cascades further argues against the possibility that anti-SR-BI is taken up nonspecifically as a result of cell damage during the isolation procedure. Finally, it is possible that the cytoplasmic carboxy tail of SR-BI is in constant motion and may be transiently exposed on the cell surface, where it can bind antibody and, over time, as the number of affected receptors is increased, SR-BI function is inhibited.

In summary, the current studies show that, although theca-interstitial cells express both ABCA1 and SR-BI, the HDL-CE required for androstenedione production is preferentially obtained via the SR-BI-selective pathway. In contrast, it seems that ABCA1 has no (or only a minor) role in HDL-CE uptake and transport and/or steroidogenesis in theca-interstitial cells. However, it is possible that ABCA1 may have some yet-unrecognized functions, which may include the transport of key regulatory substances essential to cellular sterol metabolism. The present study also presents strong evidence, for the first time, supporting the obligatory role of the HDL receptor, SR-BI, for the selective uptake and utilization of HDL-derived cholesterol for androgen synthesis by the theca-interstitial cells.


   Acknowledgments
 
The authors express their appreciation to Helle Peegel for her many contributions, including experimental work, and for her assistance with manuscript preparation.


   Footnotes
 
This work was supported by NIH Grant HD-38424 (to K.M.J.M.) and NIH Grant DK-56339 and funds from the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to S.A.).

Abbreviations: ABCA1 transporter, ATP-binding cassette A1 transporter; CE, cholesteryl ester; COE, cholesteryl oleolyl ether; DLT, dilactitol tyramine; HCD, 2-hydroxypropyl-ß-cyclodextrin; hCG, human chorionic gonadotropin; HDL, high-density lipoprotein; hHDL3, human HDL3; LDL, low-density lipoprotein; PIS, preimmune serum; SR-BI, scavenger receptor, class B, type I; TCA, trichloroacetic acid.

Received October 25, 2002.

Accepted for publication February 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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