Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues

The enzyme 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3ß-HSD) catalyzes the oxidation and isomerization of 5-ene-3ß-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3ß-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3ß-HSD genes containing 4 exons were assigned by in situ hybridization to the p11–p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3ß-HSD cDNAs as well as that of one type of 3ß-HSD from bovine and macaque ovary λgt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3ß-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3ß-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3ß-HSD. Transient expression of human type I and type II as well as rat type I and type II 3ß-HSD cDNAs in Hela human cervical carcinoma cells reveals that 3ß-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3ß-HSD proteins that are capable of converting 3ß-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3ß-hydroxy and 3-keto-5-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3ß-HSD mRNA accumulation accompanied by a similar decrease in 3ß-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3ß-HSD mRNA levels in ovarian interstitial cells. In intact females, hCG exerted marked trophic effects on rat corpora lutea with an increase in total ovarian 3ß-HSD expression and activity. We have also shown that treatment with hCG for 15 days in intact male rats caused a marked increase in testicular 3ß-HSD expression and activity while glucocorticoids exerted inhibitory effects on these parameters. We have also observed that the ontogeny of 3ß-HSD expression in human and rat adrenal gland, testis and ovary is closely correlated with steroid hormone biosynthesis, thus suggesting that regulation of the expression of 3ß-HSD is a limiting step in the biosynthesis of steroids in these tissues.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Testosterone environment of splenocytes modifies the steroidogenesis of polycystic ovary in rats.

The functional relationship between the ovary and immune cells is well known. The modulation of ovarian steroidogenesis in adult rats with polycystic ovary (PCO) by secretions of cultured splenocytes treated with 10 (-6) M testosterone or 10 (-6) M testosterone plus 10 (-4) M flutamide, an androgen receptor antagonist, was investigated. Polycystic ovary was induced by estradiol valerate (2 mg/rat). Polycystic ovary splenocyte secretions decreased the release of androstenedione from PCO ovaries in contrast to the effect of non-PCO splenocyte secretions. This decrease was associated with a significant decrease in androgen receptor and IL-12 mRNA expression in PCO splenocytes. When splenocytes were treated with testosterone, their conditioned media further decreased androstenedione release from the ovary and had a greater inhibitory effect on PCO ovary compared with non-PCO ovary. This effect was reversed by flutamide. Polycystic ovary splenocytes showed a decrease in IL-1 beta mRNA expression. Their secretions scarcely affected progesterone release from non-PCO ovaries but significantly stimulated progesterone release from PCO ovary by an androgen-independent mechanism. The differential steroidogenic ability of splenocyte secretions from PCO rats is associated with the IN VITRO testosterone environment. Polycystic ovary splenocytes might exert a protective action against PCO effects through their secretions by inducing a low androstenedione response from the ovary.     

Local aromatase activity in human breast tissues

The presence of oestradiol in malignant breast cells is considered to be an important factor in the promotion of growth of the tumor. Therefore the regulation of the local high intra-tissue oestradiol concentrations, regardless of plasma concentrations, has been investigated. Experimental evidence suggests that in situ biosynthesis of oestrogens is at least partly responsible for the local accumulation of these steroids. In this paper we report further data on measurements in fatty and tumor tissues of local aromatase activities and of concentrations of substrates and products of this enzyme. Data are given on localization of aromatase and on steroid concentrations in tumors and in adipose tissues dissected from different quadrants of breasts with malignent tumors. In adipose tissues small variations in steroid concentrations in fatty tissues were found. No tumor-directed gradients in the adipose tissue-concentrations of the androgens dehydro-epiandrosterone, 5-androstene-3ß,17ß-diol, 4-androstene-3,17-dione and testosterone and of the oestrogens oestradiol, oestrone and their sulfates could be detected. Furthermore no consistent pattern could be recognized in the aromatase activities in the fatty tissues dissected from tumor-bearing and non-affected quadrants of the same breast. No correlations between aromatase activity measured in vitro and product concentrations in vivo were found. Therefore the mechanisms for regulation of the local oestradiol levels in breast tissues remain unknown.     

Ovarian steroidogenesis in Japanese patients with polycystic ovary syndrome

Gonadotropin and steroid hormone levels in both peripheral and ovarian venous blood were measured in samples obtained from 20 Japanese patients with polycystic ovary syndrome (PCOs) and 10 normal women in early follicular phase (normal women) by radioimmunoassay. The change in the amount of steroid hormone following intravenous human menopausal gonadotropin (HMG) or dexamethasone administration was investigated. The mean concentration in patients with PCOs was significantly higher than the concentrations found in normal women for LH (p less than 0.001), but not for FSH in peripheral blood. Significantly elevated ovarian venous steroid hormone levels in PCOs were found for 17 alpha-hydroxypregnenolone (p less than 0.05), progesterone (p less than 0.05), 17 alpha-hydroxyprogesterone (p less than 0.01), 4 delta-androstenedione (p less 0.01), testosterone (p less than 0.01), estrone (p less than 0.01) and estradiol (p less than 0.05), but not for dehydroepiandrosterone-sulfate (DHEAS). The ovarian dehydroepiandrosterone (DHEA) level was slightly elevated in PCOs. The concentration of ovarian 4 delta-androstenedione in PCOs reached twelve times as much as that in normal women. After the administration of HMG, all of the ovarian venus steroid hormone levels were elevated slightly and without significance in the short observation time for 10 min. The DHEAS level was suppressed while the ovarian DHEA level remained high in PCOs following dexamethasone administration. These findings seem to indicate there is no adrenal involvement and no adrenal-like component in the ovary of PCOs, and no evidence of 3 beta-hydroxysteroid dehydrogenase and/or aromatase deficiency in this study. The increase in the steroid hormone secretion in PCOs is explained by the increase in ovarian production in polycystic enlarged ovaries.     

Prevalence of ultrasonography proved polycystic ovaries in North Indian women with type 2 diabetes mellitus

Background  

Polycystic ovaries (PCO) and their clinical expression (the polycystic ovary syndrome [PCOS]) as well as type 2 diabetes mellitus (T2DM) are common medical conditions linked through insulin resistance. We studied the prevalence of PCO and PCOS in women with diet and/or oral hypoglycemic treated T2DM and non-diabetic control women.     

Cortisol and testosterone: Inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 × 10⁻⁶ and 10⁻⁴ M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 × 10⁻⁷ to 5 × 10⁻⁵ M and testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10⁻⁸ and 10⁻⁶ M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11β, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studies in our laboratory.     

Molecular mechanisms of a novel selenium-based complementary medicine which confers protection against hyperandrogenism-induced polycystic ovary

The objective was to evaluate ovarian functionality and oxidative response in hyperandrogenism-induced polycystic ovary (PCO) and the protective effects of immunomodulator drug (IMOD), an electromagnetically-treated, selenium-based, herbal medicine. Daily oral administration of letrozole (1 mg/kg) for 21 consecutive days induced ovarian cysts in female rats. An effective dose of IMOD (30 mg/kg per day) was given intraperitoneally for 21 days. Biomarkers of ovarian function, serum concentrations of estradiol, progesterone, testosterone, and ovarian prostaglandin-E (PGE), were analyzed. To determine the role of oxidative stress (OS) in hyperandrogenism-induced PCO, concentrations of cellular lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), peroxynitrite (ONOO), and tumor necrosis factor (TNF)-α as a marker of inflammation and apoptosis were measured in serum and ovaries. Letrozole-induced PCO resulted in significant increases in concentrations of lipid peroxidation and peroxynitrite in serum and ovary, but significantly decreased superoxide dismutase, catalase, and glutathione peroxidase. Serum concentrations of testosterone and TNF-α, and ovarian prostaglandin-E were increased (P < 0.001) in animals with cysts versus control, whereas estradiol and progesterone were decreased (P < 0.01 and P < 0.001, respectively). When compared with controls, letrozole induced irregular cycles and PCO characterized by a high incidence of subcapsular ovarian cysts with a diminished granulosa cell layer, luteinized granulosa cells in the cyst wall, significantly more atretic preantral and antral follicles, and absence of CL. There were almost no intact primary, secondary, and tertiary follicles in PCO rats. All end points assessed were significantly improved by IMOD and reached close to normal levels. In conclusion, the present study provided evidence that toxic free radicals and TNF-α were involved in the pathogenesis of PCO; furthermore, IMOD prevented ovarian histopathologic, endocrine, and biochemical alterations induced by hyperandrogenism.     

Effects of electro-acupuncture on nerve growth factor and ovarian morphology in rats with experimentally induced polycystic ovaries

Despite extensive research on the pathogenesis of polycystic ovary syndrome (PCOS), there is still disagreement on the underlying mechanisms. The rat model for experimentally induced polycystic ovaries (PCO)-produced by a single injection of estradiol valerate-has similarities with human PCOS, and both are associated with hyperactivity in the sympathetic nervous system. Nerve growth factor (NGF) is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types. Electro-acupuncture (EA) is known to reduce hyperactivity in the sympathetic nervous system. For these reasons, the model was used in the present study to investigate the effects of EA (12 treatments, approximately 25 min each, over 30 days) by analyzing NGF in the central nervous system and the endocrine organs, including the ovaries. The main findings in the present study were first, that significantly higher concentrations of NGF were found in the ovaries and the adrenal glands in the rats in the PCO model than in the control rats that were only injected with the vehicle (oil or NaCl). Second, that repeated EA treatments in PCO rats resulted in concentrations of NGF in the ovaries that were significantly lower than those in non-EA-treated PCO rats but were within a normal range that did not differ from those in the untreated oil and NaCl control groups. The results in the present study provide support for the theory that EA inhibits hyperactivity in the sympathetic nervous system.     

Polycystic ovary syndrome: interaction of follicle stimulating hormone and polypeptide growth factors in oestradiol production by human granulosa cells

The mechanism of the ovarian dysfunction in polycystic ovary syndrome, the most common cause of anovulatory infertility, remains obscure. Clinical data suggest that follicle stimulating hormone (FSH) action may be inhibited at the ovarian level by paracrine factors derived, presumably, from interstitial cells. The greater responsiveness to FSH of granulosa cells isolated from polycystic ovaries (PCO) compared with that seen in cells derived from normal ovaries, provides some support for this hypothesis and we present data which suggests that epidermal growth factor, or more likely transforming growth factor alpha, could be a candidate for this inhibitor. It should be emphasized, however, that the cardinal biochemical feature of the PCO is hypersecretion of androgens by interstitial cells. Stromal tissue from the PCO will secrete significant quantities of androstenedione in response to LH, whereas there is a negligible response in stroma from normal ovaries. It remains to be determined whether androgens have a direct inhibitory effect on FSH-induced oestradiol production in the human follicle, or whether they might exert an indirect effect by activating inhibitory polypeptide growth factors.     

Adiponectin and Its Receptors in the Ovary: Further Evidence for a Link between Obesity and Hyperandrogenism in Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS), characterized by ovarian androgen excess, is the commonest endocrine disorder in women. Obesity increases androgen synthesis, a phenomenon attributed to the accompanying hyperinsulinemia. Our hypothesis was that adipokines, fat cell-derived hormones, play a direct role in modulating ovarian androgen secretion. Therefore, the aims of this study were to explore the effects of adipokines (in particular, adiponectin) on ovarian steroidogenesis and compare the expression of adiponectin receptors in ovaries from women with and without PCO. Sections of archived human ovaries (nine from women with normal ovaries and 16 with PCOS, classified histologically, with reference to menstrual history and ultrasound) were analysed by quantitative morphometry and the proportion of positive-labelling cells compared. In addition, studies of androgen production in relation to adipokine function in primary bovine theca cell culture were also performed. A significantly lower proportion of theca cells expressed adiponectin receptors 1 and 2 (AdipoR1, AdipoR2) in polycystic ovaries than in normal ovaries. In cultured theca cells, adiponectin suppressed androstenedione production and gene expression of LH receptor and key enzymes in the androgen synthesis pathway. Moreover, knockdown of genes for AdipoR1 and AdipoR2 was associated with increased androstenedione secretion by bovine theca cells. These results provide evidence for a direct link between fat cell metabolism and ovarian steroidogenesis, suggesting that disruption of adiponectin and/or its receptors plays a key role in pathogenesis of hyperandrogenism in PCOS.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

In vivo β-adrenergic blockade by propranolol prevents isoproterenol-induced polycystic ovary in adult rats

Increasing evidence in animal models and in humans shows that sympathetic nerve activity controls ovarian androgen biosynthesis and follicular development. Thus, sympathetic nerve activity participates in the follicular development and the hyperandrogenism characteristics of polycystic ovary syndrome, which is the most prevalent ovarian pathology in women during their reproductive years. In this study, we mimic sympathetic nerve activity in the rat via "in vivo" stimulation with isoproterenol (ISO), a β-adrenergic receptor agonist, and test for the development of the polycystic ovary condition. We also determine whether this effect can be reversed by the administration of propranolol (PROP), a β-adrenergic receptor antagonist. Rats were treated for 10 days with 125 μg/kg ISO or with ISO plus 5 mg/kg PROP. The ovaries were examined 1 day or 30 days following drug treatment. While ISO was present, the ovaries had an increased capacity to secrete androgens; ISO + PROP reversed this effect on androgen secretory activity. 30 days after treatment, androstenedione secretion reverted to normal levels, but an increase in the intra-ovarian nerve growth factor (NGF) concentration and luteinizing hormone (LH) plasma levels was detected. ISO treatment resulted in follicular development characterized by an increased number of pre-cystic and cystic ovarian follicles; this was reversed in the ISO + PROP group. The lack of change in the plasma levels of progesterone, androstenedione, testosterone, or estradiol and the increased LH plasma levels strongly suggests a local intra-ovarian effect of ISO indicating that β-adrenergic stimulation is a definitive component in the rat polycystic ovary condition.     

Serum 5-androstene-3 beta,17 beta-diol sulphate and 5 alpha-androstane-3 alpha,17 beta-diol sulphate in hirsute females with polycystic ovarian disease

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), unconjugated androstene-dione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI), 17 alpha-hydroxyprogesterone (17OHP), luteinising hormone (LH) and follicle stimulating hormone (FSH) were measured by specific radioimmunoassay in 28 hirsute women with polycystic ovarian disease (PCO) and in normal women (n = 73). Mean levels of steroids measured were significantly elevated, and SHBG significantly depressed, in the women with PCO with values (mean +/- SE) for 5-ADIOL-S (516 +/- 51 vs 267 +/- 10 nmol/l), 3 alpha-DIOL-S (130 +/- 9 vs 52 +/- 2 nmol/l), DHEA-S (7.3 +/- 0.5 vs 4.4 +/- 0.2 mumol/l), AD (11.3 +/- 1.1 vs 3.4 +/- 0.2 nmol/l), T (3.3 +/- 0.2 vs 1.5 +/- 0.1 nmol/l) and 17OHP (5.1 +/- 0.8 vs 2.8 +/- 0.2 nmol/l). SHBG levels were 31 +/- 2.9 vs 65 +/- 2.5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by (SHBG nmol/l)] was 12.5 +/- 1.4 vs 2.4 +/- 0.1. The mean LH to FSH ratio was also elevated at 2.8 +/- 0.3. These studies suggest that the measurement of 5-ADIOL-S and DHEA-S may indicate adrenal gland involvement in PCO while 3 alpha-DIOL-S appears to be a reflection of peripheral androgen metabolism. A comprehensive biochemical profile of PCO should thus include the analysis of these sulphoconjugates as well as unconjugated steroids.     

Effect of electro-acupuncture stimulation of different frequencies and intensities on ovarian blood flow in anaesthetized rats with steroid-induced polycystic ovaries

Background  

Maintenance of ovarian blood flow (OBF) is suggested to be important for regular ovulation in women with polycystic ovaries (PCO). The purpose of the present study was to investigate whether electro-acupuncture (EA) of different frequencies and intensities can improve the OBF of anaesthetized rat in the animal model of PCO.     

Steroid-induced polycystic ovaries in rats: effect of electro-acupuncture on concentrations of endothelin-1 and nerve growth factor (NGF), and expression of NGF mRNA in the ovaries,the adrenal glands,and the central nervous system

Previous studies on the effect of repeated electro-acupuncture (EA) treatments in rats with steriod-induced polycystic ovaries (PCO), EA has been shown to modulate nerve growth factor (NGF) concentration in the ovaries as well as corticotropin releasing factor (CRF) in the median eminence (ME). In the present study we tested the hypothesis that repeated EA treatments modulates sympathetic nerve activity in rats with PCO. This was done by analysing endothelin-1 (ET-1), a potent vasoconstrictor involved in ovarian functions, as well as NGF and NGF mRNA expression involved in the pathophysiological process underlying steroid-induced PCO.The main result in the present study was that concentrations of ET-1 in the ovaries were significantly lower in the PCO group receiving EA compared with the healthy control group (p < 0.05). In the hypothalamus, however, ET-1 concentrations were found to be significantly higher in the PCO group receiving EA than in the healthy control group (p < 0.05). Concentrations of ovarian NGF protein were significantly higher in the PCO control group compared with the healthy control group (p < 0.001), and these concentrations decreased significantly after repeated EA treatments compared with those in the PCO control group (p < 0.05) and were found to be the same as those in the healthy control group. In conclusion, these results indicate that EA modulates the neuroendocrinological state of the ovaries, most likely by modulating the sympathetic nerve activity in the ovaries, which may be a factor in the maintenance of steroid-induced PCO.     

Pituitary and ovarian responses to luteinizing hormone releasing hormone in a rat with polycystic ovaries

Female rats injected with a single dose of 2 mg estradiol valerate (EV) develop anovulatory acyclicity characterized by persistent vaginal cornification and the formation of multiple large cystic follicles on the ovaries. In order to determine if these effects of EV are accompanied by changes in ovarian and/or pituitary function, the following studies were conducted. Ovarian androgen production was determined by the measurement at 4, 5 and 6 weeks after EV treatment of circulating dehydroepiandrosterone, androstenedione and testosterone. The capacity of the polycystic ovary to ovulate in response to luteinizing hormone releasing hormone (LHRH) stimulus was assessed. Ovarian histology was examined at the termination of the study (9 weeks after EV treatment). Pituitary function was assessed 9 weeks after the EV treatment by examining the acute changes in plasma luteinizing hormone (LH) concentration in response to a double pulse of LHRH. Plasma concentrations of the androgens were unchanged over the 3-week sampling period and were similar to those found in sesame-oil-treated normal cycling control rats. The ovaries from EV-treated animals were smaller than those of controls and the cystic follicles exhibited marked thecal hypertrophy and attenuation of the granulosa cell layer. The basal plasma LH concentration at 9 weeks after EV treatment were significantly lower than in proestrus controls and plasma concentrations of LH elicited by LHRH pulses was significantly lower than in controls. The relative increase in plasma LH following the LHRH stimulus was, however, greater in the EV-treated animals than in controls. In spite of the diminished LH surge elicited in response to LHRH, the EV-treated animals ovulated as indicated by the presence of fresh corpora lutea on the ovaries. These results indicate that androgens are not responsible for the polycystic ovarian condition in this system and that the polycystic ovary is capable of ovulatory function when appropriately stimulated.     

Abnormal electromyographic activity of the urethral sphincter,voiding dysfunction,and polycystic ovaries: a new syndrome?

A potential association between abnormal electromyographic activity--that is, decelerating bursts and complex repetitive discharges--of the urethral sphincter and difficulty in voiding was examined in 57 women with urinary retention. Abnormal electromyographic activity was found in 33. Ultrasonography of the ovaries in 22 of the 33 women showed that 14 had polycystic ovaries. Of the other eight women, two had had oophorectomies, one had shrunken ovaries and ovarian failure, and one had previously undergone oophorectomy and the other ovary could not be seen; in one neither ovary could be seen, and three had ovaries of normal appearance, although two of these women were taking the contraceptive pill. Thirteen of the group had endocrine symptoms and signs characteristic of the polycystic ovary syndrome. Videocystometrography in 17 of the women who were examined by ultrasonography showed low flow rates and high residual volumes of urine after micturition in 12 women who could void, the other five having chronic urinary retention. A speculative hypothesis for the observed association of impaired voiding, abnormal electromyographic activity of the urinary sphincter, and polycystic ovaries is advanced, based on the relative progesterone deficiency that characterises the polycystic ovary syndrome. Progesterone stabilises membranes, and its depletion might permit ephaptic transmission of impulses between muscle fibres in the muscle of the urethral sphincter, giving rise to the abnormal electromyographic activity. This may impair relaxation of the sphincter, resulting in low flow rates of urine, incomplete emptying of the bladder, and, finally, urinary retention.     

Heparin-releasable lipase activity of rat adrenals, ovaries and testes.

The presence of NaCl-resistant, neutral triacylglycerol hydrolase (lipase) activity in rat adrenal gland, ovary and testis was studied. Both adrenals and ovaries but not testes were found to contain such a lipase. The activity of the enzyme in the adrenal gland was lowered during cortisol treatment and hypothyroidism. An elevated adrenal lipase activity was found during hyperthyroidism. Pseudo-pregnant and lactating rats had higher ovarian lipase activities than cyclic rats. Ovarian lipase activity in lactating rats was positively correlated with the serum concentrations of progesterone and of 20 alpha-hydroxyprogesterone and negatively correlated with the high-density-lipoprotein non-esterified cholesterol concentration. The lipase activity of adrenals and of ovaries was largely releasable from these organs by heparin and could be inhibited by an antibody against heparin-releasable liver lipase. This indicated that the lipase is extracellularly located and is similar to 'liver' lipase. A possible role of this lipase in adrenals and ovaries is discussed.     

Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450

The steroid 11ß-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4–5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011ß system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011 from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11ß-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M_r 60 kD) and a NADPH-cytochrome P450 reductase (M_r 79 kD).