Chiravudh Sawetawan

Compartmentalization of type 117β-hydroxysteroid oxidoreductase in the human ovary

Abstract The steroid-metabolizing enzyme, type I 17β-hydroxysteroid oxidoreductase (17β-HSOR) also called 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a key role in ovarian synthesis of 17β-estradiol. This is the only enzyme in the steroid-metabolizing pathway which has not been localized in the human ovary by immunohistochemistry. In this study, using antibody directed against human placenta! cytosolic 17β-HSOR (type I), a single protein band with a relative molecular mass of approximately 34 kDa was demonstrated by Western analysis in both human luteinized granulosa cells and placental tissue. In placental tissue, immunoreactive type I 17β-HSOR was demonstrated within the syncytiotrophoblast using immunohistochemistry. In human ovary, immunoreactive type I 17β-HSOR was localized exclusively in granulosa cells of developing follicles, ranging from primary follicles with a single layer of cuboidal-shaped granulosa cells, preantral follicles with multiple layers of granulosa cells, and large antral follicles. No immunoreactivity was detected in spindle-shaped granulosa cells of primordial follicles, theca interna, theca externa or surrounding stroma. In the corpus luteum, type I 17β-HSOR immunoreactivity was localized solely in granulosa-lutein cells. For comparison, immunoreactive 3β-hydroxysteroid dehydrogenase (3β-HSD) was examined in the same tissues. Both theca interna and granulosa cells of preantral and antral follicles exhibited 3β-HSD staining. Primary follicles did not exhibit detectable 3β-HSD in either granulosa or theca cells. This study serves to demonstrate that in the human ovary, type I 17β-HSOR is compartmentalized in granulosa cells of the developing follicles and granulosa-lutein cells. In addition, the expression of type I 17β-HSOR appears to be present prior to that of 3β-HSD at the stage of primary follicle. With the strategic location of type I 17β-HSOR and the ability to aromatize theca-derived androstenedione to estrone, granulosa cells have the capacity to synthesize and maintain the high intrafollicular levels of 17β-estradiol, which are essential for normal follicular development.

The Effects of Insulin on 3β-Hydroxysteroid Dehydrogenase Expression in Human Luteinized Granulosa Cells

Objective: We determined the relative effects of insuling and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3β-hydroxysteroid dehydrogenase (3βHSD) in human luteinized granulosa cells. Methods: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treatd with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3βHSD were evaluated. Results: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/μL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3βHSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02) Insulin (greater than 100 nmol/L) alone increased 3βHSD protein content as measured by Western analysis 1.8-2fold over basal levels, whereas FSH alone increased protein contetn 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3βHSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3βHSD 3.2-fold. In the presence of insulin plus FSH, 3βHSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3βHSD. Conclusion: Insulin is a regular of both 3βHSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis.

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess. Because of difficulties in obtaining adequate numbers of thecal cells, the biomolecular regulation of C19 steroid production and expression of steroidogenic enzymes is not well defined. We have overcome this obstacle by developing a highly dependable and unique human ovarian thecal-like tumor (HOTT) cell culture model system from an ovarian tumor found to produce excessive amounts of C19 steroids. Aliquots of freshly dispersed tumor cells were frozen for future use. Once placed in monolayer culture, HOTT cells proliferated and could be maintained for extended periods. Acutely, cultured HOTT cells increased progesterone and cAMP production in response to 2 h of forskolin treatment. These cells were, however, unresponsive to treatment with LH. Steroid hormone production continued in cells that were maintained in culture for up to 2 months. Analysis of the steroids produced by HOTT cells was accomplished using RIA and high performance liquid chromatography. Under basal conditions, HOTT cells produced mainly 17 alpha-hydroxyprogesterone and progesterone. Treatment with forskolin or dibutyryl cAMP (dbcAMP) increased the production of progesterone and 17 alpha-hydroxyprogesterone as well as C19 steroids. Treatment of monolayer cultures of HOTT cells with forskolin (0.01 to 20 mumol/L) or dbcAMP (0.01 to 1 mmol/L) for 48 h increased the production of androstenedione (8- to 15-fold) and progesterone (2- to 5-fold). In HOTT cells chronically treated with forskolin or dbcAMP (up to 72 h), progesterone production was observed to plateau, although the amount of androstenedione continued to increase. The enzymatic activities of both 3 beta-hydroxysteroid dehydrogenase (6-fold), and 17 alpha-hydroxylase P450 (P450c17; 9-fold) were also increased by activation of the protein kinase A messenger pathway. Treatment of HOTT cells with forskolin caused a time-dependent induction of the messenger RNAs for cholesterol side-chain cleavage P450, 3 beta-hydroxysteroid dehydrogenase, and P450c17. No changes in steroidogenic enzyme expression were observed following treatment with LH. In conclusion, these data demonstrate that certain ovarian tumor cells may serve well as appropriate models to study the molecular mechanisms regulating human ovarian thecal cell C19 steroidogenesis and the expression of steroid-metabolizing enzymes.

The effect of transforming growth factor-β on steroidogenesis and expression of key steroidogenic enzymes with a human ovarian thecal-like tumor cell model

OBJECTIVE Our purpose was to determine the effects of transforming growth factor-beta on steroidogenesis and regulation of steroidogenic enzyme expression by use of a human ovarian thecal-like tumor cell culture system. STUDY DESIGN Human ovarian thecal-like tumor cells were treated in serum-free medium in the presence or absence of forskolin and transforming growth factor-beta 1. The accumulation of progesterone and androstenedione in the culture medium was evaluated by radioimmunoassay. The effects of forskolin with or without transforming growth factor-beta 1 on the enzymatic activity of P450c17 and 3 beta HSD, the expression of immunodetectable P450c17 protein, and the expression of messenger ribonucleic acid for P450scc, P450c17, and 3 beta HSD were determined. RESULTS Basal steroid secretion, steroidogenic enzyme activity, enzyme protein, and messenger ribonucleic acid expression were not affected by transforming growth factor-beta 1 alone. Forskolin treatment significantly stimulated steroid production and the enzymatic activity of P450c17 and 3 beta HSD up to 10-fold above basal levels. However, transforming growth factor-beta 1 inhibited forskolin-stimulated androstenedione production to near basal levels and increased progesterone 1.4- to 2-fold while suppressing P450c17 enzyme activity to near basal levels, but it did not affect 3 beta HSD activity. Forskolin-stimulated immunodetectable P450c17 alpha protein was markedly inhibited by transforming growth factor-beta 1. In addition, transforming growth factor-beta 1 markedly inhibited the forskolin-stimulation of P450c17 messenger ribonucleic acid, while not significantly altering P450scc or 3 beta HSD messenger ribonucleic acid expression. CONCLUSION Forskolin stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17 and 3 beta HSD activity, immunodetectable P450c17, and messenger ribonucleic acid content for P450scc, P450c17, and 3 beta HSD. Transforming growth factor-beta 1 inhibited forskolin stimulation of androstenedione production through the inhibition of P450c17 expression.

Immunohistochemical and Biochemical Analysis of a Human Sertoli-Leydig Cell Tumor: Autonomous Steroid Production Characteristic of Overian Theca Cells

Objective: To ascertain the steroidogenic profile and location of steroidogenic enzymes in a steroid-secreting Sertoli-Leydig cell tumor of the ovary. Methods: Steroid levels from peripheral, left ovarian (tumor), and right ovarian venous blood were measure. Tumor tissue was examined for the steroidogenic enzymes 17α-hydroxylase (P450c17) and 3β-hydroxysteroid dehydrogenase (3βHSD) by immunohistochemistry. Tumor cells were isolated and incubated in serum-free media. Thereafter, media were analyzed for steroid production, steroidogenic response to effectors, and metabolism of radiolabeled pregnenolone and androstenedione. Results: Levels of C19 steroids and 17-hydroxyprogesterone (17OHP) were elevated in peripheral blood. The majority (80%) of steroids in serum from the left ovarian vein (tumor) were C19 steroids (dehydroepiandrosterone [DHEA], 45%; androstenedione, 27%, testosterone, 7%, with 13% 17OHP, 7% progesterone, and less than 1% estradiol (E2). Immunoreactivity for both P450c17 and 3βHSD was identified in clusters of large cells surrounded by nonimmunoreactive cells composing cord-like structures. Of the steroids accumulated in the incubation medium of unstimulated, freshly isolated tumor cells, 84% were C19 steroids (DHEA, 44%; androstenedione, 36%; testosterone 2%, with 16% 17OHP and less than 1% progesterone and E2. Basal steroid production was not stimulated by LH or FSH. However, treatment with forskolin (10 μmol/L), dibutyryl cAMP (1 mmol/L), or steroid precursors (22-hydroxycholesterol, 1 μmol/L; pregnenolone, 1 μmol/L) increased the production of all steroids measured. Forskolin treatment increased androstenedione (fivefold), DHEA (tenfold), and 17OHP (40-fold) compared with basal levels. Incubation of freshly isolated cells with [3H]pregnenolone demonstrated the ability of these cells to metabolize this C21 steroid precursor to androstenedione, DHEA, and 17OHP. However, [3H]androstenedione was not readily metabolized by these cells to either estrone or testosterone. Conclusions: The steroidogenic properties of a steroid hormone-producing tumor were described. Cells isolated from this tumor produced steroids similar to those secreted by ovarian theca cells. These properties suggest that certain ovarian steroidogenic tumor cells may be an appropriate model for ovarian theca cells and could be used to develop steroid-secreting cell lines.

Development of a Human Thecal Tumor Cell Model: Regulation of Steroidogenesis and Enzyme Expression

Steroid hormone production by ovarian thecal cells plays a pivotal role in reproductive physiology. Thecal cell synthesis of 19 carbon (C19) steroids [i.e., dehydroepiandrosterone (DHEA), androstenedione, and testosterone] is necessary to provide substrate for estrogen biosynthesis, which occurs in the adjacent granulosa cells (1–3). The availability of human luteinized granulosa cells from in vitro fertilization patients has expedited research into the mechanisms controlling granulosa cell differentiation. However, attempts to define the molecular and biochemical mechanisms controlling C19 steroid production have been hampered by the difficulties of obtaining and maintaining sufficient numbers of steroidogenically active human thecal cells in monolayer cell culture. Indeed, a culture system in which large numbers of functional cells could be propagated is a prerequisite for many of the studies into the molecular mechanisms controlling transcription of the genes for the enzymes involved in the synthesis of steroids.

The effect of insulin and insulin-like growth factors on the expression of steroidogenic enzymes in a human ovarian thecal-like tumor cell model**Sponsored in part by the American Society for Reproductive Medicine, Birmingham, Alabama; TAP Pharmaceutical Bridge grant, Chicago, Illinois (E.A.McG.); and National Institutes of Health, Bethesda, Maryland, grant 2-T32-H207190 (C.S.).

OBJECTIVE To determine the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on steroidogenesis and steroidogenic enzyme expression in a human ovarian thecal-like tumor cell culture model system. DESIGN Human ovarian thecal-like tumor cells treated with forskolin and insulin IGF-I or IGF-II were evaluated for media accumulation of P and androstenedione (A) as well as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and cytochrome P450 17 alpha-hydroxylase (P450c17) enzyme activity. Northern analysis of cytochrome P450 side chain cleavage (P450scc), P450c17, and 3 beta HSD messenger RNA (mRNA) also was performed. RESULTS Basal hormone secretion, enzyme activity, and mRNA levels were not affected by treatment with insulin or the IGFs. Forskolin treatment stimulated steroid production, enzyme activity, and mRNA content. Forskolin-stimulated P secretion was augmented 30% by treatment with insulin and IGFs, whereas 3 beta HSD activity was augmented twofold to threefold. Forskolin stimulated A and P450c17 activity were enhanced by treatment with insulin and the IGFs. In forskolin-treated cells. P450c17 and P450scc mRNA levels were not affected by insulin (100 nM) or IGF (10 nM) treatment; however, 3 beta HSD mRNA levels were augmented by treatment with insulin and IGFs. CONCLUSIONS We observed that forskolin-stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17, and 3 beta HSD activity, as well as mRNA content for P450scc, 3 beta HSD, and P450c17. Insulin and the IGFs augmented forskolin-stimulated production of P and the expression of 3 beta HSD, with little effect on A production, P450scc, or P450c17 expression.