Augmented Androgen Production Is a Stable Steroidogenic Phenotype of Propagated Theca Cells from Polycystic Ovaries

Abstract

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17α-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17β-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17α-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3β-hydroxysteroid dehydrogenase, and 17β-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.

INTRODUCTION

Polycystic ovary syndrome (PCOS) is the most common cause of infertility in women (1). PCOS ovaries are characterized by the accumulation of small follicles 4–7 mm in diameter with hypertrophied theca interna layers (2, 3). Reproductive abnormalities include amenorrhea or oligomenorrhea, infertility, hirsutism, and acne resulting from increased ovarian androgen production (4–7). Other frequently associated abnormalities include insulin resistance and obesity. Familial clustering of PCOS suggests that genetic factors are involved in the etiology of the disorder, and it has been proposed that PCOS is an oligogenic syndrome involving genes governing steroid hormone biosynthesis as well as insulin/glucose homeostasis (8–10).

The molecular and cellular mechanisms underlying the excessive ovarian androgen production associated with PCOS remain to be elucidated (11–18). There is in vivo evidence to support the concept that excess androgen production in PCOS results from a primary abnormality in steroid production by ovarian theca cells (19). In the human follicle, the androgen-secreting theca cells express 17α-hydroxylase/17,20- desmolase (CYP17), cholesterol side chain cleavage enzyme (CYP11A), 3β-hydroxysteroid dehydrogenase (3β-HSD), and steroidogenic acute regulatory protein (StAR), each of which are required for androgen and progestin production (20–26). Gilling-Smith et al. (19) reported that after GnRH agonist (GnRHa)-induced suppression of serum LH concentrations, ovarian androgen production in PCOS patients was significantly higher than in controls. Because CYP17 is the key enzyme required for androgen production in theca cells, Rosenfield and Barnes (5, 27, 28) and Nestler et al. (29, 30) proposed that excess androgen production in PCOS results from dysregulation of CYP17 enzyme activity due to an intrinsic ovarian defect. Subsequent studies of Gilling-Smith et al. (31) suggested that androgen production per theca cell is increased in PCOS. However, it is still not known whether the increased androgen production in PCOS is caused by dysregulation of ovarian theca cell CYP17. Although it has been repeatedly proposed that CYP17 expression is elevated in PCOS theca cells, no study has directly compared CYP17 enzyme activity or the regulation of CYP17 mRNA expression in normal and PCOS theca cells. In addition, it is also possible that changes in CYP11A, 3β-HSD activity, or StAR expression contribute to increased ovarian androgen production in PCOS. Moreover, the question of whether the excessive androgen production by PCOS theca cells in culture (31) reflects an intrinsic abnormality or whether it results from the residual effects of the hormonal milieu to which the cells were exposed in vivo has not been addressed.

Although a number of theories have been proposed to explain the etiology of excess androgen production by PCOS ovaries, few studies have focused on the regulation of steroidogenic enzyme activity and expression in isolated theca interna cells that have been maintained in culture in the absence of gonadotropins. We have begun to comprehensively examine the regulation of androgen production at the metabolic and molecular level using normal and PCOS theca interna cells isolated from size-matched follicles and propagated for multiple population doublings to determine whether increased androgen production in PCOS results from abnormalities in the regulation of StAR, CYP11A, CYP17, and 3β-HSD gene expression in response to forskolin (22, 23–25, 32).

RESULTS

Normal and PCOS Theca Cells in Long-Term Culture: Steroid Accumulation in Response to Forskolin

To compare cAMP-stimulated steroid synthesis in normal and PCOS theca cells derived from follicles of comparable size and cultured under identical conditions for 22–26 population doubling (three passages), we measured progesterone (P4), 17α-hydroxyprogesterone (17OHP4), and testosterone (T) accumulation in the media of cells treated with increasing concentrations of forskolin (Fig. 1). In both normal and PCOS theca cell cultures there was a dose-dependent increase in forskolin-stimulated steroid hormone production, which was maximal 72 h after treatment with 20 μm forskolin. The ED₅₀ for forskolin-stimulated steroidogenesis was 3μ m in both normal and PCOS theca cell preparations. In normal theca cells, forskolin-stimulated P4 and 17OHP4 production were increased 40-fold and 22-fold, respectively, over basal values. Negligible amounts of T were present in the media of normal theca cell cultures. In PCOS theca cells maximally stimulated with forskolin, P4, 17OHP4, and T production increased 43-fold, 22-fold, and 12-fold, respectively, over basal values. Under basal conditions, P4, 17OHP4, and T production by PCOS theca cultures was elevated 20-fold (P < 0.05), 22-fold (P < 0.05), and 4-fold (P < 0.05), respectively, above normal theca cell values. Moreover, PCOS theca cells stimulated with 20μ m forskolin produced 10-fold, 25-fold, and 30-fold more P4, 17OHP4, and T, respectively, than forskolin-stimulated normal theca cells. Thus, while both normal and PCOS are responsive to forskolin, PCOS thecal cells consistently produce greater quantities of steroid hormone and have the capacity to produce substantial amounts of T.

Figure 1.

Accumulation of 17OHP4, P4, and T in the Medium of Normal and PCOS Theca Cell Cultures Grown for 22–26 Population Doublings Long-term cultures of normal and PCOS theca cells were grown until subconfluence and then transferred into SFM containing 5 μg/ml low density lipoprotein, with increasing concentrations of forskolin (F) for 72 h. After treatment the media were collected and analyzed by RIA. Results are presented as the means ± sd of steroid levels from quadruplicate theca cell cultures from three normal and three PCOS patients.

Metabolism of P5, P4, and Dehydroepiandrosterone (DHEA)

To further explore the biochemical differences between normal and PCOS theca cells, the time courses and patterns of[ ³H]-P5 (Figs. 2, 3, 4), [³H]-P4 (Fig. 5), and[ ³H]-DHEA (Fig. 6) metabolism were examined in normal and PCOS theca cells that had been grown for 22–26 (three passages) and for 31–38 population doublings (four passages), and treated for 72 h with a 20 μm dose of forskolin. Although the metabolism profiles of cells from individual patients were distinct, the results presented show the predominant products of metabolism that are representative of theca cells isolated from four normal and five PCOS patients that had been matched for follicle size (∼4 mm) and patient age (38–40 yr). Similar data were also obtained with theca cells isolated from multiple size-matched follicles from individual patients (data not shown).

Figure 2.

Comparison of the Time Courses of Pregnenolone (P5) Metabolism by Normal (left panel) and PCOS (right panel) Theca Cells under Control and Forskolin-Stimulated Conditions Passaged theca cells (22–26 population doublings) isolated from normal and PCOS patients were treated with forskolin (20 μm) and without (control) in SFM for 72 h and transferred into SFM containing 1.0 μm [³H] pregnenolone. Aliquots of the medium were removed at various intervals and extracted with methylene chloride, and steroid products were separated by HPLC. Disappearance of radiolabeled pregnenolone (○) and appearance of the indicated steroid metabolites is presented as a function of time. Results are presented as means ± sd from triplicate cultures from representative normal and PCOS theca cells.

Figure 3.

Time Course of Pregnenolone (P5) Metabolism by Normal (left panel) and PCOS (right panel) Theca Cells Propagated for 22–26 Population Doublings Passaged theca cells isolated from normal and PCOS patients were treated in SFM with 20 μm forskolin for 72 h and transferred into SFM containing 1.0 μm[ ³H]-pregnenolone. Aliquots of the medium were removed at various intervals and extracted with methylene chloride, and steroid products were separated by HPLC. Disappearance of radiolabeled pregnenolone (○) and appearance of the indicated steroid products is presented as a function of time. Results are presented as means ± sd from triplicate theca cell cultures from three normal and three PCOS ovaries.

Figure 4.

Time Course of Pregnenolone (P5) Metabolism by Normal-2 (left panel) and PCOS-2 (right panel) Theca Cells Propagated for 31–38 Population Doublings Passaged theca cells isolated from normal and PCOS patients were treated in SFM with 20 μm forskolin for 72 h and transferred into SFM containing 1.0 μm[ ³H]-pregnenolone. Aliquots of the medium were removed at various intervals and extracted with methylene chloride, and steroid products were separated by HPLC. Disappearance of radiolabeled pregnenolone (○) and appearance of the indicated steroid products is presented as a function of time. Results are presented as means ± sd from triplicate theca cell cultures from three normal and three PCOS ovaries.

Figure 5.

Time Course of P4 Metabolism by Normal (left panel) and PCOS (right panel) Theca Cells in Long-Term Culture Passaged theca cells (22–26 population doublings) isolated from normal and PCOS patients were treated with 20 μm forskolin for 72 h and transferred into SFM containing 1.0 μm[ ³H]-P4. Aliquots of the medium were removed at various intervals and extracted with methylene chloride, and steroid products were separated by HPLC. Disappearance of radiolabeled P4 (○), and appearance of the indicated steroid products is presented as a function of time. Results are presented as means ± sd from triplicate theca cell cultures from three normal and three PCOS ovaries.

Figure 6.

Time Course of DHEA Metabolism by Normal (left panel) and PCOS (right panel) Theca Cells in Long-Term Culture Passaged theca cells (22–26 population doublings) isolated from normal and PCOS patients were treated with 20 μm forskolin for 72 h and transferred into SFM containing 1.0 μm[ ³H]-pregnenolone. Aliquots of the medium were removed at various intervals, extracted with methylene chloride, and steroid products were separated by HPLC. Disappearance of radiolabeled DHEA (○) and appearance of the indicated steroid products is presented as a function of time. Results are presented as means ± sd from triplicate theca cell cultures from three normal and three PCOS ovaries.

As shown in Fig. 2, in theca cells isolated from normal patients and cultured under basal conditions, the products of [³H]-P5 metabolism were primarily 17α-hydroxypregnenolone (17OHP5), and DHEA, which are both products of CYP17 enzyme activity. 5-Pregnene,3β,20α-diol (20α-OHP5), a product of 20α-hydroxysteroid dehydrogenase (20α-HSD) activity, was also produced to a limited extent. After forskolin stimulation, the rate of[ ³H]-P5 metabolism to 17OHP5 and DHEA was increased, as was the production of 17,20α-dihydroprogesterone (4-pregnene-17α, 20α-diol-3-one, 17,20α-DHP4) and 5-pregnene-3β,17α,20α-triol (17,20α-P5). 17,20α-DHP4 and 17,20α-DHP5 are produced by the action of 20α-HSD on 17OHP4 and 17OHP5, respectively. In contrast, in PCOS theca cells[ ³H]-P5 was further metabolized to T, Adione, and androstenediol (Adiol) as well as to larger amounts of 17OHP4, 17,20α-DHP4, and 17,20α-DHP5. The rate of [³H]-P5 metabolism was increased after forskolin treatment in both normal and PCOS theca cells, but was more markedly enhanced in PCOS theca cells.

To determine whether increased [³H]-P5 metabolism is a property retained by PCOS theca cells over longer culture periods,[ ³H]-P5 metabolism was examined in forskolin-stimulated theca cells isolated from three normal and three PCOS patients grown for 22–26 population doublings (Fig. 3) and 31–38 population doublings (Fig. 4). The metabolism profiles were similar for cells grown for 22–26 and 31–38 population doublings but PCOS cells differed markedly from normal theca cells. In normal theca cells, 90% of labeled pregnenolone was metabolized to 17α-hydroxypregnenolone and DHEA in 8–12 h, while at 24–48 h only low levels of Adione were produced (left panel). In contrast, in PCOS theca cells (right panel), the rate of [³H]-P5 metabolism was much faster; more than 90% of labeled P5 was converted to 17OHP5 and DHEA within 3–6 h, and within 12–24 h it had been metabolized to T, Adione, and Adiol. These data suggest that 3β-HSD and/or CYP17 enzyme activities are elevated in PCOS cells as compared with normal theca cells. Although 20α-HSD activity appeared to be stimulated by forskolin in both normal and PCOS theca cells, 20α-HSD activity/cell was not elevated in PCOS theca cells.

To further explore differences in CYP17 enzyme activity in PCOS and normal theca cells, [³H]-P4 metabolism was examined in cells grown for 22–26 population doublings (Fig. 5) and 31–38 population doublings (data not shown). In theca cells isolated from normal ovaries, we previously reported that P4 is metabolized predominantly to 17OHP4, 17,20α-DHP4, and 16α-hydroxyprogesterone (16OHP4) (25). 16α-Hydroxylation of P4 to 16OHP4 was a side reaction associated with CYP17 enzyme activity. P4 was also metabolized to a limited extent to 4-pregnene-20α-hydroxy-3-one (20α-OHP4), which is a product of the 20α-HSD reaction. In the present studies utilizing normal theca cells after forskolin stimulation (left panel), 80% of[ ³H]-P4 was metabolized to 17α-hydroxylated products within 24–36 h, whereas in PCOS theca cells (right panel) 80% of [³H]-P4 was metabolized to similar products within 4–8 h. Thus, the rate of labeled P4 metabolism was accelerated and CYP17 enzyme activity/theca cell was elevated in PCOS cells as compared with normal theca cells. Moreover, the metabolism profile presented for theca cells grown for 22–26 population doublings (Fig. 5) was similar to that observed for cells grown for 31–38 population doublings. These data again indicate that CYP17 enzyme activity per theca cell is increased in PCOS, and that increased CYP17 activity is a stable property of PCOS theca cells in long-term culture.

To compare 3β-HSD activity in normal and PCOS theca cells, as well as establish the predominant intermediates of androgen biosynthesis,[ ³H]-DHEA metabolism was examined in cells grown for 22–26 population doublings (Fig. 6) and 31–38 population doublings (data not shown). In normal theca cells (left panel), 30% of[ ³H]-DHEA was converted to Adione and, to a limited extent, Adiol within 48 h. In agreement with the data presented in Fig. 1 examining de novo T production in normal theca cells, T was not a product of DHEA metabolism during the 48-h incubation period, suggesting the absence of androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity. In contrast, in PCOS theca cells (right panel), 60% of[ ³H]-DHEA was converted to Adione, Adiol, and T within 12 h. Further metabolism of T to 5α-reduced steroids was not observed during the 48 h incubation period, indicating the absence of 5α-reductase. Moreover, the metabolism profile presented for theca cells grown for 22–26 population doublings (Fig. 6) was similar to that observed for cells grown for 31–38 population doublings (data not shown). These observations confirm that increased 3β-HSD enzyme activity per theca cell is a stable property of PCOS theca cells in culture and demonstrate that normal thecal cells are relatively deficient in androgenic 17β-HSD. Labeled DHEA (Fig. 6), P4 (Fig. 5), and P5 (Figs. 2, 3, 4) were not converted into estradiol by either normal or PCOS thecal cells. The absence of detectable estradiol formation is consistent with the lack of granulosa cell contamination of our theca cell preparations.

Expression of Steroidogenic Enzyme mRNAs

To determine whether the increased production of steroids and rates of precursor metabolism characteristic of PCOS theca cells resulted from increased steady state levels of steroidogenic enzyme mRNAs, Northern analyses were performed. Total mRNA was harvested from theca cells isolated from four normal and four PCOS patients that were cultured with and without 20 μm forskolin for 48 h. In Fig. 7, a representative Northern analysis of mRNA (50 μg/lane) isolated from theca cells from two normal and two PCOS patients, hybridized with complementary probes for human CYP17, CYP11A, StAR, and 28S rRNA, is presented. Results from these experiments demonstrated that forskolin-stimulated CYP17 and CYP11A mRNA accumulation is markedly augmented in PCOS cells. As shown in Fig. 8, 48 h of forskolin treatment of control theca cells resulted in modest (1.5- to 2-fold) but significant increases in steady state levels of CYP17 and CYP11A mRNA over the basal levels. In contrast, under identical conditions CYP17 mRNA levels were increased 6-fold and CYP11A levels were increased 5-fold over basal levels in PCOS theca cells. However, StAR mRNA levels were increased to similar extents in normal and PCOS theca cells (∼4-fold) in response to forskolin treatment.

Figure 7.

cAMP-Stimulated CYP17, CYP11A, StAR, and 28S mRNA Expression in Normal and PCOS Theca Cells Normal and PCOS theca cells (22–26 population doublings) were grown until confluent and transferred into SFM in the presence (F) or absence (C) of 20 μm forskolin. At 48 h, total mRNA was harvested and Northern blot analysis was performed using 50 μg of total mRNA per lane. A riboprobe complementary to human CYP17 mRNA was used as a hybridization probe. Multiprime labeled cDNAs homologous for human CYP11A, StAR, and 28S rRNA were also used as probes. Human adrenal mRNA (hA) was used as a positive control.

Figure 8.

Relative Abundance of CYP17, CYP11A, and StAR mRNA in Normal and PCOS Theca Cells Cumulative data from Northern blot analyses for steady state CYP17, StAR, and CYP11A mRNA levels in theca cells propagated for 22–26 population doublings from four normal and four PCOS patients normalized to 28S rRNA levels. Values presented are means ± sem for fold increases above background. Forskolin treatment significantly increased (P < 0.05) CYP17, StAR, and CYP11A mRNA levels in normal and PCOS theca cells (*).

DISCUSSION

The observations of Gilling-Smith et al. (31) suggested that androgen production per cell is elevated in PCOS theca cells in primary culture. However, it was not known whether the excessive androgen production resulted from increased CYP17 expression, changes in other proteins involved in steroid synthesis, or the hormonal milieu to which the isolated thecal cells had been exposed in vivo. Yet, these observations raised the possibility that the excessive androgen production in PCOS results from intrinsic abnormalities in theca cell steroidogenesis. If the abnormalities that underlie PCOS are indeed intrinsic, then increased androgen production should persist in PCOS theca cells maintained in long-term culture. Our data provide support for the hypothesis that excessive androgen production by PCOS theca cells results from increased expression of the enzymes involved in steroid synthesis. Using culture conditions developed for normal theca cells, we established methods to passage normal and PCOS theca cells for successive population doublings with the maintenance of regulatable CYP17, CYP11A, 3β-HSD, and StAR gene expression (23, 25, 26, 32). The ovaries from which PCOS theca cells were isolated had a characteristic gross morphology. The diagnosis of PCOS was established by menstrual history (<6 menses per year) and elevated total serum T and/or bioavailable T levels. It is important to recognize that previous studies comparing steroid production by normal and PCOS theca cells used primary cultures of theca cells that were pooled from multiple follicles and/or patients (30, 31). To our knowledge, all of these studies examined steroid production within a 48–72 h period after cell isolation, under conditions in which the influence of prior in vivo stimulation may have persisted. In marked contrast, in our studies theca cells were propagated from individual follicles isolated from normal and PCOS patients, matched for follicle size and patient age. Cells from individual follicles were initially frozen and stocked in multiple vials. From each vial of frozen theca cells, second-passage cells could be propagated, frozen, and stocked, or subsequently cultured through a third passage (i.e. 22–26 populations doublings) or fourth passage (31–38 population doublings). The ability to propagate functional theca cells for successive population doublings provides a reproducible system to repetitively compare theca cells isolated from individual patients and individual follicles. Moreover, this system permits us to examine cells under conditions that are distant from their in vivo paracrine and endocrine milieu to investigate whether there are unique molecular or biochemical phenotypes in PCOS ovarian cells.

Data from our experiments clearly establish that P4, 17OHP4, and T production are markedly elevated per cell in PCOS theca cultures. In these experiments, both androgen and P4 levels were found to be increased in PCOS theca cells, and the androgen/P4 ratio was elevated, indicating that androgen production predominates. Since the ED₅₀ for forskolin-stimulated steroid accumulation in both normal and PCOS theca cells was identical (∼3 μm), it does not appear that the differences in steroidogenic activity can be attributed to differences in forskolin-stimulated adenylate cyclase activity. It is notable that the absolute amounts of P4, 17OHP4, and T produced per PCOS theca cell (passaged for 22–38 population doublings) were similar to those reported by Gilling-Smith et al. (31), which were obtained from primary (nonpassaged) PCOS theca cells. In both our study and that of Gilling-Smith et al. (31), steroid production as well as the androgen-progestin ratio were increased in PCOS theca cells. Our findings, however, demonstrate that the rates of basal and forskolin-stimulated P5, P4, and DHEA metabolism are all dramatically increased in PCOS cells. These data definitively demonstrate for the first time that CYP17 and 3β-HSD enzyme activities per theca cell are increased under both basal and forskolin-stimulated conditions in PCOS theca cells. The metabolic profiles also indicate that an androgenic 17β-HSD activity is increased in PCOS theca cells. Thus, the distinctive biochemical phenotype of PCOS theca cells in long-term culture encompasses increased activities of multiple steroidogenic enzymes. The evidence for increased androgenic 17β-HSD is of particular interest because normal human ovaries are reported not to express type III 17β-HSD, the enzyme responsible for testicular T production (33). Thus, either PCOS theca cells aberrently express the type III enzyme or an alternative activity capable of reducing the 17-keto group of C19 steroids must be activated in PCOS.

The data presented in Figs. 2, 3, 4, 5, 6 provide new and significant information about the principal intermediates and the predominant steroidogenic pathways involved in steroid biosynthesis in normal and PCOS theca cells. Specifically, normal and PCOS theca cells do not have the capacity to convert 17OHP4 to Adione. Androgen production in normal theca cells, as well as increased androgen production by PCOS theca cells, evidently involves the initial conversion of cholesterol to P5 (via CYP11A) and the subsequent conversion of P5 to DHEA (via CYP17). DHEA is then further metabolized to Adione (via 3β-HSD) and/or Adiol (via 17β-HSD), which are finally converted to T. Thus, in agreement with our previous reports (22, 24), the Δ⁵-steroid pathway is the predominant pathway used for androgen biosynthesis by both normal and PCOS theca cells. TheΔ ⁴-steroids, P4 and 17OHP4, are not precursors for Adione or T production by the normal or PCOS ovary. As we previously reported (23), estradiol was not found to be a product of steroid metabolism in normal or PCOS theca cells, substantiating the purity of the theca cell preparations used in these studies. Of importance is the fact that the steroid metabolism profiles observed in normal and PCOS theca cells grown for 22–26 population doublings were similar to those observed when cells are grown for 31–38 population doublings. These data verify that increased androgen production is a stable phenotype of the cultured PCOS theca cells.

The ability to propagate normal and PCOS theca cells in long-term culture has and will continue to facilitate the examination of the molecular basis for increased androgen production in PCOS. In this report we present, for the first time, Northern analyses on RNA obtained from theca cells isolated from individual follicles from normal and PCOS patients. Data from these experiments definitively demonstrate that the magnitude of forskolin-stimulated CYP17 and CYP11A mRNA induction is greater in PCOS than in normal theca cells. These data support the hypothesis that increased steady state levels of CYP17 and CYP11A mRNAs contribute to increased levels of steroidogenic enzymes involved in androgen production by PCOS theca cells. Although StAR mRNA levels were increased in response to forskolin, the magnitude of StAR mRNA induction by forskolin was not different between PCOS and control theca cells. These data suggest that increased androgen production in PCOS does not result from overall differences in cAMP or adenylate cyclase regulation, but rather selective alterations in steroidogenic enzyme expression.

The persistent differences in steroidogenic activity of PCOS theca cells could reflect an intrinsic (i.e. genetic) abnormality in these cells or a stable biochemical imprint resulting from the endocrine milieu experienced by the cells in vivo. Unfortunately, at present, no definitive experiment can distinguish between these two possibilities. However, there is increasing evidence for a genetic basis for the hyperandrogenemia associated with PCOS (8). If the stable biochemical phenotype in PCOS cells that we observed is the result of a genetic variation, it evidently influences the expression of multiple genes in the steroidogenic machinery, suggesting that the putative abnormality most likely involves a signal transduction pathway. PCOS theca cells may generate autocrine factors that enhance steroidogenesis. Alternatively, PCOS cells may have increased sensitivity to some component of the culture medium that stimulates expression of steroidogenic enzymes. The factor is not likely to be insulin since we have found, in unpublished experiments, that both normal and PCOS theca cells respond equivalently to insulin in terms of steroid (170HP4) production per cell and that increased forskolin-stimulated steroid secretion by PCOS cells is observed in the absence of insulin in the culture medium. The thecal cell system we have employed is well suited for the study of the biochemical locus that results in the stable steroidogenic phenotype of PCOS cells.

In conclusion, our analyses of normal and PCOS theca cells maintained in long-term culture suggest that increased expression of CYP17 and CYP11A mRNAs, as well as increased CYP17 and 3β-HSD and 17β-HSD activities per theca cell, are stable properties of PCOS theca cells. These data are consistent with the concept that increased androgen production by PCOS theca cells is an intrinsic and, possibly genetically determined, property of the cells. In future studies we will attempt to define the locus of this abnormality through a comprehensive analysis of the molecular mechanisms involved in the transcriptional and posttranscriptional regulation of CYP17, CYP11A, 3β-HSD, and 17β-HSD expression.

MATERIALS AND METHODS

Theca Cell Isolation and Propagation

Human theca interna tissue was obtained from follicles of women undergoing hysterectomy. The PCOS and normal ovarian tissue came from age-matched women 38–40 yr of age. The diagnosis of PCOS was made according to established guidelines (34) including hyperandrogenemia, oligo-ovulation, and the exclusion of 21-hydroxylase deficiency, Cushing’s syndrome, and hyperprolactinemia. All of the theca cell preparations studied came from ovaries of women with fewer than six menses per year and elevated serum total T or bioavailable T levels as we have previously described (8). Each of the PCOS ovaries contained multiple subcortical follicles of less than 10 mm in diameter. The control theca cell preparations came from ovaries of fertile women with normal menstrual histories and no clinical signs of hyperandrogenism.

Individual follicles were dissected away from ovarian stroma. The isolated follicles were size selected for diameters ranging from 3–5 mm so that theca cells derived from follicles of similar size from normal and PCOS subjects could be compared. The dissected follicles were placed into serum containing medium and bisected. Under a dissecting microscope, the theca interna was stripped from the follicle wall, and the granulosa cells were removed with a platinum loop. The cleaned theca shells were dispersed with 0.05% collagenase I, 0.05% collagenase IA, and 0.01% deoxyribonuclease in medium containing 10% FBS (23). Dispersed cells were placed in culture dishes that had been precoated with fibronectin by incubation at 37 C with culture medium containing 5 μg/ml human fibronectin. The media used for cell plating was a 1:1 mixture of DMEM and Hams F-12 medium containing 10% FBS, 10% horse serum, 2% UltroSer G, 20 nm insulin, 20 nm selenium, 1 μm vitamin E, and antibiotics (23). From each follicle twelve 35-mm dishes of primary theca interna cells were grown until confluent, removed from the dish with neutral protease (pronase-E; protease type XXIV, Sigma Chemical Co., St. Louis, MO) in DMEM/F12 (1:1), frozen, and stored in liquid nitrogen (one 35-mm dish per vial) as previously described (23) in culture medium that contained 20% FBS and 10% dimethyl sulfoxide. In all experiments, cells were thawed and propagated in the growth medium described above.

To obtain successive passages of normal and PCOS theca cells, cells were thawed, propagated, and frozen at consecutive passages. The passage conditions and split ratios for all normal and PCOS cells were identical. Experiments comparing PCOS and normal theca were performed utilizing third passage (i.e. 22–26 population doublings) and fourth passage (31–38 population doublings) theca cells isolated from size-matched follicles obtained from age-matched subjects.

At confluence the cells were transferred into serum-free medium (SFM) containing DMEM/F12, 1.0 mg/ml BSA, 100 μg/ml transferrin, 20 nm insulin, 20 nm selenium, 1.0μ m vitamin E, and antibiotics. Sera and growth factors were obtained as follows: FBS was obtained from Irvine Scientific (Irvine, CA): horse serum was obtained from Gibco BRL (Gaithersburg, MD); UltroSer G was from Reactifs IBF (Villeneuve-la-Garenne, France): other compounds were from Sigma Chemical Co. In all experiments the gas phase used was 5% O₂, 90% N₂, and 5% CO₂. Reduced oxygen tension and supplemental antioxidants (vitamin E and selenium) were employed to prevent oxidative damage to CYP17 and CYP11A (22, 23). Subculture was performed by incubation with neutral protease.

Assays for P5, P4, 17OHP4, and T

For evaluation of steroid production, normal or PCOS theca cells were grown until subconfluent and transferred into SFM with 5 μg/ml low-density lipoprotein in the presence and absence of 20μ m forskolin for 72 h to induce full steroidogenic capacity. At 72 h the media were collected. RIAs for 17OHP4, P, and T were then performed without organic solvent extraction using RIA kits from ICN Biochemicals, Inc. (Irvine, CA).

Steroid Metabolism Assays

Long-term theca cultures (normal and PCOS) were grown until subconfluent and transferred into SFM in the presence or absence of 20μ m forskolin for 72 h to induce full steroidogenic capacity. The cells were then transferred into medium containing saturating concentrations of [³H]-pregnenolone (1.0μ m), [³H]-P4 (1.0 μm), or[ ³H]-DHEA (1.0 μm). Aliquots of the medium were obtained at various time intervals (i.e. 3, 6, 12, 24, 36, 48, and 72 h). Steroids were extracted from the medium with 4 vol dichloromethane (HPLC grade) with an extraction efficiency greater than 90%. The dichloromethane phase containing unconjugated steroids was evaporated. The residue was dissolved in methanol and subjected to reverse-phase HPLC. HPLC was conducted on a computer-controlled automated chromatogram (Gilson Medical Electronics, Inc., Middleton, WI) using a Phenomenex 25-cm 5μ m Prodigy C18 column (Milford, MA). The gradient solvent delivery system consisted of 1:1 acetonitrile/methanol (A/M) and water (50:50) for 10 min, followed by a 10-min linear gradient to 57% A/M, and an additional 4-min linear gradient to 73% A/M for 9 min, and then a 2 min linear gradient to 100% A/M. Radioactive material was detected by an in-line liquid scintillation spectrophotometer (IN/US System Inc., Tampa, FL). The retention times of authentic steroid standards were established for the nonreduced and reduced steroids at 240 and 200 nm, respectively.

Determination of CYP17, CYP11A, StAR, and 3β-HSD mRNA Levels

Total RNA was extracted from theca cells as previously described (23). Specific mRNA levels were quantitated using standard Northern techniques. Human CYP17, CYP11A, 3β-HSD, and StAR cDNAs were used as hybridization probes. Hybridizable mRNA species were identified by autoradiography and normalized using 28S ribosomal RNA.

Statistical Analysis

Statistical analysis was performed using unpaired two-tailed t tests after combining the results from individual patients. Each experiment was performed using triplicate or quadruplicate replicate dishes. Experiments were repeated several times with cells obtained from various PCOS and normal patients, that had been thawed and grown to the appropriate passage.

Acknowledgments

We would like to thank Drs. Ronald E. Estabrook and John M. Trant for their valuable advice in the establishment of our HPLC separation technique for identifying the numerous metabolic products of steroid metabolism in normal and PCOS theca cells. We also thank Jessica Wickenheisser for collecting media samples at various time points throughout the steroid metabolism experiments.

This work was supported by NIH Grants U54 HD-34449 (J.F.S. and J.M.M.) and R01 HD-33852 (J.M.M.)