In <em>4</em>3 amenorrheic women with polycystic ovary syndrome (PCOS), 31 (7<em>4</em>%) with fasting hyperinsulinemia >> or =20 microU/mL), our aim was to determine whether Metformin (Bristol-Myers Squibb, Princeton, NJ), which reduces hyperinsulinemia, would reverse the endocrinopathy of PCOS, allowing resumption of regular normal menses. A second aim was to assess the effects of weight loss versus other Metformin-induced effects on ovarian function, and to determine if there were different responses to Metformin between those who lost weight and those who did not. A third aim was to assess associations between PCOS, <em>4</em>G/5G polymorphism in the promoter sequence of the plasminogen activator inhibitor-1 gene (PAI-1 gene), and PAI activity (PAI-Fx). Of the <em>4</em>3 women, <em>4</em>0 (93%) had normal fasting blood glucose and 37 had normal hemoglobin A1C (HgA1C); onlythree (7%) had type 2 diabetes mellitus. Metformin (1.5 to 2.25 g/d) was given for 6.1+/-5.1 months (range, 1.5 to 2<em>4</em>), to 16 patients for less than 3 months, to 12 for 3 to 6 months, and to 15 for at least 6 months. On Metformin, 39 of <em>4</em>3 patients (91%) resumed normal menses. The percentage of women resuming normal menses did not differ among treatment duration groups (P<.1) or among dose groups (P>.1). The body mass index (BMI) decreased from 36.<em>4</em> + 7 Kg/m2 at study entry to 35.1+/-6.7 on Metformin (P=.0008). Of <em>4</em>3 patients, 28 (67%) lost weight (1 to 69 pounds), with nine (21%) losing at least 12 pounds. On Metformin, the median fasting serum insulin decreased from 26 microU/mL to 22 (P=.019), testosterone decreased from 61 ng/dL to <em>4</em>7 (P=.003), and estradiol increased from <em>4</em>1 pg/mL to 71 (P=.0001). Metformin-induced improvements in ovarian function were independent of weight loss (testosterone decrease, P<.002; estradiol increase, P<.000<em>4</em>). The change in response variables on Metformin did not differ (P>.05) between those who lost weight and those who did not, excepting Lp(a), which increased <em>4</em> mg/dL in those who lost weight and decreased 9 mg/dL in those who did not (P = .003). The change in response variables on Metformin did not differ among the five quintiles of weight loss, excepting fasting glucose (P<.05), which increased 6 mg/dL in those who lost the least weight on Metformin versus those in the 60th to 80th percentile for weight loss, in whom glucose decreased 33 mg/dL. Although the pretreatment fasting serum insulin was not significantly correlated with testosterone (r=.2<em>4</em>, P=.13) or <em>androstenedione</em> (r=.27, P=.09), on Metformin, the change in insulin correlated positively with the change in testosterone (r=.35, P=.0<em>4</em>7) and with the change in <em>androstenedione</em> (r=.<em>4</em>8, P=.01). Patients were more likely than normal controls (83% v 6<em>4</em>%, P=.016) to be heterozygous or homozygous for <em>4</em>G polymorphism of the PAI-1 gene and were also more likely to have high PAI-Fx >> or =22 U/mL, 28% v3%, chi(2)=10.1, P=.001). Metformin reduces the endocrinopathy of PCOS, allowing resumption of normal menses in most (91%) previously amenorrheic women with PCOS.

Levels of norepinephrine (NE), dopamine (DA), serotonin and the latter's primary metabolite, 5-hydroxyindoleacetic acid, were measured in two hypothalamic and 6 vocal control areas in the brains of male zebra finches. NE and DA turnover in these areas were also estimated using alpha-methylparathyrosine. Males were castrated for at least 3 weeks and then received an implant of <em>androstenedione</em> (AE) or a control implant of cholesterol. Each male was then housed with a female for at least one week. significant quantities of the <em>4</em> monoamines were found in all brain areas examined; steady-state levels varied significantly across brain areas. Hormone treatment affected steady-state monoamine levels in the preoptic area (POA), the magnocellular paraventricular nucleus and the vocal control area, area X. Hormone treatment altered both NE and DA turnover in the POA, area X and a second vocal control area, nucleus robustus archistriatalis. NE turnover as altered by hormone treatment ina third vocal area, the dorsomedial portion of the intercollicular nucleus. These data suggest that monoaminergic neurotransmitters may be involved in the mediation of steroid-dependent changes in singing behavior in passerine birds.

According to the 2-cell theory, ovarian steroidogenesis requires the coordinate action of both FSH and LH. To evaluate the relative importance of these hormones in follicular maturation, a randomized cross-over study was performed in 10 women with complete gonadotropin deficiency (absence of pulsatile LH secretion and no LH response to LHRH). Five women were treated with highly purified FSH (LH bioactivity, 0.09%) and 3 months later with human menopausal gonadotropin (hMG; LH bioactivity, 65%), each given for 10 days at a daily dose of 225 IU FSH, im. The sequence was reversed in the other 5 women. hCG (5000 IU) was administered im 2<em>4</em> h after the last injection of FSH or hMG. Plasma estradiol (E2), estrone (E1), <em>androstenedione</em> (A), testosterone, LH, and FSH concentrations and urinary LH and FSH were measured daily by RIA. Ultrasonography was performed during each treatment and 2 days after each hCG injection. After FSH treatment, mean plasma and urinary FSH levels increased, mean plasma LH did not change, and urinary LH increased slightly but not significantly from 91 +/- 32 (SE) to 16<em>4</em> +/- 55 mIU/2<em>4</em> h (10(-3) IU/2<em>4</em> h). After hMG treatment, mean plasma and urinary LH and FSH levels increased accordingly. The mean basal plasma E2 [11 +/- 1 pg/mL (<em>4</em>0 +/- <em>4</em> pmol/L)] and E1 [1<em>4</em> +/- <em>4</em> pg/mL (52 +/- 15 pmol/L)] levels increased after FSH treatment to 207 +/- 69 pg/mL (760 +/- 253 pmol/L) and 82 +/- 21 pg/mL (303 +/- 78 pmol/L), respectively (P less than 0.01), but plasma A did not change. In response to hMG, the mean plasma E2, E1, A, and testosterone levels increased more than during FSH treatment. Ultrasonography revealed multiple preovulatory follicles (greater than or equal to 16 mm) in 2 women after hMG and 1 woman after FSH treatment; therefore, hCG was not administered. In 3 women given FSH, hCG did not induce ovulation. hCG induced ovulation in 8 women given hMG and in 6 women given FSH, based on ultrasonography and plasma progesterone levels. Thus, in the presence of profound gonadotropin deficiency pharmacological doses of FSH, with minute LH contamination, are capable of stimulating ovarian follicular maturation, underlining the key role of FSH in folliculogenesis.

Thirty late-onset adrenal hyperplasia patients consulting for isolated hirsutism were randomly divided into two groups; group 1 (n = 16) was treated with hydrocortisone in order to suppress androgen adrenal secretion, and group 2 (n = 1<em>4</em>) received cyproterone acetate (CPA) antiandrogen therapy to inhibit peripheral androgen activity. The clinical and hormonal effects of each type of treatment were evaluated. Before treatment, the clinical and hormonal profiles of the two patient groups did not differ significantly. Excellent clinical evolution in terms of the regression of hirsutism was observed in the CPA-treated patients (5<em>4</em>% decrease in the clinical score in 1 yr), in contrast with the slight decrease in hirsutism (26%) after hydrocortisone treatment. In hydrocortisone-treated patients, plasma androgen decreased to normal levels: testosterone from 3.05 +/- 1.<em>4</em>5 to 1.<em>4</em>6 +/- 0.<em>4</em>2 nmol/L and delta <em>4</em>-<em>androstenedione</em> from 13.6 +/- <em>4</em>.1 to 6.33 +/- 1.<em>4</em>7 nmol/L. Conversely, in CPA-treated patients, only a slight decrease in testosterone from 2.98 +/- 1.98 to 2.29 +/- 0.6<em>4</em> nmol/L and in delta <em>4</em>-<em>androstenedione</em> from 12.9 +/- 5.9 to 9.86 +/- 2.23 nmol/L was observed. This slight decrease in plasma androgens contrasts with the rapid clinical improvement after CPA. These results emphasize the importance of peripheral receptivity to androgens in the clinical expression of hyperandrogenism. Moreover, they indicate that peripheral antiandrogen therapy may be more appropriate in late-onset adrenal hyperplasia patients than conventional adrenal inhibition using cortisone therapy.

The association between hyperinsulinaemia and hyperandrogenism in many women with polycystic ovarian syndrome (PCOS) implies roles for insulin and insulin-like growth factors (IGFs) in the regulation of ovarian androgen production. The aim of the present study was to compare the abilities of insulin, IGF-I and IGF-II to stimulate androgen production by human thecal cells in vitro. Serum-free monolayer cell cultures were established from the ovaries of euandrogenic women undergoing hysterectomy with oophorectomy for non-ovarian indications. Androgen (<em>androstenedione</em>) production was determined after <em>4</em> days of culture in the presence of insulin or either of the IGFs (10-100 ng/ml), with and without a maximal stimulatory dose of luteinizing hormone (LH; 10 ng/ml). Interactions with inhibin (30 ng/ml), a putative paracrine regulator of ovarian androgen synthesis, were also tested. The three metabolic hormones exerted similar dose-related effects on androgen production (ED50 < or = 10 ng/ml), which were augmented 2- to 3-fold in the presence of LH and further increased several-fold by the additional presence of inhibin. No treatment with insulin or either IGF stimulated thecal cell growth, but all treatments caused striking morphological changes consistent with enhanced steroidogenesis. These results reveal potent regulatory effects of metabolic hormones on human thecal androgen synthesis, which imply (i) 'progonadotrophic' roles for insulin and IGF-I in regulating normal ovarian androgen production, (ii) a role for insulin in the aetiology of hyperandrogenism (both with and without hyperinsulinism) in PCOS and (iii) paracrine roles for granulosa-derived IGF-II and inhibin in regulating ovarian androgen production.

An isotopic infusion technique has been used in an attempt to determine the contribution that local, in situ, oestrone synthesis makes to the oestrogen content of breast tumours. 3H-<em>Androstenedione</em> and 1<em>4</em>C-oestrone were infused into women with advanced breast cancer for 12 hr before operation. At surgery, normal breast and breast tumour biopsy samples were obtained and 3H-<em>androstenedione</em>, 3H-oestrone derived from 3H-<em>androstenedione</em> and 1<em>4</em>C-oestrone were isolated and measured. DNA polymerase alpha activity, a marker of cellular proliferation, was also measured to examine whether local synthesis of oestrone exerted a biological effect. The study was repeated after patients had been treated with the aromatase inhibitor, <em>4</em>-hydroxy<em>androstenedione</em>, before undergoing further surgery for removal of their tumours. In <em>4</em>/6 tumours examined, in situ synthesis of 3H-oestrone from 3H-<em>androstenedione</em> accounted for the major part (8<em>4</em>.3 +/- 9.0%) of the 3H-oestrone detected, while no significant in situ synthesis occurred in 2 other tumours. Although treatment with <em>4</em>-hydroxy<em>androstenedione</em> did not significantly alter the uptake of 3H-<em>androstenedione</em> or 1<em>4</em>C-oestrone into breast tissues, in situ formation of 3H-oestrone was only detected in one tumour sample after treatment. DNA polymerase alpha activity decreased in <em>4</em>/6 tumours after treatment with <em>4</em>-hydroxy<em>androstenedione</em>. Overall, however, there was no significant correlation between the level of 3H-oestrone formed in situ and DNA polymerase alpha activity (r = 0.38, NS). It is concluded that in some, but not all, breast tumours in situ formation of oestrone can make an important contribution to the oestrogen content of breast tumours.

In the present study, we investigated the effects of 1<em>4</em> endogenous steroids on the CYP3A<em>4</em>-mediated drug metabolism by human liver microsomes in vitro. Nevirapine (NVP) 2-, 12-hydroxylations, carbamazepine (CBZ) 10,11-epoxidation, triazolam (TZM) 1'-, <em>4</em>-hydroxylations, erythromycin (EM) N-demethylation, and 2-sulphamoylacetylphenol (SMAP) formation from zonisamide (ZNS) were investigated. The activities of the NVP 2-, 12-hydroxylations, the CBZ 10,11-epoxidation, and the TZM <em>4</em>-hydroxylation were activated by endogenous androgens, such as <em>androstenedione</em> (AND), testosterone, and dehydroepiandrosterone. However, these androgens inhibited EM N-demethylation, TZM 1'-hydroxylation, and SMAP formation. To understand the mechanisms of these effects of androgens on CYP3A<em>4</em> activities, we performed a kinetic analysis of the metabolism of CBZ and ZNS in the presence or absence of AND using the modified two-site equation model. The addition of AND to the reaction mixture caused a drastic increase in the activity of CBZ 10,11-epoxidase, especially at a low substrate concentration, and resulted in a change in the kinetics from the sigmoid to Michaelis-Menten type. On the other hand, the metabolism of ZNS was strongly inhibited by AND, although no allosteric change was observed in this case. These data demonstrate that endogenous steroids, especially androgens, strongly affect CYP3A<em>4</em>-mediated drug metabolism in vitro. The postulated mechanisms of the interactions between AND and CBZ or ZNS are discussed.

In this study, we investigated androgen metabolism in two different human prostate cancer cell lines, the androgen-responsive LNCaP cells and the nonresponsive PC3 cells. Following 2<em>4</em>-h and 72-h incubation with either testosterone (T) or <em>androstenedione</em> (Ad) used as precursor, divergent patterns and rates of androgen metabolism were observed. Given the recent interest in the multiple uses of embryonic and adult stem cells for basic and applied research, we compared the expression of three presumptive stem cell markers (Oct-<em>4</em>, SUZ-12, and Cripto-1), along with connexin <em>4</em>3 (Cx<em>4</em>3), Cx32, and androgen receptor (AR), used as cell differentiation gene markers. In anchorage-independent cell growth conditions, the expression levels of candidate markers of cancer stem cells initially increased (days 2-<em>4</em>) but drastically fell thereafter (day 6) in both cell lines. Results of immunocytochemical assay (ICA) largely confirmed those obtained by RT-PCR. Interestingly, both symmetrical and asymmetrical cell divisions were revealed in PC3 cells using Oct-<em>4</em> immunostaining. Our data suggest that both androgen-responsive and androgen-nonresponsive prostate tumor cell lines contain a presumptive cancer stem cell population that can be identified using a panel of selected gene markers, including Oct-<em>4</em>, SUZ-12, and Cripto-1.

Aromatase, a cytochrome P-<em>4</em>50 enzyme that catalyzes the conversion of androgens to estrogens, is the major mechanism of estrogen synthesis in the post-menopausal woman. We review some of the recent scientific advances which shed light on the biologic significance, physiology, expression and regulation of aromatase in breast tissue. Inhibition of aromatase, the terminal step in estrogen biosynthesis, provides a way of treating hormone-dependent breast cancer in older patients. Aminoglutethimide was the first widely used aromatase inhibitor but had several clinical drawbacks. Newer agents are considerably more selective, more potent, less toxic and easier to use in the clinical setting. This article reviews the clinical data supporting the use of the potent, oral competitive aromatase inhibitors anastrozole, letrozole and vorozole and the irreversible inhibitors <em>4</em>-OH <em>androstenedione</em> and exemestane. The more potent compounds inhibit both peripheral and intra-tumoral aromatase. We discuss the evidence supporting the notion that aromatase inhibitors lack cross-resistance with antiestrogens and suggest that the newer, more potent compounds may have a particular application in breast cancer treatment in a setting of adaptive hypersensitivity to estrogens. Currently available aromatase inhibitors are safe and effective in the management of hormone-dependent breast cancer in post-menopausal women failing antiestrogen therapy and should now be used before progestational agents. There is abundant evidence to support testing these compounds as first-line hormonal therapy for metastatic breast cancer as well as part of adjuvant regimens in older patients and quite possibly in chemoprevention trials of breast cancer.

Estrogens produced within breast tumors may play a pivotal role in growth stimulation of the breast cancer cells. However, it is elusive whether the epithelial breast cancer cells themselves synthesize estrogens, or whether the surrounding tumor stromal cells synthesize and supply the cancer cells with estrogen. The aromatase enzyme catalyzes the estrogen production, aromatizing circulating androgens into estrogens. The aim of this study was to investigate aromatase expression and function in a model system of human breast cancer, using the estrogen responsive human MCF-7 breast cancer cell line. Cells were cultured in a low estrogen milieu and treated with estrogens, aromatizable androgens or non-aromatizable androgens. Cell proliferation, expression of estrogen-regulated proteins and aromatase activity were investigated. The MCF-7 cell line was observed to express sufficient aromatase enzyme activity in order to aromatize the androgen testosterone, resulting in a significant cell growth stimulation. The testosterone-mediated growth effect was completely inhibited by the aromatase inhibitors letrozole and <em>4</em>-hydroxy-<em>androstenedione</em>. Expression studies of estrogen-regulated proteins confirmed that testosterone was aromatized to estrogen in the MCF-7 cells. Thus, the results indicate that epithelial breast cancer cells possess the ability to aromatize circulating androgens to estrogens.

The present study concerned the seasonal and acute effects of heat stress on steroid concentrations in follicular fluid and on steroid production by granulosa and theca interna cells, in bovine dominant follicles. Three groups of cows were studied: summer (n = 5), autumn (n = 5) and winter (n = 9) cows. During the winter season, another group of cows was acutely heat-stressed from days 3 through 5 of the estrous cycle (n = 5). On day 7 of the estrous cycle, follicular fluid from first-wave dominant follicles was aspirated, and dispersed granulosa and theca cells from each seasonal group were incubated for 18 h at normothermic (37.5 degrees C) or high (<em>4</em>0.5 degrees C) temperatures. Cells were incubated in media only or in media containing testosterone (300 ng ml-1, for granulosa cells) or forskolin (<em>4</em> micrograms ml-1, for theca cells). In follicular fluid the 17 beta-estradiol concentration was high (P < 0.05) in winter and low in autumn, and summer, the <em>androstenedione</em> concentration was high in summer (P < 0.05), low in autumn, and intermediate in winter. During the winter season, acute in vivo heat stress increased follicular fluid <em>androstenedione</em> and decreased estradiol to levels comparable with those prevailing in summer. Basal and forskolin-stimulated <em>androstenedione</em> production by theca cells was higher (P < 0.05) in the winter group than in the summer and autumn groups, and also higher than in the cows that were heat-stressed during winter, which suggests that theca cell function is susceptible to chronic (summer), short-term (winter) and delayed (autumn) heat stresses. In vitro incubation at high temperature (<em>4</em>0.5 degrees C) reduced the high, forskolin-stimulated <em>androstenedione</em> production in winter (P < 0.05). Estradiol production by granulosa cells was high in winter and autumn, and low in summer (P < 0.05). Acute heat stress in winter did not alter estradiol production relative to winter controls, whereas a high incubation temperature (<em>4</em>0.5 degrees C) reduced (P < 0.05) estradiol production only in the autumn, when the highest production rate was recorded. The results indicate a differential effect of heat stress on the functions of granulosa and theca cells. Both concurrent and delayed effects of heat stress on the steroidogenic capacity of ovarian follicles in cattle are presented.

We previously showed that in innately resistant tumors, silencing of the estrogen receptor (ER) could be reversed by treatment with a histone deacetylase (HDAC) inhibitor, entinostat. Tumors were then responsive to aromatase inhibitor (AI) letrozole. Here, we investigated whether ER in the acquired letrozole-resistant tumors could be restored with entinostat. Ovariectomized athymic mice were inoculated with MCF-7Ca cells, supplemented with <em>androstenedione</em> (Δ(<em>4</em>)A), the aromatizable substrate. When the tumors reached about 300 mm(3), the mice were treated with letrozole. After initial response to letrozole, the tumors eventually became resistant (doubled their initial volume). The mice then were grouped to receive letrozole, exemestane (250 μg/d), entinostat (50 μg/d), or the combination of entinostat with letrozole or exemestane for 26 weeks. The growth rates of tumors of mice treated with the combination of entinostat with letrozole or exemestane were significantly slower than with the single agent (P < 0.05). Analysis of the letrozole-resistant tumors showed entinostat increased ERα expression and aromatase activity but downregulated Her-2, p-Her-2, p-MAPK, and p-Akt. However, the mechanism of action of entinostat in reversing acquired resistance did not involve epigenetic silencing but rather included posttranslational as well as transcriptional modulation of Her-2. Entinostat treatment reduced the association of the Her-2 protein with HSP-90, possibly by reducing the stability of Her-2 protein. In addition, entinostat also reduced Her-2 mRNA levels and its stability. Our results suggest that the HDAC inhibitor may reverse letrozole resistance in cells and tumors by modulating Her-2 expression and activity.

Female social dominance characterizes many strepsirrhine primates endemic to Madagascar, but currently there is no comprehensive explanation for how or why female lemurs routinely dominate males. Reconstructing the evolutionary pressures that may have shaped female dominance depends on better understanding the mechanism of inheritance, variation in trait expression, and correlating variables. Indeed, relative to males, many female lemurs also display delayed puberty, size monomorphism, and 'masculinized' external genitalia. As in the spotted hyena (Crocuta crocuta), a species characterized by extreme masculinization of the female, this array of traits focuses attention on the role of androgens in female development. Consequently, I examined endocrine profiles and social interaction in the ringtailed lemur (Lemur catta) to search for a potential source of circulating androgen in adult females and an endocrine correlate of female dominance or its proxy, aggression. I measured serum <em>androstenedione</em> (A(<em>4</em>)), testosterone (T), and estradiol (E(2)) in reproductively intact, adult lemurs (10 females; 12 males) over four annual cycles. Whereas T concentrations in males far exceeded those in females, A(<em>4</em>) concentrations were only slightly greater in males than in females. In both sexes, A(<em>4</em>) and T were positively correlated, implicating the Delta(<em>4</em>)-biosynthetic pathway. Moreover, seasonal changes in reproductive function in both sexes coincided with seasonal changes in behavior, with A(<em>4</em>) and T in males versus A(<em>4</em>) and E(2) in females increasing during periods marked by heightened aggression. Therefore, A(<em>4</em>) and/or E(2) may be potentially important steroidal sources in female lemurs that could modulate aggression and underlie a suite of masculinized features.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have important regulatory functions in ovarian follicular development. Although most studies have investigated the IGF system in ovarian cells in vitro, investigation of the IGF system in the peripheral circulation and in follicles of varying sizes throughout the menstrual cycle in large numbers of subjects has been lacking. In the current study we performed daily IGF-I, IGF-II, IGFBP-1, and IGFBP-3 measurements in 9 healthy regularly cycling volunteers throughout the menstrual cycle. In addition, we investigated IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels in 13 samples of androgen-dominant follicular fluid [FFa <em>androstenedione</em> to estradiol (AD:E2) ratio,>> <em>4</em>] and 19 samples of estrogen-dominant follicular fluid (FFe; AD:E2 ratio, <em>4</em>) obtained from 21 regularly cycling subjects and in 18 samples of fluid from luteinizing follicles obtained from patients undergoing in vitro fertilization (IVF) treatment (FFivf). IGF-I, IGF-II, IGFBP-1, and IGFBP-3 were measured using two-site immunoradiometric assays. No significant day to day differences were observed in IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels across the menstrual cycle. Median IGF-II levels in FFe (630 ng/mL; range, 212-1000) were significantly higher compared to those in FFa (<em>4</em>7<em>4</em> ng/mL; range, 272-603; P = 0.002). Median IGFBP-3 levels in FFe (2955 ng/mL; range, 388-3<em>4</em><em>4</em>8) were also significantly higher than those in FFa (2352 ng/mL; range, 756-260<em>4</em>; P = 0.003). Median IGF-I (192 ng/mL; range, 29-256) and IGFBP-1 (12 ng/mL; range, 2-281) levels in FFe were not significantly different from those in FFa [1<em>4</em>9 (range, 22-232) and 21 (range, 5-32) ng/mL, respectively). In contrast, significantly lower IGFBP-1 levels were found in FFe compared to FFivf (79 ng/mL; range, 57-23<em>4</em>; P = 0.002), whereas there was no significant difference between FFe and FFivfe IGF-I, IGF-II, or IGFBP-3 levels, respectively. IGF-II levels were correlated with follicle diameter (r = 0.52; P = 0.002), cycle day (r = 0.<em>4</em>7; P = 0.0065), E2 levels (r = 0.53; P = 0.003), AD:E2 ratio (r = -0.58; P = 0.001), and P concentrations (r = 0.60; P = 0.001) in all follicles, whereas no such correlations were found with IGF-I. In conclusion, as circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3 are not menstrual cycle dependent, it is unlikely that these growth factors and these binding proteins play an endocrine role in cyclic ovarian follicle development, although both cycle-dependent delivery to the ovary and modification of their actions locally within the ovary cannot be excluded. With regard to FF1 the findings that IGF-II levels in FF1 are elevated compared to those in FFa and correlate with follicular functional status support a role for IGF-II during development of the dominant follicle. In addition, as IGFBP-3 in estrogen-dominant follicles mirrors the rise of IGF-II, this IGFBP may be a primary regulator of IGF-II action within the estrogen-dominant follicle. Finally, the finding of elevated levels of IGFBP-1 in luteinizing (IVF) follicles suggests an important role for this peptide in corpus luteum regulation.

This report is concerned with hormone concentrations accompanying sexual maturation in a highly 'masculinized' female mammal, the spotted hyaena, Crocuta crocuta. Plasma concentrations of testosterone, <em>androstenedione</em> and oestrogen were determined by radioimmunoassay in a longitudinal study of 12 female and eight male hyaenas 2.5-62.5 months old. Concentrations of testosterone were significantly higher in males than in females after 26.5 months of age, but earlier measurements did not differ between sexes. Mean testosterone concentrations in adult female hyaenas (0.<em>4</em>-0.5 ng ml-1) were similar to those in several other female mammals that do not display a 'masculine' profile, but mean concentrations of <em>androstenedione</em> (2.5-5.5 ng ml-1) in female hyaenas were significantly higher than in males (1.0-2.0 ng ml-1), at most ages. Oestrogen could not be detected (less than 0.03 ng ml-1) in females until about 1<em>4</em> months of age and then increased (to approximately 0.13 ng ml-1) between 18 and 30 months; oestrogen remained undetectable in males. This rise in oestrogen in females corresponded to nipple enlargement and to changes in the size and elasticity of the urogenital meatus, permitting copulation and parturition through the clitoris. Gonadectomy (two males and four females) at <em>4</em>-7 months resulted in nondetectable concentrations of testosterone and oestrogen and a marked attenuation in <em>androstenedione</em> (to approximately 0.39 ng ml-1), indicating that the gonads are the major source of these three steroids. Gonadectomy also eliminated sex differences in weight, nipple development and elasticity of the urogenital meatus.

Spironolactone (S) has been used successfully for the treatment of hirsutism. We evaluated whether the effects of S on serum androgens and hair growth are dose-related and whether S affects secreted androgens to the same degree as peripherally derived androgens. Two groups of 15 hirsute patients, similarly matched, received either 100 or 200 mg S daily for 3 months. Serum total testosterone (T) decreased significantly (P less than 0.05) and to a similar degree with both dosages, whereas unbound T was unaltered. Dehydroepiandrosterone sulfate was unaltered, whereas <em>androstenedione</em> decreased with 200 mg S (P less than 0.05). Peripherally derived serum dihydrotestosterone decreased to a similar degree with 100 and 200 mg S (P less than 0.05), whereas 5 alpha-androstane-3 alpha-17 beta-diol (3 alpha-diol) increased (P less than 0.05) similarly with both dosages. Serum 3 alpha-diol glucuronide (3 alpha-diol-G) increased with both dosages, but not significantly. Anagen hair shaft diameters decreased significantly in both groups by 19% +/- 8% and 30% +/- <em>4</em>% (P less than 0.05). No correlation was found between hair growth and serum androgens. Because serum unbound T was largely unaltered by S, it is suggested that the antiandrogenic effects of S are primarily related to its peripheral effect. However, there is no good clinical marker for this effect as levels of 3 alpha-diol and 3 alpha-diol-G increase.

From a longitudinal prospective study, 160 women with spontaneous menopause and without steroid medication were followed during the transition from pre- to postmenopause. After 12 years 152 women were still participating in the study. Blood samples were drawn every 6 months until 1 year after the menopause and every 12 months thereafter. Measurements of bone mineral density (BMD) on the forearm were performed every second year. All women routinely completed a questionnaire concerning symptoms frequently attributed to the climacteric period. All data were grouped around the onset of the menopause, thereby allowing longitudinal evaluation of the changes in the variables from the premenopausal to the postmenopausal period. The beginning of the perimenopausal period was characterized by transitory elevations of follicle-stimulating hormone (FSH). A significant increase in serum levels of gonadotropins was observed for both FSH and luteinizing hormone (LH) from about 5 years before the menopause. Within the 6 month period around the menopause there was a further increase which culminated within the first postmenopausal year for LH and 2-3 years postmenopause for FSH. Thereafter, a continuous decrease in LH occurred over the following 8 years. With respect to FSH, there was a slight decline starting about <em>4</em> years postmenopause. During the premenopausal period an increasing frequency of inadequate luteal function or anovulation occurred and, in the postmenopausal years, the serum levels of progesterone (P) were invariably low. Gradually, the ratio between estrone (E1) and 17-beta-estradiol (E2) increased, reflecting the declining follicular steroidogenesis. A marked decrease in estrogen levels occurred during the 6 month period around the menopause, most pronounced in E2. During the next 3 years, the levels of E2 and E1 showed an essentially parallel, moderate decline. Around the menopause, serum levels of testosterone (T), delta<em>4</em>-<em>androstenedione</em> (A) and sex hormone-binding globulin (SHBG) showed small but significant decreases. From about 3 years postmenopause, the levels were relatively constant over the following 5 years. A decrease in BMD was observed in the postmenopause, and from about 3 years postmenopause, estradiol correlated positively with BMD. Before, as well as after the menopause, body mass index (BMI) showed an inverse correlation with SHBG. Postmenopausal <em>androstenedione</em> correlated positively with E1, E2 and T. BMI correlated positively with E1 and E2. The concentrations of the free fraction of E2 and T are dependent on the levels of SHBG, which in turn has a negative correlation with BMI. The impact of this will influence the severity of symptoms, the degree of bone loss and the need for supplementary therapy.

The brain traditionally has been considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have shown that the brain itself also has the capability of forming steroids de novo, the so-called "neurosteroids". De novo neurosteroidogenesis in the brain from cholesterol is a conserved property of vertebrates. Our studies using the quail, as an excellent animal model, have demonstrated that the avian brain possesses cytochrome P<em>4</em>50 side-chain cleavage enzyme (P<em>4</em>50scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(<em>4</em>)-isomerase (3beta-HSD), cytochrome P<em>4</em>50 17alpha-hydroxylase/c17,20-lyase (P<em>4</em>50(17alpha,lyase)), 17beta-HSD, etc., and produces pregnenolone, progesterone, 3beta, 5beta-tetrahydroprogesterone, <em>androstenedione</em>, testosterone and estradiol from cholesterol. However, the biosynthetic pathway of neurosteroids in the avian brain from cholesterol may be still incomplete, because we recently found that the quail brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid. This paper summarize the advances made in our understanding of biosynthesis of neurosteroids in the avian brain.

Dehyroepiandrosterone (DHEA), an adrenal-derived steroid, has been clinically implicated in protection against coronary artery disease and experimentally in inhibition of atherosclerosis and plaque progression. Because DHEA is enzymatically metabolized to androgens or estrogens, it is not clear whether DHEA exerts effects directly or after conversion to these hormones, both of which are associated with well-characterized pathways of action. We therefore examined the effects of DHEA on proliferation of human vascular smooth muscle cells (VSMCs) in culture in the presence or absence of the ER antagonist ICI 182,780 and the AR antagonist flutamide and compared them with the effects of 17beta-estradiol, <em>androstenedione</em>, and T. We also determined the affinity of DHEA for ERs and ARs in VSMC and its specific binding in intact cells. To explore a possible mechanism for DHEA action in these cells, we measured the phosphorylation of ERK-1, c-jun N-terminal protein kinase, and p38 (three members of the MAPK superfamily). Both DHEA and 17beta-estradiol significantly inhibited platelet derived growth factor (PDGF)-BB-induced increases in VSMC proliferation, whereas <em>androstenedione</em> and T increased proliferation. Although E2-induced inhibition of the PDGF effect was abolished by ICI 182,780 and T-induced stimulation was abolished by flutamide, neither receptor antagonist altered the inhibitory effect of DHEA. Binding studies confirmed the presence of both ERs and ARs; DHEA showed minimal affinity for either receptor but bound specifically and with high affinity to putative receptors in intact cells. Following <em>4</em>-h incubation with DHEA (1-100 nM), ERK1 phosphorylation was significantly reduced in a dose-dependent manner, whereas neither c-jun N-terminal protein kinase nor p38 kinase activity was altered by either PDGF-BB or DHEA. DHEA inhibits human VSMC proliferation by a mechanism independent of either ARs or ERs, presumably via a DHEA-specific receptor that involves ERK1 signaling pathways.

LHRH synthesis and release are modulated in vivo by gonadal steroids. Although immunocytochemical and autoradiographic studies failed to detect appreciable amounts of estrogen or androgen receptor in LHRH-producing neurons, the recent finding that the promoter region of the LHRH gene contains several steroid hormone-responsive elements indicates a possible direct effect of sex steroids on these specialized neurons. The immortalized LHRH-producing neuronal cell line, GT1, which became recently available, may allow the study of LHRH dynamics. The presence of specific binding sites for estrogen and androgens as well as the presence of the two major enzymatic pathways involved in modulation of androgen action (the 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase and the aromatase) have been studied in the GT1-1 clone. High affinity, low capacity binding sites for [3H]estradiol (Kd, 0.11 nM; binding capacity, 6.2 fmol/mg protein) and for a ligand of the androgen receptor, [3H]R1881 (Kd, 0.05<em>4</em> nM; binding capacity, 9.58 fmol/mg protein), have been identified in this cell line. A 2-fold induction of androgen-binding sites has been observed after 3 days of treatment of GT1-1 cells with estradiol (1 microM), indicating that the estradiol binding is probably linked to a functional estrogen receptor. Aromatase and 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase activities have been also tested in GT1-1 cells. Under the culture conditions adopted, no detectable aromatization of [1 beta 3H]delta <em>4</em>-<em>androstenedione</em> to estrone was observed using the tritiated water method. On the other hand, GT1-1 cells efficiently converted testosterone into dihydrotestosterone and subsequently into 5 alpha-androstan-3 alpha,17 beta-diol. In conclusion, GT1-1 cells possess several elements of the machinery through which sex steroids may influence LHRH dynamics.

High-dose ketoconazole (<em>4</em>00 mg orally three times a day) and physiologic replacement doses of glucocorticoids (hydrocortisone, 20 mg 8 AM, 10 mg <em>4</em> PM, and 8 PM) were administered to 38 patients with advanced prostatic cancer, refractory to at least initial testicular androgen deprivation. Thirty patients were completely evaluable; six were withdrawn due to possible ketoconazole-related toxicity and were considered drug failures. Two patients were unevaluable due to intercurrent therapy or inability to maintain follow-up. Ketoconazole was generally well tolerated. Mild or moderate nausea and vomiting occurred in 37% of patients, but required dose modification or discontinuation in only three patients; no hepatic damage was seen. Five of 36 patients (1<em>4</em>%) responded to ketoconazole as determined by palpable or radiographic tumor mass reduction of 50% or greater and normalization of acid phosphatase or bone scan. Fifty percent of patients entered were stable at 90 days. Plasma <em>androstenedione</em> and dehydroepiandrosterone sulfate (DHEAS) were reduced markedly in almost all patients. Plasma testosterone (T) levels were low and remained unchanged, while gonadotropins were persistently elevated. Mean plasma ketoconazole content was 6.6 micrograms/mL after 28 days of therapy. While ketoconazole with hydrocortisone does suppress plasma androgens in advanced prostatic cancer patients, this infrequently causes regression of cancer that has progressed despite adequate testicular androgen ablation.

Using routine stable isotope dilution/gas chromatography-mass spectrometry, 17-hydroxyprogesterone, <em>androstenedione</em>, testosterone, dehydroepiandrosterone, androstanediol, and 5alpha-dihydrotestosterone have been profiled in amniotic fluid of midgestation in 77 normal fetuses and 38 untreated or dexamethasone-treated fetuses at risk for 21-hydroxylase deficiency. Dexamethasone was suspended 5-7 days before amniocentesis. In normal fetuses, amniotic fluid concentrations (median, range; nanograms per mL) of 17-hydroxyprogesterone did not reveal a sex difference (1.<em>4</em>8, 0.21-<em>4</em>.96), whereas those of <em>androstenedione</em> were lower in females (0.53, 0.00-2.71) than in males (0.93, 0.29-1.98). Testosterone levels were higher in males (0.2<em>4</em>, 0.00-0.50) than in females (0.00, 0.00-0.27). No sex difference was found for dehydroepiandrosterone (0.<em>4</em>7, 0.19-1.77). Levels of androstanediol and 5alpha-dihydrotestosterone were below the detection limit of our method in most cases. Regarding prenatal diagnosis of 21-hydroxylase deficiency, 17-hydroxyprogesterone and <em>androstenedione</em> presented the diagnostically most valuable steroids and were of equal diagnostic potential. They permitted successful diagnosis in 36 of 37 fetuses at risk: 12 were untreated and unaffected, 13 were treated and unaffected, <em>4</em> were untreated and affected (3 salt wasters and 1 simple virilizer), and 8 were treated and affected (5 salt wasters and 3 simple virilizers). In the latter group, one simple virilizer revealed normal steroid concentrations. Isotope dilution/gas chromatography-mass spectrometry, providing the highest specificity in steroid analysis, is proposed for routine use in clinical steroid analysis whenever maximal reliability is requested. Our study provides the first mass spectrometric reference data on amniotic fluid steroid concentrations and underscores the high accuracy of prenatal hormonal diagnosis of 21-hydroxylase deficiency.

to examine the possible effects of aging on circulating steroid hormones in postmenopausal women, blood samples were drawn from 155 women, aged 3<em>4</em> to 83 years, with spontaneous ovarian failure. The C-21 steroids, pregnenolone and 17-hydroxypregnenolone; the delta <em>4</em> progestins, progesterone and 17-hydroxyprogesterone; and cortisol did not change with age and were similar in concentration to the levels measured during the follicular phase of premenopausal women. The delta 5 androgens, dehydropiandrosterone and dehydroepiandrosterone sulfate, declined significantly (P less than .001) with age, whereas no change was noted in the delta <em>4</em> androgens, <em>androstenedione</em> and testosterone. The levels of estradiol (E2) and estrone (E1) were strongly correlated with percent ideal weight but did not change with age. The authors conclude that 1) The production of progestins does not change with age in normal adult women, other than that resulting from the loss of secretion associated with ovarian corpus luteum function. 2) The decline of delta 5 androgens without corresponding changes in their precursors suggests an age-related change of adrenal 17,20 desmolase activity. 3) The levels of E2 and E1 reflect an effect of body size but not of age on peripheral aromatization of precursor androgens.

Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant found in consumer products that causes ovarian toxicity. Antral follicles are the functional ovarian units and must undergo growth, survival from atresia, and proper regulation of steroidogenesis to ovulate and produce hormones. Previous studies have determined that DEHP inhibits antral follicle growth and decreases estradiol levels in vitro; however, the mechanism by which DEHP elicits these effects is unknown. The present study tested the hypothesis that DEHP directly alters regulators of the cell cycle, apoptosis, and steroidogenesis to inhibit antral follicle functionality. Antral follicles from adult CD-1 mice were cultured with vehicle control or DEHP (1-100 μg/ml) for 2<em>4</em>-96 h to establish the temporal effects of DEHP on the follicle. Following 2<em>4</em>-96 h of culture, antral follicles were subjected to gene expression analysis, and media were subjected to measurements of hormone levels. DEHP increased the mRNA levels of cyclin D2, cyclin dependent kinase <em>4</em>, cyclin E1, cyclin A2, and cyclin B1 and decreased the levels of cyclin-dependent kinase inhibitor 1A prior to growth inhibition. Additionally, DEHP increased the mRNA levels of BCL2-associated agonist of cell death, BCL2-associated X protein, BCL2-related ovarian killer protein, B-cell leukemia/lymphoma 2, and Bcl2-like 10, leading to an increase in atresia. Further, DEHP decreased the levels of progesterone, <em>androstenedione</em>, and testosterone prior to the decrease in estradiol levels, with decreased mRNA levels of side-chain cleavage, 17α-hydroxylase-17,20-desmolase, 17β-hydroxysteroid dehydrogenase, and aromatase. Collectively, DEHP directly alters antral follicle functionality by inhibiting growth, inducing atresia, and inhibiting steroidogenesis.