Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: immunohistochemical expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450.

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries.

The mechanism for protein kinase C inhibition of androgen production and 17alpha-hydroxylase expression in a theca cell tumor model.

INTRODUCTION: In polycystic ovary syndrome (PCOS), there is increased formation of androgens by thecal cells. Moreover, PCOS ovaries have been shown to have decreased levels of c-fos transcription factor. We hypothesize that c-fos expression inhibits 17alpha-hydroxylase 17,20 lyase (CYP17) activity in the human ovary, and its decreased expression seen in PCOS may lead to elevated CYP17 transcription, resulting in increased androgen production. OBJECTIVE: Our objective was to define the role of the activator protein-1 transcription factors, namely c-fos, in the regulation of CYP17 expression in theca cells. METHODS: Human ovarian thecal-like tumor cells were used for all experiments. The following techniques were used: steroid quantification, mRNA extraction, microarray analysis, transfection, small interfering RNA, and immunohistochemistry. RESULTS: Stimulation of human ovarian thecal-like tumor cells with the protein kinase A pathway activator forskolin resulted in stimulation of C19 androgen production. In contrast, treatment with the protein kinase C pathway activator tetradecanoylphorbol acetate (TPA) resulted in decreased androgen production with a shift toward C21 progesterone production. TPA also led to complete inhibition of CYP17. Microarray data showed a 37-fold increase in c-fos after treatment with TPA. Transfection with steroidogenic factor 1 resulted in an increase in CYP17 promoter activity, which was significantly inhibited in the presence of c-fos. c-fos gene silencing led to an increase in CYP17 mRNA levels. Immunohistochemical staining for c-fos in ovaries demonstrated strong staining in granulosa cells, but not theca. CONCLUSIONS: The activator protein-1 transcription factor c-fos plays a role in the inhibition of CYP17 expression. The decreased levels of c-fos expression in polycystic ovaries may be responsible for increased CYP17 levels in PCOS.

Bone morphogenetic protein inhibits ovarian androgen production.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta 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 17alpha-hydroxyprogesterone (17OHP) in the incubation medium was measured by RIA. The expression of 17alpha-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 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, 3betaHSD, and StAR protein expression. BMP-4 markedly inhibited forskolin stimulation of CYP17 expression but had little effect on 3betaHSD, 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 beta 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 factor-beta superfamily of proteins.

Sex steroid biosynthesis enzymes in ovarian sex-cord stromal tumors.

Previous studies on neoplastic and hyperplastic ovarian lesions using paraffin-embedded material have demonstrated immunolocalization of sex steroid biosynthetic enzymes (SSBEs): P-450 side chain cleavage (P-450 SCC), which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), which converts pregnenolone to progesterone; P-450 17 alpha-hydroxylase and lyase (P-450 17A), which convert progesterone to 17 alpha-hydroxyprogesterone and 4-androstene-3,17-dione; and P-450 aromatase (P-450 AR), which converts 4-androstene-3,17-dione to estradiol. To investigate the utility of immunohistochemical staining for SSBEs, we studied a series of 45 sex cord-stromal tumors of the ovary. P-450 SCC was present in 9 of 11 Sertoli-stromal cell tumors, 3 of 12 granulosa cell tumors, 2 of 7 thecomas, and 1 of 1 stromal luteomas; 3 beta-HSD was present in 5 of 11 Sertoli-stromal cell tumors, 2 of 12 granulosa cell tumors, 2 of 7 thecomas, and 1 of 1 stromal luteoma; P-450 17A was present in 5 of 11 Sertoli-stromal cell tumors, 2 of 12 granulosa cell tumors, 2 of 6 thecomas, and 1 of 1 stromal luteomas; P-450 AR was present in 6 of 11 Sertoli-stromal cell tumors, 2 of 12 granulosa cell tumors, none of 7 thecomas, and 1 of 1 stromal luteoma. SSBEs were not present in 12 fibromas, one sclerosing stromal tumor, and one myxoma. Five of 45 patients with sex cord-stromal tumors showed androgenic effects; 4 of 11 patients with Sertoli-stromal cell tumors and the patient with a stromal luteoma. These five sex cord-stromal tumors contained P-450 SCC, and three of four of the Sertoli-stromal cell tumors contained 3 beta-HSD, P-450 17A, and P-450 AR. Concurrent endometrial histology was available in 25 of 45 sex cord-stromal tumor patients. None of the five sex cord-stromal tumors arising in patients with endometria that showed hyperplasia or adenocarcinoma showed immunoreactivity for SSBEs. Eight patients' endometria were unremarkable, but their sex cord-stromal tumor contained SSBEs. SSBEs were present in areas showing Leydig cell, Sertoli cell, or steroid cell differentiation or luteinized areas; however, the results did not significantly add to the histologic classification of sex-cord stromal tumors. Androgenic hormonal effects could always be explained by synthesis of hormones by SSBEs present in the patient's sex cord-stromal tumor.(ABSTRACT TRUNCATED AT 400 WORDS)

[The role of the system of proteolytic enzymes and their inhibitors in the pathogenesis of ovarian tumors in Wistar rats and the possibilities for correcting the disorders detected]. / Rol' sistemy proteoliticheskikh fermentov i ikh ingibitorov v patogeneze opukholei iaichnika u krys Wistar i vozmozhnosti korrektsii vyiavlennykh narushenii.

The system of proteolytic enzymes and their inhibitors was studied in tissues of ovarian tumours of Wistar rats. It has been revealed that activation of proteolysis enzymes is observed in the genesis of tumour growth against a background of the absence of protease inhibitors. The correction of revealed disturbances by introducing the inhibitors of proteolytic enzymes and hormone preparations has shown that contrical and norcolute are the most effective. The results obtained suggest the further search of preparation normalizing the condition of the given system.

Immunohistochemical study of 3 beta-hydroxysteroid dehydrogenase in sex cord-stromal tumors of the ovary.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD), which converts pregnenolone to progesterone, was localized immunohistochemically in 18 thecomas, 23 fibromas, 5 granulosa-cell tumors, 5 sclerosing stromal tumors, and 2 steroid-cell tumors. Immunohistochemical study of estrogen, progesterone, and testosterone was also performed in serial sections of thecomas and fibromas. In thecomas, immunoreactivity of 3 beta-HSD was observed only in luteinized theca cells and thecomatous tumor cells with abundant pale to vacuolated cytoplasm but not in spindled tumor cells and thecomatous tumor cells with small to moderate amounts of pale to vacuolated cytoplasm. Immunoreactivity of steroids was not observed in thecomas except for testosterone immunoreactivity in one case. No immunoreactivity of steroids or the enzyme was present in fibromas. No tumor cells were positive for 3 beta-HSD in any of the cases of granulosa-cell tumor examined. Immunoreactivity of 3 beta-HSD was present in cells in steroid-cell tumors and polygonal tumor cells with prominent cytoplasmic vacuoles in two cases of sclerosing stromal tumor. Thus, 3 beta-HSD can be a good immunohistochemical marker of steroidogenesis in functioning ovarian neoplasms.

Immunohistochemical studies of steroidogenic enzymes (aromatase, 17 alpha-hydroxylase and cholesterol side-chain cleavage cytochromes P-450) in sex cord-stromal tumors of the ovary.

Aromatase, 17 alpha-hydroxylase, and cholesterol side-chain cleavage P-450 cytochromes (P-450AROM, P-450(17 alpha,) and P-450SCC, respectively) were immunohistochemically localized in nine granulosa cell tumors, 15 thecomas, ten Sertoli-Leydig cell tumors, two steroid cell tumors, five fibromas, and five sclerosing stromal tumors. In the thecomas, P-450SCC and P-450(17 alpha) were positive in luteinized theca cells and in cells with vacuolated cytoplasm, while P-450AROM was not observed. In the steroid cell tumors, all the P-450 cytochromes were intensely stained. P-450SCC and P-450(17 alpha) were present in cells with vacuolated cytoplasm in two cases of sclerosing stromal tumor. P-450AROM was weakly demonstrated in one of the granulosa cell tumors. P-450(17 alpha,) P-450SCC, and P-450AROM were all faintly stained in the Sertoli-Leydig cell tumors. No P-450 cytochrome immunoreactivity was observed in any fibroma.

Gonadotropin receptor of a mouse luteoma: interactions with luteinizing hormone (LH) and its and subunits.

PIP: A radioligand-receptor system for luteinizing hormone (LH), USING transplantable mouse luteoma, was used to investigate the interactions of LH, other peptide hormones, and LH subunits. Since tumor size decreased as did production of androgenic hormones following hypophysectomy, the luteoma is believed to have been dependent on pituitary tropic hormones; posthypophysectomy histologic changes supported this conclusion. An homogenate was prepared from 1-4 gm luteomas, which had been borne by mice for 4-10 months. Ovine LH, bovine LH, and human chorionic gonadotrophin reduced the binding of iodine-125 human luteinizing hormone (125-I-hLH). Growth hormone, adrenocorticotrophic hormone, and prolactin had no capacity to interfere with binding of 125-I-hLH. Though follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) reduced the binding somewhat, the reductions were consistent with the known presence of contaminating amounts of LH in the FSH and TSH. The accumulated results of a number of experiments suggest that binding to the luteoma LH receptor requires a particular polypeptide structural conformation, one found in the native hormone but found in neither alpha nor beta subunit alone.^ieng