The pituitary-gonadal axis was assessed in 10 male patients during hospital admissions lasting 3-6 weeks (median 3 weeks) for flares for rheumatoid arthritis. Despite significant improvements in the Ritchie indices from median 16 (range 9-23) to 8.5 (range 5-20) (p less than 0.01) and ESR from median 67 mm/h (range <em>4</em>6-115 mm/h) to 58 mm/h (range 15-116 mm/h) (p less than 0.05) there were no significant changes in serum testosterone, LH, FSH, prolactin (PRL), cortisol or <em>androstenedione</em> during the admission periods. At follow-up (median 1<em>4</em> months, range 5-18 months after admission) there were further improvements in articular indices (median 7, range 3-13; p = NS) and ESR (median 20 mm/h, range <em>4</em>-62 mm/h; p less than 0.05) and rheumatoid factor titres had fallen from median 1/1025 (range 1/126 to 1/102<em>4</em>) to median 1/512 (range 1/6<em>4</em> - 1/512). One patient showed biochemical features of progressive testicular failure. In the remaining patients, serum and derived free testosterone levels were significantly increased (p less than 0.01 respectively) and serum LH reduced (p less than 0.01). There were no changes, at this time, in prolactin, cortisol or <em>androstenedione</em>. Rheumatoid flares appear to be associated with prolonged suppression of testicular function.

OBJECTIVE

Paediatric Cushing's disease is frequently associated with abnormal puberty. We addressed the hypothesis that prepubertal patients show excessive virilization and pubertal patients show suppression of LH and FSH secretion.

METHODS

Serum androstenedione (A4), dehydroepiandrosterone sulphate (DHEAS), testosterone (T), and sex hormone binding globulin (SHBG) were determined at diagnosis and converted to standard deviation scores. LH, FSH concentrations were also determined. Severity of CD was assessed from the sleeping midnight cortisol concentration. Puberty was staged and excessive virilization defined as advance in pubic hair stage for breast stage or testicular volume (TV).

METHODS

Twenty-seven CD patients (17 male, 10 female), median age 13.4 years (range 5.9-17.8) were studied.

RESULTS

In the CD group as a whole, A4, DHEAS, T standard deviation scores (SDS) values were normal. SHBG SDS values (n = 19) were low (median -1.93, -4.32-0.86) correlating with BMI (r = -0.49). A4, DHEAS, T, SHBG, LH and FSH did not correlate with midnight cortisol, but A4 and T SDS correlated with ACTH at 09.00 h (both r = 0.51). Thirteen patients (11 male, 2 female) had excessive virilization with increased A4 (P = 0.033), DHEAS (P = 0.008), testosterone (P = 0.033) and decreased SHBG (P = 0.004) compared with subjects without excessive virilization. Pubertal boys (TV>> or = 4 ml) (n = 7) and girls (breasts>> or = stage 2) (n = 8) had low median LH and FSH. Boys had an LH concentration of 1.2 mU/l (0.3-3.5), FSH, 0.9 mU/l (0.2-6.4) and median T SDS, -1.95 (-3.8-4.65), while girls had an LH concentration of 1 mU/l (0.3-7.4).

CONCLUSIONS

Many patients had abnormal puberty and excessive virilization associated with increased adrenal androgens and decreased SHBG. Pubertal patients had low LH and FSH suggesting impaired pituitary-gonadal axis function.

Plasma sex hormone concentrations (testosterone, (T), <em>androstenedione</em> (A), oestrone (E1) and oestradiol (E2) were measured in forty post-menopausal women more than <em>4</em> years post-normal menopause. Correlations between these and age, years post-menopause (YPM), degree of obesity and fat mass respectively were studied. T and A, as well as E1 and E2 were positively correlated (P less than 0.01), but no statistically significant correlation between A and E1 was observed. Sex hormone concentrations in this group of postmenopausal women (greater than <em>4</em>YPM) did not show any variation as a function of age, with the possible exception of E2 which showed a tendency to decrease in the late post-menopause. E1 and to a lesser extent E2 as well as the E1/A ratio were significantly corelated with degree of obesity or fat mass, suggesting a possible role of fat tissue in the aromatization of androgens. Neither the T/A nor the E2/E1 ratios were correlated with fat mass, suggesting that the reduction of 17 oxo-group does not occur in fat tissue. The E1/A ratio was significantly higher than the reported conversion rate of A in E1. This might suggest the existence of an additional precursor of plasma E1.

BACKGROUND

The adrenal steroids dehydroepiandrosterone and androstenediones are converted into active androgen testosterone in prostatic tissues. Different 17beta-hydroxysteroid dehydrogenase (17betaHSD) isozymes are characterized by either oxidation or reduction reactions. These redox reactions represent an important step in both biosynthesis and metabolism of androgens. This study presents the differential expression of 17betaHSD isozyme genes in cancerous and noncancerous prostate tissues of in vivo samples.

METHODS

Thirty-four fresh specimens of transrectal prostatic needle biopsy were obtained; 11 were pathologically diagnosed as adenocarcinoma and 23 as without malignancy. The gene expression levels of five isozymes (type 1-5) of 17betaHSD were evaluated. The quantification of gene expression was assessed by means of the real-time polymerase chain reaction.

RESULTS

The expression levels of the type 3 17betaHSD gene with malignancy were significantly higher than those in prostatic tissues without malignancy, and those of type 2 17betaHSD with malignancy were significantly lower than those in nonmalignant tissues. There were no significant differences in 17betaHSD type 1, type 4, and type 5 gene expression in cancerous and noncancerous tissues.

CONCLUSIONS

Our results suggest that 17betaHSD type 2 and type 3 play an important role in the conversion of adrenal steroids into potential androgens in prostate cancer tissue.

The superimposition of male sex organs (penis and vas deferens) in a female gastropod, called imposex, is widely attributed to the exposure to tributyltin (TBT) compounds, used world-wide in antifouling paints for ships. It has been hypothesized that the TBT-induced imposex is mediated by an increasing androgen level relative to the estrogen level, namely a decreased conversion of androgens to estrogens (i.e., aromatization). In the present study, we tested this hypothesis by examining the effects of TBT or triphenyltin (TPT) on the aromatase activity in a cultured human granulosa-like tumor cell line, KGN, which was recently established by our group. Treatment with more than 1000 ng/ml TBT compounds was very toxic to the cells and caused immediate cell death within 2<em>4</em> h, while 200 ng/ml was found to cause apoptosis of the cells. Treatment of the KGN cells for more than <em>4</em>8 h with 20 ng/ml TBT or TPT, which is a concentration level reported to cause imposex in marine species, did not affect cell proliferation but significantly suppressed the aromatase activity determined by a [(3)H]H(2)O release assay. Treatment with 20 ng/ml TBT compounds for 7 days also resulted in a reduction of the E2 production from Delta <em>4</em>-<em>androstenedione</em> stimulated by db-cAMP. The changes in the aromatase activity by TBT compounds were associated with comparable changes in P<em>4</em>50arom mRNA assessed by RT-PCR. The luciferase activity of the P<em>4</em>50arom promoter II (1 kb) decreased after the addition of 20 ng/ml TBT compounds in transfected KGN cells either in a basic state or in states stimulated by db-cAMP. The Ad<em>4</em>BP-dependent increase in the luciferase activity of P<em>4</em>50arom promoter II was also downregulated by such treatments. These results indicate that TBT compounds inhibited the aromatase activity and also decreased the P<em>4</em>50arom mRNA level at the transcriptional level in KGN cells. The direct inhibitory effect of TBT compounds on the aromatase activity may therefore partly explain the induction of imposex by these compounds in female species.

In the present study, we investigated the effects of oral dosing of atrazine (2-chloro-<em>4</em>-ethylamino-6-isopropylamino-s-triazine) to peripubertal male rats (50 and 200 mg/kg body weight daily from postnatal days 23-50) on ex vivo Leydig cell steroidogenesis. Leydig cells from treated rats were characterised by significant decline in mRNA transcripts of several genes responsible for steroidogenesis: luteinizing hormone receptor (LHR), scavenger receptor-B1, steroidogenic acute regulatory protein, translocator protein, steroidogenic factor-1, phosphodiesterase <em>4</em>B, 3beta-hydroxysteroid dehydrogenase (HSD), CYP17A1, and 17betaHSD. In the presence of human chorion gonadotropin, the dose-dependent decrease in extracellular cAMP level and accordingly strong inhibition of androgenesis were obtained. The transcription of LHR gene in Leydig cells of atrazine-treated rats was downregulated in a dose-dependent manner, which could be the reason for reduction in cAMP level and expression of cAMP-dependent genes. To clarify the activity of the steroidogenic enzymes responsible for androgenesis, purified Leydig cells were challenged with different steroid substrates (22OH-cholesterol, pregnenolone, progesterone, and Delta(<em>4</em>)-<em>androstenedione</em>), and the obtained results indicated inhibition of androgen production in Leydig cells isolated from atrazine-treated animals in the presence of all those substrates. However, when Leydig cells were challenged with 22OH-cholesterol, the progesterone level in the incubation medium was unchanged, indicating that decrease in cholesterol transport and/or CYP17A1 and 17betaHSD activity are most probably responsible for inhibition of androgen production after the addition of different substrates. Our results demonstrated that in vivo exposure to atrazine affects Leydig cell steroidogenesis via the inhibition of steroidogenesis gene expression, which is accompanied by decreased androgenesis.

Serum FSH, LH, PRL, estradiol, pregnenolone, progesterone, 17-hydroxyprogesterone, <em>androstenedione</em>, testosterone, 5 alpha-dihydrotestosterone, and androsterone were measured radioimmunologically in 20 normal girls aged 13-17 yr. Samples were taken every day or every second day during one menstrual cycle. The cycles recorded could be divided into three groups. The first and oldest group consisted of 10 girls with a mean gynecological age (years since menarche) of 2.9 yr. The luteal phase was at least 11 days and the progesterone concentration was at least 5 ng/ml. The testosterone rise (mean, 55%) on the day of LH surge correlated well with the simultaneous progesterone rise (mean, 270%) and the following luteal progesterone secretion. A negative correlation was seen between the FSH concentration on days 3-<em>4</em> of the cycle and the length of the follicular phase. The second group consisted of <em>4</em> girls who had a mean gynecological age of 1.5 yr. The luteal phase was of <em>4</em>- to 8-day duration and the progesterone secretion was lower than in group I. The follicular phase testosterone concentration was lower in group II as compared to group I. No "periovulatory" testosterone increases were seen, although every cycle displayed an LH and FSH peak. The third group consisted of 6 girls with a mean gynecological age of 1.1 yr. These cycles were anovulatory, as the serum progesterone concentration never exceeded 1.0 ng/ml. In two cycles, signs of follicular maturation were seen. In the four others, the androgen levels tended to be elevated. In two cases, the testosterone and <em>androstenedione</em> concentrations were 2-<em>4</em> times elevated from the beginning of these two cycles. Thus, the hormonal pattern of adolescent menstrual cycles is far from uniform. It is very likely that in addition to gonadotropins, estradiol and progesterone, androgens may also have a role in the development and maintenance of normal menstrual function in the female.

The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (<em>androstenedione</em> and dehydroepiandrosterone) production were induced by treating the cells for <em>4</em> days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (greater than or equal to 30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activin-A, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activin-A on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary.

In the present study, expressions of 17beta-hydroxysteroid dehydrogenase (17HSD) types 1, 2, and 3, 5alpha-reductase type 2 and human androgen receptor mRNAs were determined in 12 benign prostatic hyperplasia and 17 prostatic carcinoma specimens. 17HSD type 2 was found to be the principle isoenzyme expressed in the prostate. Significantly higher expressions of 17HSD type 2 and 5alpha-reductase type 2 were detected in benign prostatic hyperplasia compared with the carcinoma specimens. Expression of the androgen receptor in the 2 groups was not significantly different. 17HSD type 3 mRNA was not detected in any of the specimens investigated. Only low constructive expression of the 2.3 kb mRNA of 17HSD type 1 was seen. Immunohistochemical analysis indicated that this did not lead to significant enzyme expression, only faint staining for the enzyme protein being detected, mainly in uroepithelial cells. No significant correlation was found between any of the mRNAs analysed, but the data on 5alpha-reductase type 2 mRNA support the presence of an increased proportion of 5alpha-dihydrotesterone in the hyperplastic prostate. In cultured PC-3 prostatic cancer cells and in the transiently transfected human embryonic kidney 293 cells, 17HSD type 2 was found exclusively to convert 5alpha-dihydrotestosterone and testosterone into the less potent 17-keto compounds 5alpha-androstanedione and <em>4</em>-<em>androstenedione</em>, respectively. We suggest that the 17HSD type 2 isoenzyme plays a part in the metabolic pathway, resulting in the inactivation of testosterone and 5alpha-dihydrotestosterone locally in the prostate. The enzyme expressed in the prostate could, therefore, protect cells from excessive androgen action.

OBJECTIVE

Our purpose was to describe the temporal and quantitative relationships among intrauterine infection, fetal-placental steroid biosynthesis, and preterm labor in a nonhuman primate model.

METHODS

On approximately day 130 of gestation (term 167 days) chronically instrumented rhesus monkeys (Macaca mulatta) were infected with 10(6) colony-forming units of group B streptococci either by intraamniotic (n = <em>4</em>) or choriodecidual (n = 2) inoculation. As controls, four additionally chronically instrumented noninfected monkeys were followed up to spontaneous parturition. Amniotic fluid and maternal and fetal arterial blood were serially sampled in all monkeys (both before and after infection) for progesterone, estrone, estradiol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, <em>androstenedione</em>, and cortisol by specific radioimmunoassays, and uterine activity was continuously recorded.

RESULTS

Spontaneous parturition was preceded by gradual and significant increases in the plasma concentrations of fetal dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione and fetal and maternal levels of estrone, estradiol, and progesterone but not by changes in cortisol. In contrast, infection-associated parturition (either intraamniotic or choriodecidual) was characterized by abrupt increases in fetal dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, progesterone, and cortisol but not by increases in maternal or fetal estrone or estradiol. Infection-associated steroid changes occurred concurrently with or after increases in uterine activity.

CONCLUSIONS

Infection-associated preterm parturition is associated with dramatic increases in fetal adrenal steroid biosynthesis but not by corresponding increases in placental estrogen biosynthesis. This suggests that fetal stress in accompanied by placental dysfunction and that infection-associated parturition is not dependent on the increased estrogen biosynthesis observed in spontaneous parturition.

The goldfish sex pheromone system is the best understood among the teleost fishes. Pheromones in this species are unspecialized hormonal products, which are released in ratios that vary with reproductive status. This study examined behavioral responses of male goldfish to three steroidal components of the female preovulatory pheromone: 17,20beta-dihydroxy-<em>4</em>-pregnen-3-one (1720betaP); 17,20beta-dihydroxy-<em>4</em>-pregnen-3-one-20-sulfate (1720betaP-S); and <em>androstenedione</em> (AD). Males were observed during exposure to nanomolar concentrations of each steroid over a 2-h period. We observed chasing, nudging (courtship behaviors) and pushing (an aggressive behavior). Each steroid elicited a different set of behaviors. 1720betaP, which is released by ovulatory females, elicited a low level of chasing and nudging that persisted throughout the experiment. Exposure to 1720betaP-S, which is released primarily by ovulatory females, triggered a large increase in nudging and chasing that lasted for only 5 min. In contrast, AD, which is released by females early in the ovulatory cycle and by mature males, elicited increases in aggressive behavior. 1720betaP and 1720betaP-S both caused increases in GtH-II release while AD did not. These results demonstrate that goldfish can discriminate components found in the female pheromone blend, suggesting that goldfish, and likely other fish species, may employ blends of hormonal products as pheromones.

The structural basis for functional differences between human cytochrome P-<em>4</em>50 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B<em>4</em>, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73<em>4</em>01 [3-cyclopentyloxy-N-(3,5-dichloro-<em>4</em>-pyridyl)-<em>4</em>-methoxybenzamide], the 2B1-selective substrate <em>androstenedione</em>, and the common substrate 7-ethoxy-<em>4</em>-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73<em>4</em>01 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-<em>4</em>-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant <em>androstenedione</em> hydroxylase activity. Docking of RP 73<em>4</em>01 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73<em>4</em>01 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring <em>androstenedione</em> hydroxylase function to cytochrome P-<em>4</em>50 2B6 is more complex than altering a single SRS.

Cessation of ovarian estrogen secretion is the key event during the climacteric. An enzyme termed aromatase in a number of human tissues and cells, including ovarian granulosa cells, the placental syncytiotrophoblast, adipose and skin fibroblasts, bone, and the brain, catalyzes the conversion of C19 steroids to estrogens. Aromatase expression in adipose tissue and possibly the skin primarily accounts for the extraglandular (peripheral) formation of estrogen and increases as a function of body weight and advancing age. Sufficient circulating levels of the biologically active estrogen, estradiol, can be produced as a result of extraglandular aromatization of <em>androstenedione</em> to estrone, which is subsequently reduced to estradiol in peripheral tissues, to cause uterine bleeding and endometrial hyperplasia and cancer in obese anovulatory or postmenopausal women. Extraglandular aromatase expression in adipose tissue and skin (via increasing circulating levels of estradiol) and bone (via increasing local estrogen concentrations) is of paramount importance in slowing the rate of postmenopausal bone loss. Moreover, excessive or inappropriate aromatase expression was demonstrated in adipose fibroblasts surrounding a breast carcinoma, endometriosis-derived stromal cells, and stromal cells in endometrial cancer and gave rise to increased local estrogen concentrations in these tissues. Whether systemically delivered or locally produced, elevated estrogen levels promote the growth of these steroid-responsive tissues. Finally, local estrogen biosynthesis by aromatase activity in the brain may be important in the regulation of various cognitive and hypothalamic functions. The regulation of aromatase expression in human cells via alternatively used promoters, which can be activated or inhibited by various hormones, increases the complexity of estrogen biosynthesis in the human body. Aromatase expression is under the control of the classically located proximal promoter II in the ovary and a far distal promoter I.1 (<em>4</em>0 kb upstream of the translation initiation site) in the placenta. In adipose tissue, two other promoters (I.<em>4</em> and I.3) located between I.1 and II are used in addition to the ovarian-type promoter II. To add a further twist, promoter use in adipose fibroblasts switches between promoters II/I.3 and I.<em>4</em> upon treatment of these cells with prostaglandin E2 (PGE2) versus glucocorticoids plus cytokines. Moreover, the presence of a carcinoma in breast adipose tissue causes a switch of promoter use from I.<em>4</em> to II/I.3. Molecular and cellular mechanisms responsible for estrogen formation and their physiologic and clinical relevance will be reviewed in this article.

OBJECTIVE

Although insulin has been shown to stimulate ovarian steroidogenesis and hyperinsulinaemia has been implicated in the raised androgen levels found in diseases associated with significant insulin resistance, ovarian function has not been studied so far in women with NIDDM. We have assessed ovarian function in women with NIDDM at the early (hyperinsulinaemic) and late (relative insulinopaenic) stages of evolution of the disease after strong stimulation with buserelin, a long-acting GnRH analogue. Significant differences in ovarian function would be expected, depending on the stage of evolution of NIDDM.

METHODS

Following an overnight fast, a standard OGTT (75 g, orally) was performed (0830 h) in all diabetic and control women. Blood samples were obtained for blood glucose, insulin and C-peptide measurements before and at 30-minute intervals for 2 hours. On the termination of the OGTT, a buserelin test (100 micrograms, s.c.) was performed (1030 h) and blood samples were obtained for FSH, LH, delta <em>4</em>-<em>androstenedione</em>, total testosterone, free testosterone and oestradiol measurements before and then at <em>4</em>-hour intervals for 20 hours.

METHODS

Thirty-one women with NIDDM (13 hyperinsulinaemic and 18 with relative insulinopaenia), 12 obese and 11 normally menstruating non-obese, non-diabetic women, aged 29-39 years, were studied.

RESULTS

The integrated response (AUC) of oestradiol to buserelin was found to be normal in hyperinsulinaemic NIDDM and obese non-diabetic women in the face of an increased free testosterone response, while in relatively insulinopaenic NIDDM women the oestradiol response was significantly reduced in the face of a normal free testosterone response.

CONCLUSIONS

The results suggest that in women with NIDDM the ovaries have a reduced ability to convert androgen to oestrogen, probably due to a reduction of ovarian aromatase activity. As oestrogens protect against atherogenesis, it is speculated that the relative inability of the ovaries to produce oestradiol in NIDDM women with relative insulinopaenia might be involved in the development of the macroangiopathy, which often complicates this disease.

1. Adrenal and gonadal steroids are derived from cholesterol, which may be derived from plasma lipoproteins or de novo synthesis. 2. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme in cholesterol synthesis, may therefore affect steroidogenesis when used as lipid-lowering agents in hypercholesterolaemia. 3. We have assessed gonadal and adrenal function in subjects with heterozygous familial hypercholesterolaemia (FH) before and after 12 weeks treatment with pravastatin, an HMG CoA reductase inhibitor, or cholestyramine as a control in maximal recommended doses. <em>4</em>. No changes in measured plasma cortisol responses to tetracosactrin injection were seen in 11 patients on cholestyramine or 12 on pravastatin. 5. No changes were seen in testosterone, sex hormone binding globulin, <em>androstenedione</em>, dehydroepiandrosterone sulphate, oestradiol or 17 alpha-hydroxyprogesterone. 6. Gonadotrophin levels were unaffected in 10 male subjects on cholestyramine and 7 on pravastatin. 7. Measurements on a subset of subjects continuing to 2<em>4</em> weeks treatment also showed no changes. 8. No adverse effect on adrenal or gonadal function could be demonstrated in patients with familial hypercholesterolaemia on maximal recommended doses of pravastatin.

OBJECTIVE

To study the gonadal steroid responses to FSH and hCG in individuals with the inherited Finnish-type inactivating Ala189Val mutation of the FSH receptor gene.

METHODS

Prospective clinical and descriptive study.

METHODS

University hospital.

METHODS

Two women and one man homozygous for the Ala189Val mutation of the FSH receptor gene, and ovarian biopsies from four affected and four healthy women, and four normal fetuses.

METHODS

Individuals were treated with increasing doses of recombinant FSH (300 IU/day start, 900 IU/day final) and/or a single dose of hCG (5000 IU). Ovarian biopsies were used in immunohistochemical analyses for detection of aromatase cytochrome P<em>4</em>50 and transcription factor GATA-<em>4</em>. In situ 3'-end labeling analyses were used for detection of apoptosis.

METHODS

Measurements of serum concentrations of follicle-stimulating hormone, leuteinizing hormone, inhibin A and B, estradiol, testosterone (T), androstenedione, and prolactin, immunostaining for ovarian aromatase, GATA-<em>4</em>, and apoptosis.

RESULTS

Administration of FSH had no effect on production of the steroids. Similarly, human chorionic gonadotropin (hCG) treatment, alone or after FSH administration, failed to raise serum steroid concentrations. Ovarian apoptosis was absent, and the expression of transcription factor GATA-<em>4</em> and aromatase was negligible in the ovarian biopsies from Ala189Val homozygous individuals.

CONCLUSIONS

The Ala189Val mutation of the FSH receptor gene results in a complete block of FSH action in vivo. Furthermore, the failure of hCG to increase both ovarian estradiol and testosterone secretion emphasizes the possible contribution of FSH in regulating ovarian androgen synthesis, and supports the concept that both gonadotropins are necessary for appropriate ovarian steroidogenesis in humans.

Classically, the estrogen signaling system has two core components: cytochrome P<em>4</em>50 aromatase (CYP19), the enzyme complex that catalyzes the rate limiting step in estrogen biosynthesis; and estrogen receptors (ERs), ligand activated transcription factors that interact with the regulatory region of target genes to mediate the biological effects of estrogen. While the importance of estrogens for regulation of reproduction, development and physiology has been well-documented in gnathostome vertebrates, the evolutionary origins of estrogen as a hormone are still unclear. As invertebrates within the phylum Chordata, cephalochordates (e.g., the amphioxus of the genus Branchiostoma) are among the closest invertebrate relatives of the vertebrates and can provide critical insight into the evolution of vertebrate-specific molecules and pathways. To address this question, this paper briefly reviews relevant earlier studies that help to illuminate the history of the aromatase and ER genes, with a particular emphasis on insights from amphioxus and other invertebrates. We then present new analyses of amphioxus aromatase and ER sequence and function, including an in silico model of the amphioxus aromatase protein, and CYP19 gene analysis. CYP19 shares a conserved gene structure with vertebrates (9 coding exons) and moderate sequence conservation (<em>4</em>0% amino acid identity with human CYP19). Modeling of the amphioxus aromatase substrate binding site and simulated docking of <em>androstenedione</em> in comparison to the human aromatase shows that the substrate binding site is conserved and predicts that <em>androstenedione</em> could be a substrate for amphioxus CYP19. The amphioxus ER is structurally similar to vertebrate ERs, but differs in sequence and key residues of the ligand binding domain. Consistent with results from other laboratories, amphioxus ER did not bind radiolabeled estradiol, nor did it modulate gene expression on an estrogen-responsive element (ERE) in the presence of estradiol, <em>4</em>-hydroxytamoxifen, diethylstilbestrol, bisphenol A or genistein. Interestingly, it has been shown that a related gene, the amphioxus "steroid receptor" (SR), can be activated by estrogens and that amphioxus ER can repress this activation. CYP19, ER and SR are all primarily expressed in gonadal tissue, suggesting an ancient paracrine/autocrine signaling role, but it is not yet known how their expression is regulated and, if estrogen is actually synthesized in amphioxus, whether it has a role in mediating any biological effects. Functional studies are clearly needed to link emerging bioinformatics and in vitro molecular biology results with organismal physiology to develop an understanding of the evolution of estrogen signaling. This article is part of a Special Issue entitled 'Marine organisms'.

Sixty-six hirsute women were randomized and treated with 1) flutamide (n = 15), 250 mg/day; 2) finasteride (n = 15), 5 mg/day; 3) ketoconazole (n = 16), 300 mg/day; and <em>4</em>) ethinyl estradiol (EE)-cyproterone acetate (CPA; n = 20), 0.01 mg EE/day for the first week, 0.02 mg EE/day for the second week, and 0.01 mg EE/day for the third week, followed by a pause of 7 days, then 12.5 mg CPA/day added during the first 10 days of every month for 12 months. Hirsutism was evaluated by the Ferriman-Gallwey score, and hair diameter and hair growth rate were determined by a special image analysis processor in basal conditions and after 90, 180, 270, and 360 days of treatment. All treatments produced a significant decrease in the hirsutism score, hair diameter, and daily hair growth rate: flutamide, -55 +/- 13%, -21 +/- 1<em>4</em>%, and -37 +/- 18%; finasteride, -<em>4</em><em>4</em> +/- 13%, -16 +/- 12%, and -27 +/- 1<em>4</em>%; ketoconazole, -53 +/- 18%, -1<em>4</em> +/- 12%, and -30 +/- 21%; and EE-CPA, -60 +/- 18%, -20 +/- 11%, and -28 +/- 21%. Some differences existed among treatments with regard to effectiveness; EE-CPA and flutamide seem to be the most efficacious in improving hirsutism. For the hirsutism score, a greater decrease was seen with EE-CPA (-60 +/- 18%) than with finasteride (-<em>4</em><em>4</em> +/- 13%; P < 0.01) and a greater decrease was seen with flutamide (-58 +/- 18%) than with finasteride (-<em>4</em><em>4</em> +/- 13%; P < 0.05). Flutamide is the fastest in decreasing hair diameter; EE-CPA is the fastest in slowing down hair growth, even though at the end of the treatment there was a significant difference between flutamide and finasteride only (-<em>4</em>1 +/- 18% vs. -27 +/- 1<em>4</em>%; P < 0.05). Flutamide, ketoconazole, and EE-CPA induced a significant decrease in total and free testosterone, 5alpha-dihydrotestosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and <em>androstenedione</em> plasma levels. During the EE-CPA treatment, gonadotropins were suppressed, and the sex hormone-binding globulin level increased. Finasteride induced a decrease in dehydroepiandrosterone sulfate and 5alpha-dihydrotestosterone and an increase in testosterone levels. Very few side-effects were observed during treatment with low doses of flutamide, EE-CPA, and particularly finasteride. Flutamide induced a decrease whereas EE-CPA induced an increase in triglycerides and cholesterol, showing higher values within the normal range. Ketoconazole induced several side-effects and complications, and several people dropped out of the study. Despite different modalities of action and significantly different effects on androgen levels, low doses of flutamide, finasteride, and EE-CPA constitute very satisfactory alternative therapeutic regimens in the treatment of hirsutism.

The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and <em>4</em> were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of <em>androstenedione</em> (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert <em>androstenedione</em> and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.

Studies were performed to investigate the effect of microbial culture supernatants of periodontal pathogens on the metabolism of radiolabelled testosterone in the presence or absence of human gingival fibroblasts. Subgingival plaque samples were obtained on paper points from 3 sites with probing depth values of 6-8 mm. Samples were incubated with 1<em>4</em>C-testosterone for 2<em>4</em> h in brain heart infusion (BHI) broth. Similar incubations were also carried out with strains of A. actinomycetemcomitans, P. Intermedius and P. gingivalis to study the metabolism of radiolabelled testosterone by these periodontal pathogens. At the end of a 2<em>4</em> h incubation period with fibroblasts and supernatants or sonicates, the radioactive metabolites were extracted with ethyl acetate, evaporated and subjected to thin layer chromatography. The separated metabolites were quantified by scanning the radioactive plates using a Berthold linear analyser. When three sub-gingival plaque samples were incubated with radiolabelled testosterone there were 50-fold, 10-12-fold and 15-17-fold increases in 5 alpha-dihydrotestosterone (DHT) synthesis over <em>4</em>-<em>androstenedione</em> production in these mixed microbial cultures. The two strains of P. intermedius produced 3- and 20-fold increases in <em>4</em>-<em>androstenedione</em> production and DHT synthesis respectively. Both strains of A. actinomycetemcomitans and P. gingivalis showed 3-<em>4</em>-fold and 12-28-fold increases respectively in <em>4</em>-<em>androstenedione</em> synthesis over that of DHT. Culture supernatants of P. intermedius and P. gingivalis caused 3-fold and 2-fold increases in DHT synthesis by fibroblasts over controls. There was little change in the case of the third pathogen. Since DHT has implications on matrix synthesis by fibroblasts in the environment of plaque associated inflammatory periodontal disease, bacterial metabolism and the effect of bacterial supernatants on human gingival fibroblasts can influence the degree of inflammatory repair.

The possible effect of dietary fat content and the ratio of polyunsaturated to saturated fatty acids (P/S-ratio) on serum sex hormones was studied in 30 healthy male volunteers. The customary diet of the subjects, which supplied <em>4</em>0% of energy as fat (mainly from animal sources, P/S-ratio 0.15) was replaced for a 6 weeks period by a practically isocaloric experimental diet containing significantly less fat (25% of energy) with a higher P/S-ratio (1.22) and other environmental factors were stabilized. Serum testosterone and <em>4</em>-<em>androstenedione</em> decreased from 22.7 +/- 1.1 nmol/l to 19.3 +/- 1.2 nmol/l, (SEM, P less than 0.001) and from <em>4</em>.6 +/- 0.2 nmol/l to <em>4</em>.3 +/- 0.2 nmol/l (SEM, P less than 0.01), respectively. These changes were paralleled by a reduction in serum free (non-protein bound) testosterone (P less than 0.01) suggesting a possible change in biological activity. During the low fat period a significant negative correlation between serum prolactin and androgens was observed. All the changes in androgen levels were reversible. With the exception of a small but non-significant decrease in serum estradiol-17 beta, the other hormone parameters were practically unaffected by the dietary manipulation. Our results indicate that in men a decrease in dietary fat content and an increase in the degree of unsaturation of fatty acids reduces the serum concentrations of <em>androstenedione</em>, testosterone and free testosterone. The mechanism and importance of this phenomenon is discussed in the light of epidemiological and experimental data.

This study characterizes the effect of an excess-calorie, high-fat, high-cholesterol, high-fructose diet on metabolic parameters and reproductive function in female Ossabaw minipigs. Cycling sows were fed a hypercaloric, high-fat, high-cholesterol, and high-fructose diet (obese, n = <em>4</em>) or a control diet (control, n = 5) for 13 mo. During the final <em>4</em> mo, ovarian ultrasonography was done, blood was collected, and weights and measures were taken. Pigs then underwent ovarian stimulation. Cycle length and <em>androstenedione</em>, total testosterone, progesterone, estradiol, follicle-stimulating hormone, luteinizing hormone, insulin, fructosamine, lipid, and glucose levels were measured. In addition, adipose tissue aromatase gene expression was assessed. As compared with control pigs, obese pigs were hyperglycemic and hyperinsulinemic; had elevated total cholesterol, triglyceride, and leptin levels, and demonstrated abdominal adiposity. Visceral adipose tissue of obese pigs, as compared with control pigs, showed increased aromatase gene expression. Obese pigs had longer estrous cycles, higher serum <em>androstenedione</em>, and higher luteal phase serum luteinizing hormone, compared with control pigs. During the luteal phase, obese pigs had more medium, ovulatory, and cystic ovarian follicles, whereas control pigs had more small ovarian follicles. When fed an excess-calorie, high-fat, high-cholesterol, high-fructose diet, female Ossabaw minipigs develop obesity, metabolic syndrome, and abnormal reproductive function. This animal model may be applicable to studies of the effects of obesity on fertility in women.

BACKGROUND

The measurement of adrenal and ovarian androgens in women with PCOS has been difficult based on poor specificity and sensitivity of assays in the female range.

METHODS

Women with PCOS (NIH criteria; n = 52) and control subjects with 25-35 day menstrual cycles, no evidence of hyperandrogenism and matched for BMI (n = <em>4</em>2) underwent morning blood sampling. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to simultaneously measure 13 steroids from a single blood sample to measure adrenal and ovarian steroids. Androgen and progesterone results were compared in the same samples using RIA.

RESULTS

Testosterone, <em>androstenedione</em>, progesterone and 17OH progesterone levels were higher when measured using RIA compared to LC-MS/MS, although the testosterone RIA demonstrated the best agreement with the LC-MS/MS using a Bland-Altman analysis. Results using LC-MS/MS demonstrated that the concentration of androgens and their precursors were higher in women with PCOS than controls [median (2.5, 97.5th %ile); 1607 (638, 3085) vs. 11<em>4</em>3 (511, <em>4</em>78<em>4</em>) ng/dL; p = 0.03]. Women with PCOS had higher testosterone [<em>4</em>9 (16, 125) vs. 2<em>4</em> (10, 59) ng/dL], <em>androstenedione</em> [203 (98, <em>4</em>76) vs. 106 (69, 223) ng/dL] and 17OH progesterone levels [80 (17, 176) vs. <em>4</em><em>4</em> (17, 1<em>4</em>2) ng/dL] compared to controls (all P<0.02), but no differences in serum concentrations of the adrenal steroids DHEAS, cortisol, corticosterone and their 11 deoxy precursors. Women with PCOS also had an increase in the product:precursor ratio for 3β-hydroxysteroid dehydrogenase [22% (6, 92) vs. 20% (<em>4</em>, <em>4</em>3); p = 0.009].

CONCLUSIONS

LC-MS/MS was superior to RIA in measuring androstenedione, progesterone and 17OH progesterone levels, while testosterone measurements were better matched in the two assays. Androgen levels were higher in women with PCOS in the absence of a difference in adrenal-predominant steroids. These data support previous findings that the ovary is an important source for the androgen excess in women with PCOS.

A cDNA clone encoding the complete rat 17 alpha-hydroxylase (P<em>4</em>50(17 alpha] from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P<em>4</em>50(17 alpha), 6<em>4</em>% similarity with bovine P<em>4</em>50(17 alpha), and <em>4</em>7% similarity with chicken P<em>4</em>50(17 alpha). The protein contains 507 amino acids being one amino acid shorter than the human P<em>4</em>50(17 alpha) as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (approximately 2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and <em>androstenedione</em>, respectively. This activity profile is distinct from that of either the human or bovine forms of P<em>4</em>50(17 alpha) which are unable to catalyze 17,20-lyase conversion of delta <em>4</em>-C21 steroids to delta <em>4</em>-C19 steroids at significant rates.