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Bone Morphogenetic Protein Inhibits Ovarian Androgen Production

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9032

Address correspondence and requests for reprints to: Bruce R. Carr, M.D., Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9032.

	Abstract

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Bone morphogenetic proteins (BMPs), members of the transforming growth factor ß superfamily, were recently shown to be expressed and to regulate steroidogenesis in rat ovarian tissue. The purpose of this study was to investigate the effect of BMP-4 on androgen production in a human ovarian theca-like tumor (HOTT) cell culture model. We have previously demonstrated the usefulness of these cells as a model for human thecal cells. HOTT cells respond to protein kinase A agonists by increased production of androstenedione and with an induction of steroid-metabolizing enzymes. In this investigation, HOTT cells were treated with forskolin or dibutyryl cyclic AMP (dbcAMP) in the presence or absence of various concentrations of BMP-4. The accumulation of androstenedione, progesterone, and 17-hydroxyprogesterone (17OHP) in the incubation medium was measured by RIA. The expression of 17-hydroxylase (CYP17), 3ß-hydroxysteroid dehydrogenase (3ßHSD), cholesterol side-chain cleavage (CYP11A1), and steroidogenic acute regulatory (StAR) protein was determined by protein immunoblotting analysis using specific rabbit polyclonal antibodies. We also examined the expression of BMP receptor subtypes in our HOTT cells using RT-PCR. In cells treated with medium alone, steroid accumulation and steroid enzyme expression was unchanged. In cells treated with BMP alone there was a modest decrease in androstenedione secretion. In the presence of forskolin, HOTT cell production of androstenedione, 17OHP, and progesterone increased by approximately 4.5-, 35-, and 3-fold, respectively. In contrast, BMP-4 decreased forskolin-stimulated HOTT cell secretion of androstenedione and 17OHP by 50% but increased progesterone production 3-fold above forskolin treatment alone. Forskolin treatment led to an increase in CYP17, CYP11A1, 3ßHSD, and StAR protein expression. BMP-4 markedly inhibited forskolin stimulation of CYP17 expression but had little effect on 3ßHSD, CYP11A1, or StAR protein levels. Similar results were observed with the cAMP analog dbcAMP. In addition, BMP-4 inhibited basal and forskolin stimulation of CYP17 messenger RNA expression as determined by RNase protection assay. Other members of the transforming growth factor ß superfamily, including activin and inhibin, had minimal effect on androstenedione production in the absence of forskolin. In the presence of forskolin, activin inhibited androstenedione production by 80%. Activin also inhibited forskolin induction of CYP17 protein expression as determined by Western analysis. We identified the presence of messenger RNA for three BMP receptors (BMP-IA, BMP-IB, and BMP-II) in the HOTT cells model. In conclusion, BMP-4 inhibits HOTT cell expression of CYP17, leading to an alteration of steroidogenic pathway resulting in reduced androstenedione accumulation and increased progesterone production. These effects of BMP-4 seem similar to those caused by activin, another member of the transforming growth factorß superfamily of proteins.

	Introduction

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ESTRADIOL FORMATION by developing follicles is critical for normal reproductive function in women. Estrogen biosynthesis relies on the intricate interaction of granulosa and thecal cells due to compartmentalization of 17-hydroxylase (CYP17) in the theca and its absence in the granulosa. This allows follicular thecal cells to secrete and provide 19 carbon (C₁₉) steroids to the adjacent granulosa cells, for conversion to estrogens (1, 2). Thus, the regulation of C₁₉ steroid production in the thecal cell is a key component of estrogen biosynthesis.

There is growing evidence that locally produced growth factors play an important role in thecal cell steroid production, either alone or in combination with gonadotropins (3). Among these growth factors is the transforming growth factor-ß (TGF-ß) superfamily, which includes TGF-ß, mullerian-inhibiting substance, inhibin, activin, and the bone morphogenetic proteins (BMPs) (4, 5, 6).

BMPs comprise one of the largest subgroups in the TGF-ß superfamily. Fifteen BMPs have been described, and seven BMPs (2, 3, 3b, 4, 6, 7, 15) have been localized in mammalian ovaries (7, 8, 9, 10, 11, 12, 13). Additionally, in situ hybridization has demonstrated BMP-4 and BMP-7 messenger RNA (mRNA) in rat thecal cells (14).

Attempts to define the molecular and biochemical mechanisms controlling human thecal cell steroidogenesis have been hampered by the difficulty in obtaining and maintaining sufficient numbers of human thecal cells in monolayer culture. Our laboratory has developed a human ovarian thecal-like tumor (HOTT) cell culture model that seems to act as an appropriate model system for thecal cell steroidogenesis (15, 16). These cells continue to produce C₁₉ steroids and express the enzymes involved in normal thecal cell steroidogeneisis. In the current study, the HOTT cell culture model system was used to investigate the effects of BMP-4 on androgen production.

	Materials and Methods

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Cell isolation and culture

The descriptions of the in vitro HOTT cell culture model have been reported previously (15, 16, 17). Briefly, a portion of the tumor was dispersed into single cells using constant gentle agitation in 0.025% trypsin in Dulbecco’s Modified Eagle’s (DME)/F-12 (Life Technologies, Inc., Grand Island, NY) and antibiotic (37 C, 30 min x 8). After each time point the cell suspension was collected, pooled, pelleted, and resuspended in DME/F-12 medium containing 5% FBS to inactivate the trypsin. Aliquots of these cells were frozen and used as needed for the study. For monolayer culture, HOTT cells were thawed and maintained in DME/F-12 medium supplemented with insulin (6.25 µg/mL), transferrin (6.25 µg/mL), selenious acid (6.25 ng/mL), BSA (1.25 mg/mL), and linoleic acid (5.35 µg/mL) added in the form of 1% ITS plus (Collaborative Research, Waltham, MA), 2% Ultroser G (BioSepra, Villeneuve, France), and antibiotics. Cells were maintained and grown on 75-cm² flasks at 37 C under an atmosphere of 5% CO₂/95% air. Cells were routinely subcultured using 0.05% trypsin and replated at a 1:4 split. All experiments described in this study were accomplished using cells in culture for 2–8 weeks. For the experiments, growth medium was removed and replaced with a low-serum medium (DME/F-12 medium containing antibiotics and 0.01% Ultroser G) for 24 h. Cells were then rinsed and experimentally treated in the same low-serum medium. At the end of the treatment period, the medium was removed and the cells and medium were stored at -20 C for subsequent assay.

Experimental treatments

Specifics of various treatments are detailed in the figure legends. Reagents included: forskolin (Sigma, St. Louis, MO), dbcAMP (Sigma), and recombinant human BMP-4 (Research Diagnostics Inc., Flanders, NJ). Human recombinant inhibin-A and human recombinant activin-A were graciously supplied by Dr. A. F. Parlow, the scientific director of the National Hormone and Pituitary Program (Torrance, CA).

Measurement of steroid content

The steroid contents of culture medium were assayed after 36 h. This time was chosen as optimal for inhibition of androstenedione by BMP on forskolin-stimulated cells. Steroid contents was measured using standard RIA kits [androstenedione, 17OHP, and progesterone from Diagnostic Systems Laboratory (Webster, TX)]. The amount of steroid measured was expressed as picomoles steroid per milligram of cellular protein.

Protein determination

Cells were solubilized in Tris-HCl (50 mM/pH 7.4) containing NaCl (150 mM), deoxycholic acid (0.1%), EGTA (5 mM), MgCl₂ (0.5 mM), and phenylmethylsulfonylfluoride (0.2 mM) and stored frozen at -20 C. Subsequently, protein content was determined by bicinchonic acid protein assay, using the BCA assay kit (Pierce, Rockford, IL).

Western analysis

Using a Novex Xcell system (Novel Experimental Technology, San Diego, CA), one dimensional electrophoresis (200 V, 35 min) was performed in a 4–12% polyacrylamide pre-cast gel in a NuPAGE MES SDS running buffer [50 mM 2-(N-morpholino) ethane sulfonic acid, 50 mM Tris base, 3.5 mM SDS, and 1 mM EDTA (pH 7.2)] under reducing conditions. Proteins were transferred to nylon membrane (25 V, 1 h) in NuPAGE transfer buffer [25 mM Bicine, 25 mM Bis-tris, 1 mM EDTA, 0.05 mM chlorobutanol, and 20% methanol (pH 7.2)]. Immunoblotting was performed with rabbit polyclonal antibodies. Antibody sources were: human CYP17 antibody (M. Waterman, Vanderbilt University Medical Center, Nashville, TN), StAR antibody (D. Stucco, Texas Tech University Health Sciences Center, Lubbock, TX), human 3ßHSD antibody (J. Ian Mason, University of Edinburgh, Edinburgh, Scotland), and human CYP11A1 (Bon-Chu Chung, Institute of Molecular Biology Taiwan, Taiwan, China). The primary antibody incubation was followed by incubation with an antirabbit Ig, and horseradish peroxidase-linked F (ab')₂ fragment from donkey (Amersham Life Sciences, Buckinghamshire, England). Proteins were detected with enhanced chemiluminescence reagents (Amersham Life Sciences) on X-Omat Blue XB-1 Scientific Imaging Film (Kodak, Rochester, NY).

RNA isolation

Total RNA was isolated from a 15-mm follicle obtained from a patient undergoing oophrecetomy for a benign gynecological condition, as well as from HOTT cells that were maintained in low-serum medium for 48 h. RNA was isolated using Ultraspec RNA reagent according to the manufacturer’s suggested protocol. Frozen follicle and HOTT cells were homogenized in Ultraspec RNA reagent. The homogenate was kept on ice for at least 5 min to permit the complete dissociation of nucleoprotein complexes. Chloroform (0.2 mL/1 mL Ultraspec RNA reagent) was added while vigorously shaking, incubated at 4 C for 5 min, and centrifuged at 12,000 x g for 15 min at 4 C. The aqueous phase was carefully transferred to a fresh tube without disturbing the interphase. An equal volume of isopropanol was added, and the tube was stored at 4C for 1 h or longer. RNA precipitate was formed as a pellet at the bottom of the tube after centrifugation for 10 min at 12,000 x g (4 C). The pellet was washed twice with 75% ethanol by vortexing and subsequent centrifugation for 5 min at 12,000 x g (4 C) and briefly dried. The pellet was then dissolved in 50 mL diethylprocarbonate-treated water.

RNase Protection Assay (RPA)

A 295-bp fragment was obtained from CYP17 cDNA using Sac1 and Xba 1. This fragment, containing the nucleotides between +6 and +300, was inserted in pBluescript KS vector and sequenced to confirm its identity and orientation. A ³²P-labeled probe was prepared from the linearized plasmid using Maxiscript T7/T3 polymerase kit (Ambion, Inc., Austin, TX). A 125-bp ³²P-labeled riboprobe for ß-actin was also prepared using Maxiscript T7/T3 polymerase kit and was used as internal control. Because ß-actin is more abundant than CYP17 mRNA, the ³²P-labeled ß-actin probe was prepared at 100-fold lower specific activity. This ensured that the ß-actin probe could be used in molar excess to its target and a similar exposure time could be used for both probes. The labeled probes were purified on a 6% acrylamide gel. The labeled CYP17 and ß-actin probes were added to each of the RNA sample (10 µg). RPA was performed according to the manufacturer-suggested protocol (Ambion). Following hybridization and RNase digestion the protected fragments were separated on a 6% polyacrylamide gel. The gel was placed in a phosophoimager caste and left overnight. A phosphoimager counter was used to measure the density of each band.

RT-PCR

The first-strand cDNA synthesis from 4 µg total RNA was catalyzed by superscript II RT using random hexamer primers according to the manufacturer’s protocol. The 20-L of reaction mixture consisted of 4 µg total RNA, extracted either from ovarian follicles or HOTT cells, 125 ng random hexamers, 50 mM Mg Cl₂, 0.5 mM dNTP, 10 mM dithiothreitol, and 200 U Superscript II RT in 20 mM Tris-HCl buffer (pH 8.4). Two microliters of the first-strand cDNA reaction was used for PCR reaction to amplify the three different subtypes of BMP receptors. Primers used for PCR reaction are listed in Table 1. The PCR condition was 94 C for 3 min to denature the RNA/cDNA hybrid, then 30 cycles for 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min. PCR products were examined on a 1% agarose gel.

Statistical comparison of means of three or more samples was accomplished by ANOVA with Newman-Keuls post hoc testing. Significance was accepted at the 0.05 level of P.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Steroid production in HOTT cells

Medium accumulation of androstenedione, 17hydroxyprogesterone (17OHP), and progesterone was evaluated after 36 h of treatment. Treatment groups consisted of control (basal), forskolin (10 µM), BMP-4 (50 ng/mL), and forskolin (10 µM) plus BMP-4 (50 ng/mL). In cells treated with control medium or BMP-4, steroid accumulation was unchanged. In the presence of forskolin, HOTT cell production of androstenedione, 17OHP, and progesterone increased by approximately 4.5-, 35-, and 3-fold, respectively (Fig. 1, A, B, and C, respectively). In contrast, BMP-4 decreased forskolin-stimulated HOTT cell secretion of androstenedione and 17OHP by 50% but increased progesterone 3-fold above forskolin treatment alone (P < 0.001).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of BMP-4 with and without forskolin on steroid production in HOTT cells. Cells were treated for 36 h with medium alone, forskolin (10 µM), BMP-4 (50 ng/mL), or forskolin (F) (10 µM) plus BMP-4 (50 ng/mL). A, Androstenedione production. B, 17OHP production. C, Progesterone production. All are expressed as a percentage of steroid production in control (medium only treatment). The basal values: A, 69.13 pmol/mg protein; B, 75.54 pmol/mg protein; C, 1747.44 pmol/mg protein. Each data point represents ± SE of replicate dishes (n = 3). Data are from a representative experiment that was run three times. *, P < 0.001, compared to forskolin alone.

View larger version (19K): [in this window] [in a new window]   Figure 2. Concentration-dependent effect of BMP-4 on forskolin stimulation of androstenedione production in HOTT cells. Cells were treated for 36 h with medium alone, forskolin (10 µM), BMP-4 (50 ng/mL), or forskolin (F) (10 µM) plus BMP-4 in increasing doses (1–50 ng/mL). Data are expressed as a percentage of control steroid production (medium only treatment). Each data point represents the mean ± SE of replicate dishes (n = 3). The basal value is 69.13 pmol/mg protein. Data are for a representative experiment that was run twice. *P < 0.001 and **P < 0.025, compared with forskolin alone.

Cells were treated for 36 h with control media, forskolin (10 µM), BMP-4 (50 ng/mL), inhibin-A (50 ng/mL), activin-A (50 ng/mL), or forskolin (10 µM) plus BMP-4 (50 ng/mL), inhibin-A (50 ng/mL), or activin-A (50 ng/mL) (Fig. 3). Androstenedione levels were evaluated using RIA. In cells treated with medium only, BMP-4 modestly inhibited steroid accumulation. In the presence of forskolin, HOTT cell production of androstenedione, increased by approximately 4-fold and was not affected by the presence of inhibin. In contrast, BMP-4 and activin significantly decreased forskolin-stimulated HOTT cell secretion of androstenedione (BMP-4, P < 0.001; activin-A, P < 0.001).

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of BMP-4, inhibin-A, and activin-A with and without forskolin treatment on androstenedione production in HOTT cells. Cells were treated for 36 h with medium only, forskolin (10 µM), BMP-4 (50 ng/mL), inhibin-A (50 ng/mL), activin-A (50 ng/mL), or forskolin (F) (10 µM) plus BMP-4 (50 ng/mL) inhibin-A (50 ng/mL), and activin-A (50 ng/mL). Each data point represents the mean ± SE of replicate dishes (n = 3). Basal value is 60.29 pmol/mg protein. Data are from a representative experiment that was run three times. *P < 0.001 compared to forskolin alone.

We sought to elucidate the effect of BMP-4 on HOTT cell expression of immuno-detectable steroidogenic enzymes using Western analysis. Cells were treated for 36 h with medium only, forskolin (10 µM), BMP-4 (50 ng/mL), or forskolin (10 µM) plus BMP-4 (50 ng/mL). Additionally, cells were treated for 36 h with dbcAMP (1 mM) or dbcAMP (1 mM) plus BMP-4 (50 ng/mL). Western analysis was performed as described in Materials and Methods. The results are presented in Fig. 4. In cells treated with medium alone or BMP-4, steroidogenic enzyme expression was unchanged. Forskolin treatment increased CYP17, CYP11A1, and StAR protein expression. In contrast, BMP-4 markedly inhibited forskolin-stimulated CYP17 expression but had little effect on 3ßHSD, CYP11A1, or StAR protein. Similar results were observed with the cAMP analog dbcAMP.

View larger version (59K): [in this window] [in a new window]   Figure 4. Effect of BMP-4 with and without forskolin treatment on steroidogenic enzyme expression in HOTT cells. Cells were treated for 36 h with medium only, forskolin (10 µM), BMP-4 (50 ng/mL) or forskolin (10 µM) plus BMP-4 (50 ng/mL). Additionally, cells were treated for 36 h with dbcAMP (1 mM) or dbcAMP (1 mM) plus BMP-4 (50 ng/mL). Western analysis was performed as described in Materials and Methods with 20 µg cellular protein per lane. Lanes corresponding to each experimental condition are listed above the figure. Molecular weight markers are indicated with a dash.

View larger version (54K): [in this window] [in a new window]   Figure 5. Effect of BMP-4, inhibin-A, and activin-A with and without forskolin on steroidogenic enzyme expression in HOTT cells. Cells were treated for 36 h with medium only, forskolin (10 µM), BMP-4 (50 ng/mL), inhibin-A (50 ng/mL), activin-A (50 ng/mL), or forskolin (10 µM) plus BMP-4 (50 ng/mL) inhibin-A (50 ng/mL) or activin-A (50 ng/mL). Western analysis was performed as described in Materials and Methods with 20 µg cellular protein per lane. Lanes corresponding to each experimental condition are listed above the figure. Molecular weight markers are indicated with a dash.

To better define the mechanism for BMP-4 inhibition of CYP17 protein expression, we sought to evaluate CYP17 mRNA using a RPA for CYP17 as discussed in Materials and Methods. Cells were incubated for 24 h in basal media, BMP-4 (10 ng/mL), forskolin (10 µM) or forskolin (10 µM) plus BMP-4 (10 ng/mL). The results of the experiment are shown in Fig. 6, A and B. Compared with basal levels, BMP-4 alone inhibited CYP17 mRNA expression by 60%. Forskolin treatment caused a 20-fold increase in CYP17 mRNA levels, but BMP-4 inhibited forskolin stimulated CYP17 mRNA expression by 40%. These data suggest BMP-4 action on CYP17 protein expression occurs through alterations in mRNA levels.

View larger version (26K): [in this window] [in a new window]   Figure 6. Effect of BMP-4 and forskolin on CYP17 mRNA expression in HOTT cells. A, RPA for CYP17. Cells were treated without (basal) or with BMP-4 (10 ng/mL), forskolin (10 µM) or BMP-4 (10 ng/mL) plus forskolin (10 µM) for 24 h. RNA was then extracted, and the RPA was performed as described in Materials and Methods. Yeast was used as a negative control, and ß-actin was used to standardize results from each lane. B, The data from panel A is expressed as a percentage of CYP17 mRNA expression compared to basal (control) cells.

To better define the molecular action of BMP-4 on steroidogensis, we examined for the presence of BMP receptors subtypes in HOTT cells maintained in low serum medium as described in Materials and Methods. As seen in Fig. 7, the expression of BMP-IA, BMP-IB, and BMP-II was observed in a human ovarian follicle and in HOTT cells.

View larger version (37K): [in this window] [in a new window]   Figure 7. Expression on BMP receptors RNA in HOTT cells. RT-PCR was performed to demonstrate the presence of the three different subtypes of BMP receptors in HOTT cells. A, BMP receptor IA (BMPRIA). B, BMP receptor IB (BMPRIB). C, BMP receptor II (BMPRII). Follicular RNA was used as positive control for all three reactions. -RT was used as a negative control in which the RT reaction was performed in the absence of Superscript II enzyme. -RNA was performed in the absence of RNA in the RT reaction. Molecular weight markers are noted by the arrows on the right.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

BMPs are multifunctional cytokines that regulate cellular proliferation, differentiation, and apoptosis of various cell types, including osteoblasts and chondroblasts (7, 8, 9, 10, 11, 12, 13). These functions are related to various biological functions in vivo (e.g. formation of bone and cartilage, embryogenesis, and organogenesis). There are at least 15 structurally related BMPs, all of which have structural similarities that place them in the TGFß superfamily of proteins. Recently, the expression of BMPs was demonstrated in rat ovarian tissues, and predominantly in thecal cells (14). Thecal cells expressed both BMP-4 and BMP-7 mRNA as determined by in situ hybridization. BMP type IA, IB, and II receptors were also studied and shown to be predominantly expressed in the granulosa cells (14). Granulosa cell steroidogenesis was also influenced by BMP-4 and BMP-7 treatment. In addition, human granulosa cells obtained from in vitro fertilization cycles seem to express BMP-3, and its expression was regulated negatively by human chorionic gonadotropin (hCG) (11). The production of BMPs in granulosa cells suggests the possibility of an autocrine role on granulosa cells or a paracrine effect on thecal cells.

The purpose of this study was to investigate the role of one of the BMP family members, BMP-4, on the regulation of steroidogenesis as well as evaluating protein and mRNA expression of the principal enzymes involved in human ovarian thecal cell steroidogenesis. The difficulty in obtaining human thecal cell in sufficient quantities has slowed progress directed at defining the mechanism regulating of steroidogenesis. Our laboratory has isolated cells from ovarian tumors and place them in monolayer cell culture (15, 16). These HOTT cells have retained many of the characteristics of normal human thecal cells maintained in primary culture. These include the production of C₁₉ steroids and expression of steroid-metabolizing enzymes, each of which is under the control of cAMP. In addition, we have demonstrated that the HOTT cell model responds to TGFß, activin, and inhibin with similar responses to those seen in primary cultures of human thecal cells (5, 6, 21, 22).

Treatment of HOTT cells with BMP-4 caused a concentration-dependent inhibition of forskolin-stimulated androstenedione production. This effect was also observed on the stimulation of androstenedione by dbcAMP, an analog of cAMP. The effect was not due to an overall inhibition of steroid hormone biosynthesis because the level of progesterone production actually increased following BMP-4 treatment. This effect (decreased androgen production and increased progestin formation) is similar to that we reported previously for activin treatment of HOTT cells (6). In vivo such a shift in thecal cell androgen production is observed at ovulation when the cells start to luteinize. Thus, one interpretation of the effects of BMP and activin would be that they act to promote a luteinized phenotype or alternatively that they act as a negative regulator of androgen production to prevent excessive androstenedione production by thecal cells.

BMPs transduce signals through binding to specific BMP receptors that have been grouped as type IA, type IB, and type II BMP receptors, each of which has serine/threonine kinase activity (7, 8, 9, 10). The BMPs can also bind and activate acitivin receptors (24, 25, 26). Therefore, the effects on thecal cell androgen production could result from BMP-4 activation of specific BMP receptors or through activin receptors. The observation that activin and BMP-4 have the similar effects on enzyme expression and androgen production support this hypothesis. However, we observed that the HOTT cells model expresses receptors for BMP; therefore, we suggest that the effect of BMP could be mediated through its BMP receptors or to a lesser degree through activin receptors.

The production of thecal cell androstenedione relies on the activities of CYP11A1, 17-hydroxylase; CYP17, and 3ßHSD (27). In addition, the production of androstenedione can be regulated by the expression of StAR protein that is needed for cholesterol to enter the mitochondria (28). As mentioned above, BMP-4 inhibited androstenedione production but increased the production of progesterone, suggesting that the enzymes involved in progesterone biosynthesis are not effected. Indeed, Western analysis revealed that BMP-4 exhibited specific effects on CYP17 without an inhibition of StAR, CYP11A1, or 3ßHSD. Human CYP17 and 3ßHSD represent key regulatory enzymes at branchpoints in the production of C₂₁ steroids (progesterone and 17-OHP) vs. the production of C₁₉ steroids (dehydroepiandrosterone and androstenedione). CYP17, through its 17-hydroxylase activity, will readily bind and metabolize pregnenolone or progesterone to 17-hydroxypregnenolone or 17-OHP, respectively. By virtue of its broad substrate specificity 3ßHSD will complete with CYP17 for the metabolism of pregnenolone and 17-hydroxypregnenolone (29, 30). Therefore, the relative expression of CYP17 and 3ßHSD will have direct effects on the amount of androstenedione produced by thecal tissue. Activin inhibited CYP17 in a similar manner and had little effect on StAR, CYP11A1, or 3ßHSD. Taken together, these data suggest that activin and BMP-4 decrease thecal cell androgen production by modifying the ratio of CYP17 to 3ßHSD within the cell. This results in the production of more progesterone and less androstenedione.

It could be hypothesized that such an effect could inhibit overall estrogen production in vivo by decreasing production of C₁₉ steroids in the thecal cells. Such an effect could be considered luteotropic because the production of estrogen does decrease following ovulation as the thecal cells luteinize. We have previously observed similar effects using activin, another member of the TGFß family of proteins (6). Activin also lead to an increase in progesterone production and an inhibition of androstenedione production. Activin and BMP-4 may well share signaling pathways and, therefore, act through a similar manner to regulate thecal cell steroidogenesis.

Received March 13, 2000.

Revised June 8, 2000.

Accepted June 15, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Short RV. 1962 Steroids in the follicular fluid and the corpus luteum of the mare: a "two-cell type" theory of ovarian steroid synthesis. J Endocrinol. 24:59–63.
2. McNatty KP, Makris A, DeGrazia C, Osathanondh R, Ryan K. 1979 The production of progesterone, androgens, and estrogens by granulosa cells, theca tissue and stroma tissue from human ovaries in vitro. J Clin Endocrinol Metab. 49:687–699.[Abstract/Free Full Text]
3. Ruutiainen K, Adashi EY. 1993 Intraovarian factors in hyperandrogenism. Semin Reprod Endocrinol. 11:324–328.[CrossRef]
4. Koppa SD. 1994 TGF-ß: what’s in a name? Drug Ther. 24:34–37.
5. Carr BR, McGee EA, Sawetawan C, Clyne CD, Rainey WE. 1996 The effect of transforming growth factor-ß on steroidogenesis and expression of key steroidogenic enzymes with a human ovarian thecal-like tumor cell model. Am J Obstet Gynecol. 174:1109–1117.[CrossRef][Medline]
6. Sawetawan C, Carr BR, McGee E, Bird IM, Hone TL, Rainey WE. 1996 Inhibin and activin differentially regulate androgen production and 17-hydroxylase expression in human ovarian thecal-like tumor cells. J Endocrinol. 148:213–221.[Abstract/Free Full Text]
7. Hogan BL. 1996 Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev. 10:1580–1594.[Free Full Text]
8. Wozney JM, Rosen V. 1998 Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop. 346:26–37.
9. Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM. 1998 The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol. 12:1809–1817.[Abstract/Free Full Text]
10. Leong LM, Brickell PM. 1996 Molecules in focus. Bone morphogenetic protein-4. Int J Biochem Cell Biol. 28:1293–1296.[CrossRef][Medline]
11. Jaatinen R, Rosen V, Tuuri T, Ritvos O. 1996 Identification of ovarian granulosa cells as a novel site of expression for bone morphogenetic protein-3 (BMP-3/osteogenin) and regulation of BMP-3 messenger ribonucleic acids by chorionic gonadotropin in cultured human granulosa-luteal cells. J Clin Endocrinol Metab. 81:3877–3882.[Abstract/Free Full Text]
12. Ring CJ, Cho KWY. 1996 Insights from model systems. Specificity in transforming growth factor-ß signaling pathways. Am J Hum Genet. 64:691–697.[CrossRef]
13. Shimizu K, Yoshikawa H, Matsui M, Masuhara K, Takaoka K. 1994 Periosteal and intratumorous bone formation in a thymic nude mice by Chinese hamster ovary tumors expressing murine bone morphogenetic protein-4. Clin Orthop Relat Res. 300:274–280.
14. Shimasaki S, Zachow RJ, Li D, et al. 1999 A functional bone morphogenetic protein system in the ovary. Proc Natl Acad Sci USA. 96:7282–7287.[Abstract/Free Full Text]
15. Rainey WE, Sawetawan C, McGee EA, Bird IM, Word RA, Carr BR. 1996 Human ovarian tumor cells: a potential model for theca cell steroidogenesis. J Clin Endocrinol Metab. 80:257–263.
16. Carr BR, McGee EA, Sawetawan C, Rainey WE. 1996 Development of a human thecal tumor cell model: regulation of steroidogenesis and enzyme expression. Proceedings of the Conference on Polycystic Ovarian Syndrome. Serono Symp (USA). 165–195.
17. Sawetawan C, Rainey WE, Word RA, Carr BR. 1995 Immunohistochemical and biochemical analysis of a human Sertoli-Leydig cell tumor: autonomous steroid production characteristic of ovarian theca cells. J Soc Gynecol Invest. 2:30–37.[Medline]
18. Chegini N, Flanders KC. 1992 Presence of transforming growth factor-ß and their selective localization in human ovarian tissue of various reproductive stages. Endocrinology. 130:1707–1715.[Abstract/Free Full Text]
19. Chegini N, Williams RS. 1992 Immunocytochemical localization of transforming growth factors (TGF_s) TGF- and TGF-ß in human ovarian tissues. J Clin Endocrinol Metab. 74:973–980.[Abstract]
20. Magoffin DA, Gancedo B, Erickson GF. 1989 Transforming growth factor-ß promotes differentiation of ovarian thecal-interstitial cells but inhibits androgen production. Endocrinology. 125:1951–1958.[Abstract/Free Full Text]
21. Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall RH, Mason AJ. 1996 Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells. Mol Cell Endocrinol. 157:R1–R6.
22. Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall RH, Mason AJ. 1992 Effect of recombinant activin on androgen synthesis in cultured human thecal cells. J Clin Endocrinol Metab. 72:1206–1211.[Abstract/Free Full Text]
23. McAllister JM, Byrd W, Simpson ER. 1994 The effects of growth factors and phorbol esters on steroid biosynthesis in isolated human theca interna and granulosa-lutein cells in long term culture. Endocrinology. 79:106–112.[Abstract/Free Full Text]
24. Peng C, Ohno T, Koh LY Chen VT, Leung PC. 1999 Human ovary and placenta express messenger RNA for multiple activin receptors. Life Sci. 64:983–994.[CrossRef][Medline]
25. Miyazono K. 1999 Signal transduction by bone morphogenetic protein receptors: function roles of Smad proteins. Bone. 25:91–93.[Medline]
26. Yamashita H, Miyazono K. 1999 Bone morphogenetic protein (BMP) receptors and transduction. Nippon Rinsho. 57:220–226.
27. Carr BR. 1998 Disorders of the ovaries and female reproductive tract. In: Wilson JD, Foster DW, Kronenbert HM, Larsen PR, eds. Williams Textbook of Endocrinology, ed 9. Philadelphia: WB Saunders Co; 751–817.
28. Orly J, Stocco DM. 1999 The role of the steroidogenic acute regulatory (StAR) protein in reproductive tissues. Horm Metabol Res. 31:389–398.[Medline]
29. Barnes HJ, Arlotto MP, and Waterman MR. 1991 Expression and enzymatic activity of recombinant cytochrome P450 17-hydroxylase in Escherichia coli. Proc Natl Acad Sci USA. 88:5597–5601.[Abstract/Free Full Text]
30. Brock BJ, Waterman MR. 1999 Biochemical differences between rat and human cytochrome P450c17 support the different steroidogenic needs of these two species. Biochemistry. 38:1598–1606.[CrossRef][Medline]

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