Microsomal estrogen synthetase (aromatase) cytochrome P-<em>4</em>50 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-<em>4</em>50 antibody-Sepharose <em>4</em>B chromatography. The purified P-<em>4</em>50 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-<em>4</em>50 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = <em>4</em>3 nM for <em>androstenedione</em> aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at <em>4</em>50 nm and a specific content of 9.1 nmol of P-<em>4</em>50/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a <em>4</em>90-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, <em>4</em>(-1<em>4</em>)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]<em>androstenedione</em> and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/<em>4</em>0 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-<em>4</em>50 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-<em>4</em>50.

We have shown that traditional herbal medicine, Shakuyaku-Kanzo-To consisted of Shakuyaku and Kanzo decreased serum testosterone levels in woman and rat. Therefore, paeoniflorin and glycyrrhizin, a main component of Shakuyaku and Kanzo, respectively, and glycyrrhetic acid, a metabolite of glycyrrhizin in vivo, were investigated for the steroid production in the rat ovary on the morning of proestrus. The homogenized tissues of one ovary were incubated in the Dulbecco's modified Eagle medium (pH 7.5) with 100 micrograms/ml of paeoniflorin, glycyrrhetic acid and glycyrrhizin and the medium only (the control) at 37 degrees C for 270 min. After the centrifugation, the concentrations of delta <em>4</em>-<em>androstenedione</em>, testosterone and estradiol in the supernatants were determined by RIA. The production of the hormones expressed by [concentration x supernatant volume/weight of the ovary] was compared to the control. Paeoniflorin, glycyrrhetic acid and glycyrrhizin decreased significantly the testosterone production but did not change that of delta <em>4</em>-<em>androstenedione</em> and estradiol. Testosterone/delta <em>4</em>-<em>androstenedione</em> production ratio was lowered significantly by paeoniflorin, glycyrrhetic acid and glycyrrhizin. Estradiol/testosterone production ratio was increased significantly by glycyrrhetic acid and not changed by paeoniflorin and glycyrrhizin. These results suggest that paeoniflorin, glycyrrhetic acid and glycyrrhizin affect the conversion between delta <em>4</em>-<em>androstenedione</em> and testosterone to inhibit testosterone synthesis and stimulate the aromatase activity to promote estradiol synthesis by the direct action on the rat proestrous ovary.

According to common understanding of sexual differentiation, the formation and development of a penile clitoris in female spotted hyaenas requires the presence of naturally circulating androgens during fetal life. The purpose of the present study was to determine potential source(s) of such fetal androgens by investigating the timing of urogenital development and placental production of androgen during early and mid-gestation. Fetuses determined to be female by molecular techniques (lack of SRY gene) at days 33 and <em>4</em>8 of gestation had undifferentiated gonads, but the clitoris was already 'masculinized' and was generally similar to the phallus of a 50-day-old male fetus. Wolffian and Müllerian ducts terminated at the urogenital sinus in both sexes and a urethra was present along the entire length of the clitoris and penis. The adrenal gland was large and histologically differentiated at 33 days. Steroid gradients across the uterus (a drop in delta <em>4</em>-<em>androstenedione</em>, with increases in oestrogen and androgen), and high <em>androstenedione</em> in ovarian veins indicated that ovarian <em>androstenedione</em> was metabolized and secreted as testosterone by the placenta throughout gestation. In vitro, whole or homogenized placentae at days <em>4</em>8 and 58 of gestation (110 days total) metabolized radiolabelled <em>androstenedione</em> into testosterone and oestradiol; the specific enzymatic activity of early placental tissues was higher than at later stages. A human placental homogenate had higher aromatase activity but did not produce testosterone unless aromatase was inhibited. Infusion of labelled <em>androstenedione</em> into the uterine arteries of hyaenas demonstrated the conversion of this substrate into testosterone and oestradiol and their secretion into the fetal circulation. Evidently, androgen is produced by the placenta and secreted into the fetal circulation from early in pregnancy when masculinization is first evident, before differentiation of the fetal ovary.

The plasma or serum concentrations of testosterone, LH, FSH, PRL, cortisol, 17-hydroxyprogesterone, <em>androstenedione</em>, dehydroepiandrosterone, estrone and estradiol were monitored in 8 healthy male volunteers for a period of <em>4</em>8 hr after administration of one large dose of ethanol (1.75 g/kg BW) within the first 3 hr of the experiment. Each subject served as his own control in an identical experiment without ethanol. Blood alcohol concentration reached a maximum of 1.51 +/- 0.08 g/l (mean +/- SEM) <em>4</em> hr after the start of drinking. The maximum decrease in serum testosterone was observed at 12 hr when the serum concentrations of gonadotropins were still unchanged. The decrease in serum testosterone persisted at 2<em>4</em> hr despite increases in the serum levels of LH and FSH. The serum or plasma concentrations of PRL, cortisol, 17-hydroxyprogesterone, <em>androstenedione</em> and dehydroepiandrosterone were clearly increased <em>4</em> hr after the start of drinking. The increase in serum cortisol lasted as long as the decrease in serum testosterone. No significant changes were found in plasma concentrations of estrone and estradiol. Our results suggest that in addition to direct testicular effects of alcohol, increased adrenal secretion of cortisol may contribute to the decrease in serum testosterone in men acutely intoxicated with ethanol.

We studied 13 adolescents (mean gynecological age 29.2 +/- 1<em>4</em>.1 months) with anovulatory cycles and 7 women with ovulatory cycles (mean gynecological age 33.1 +/- 15.3 months) as a control group. Adolescents with anovulatory cycles were grouped on the basis of mean plasma LH values: group 1 (n = 7) with high LH values, and group 2 (n = 6) with normal LH values. In all women plasma gonadotropin concentrations were measured at 10-min intervals for 8 h on day <em>4</em> of the cycle. Pulsatile gonadotropin secretion was also studied in each subject a second time <em>4</em>0 months later, to establish the outcome of the different pulsatile patterns. Group 1 had more frequent and greater LH pulses than the other two groups (which were similar) and had the highest plasma 17 beta estradiol, testosterone, <em>androstenedione</em>, and 17 hydroxyprogesterone concentrations. Longitudinal control showed that: in group 1, three subjects out of seven acquired ovulatory cycles and there was a fall in mean LH plasma levels (30 +/- 5 vs. 9 +/- <em>4</em> IU/L; P less than 0.01), number of pulses (8.3 +/- 1.5 vs. 5 +/- 0; P less than 0.025), mean amplitude (13 +/- 3 vs. 5 +/- 2 IU/L; P less than 0.02) and an increase in interpulse interval (56 +/- 10 vs. 91 +/- 6 min; P less than 0.01). In four subjects anovulatory cycles persisted and the LH pulsatile profile remained unchanged. In group 2, five subjects out of six acquired ovulatory cycles, but there were no significant changes in the number of pulses (6 +/- 1 vs. 6 +/- 2; P = NS), interpulse interval (97 +/- 30 vs. 85 +/- 30 min; P = NS), or amplitude (5 +/- 2 vs. <em>4</em> +/- 2 IU/L; P = NS). The results indicate that: 1) anovulatory young women with early normal plasma LH values have an adequate GnRh pulsatile pattern which will easily lead to ovulation; 2) anovulatory young women with high LH plasma values may have a reproductive system blocked in a pathological condition, similar to that observed in polycystic ovary syndrome; 3) only few subjects with high plasma LH values are able to achieve ovulation and normalize LH pulsatile pattern as a consequence of a new mode of GnRh release.

BACKGROUND

To evaluate the metabolic changes in urinary steroids in pre- and post-menopausal women and men with papillary thyroid carcinoma (PTC).

METHODS

Quantitative steroid profiling combined with gas chromatography-mass spectrometry was used to measure the urinary concentrations of 8<em>4</em> steroids in both pre- (n = 21, age: 36.95 ± 7.19 yr) and post-menopausal female (n = 19, age: 52.79 ± 7.66 yr), and male (n = 16, age: <em>4</em>1.88 ± 8.<em>4</em>8 yr) patients with PTC. After comparing the quantitative data of the patients with their corresponding controls (pre-menopause women: n = 2<em>4</em>, age: 33.21 ± 10.<em>4</em>8 yr, post-menopause women: n = 16, age: <em>4</em>9.67 ± 8.9<em>4</em> yr, male: n = 20, age: <em>4</em>2.75 ± <em>4</em>.22 yr), the levels of steroids in the patients were normalized to the mean concentration of the controls to exclude gender and menopausal variations.

RESULTS

Many urinary steroids were up-regulated in all PTC patients compared to the controls. Among them, the levels of three active androgens, <em>androstenedione</em>, androstenediol and 16α-hydroxy DHEA, were significantly higher in the pre-menopausal women and men with PTC. The corticoid levels were increased slightly in the PTC men, while progestins were not altered in the post-menopausal PTC women. Estrogens were up-regulated in all PTC patients but 2-hydroxyestrone and 2-hydroxy-17β-estradiol were remarkably changed in both pre-menopausal women and men with PTC. For both menopausal and gender differences, the 2-hydroxylation, <em>4</em>-hydroxylation, 2-methoxylation, and <em>4</em>-methoxylation of estrogens and 16α-hydroxylation of DHEA were differentiated between pre- and post-menopausal PTC women (P < 0.001). In particular, the metabolic ratio of 2-hydroxyestrone to 2-hydroxy-17β-estradiol, which could reveal the enzyme activity of 17β-hydroxysteroid dehydrogenase, showed gender differences in PTC patients (P < 1 × 10-7).

CONCLUSIONS

These results are expected be helpful for better understanding the pathogenic differences in PTC according to gender and menopausal conditions.

This study evaluates a recent report indicating that <em>androstenedione</em> (<em>4</em>-androsten-3, 17-dione) contributes to the androgenicity of water downstream of a pulp and paper mill discharge on the Fenholloway River (FL, USA). Extraction and concentration of Fenholloway water with C18 solid-phase extraction columns followed by reverse-phase high-pressure liquid chromatography resulted in clearly defined fractions with in vitro androgenic activity in CV-1 cells that had been transiently cotransfected with human androgen receptor and reporter gene constructs. However, we were unable to detect <em>androstenedione</em> in the active fractions by gas chromatography/mass spectrometry. Mass spectrometry analyses of deionized and Fenholloway River water samples that had been spiked with <em>androstenedione</em>, then extracted and fractionated, revealed that the androgen was found only in inactive fractions. We conclude that, although <em>androstenedione</em> was present at easily detectable concentrations in the river water >> 100 ng/L), this compound is not associated with androgenic activity of water from the site.

BACKGROUND

Phytoestrogens, including genistein and other inhibitors of tyrosine kinases (TKs), inhibit specific steroidogenic enzymes. This study was designed to compare the effects of genistein, with two other TK inhibitors, on steroid synthesis in human granulosa luteal (GL) cells and to identify which steroidogenic enzymes they may affect.

METHODS

GL cells, obtained from women undergoing IVF procedures, were cultured for various periods of time with and without substrates for progesterone and estradiol synthesis, in the presence or absence of the TK inhibitors.

RESULTS

The TK inhibitors significantly suppressed progesterone and estradiol synthesis in a dose-dependent manner over a <em>4</em>8 h culture period. Progesterone production in the presence of 10(-7) mol/l pregnenolone during a <em>4</em> h period was inhibited by both acute (<em>4</em> h) and chronic (2<em>4</em> h) exposure of GL cells to 50 micromol/l genistein (P < 0.05) whilst no significant effects of 50 micromol/l tyrphostin A23 were observed. Genistein (<em>4</em> and 2<em>4</em> h exposure) inhibited the production of estradiol using 10(-7) mol/l estrone as a substrate, but inhibition of estradiol synthesis using <em>androstenedione</em> or testosterone as substrates was only observed after a 2<em>4</em> h exposure. In contrast, tyrphostin acutely stimulated estradiol synthesis when <em>androstenedione</em> and testosterone were used as substrates (P < 0.05) but not estrone.

CONCLUSIONS

Genistein directly inhibits 3 and 17beta-hydroxysteroid dehydrogenase activity, whilst tyrphostin has an acute stimulatory effect on aromatase activity. Over a longer time (2<em>4</em> and/or <em>4</em>8 h period), both TK inhibitors suppress steroid synthesis.

Thiazolidinediones have gained widespread use for the treatment of type 2 diabetes mellitus and other insulin resistance states, including polycystic ovary syndrome (PCOS). In thiazolidinedione-treated patients a small reduction in hemoglobin and hematocrit levels often is observed, and this generally has been attributed to fluid retention. Because testosterone is a hematopoietic hormone, we investigated whether a reduction in plasma free testosterone concentration was associated with the decrease in hemoglobin and hematocrit levels in 22 nondiabetic women (9 with normal glucose tolerance and 13 with impaired glucose tolerance; mean age, 29 +/- 5 years; mean body mass index, 35.6 +/- 5.8 kg/m2) with PCOS who were treated with pioglitazone, <em>4</em>5 mg/d. Before treatment and after <em>4</em> months, subjects underwent an oral glucose tolerance test and measurement of total body water content with bioimpedance. Plasma testosterone, <em>androstenedione</em>, dehydroepiandrosterone sulfate, hemoglobin, and hematocrit levels were evaluated at baseline and every month for <em>4</em> months. The fasting plasma glucose concentration (98 +/- 9 mg/dL) was unchanged after pioglitazone treatment, whereas the 2-hour plasma glucose concentration declined from 1<em>4</em>6 +/- <em>4</em>1 to 119 +/- 20 mg/dL (P = .002). Both the free androgen index and the free testosterone levels calculated according to Vermeulen et al decreased significantly (from 1<em>4</em>.<em>4</em> +/- 7.1 to 10.6 +/- 7.8 [P = .02] and from 59.<em>4</em> +/- 23.<em>4</em> to <em>4</em>6.6 +/- 23.3 [P = .03], respectively). The plasma <em>androstenedione</em> level declined from 259 +/- 13<em>4</em> to 190 +/- 109 ng/dL (P = .01), whereas the dehydroepiandrosterone sulfate level did not change significantly (from 139 +/- 90 to 127 +/- 8<em>4</em> mug/dL, P = .2 [not significant]). The levels of both hemoglobin (from 13.6 +/- 1.0 to 12.8 +/- 1.1 g/dL, P = .0002) and hematocrit (from 39.7% +/- 2.2% to 37.9% +/- 2.7%, P = .002) fell slightly after <em>4</em> months of pioglitazone administration. Collectively, before and after pioglitazone administration, the plasma free testosterone level according to Vermeulen et al correlated positively with the levels of hemoglobin (r = 0.<em>4</em>9, P < .0001) and hematocrit (r = 0.<em>4</em>0, P < .0001), as well as the free androgen index (r = 0.38 [P < .0003] with hemoglobin and r = 0.29 [P < .006] with hematocrit); the decrement in plasma free testosterone level and free androgen index also correlated with the decrements in the levels of both hemoglobin (r = 0.51 [P = .01] and r = 0.5<em>4</em> [P = .01], respectively) and hematocrit (r = 0.<em>4</em>2 [P = .05] and r = 0.50 [P = .02], respectively). Body weight increased from 90.5 +/- 17.3 to 92.<em>4</em> +/- 18.8 kg after pioglitazone administration (P = .05), as did body fat content (from <em>4</em>2.7 +/- 15.3 to <em>4</em><em>4</em>.8 +/- 17.1 kg, P = .03), which could explain the increase in weight, because edema did not develop in any of the subjects. Total body water content did not change significantly after pioglitazone administration (from 37.7 +/- 5.0 to 37.8 +/- <em>4</em>.9 L, P = .68 [not significant]). In summary, pioglitazone treatment is associated with a mild decline in hematocrit or hemoglobin level, which is correlated with the reduction in plasma testosterone level. These results suggest that increased body water content cannot explain the reduction in hematocrit or hemoglobin level in women with PCOS. Further studies are necessary to evaluate whether the same scenario is applicable to normoandrogenic women and individuals with type 2 diabetes mellitus.

The aromatase cytochrome P<em>4</em>50 complex is responsible for the in vivo conversion of androgens to estrogens. Although breast cancer epithelial cells have been reported to have appreciable aromatase activity, its biologic significance remains uncertain. To address this, the effect of androgens on the expression of the estrogen-regulated gene pS2 in hormone-dependent human breast carcinoma cells in vitro was examined. Steroid-deprived MCF-7 cells were exposed to varying concentrations (1 nM, 10 nM, and 100 nM of <em>androstenedione</em> or testosterone for 2,<em>4</em>, and 6 days. Baseline aromatase activity was <em>4</em>.9 (+/-3.1) fmol 3H2O/hour/microgram DNA [3<em>4</em>.3 (+/-21.3) fmol/hr/10(6) cells] and was not influenced by the androgens. As an indication of estrogen biosynthesis, northern analysis was performed to quantitate pS2 mRNA expression. Although no significant pS2 induction was observed at 2 days, both <em>4</em> and 6 day exposure to 100 nM testosterone resulted in a 3-fold increase in pS2 mRNA expression. 5 alpha-dihydrotestosterone (5 alpha-DHT) failed to elicit a similar pS2 response. This testosterone-induced response was inhibited with the aromatase inhibitor 7 alpha (<em>4</em>'-amino) phenylthio-1,<em>4</em>-androstadiene-3,17-dione (7 alpha-APTADD) and with 10 microM tamoxifen. MCF-7 breast cancer cells possess endogenous aromatase activity at high enough levels to convert androgens to estrogens and elicit an estrogen-induced response. The expression of aromatase may offer a potential advantage to hormone-responsive cells, providing an additional autocrine growth pathway which may be exploited.

Androgen deficiency in men is associated with severe osteopenia, alterations in body composition including an increase in fat mass, and decreased libido. Little is known about the pathophysiology, metabolic consequences, or gender-specific effects of androgen deficiency in women. Acquired hypopituitarism in women is characterized by central hypogonadism and/or hypoadrenalism and therefore may affect critical sources of androgen production in women. We hypothesized that serum androgen levels would be decreased in women with hypopituitarism. We therefore determined serum androgen levels in 55 women with hypopituitarism and 92 controls. This included <em>4</em> subsets of hypopituitary women: 1) women less than 50 yr old not receiving estrogen, 2) women less than 50 yr old receiving estrogen, 3) women more than 50 yr old not receiving estrogen, and <em>4</em>) women more than 50 yr old receiving estrogen. Premenopausal controls with regular menstrual cycles were studied in the early follicular phase, midcycle, and luteal phase during one cycle. All other subjects were studied 3 times during the month at comparable intervals to mimic these 3 time points of the normal menstrual cycle. Serum testosterone, free testosterone, <em>androstenedione</em>, and dehydroepiandrosterone sulfate levels were markedly reduced in all <em>4</em> groups of women with hypopituitarism compared with controls (P < 0.0001). Moreover, serum testosterone, free testosterone, and <em>androstenedione</em> levels were lower in women with central hypoadrenalism and hypogonadism than in subjects with hypoadrenalism or hypogonadism alone (P < 0.025). Mean DHEAS levels were decreased in hypopituitary women with both hypogonadism and hypoadrenalism compared with those in women with hypogonadism alone (P < 0.0001). These data demonstrate hypoandrogenemia in women with hypopituitarism. The physiological consequences of low androgen levels in this population remain to be determined.

OBJECTIVE

Patients with primary adrenocortical failure (Addison's disease) have abnormally low levels of DHEA and androgens relative to age. To define a suitable dose, the effect of oral dehydroepiandrosterone (DHEA) replacement therapy in women with Addison's disease (n = 9) was evaluated.

METHODS

DHEA was administered as a daily oral dose of either 50 mg (n = 5) or 200 mg (n = <em>4</em>). Blood sampling and measurements of insulin sensitivity (as measured with euglycemic insulin clamp technique) and body composition (as measured by dual energy X-ray absorptiometry) were performed before and during DHEA treatment and at a 3-month follow up.

RESULTS

DHEA and DHEA(S) levels were restored to normal in those patients receiving 50 mg whereas DHEA(S) level was slightly above the normal reference value in those receiving 200 mg. Circulating levels of androgens (androstenedione, testosterone and testosterone/SHBG ratio) were normalized in all patients. A slight rise in IGF-1 levels was seen in both groups as was a decrease in the levels of low and high density lipoproteins. No effect on blood glucose levels or insulin sensitivity was seen and no change of body composition was observed. No serious side-effects were seen, but some of the patients experienced increased apocrine sweat secretion (n = 7), itchy scalp (n = 2) and acne (n = 7), all of which were reversed when DHEA was discontinued.

CONCLUSIONS

A daily replacement dose of 50 mg of DHEA results in near physiological levels of DHEA, DHEA(S) androstenedione and testosterone in women with Addison's disease, without severe side-effects.

The mechanism of cytochrome P-<em>4</em>50 catalyzed steroid hydroxylations in rat liver microsomes has been investigated by employing derivatives of iodosylbenzene as oxygen donors. The model steroid substrate <em>androstenedione</em> which was hydroxylated in positions 7 alpha, 6 beta, and 16 alpha was used in reactions supported by NADPH, iodosylbenzene, and iodosylbenzene derivatives. Evidence for cytochrome P-<em>4</em>50 involvement in iodosylbenzene-sustained <em>androstenedione</em> hydroxylation included inhibition by substrates and modifiers of cytochrome P-<em>4</em>50. The most efficient oxygen donors were (diacetoxyiodo)-2-nitrobenzene greater than (diacetoxyiodo)-2-chlorobenzene greater than 2-nitroiodosylbenzene greater than (dinitratoiodo)-2-nitrobenzene greater than (diacetoxyiodo)benzene greater than (diacetoxyiodo)-2-methoxybenzene greater than <em>4</em>-(diacetoxyiodo)toluene greater than iodosylbenzene. The capacity of the oxidation agents to serve as oxygen donors in cytochrome P-<em>4</em>50 dependent steroid hydroxylation is probably dependent upon several factors such as the tendency of iodosyl compounds to associate, which decreases coordination with the heme iron, the presence of bulky substituents in the 2 position (decreases association), and the presence of electron-withdrawing substituents (tends to decrease coordination with the heme iron). The rates of 7 alpha, 6 beta, and i6 alpha hydroxylation of <em>androstenedione</em> catalyzed by (diacetoxyiodo)-2-nitrobenzene were 108-, 130-, and 167-fold higher, respectively, than the rates of the NADPH-supported reactions. These results strongly suggest that the rate-limiting step in NADPH-sustained cytochrome P-<em>4</em>50 catalyzed reactions is the rate of reduction of cytochrome P-<em>4</em>50.

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P<em>4</em>50 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [(3)H]-<em>androstenedione</em> to estrone and labeled water was dose-dependently inhibited by <em>4</em>-hydroxy<em>androstenedione</em> and obeyed Michaelis-Menten kinetic with apparent Km values of approximately 20 nM and V(max) values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 2<em>4</em> h incubation, genistein (1 microM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 microM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.

Ovarian follicular growth and development are regulated by extraovarian and intraovarian factors, which influence granulosa cell proliferation and differentiation. However, the molecular mechanisms that drive follicular growth are not completely understood. Ovarian follicular cysts are one of the most common causes of reproductive failure in dairy cattle. Nevertheless, the primary cause of cyst formation has not been clearly established. A gene expression comparison may aid in elucidating the causes of ovarian cyst disease. Our objective was to identify differentially expressed genes in ovarian granulosa cells between normal dominant and cystic follicles of cattle. Granulosa cells and follicular fluid were isolated from dominant and cystic follicles collected via either ultrasound-guided aspiration from dairy cows (n = 2<em>4</em>) or slaughterhouse ovaries from beef cows (n = 23). Hormonal analysis for progesterone, estradiol, and <em>androstenedione</em> in follicular fluid was performed by RIA. Total RNA was extracted and hybridized to 6 Affymetrix GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA). Abundance of mRNA for differentially expressed selected genes was determined through quantitative real-time reverse-transcription PCR. Follicular cysts showed greater (P < 0.05) progesterone, lesser (P < 0.05) estradiol, and no differences (P>> 0.10) in <em>androstenedione</em> concentrations compared with noncystic follicles. A total of 163 gene sequences were differentially expressed (P < 0.01), with 19 upregulated and 1<em>4</em><em>4</em> downregulated. From selected target genes, quantitative real-time reverse-transcription PCR confirmed angiogenin, PGE(2) receptor <em>4</em>, and G-protein coupled receptor 3<em>4</em> genes as upregulated in cystic follicles, and Indian hedgehog protein precursor and secreted frizzled-related protein <em>4</em> genes as downregulated in cystic follicles. Further research is required to elucidate the role of these factors in follicular development and cyst formation.

We have described the responses of selected hormones [luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), progesterone (P), <em>androstenedione</em> (delta <em>4</em>-A), testosterone (T)] and substrates (glucose, lactate) before, during, and after a marathon run in five women of varying gynecological status. Blood samples (15 ml) were obtained 60 and 30 min before the race, during the race at 10, 20, and 30 km, and after the race at min 5, 60, and 120. The relative intensity of running varied between 60% and 85% VO2 max, and weight loss ranged from 2.6 to 3.0 kg. The new observations from our data include the fact that FSH was increased during exercise, whereas this was not observed for LH. This latter hormone appears to be linked more closely to the exercise intensity as defined by blood lactate increments. Increments occurred in all steroid hormones and paralleled those observed in controlled laboratory studies, although the magnitude of these changes was larger at the end of the marathon run. Except for dehydroepiandrosterone sulfate (DHEA-S), most of the hormones returned to basal levels in the 2 h after the marathon. DHEA-S exhibited no change until the 30-km point, when a sharp increase was observed, and then remained elevated for 2 h after exercise, suggesting that a) adrenocortical production of steroids continues for a long time after exercise, b) there is a marked reduction in hepatic clearance of this hormone. In summary, the present study shows that prolonged exercise has a marked effect on both steroid and pituitary hormones.

The components of a carrier formulation can interact with an added drug as well as with the membrane surface they were applied on. Therefore, they can influence permeability of the membrane and permeation of the drug. The particular membrane structure might lead to different drug permeation out of one and the same carrier formulation. In this study, in vitro permeability of <em>androstenedione</em> (AD) as a highly lipophilic substance was investigated in excised bovine nasal mucosa, porcine cornea and the artificial cellulose membrane Nephrophan. Two microemulsions (ME) with either the addition of the co-surfactants hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD; ME-CD) or propylene glycol (PG, ME-PG) were tested in order to be used as carrier systems. Both MEs also consisted of 5% isopropyl myristate (IPM), 20% Cremophor EL (CrEL), and water. Buffer solution (EBS) with 0.0025% AD served as control solution and was furthermore compared to 0.0025% AD/buffer-solutions containing the ME components HP-gamma-CD in different concentrations (0.012, 0.02<em>4</em>, 9%) as well as 20% CrEL. The AD-permeation behaviour through the three tissues was differently influenced by the MEs. The apparent permeability coefficients (Papp) of nasal mucosa for both ME systems did not differ from the Papp of the AD/buffer solution. In case of the other two barriers (cornea, Nephrophan, ME-PG as well as ME-CD provoked extended time lags for AD to permeate, so the Papp could not be calculated or declined to zero. Papp of AD/buffer solution without any additives resulted for cornea, nasal mucosa and Nephrophan in a ratio of 1:3:<em>4</em>. CrEL and 9% HP-gamma-CD diminished the Papp, except HP-gamma-CD in molar AD/HP-gamma-CD-ratios of 1:1 (0.012%) and 1:2 (0.02<em>4</em>%). It seems that the composition of lipophilic and hydrophilic structures of the carrier systems or the additives had a higher impact on the Papp of cornea than on the Papp of the other tissues. Structure and character of the different membranes are considered to be mainly responsible for the differentiated permeation behaviour.

Aromatase, a cytochrome P<em>4</em>50, catalyzes the formation of aromatic C-18 estrogenic steroids from C-19 androgens. Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using [1 beta,2 beta-3H]<em>androstenedione</em>, [19-1<em>4</em>C]<em>androstenedione</em>, and [1 beta,2 beta-3H]testosterone as substrates. The mutant Phe-<em>4</em>06----Arg was completely inactive. Since there were only small changes in the Km and Vmax values for all substrates for mutants Tyr-361----Phe and Tyr-361----Leu, the residue Tyr-361 appears not to be directly involved in the substrate binding. The mutant Pro-308----Phe had altered catalytic properties; the Km values for <em>androstenedione</em>, but not testosterone, decreased significantly. These results, along with those obtained from inhibition studies with aromatase inhibitors, <em>4</em>-hydroxy<em>androstenedione</em> and aminoglutethimide, suggest that Pro-308 is probably situated in the active site of the enzyme and may be interacting with the D ring of the steroids.

OBJECTIVE

As part of a study on hormones and bone density in older women, we measured a number of steroid and polypeptide hormones at <em>4</em>- to 6-month intervals in 32 women over a 10-year period.

METHODS

All women were postmenopausal during this time and all measurements from 12 months after the menopause to the end were used in the analysis. To determine whether there was a significant trend in steroid or polypeptide hormones over the years, the data were analyzed by random coefficient linear regression against number of months since menopause.

RESULTS

There was no significant trend in the concentrations of estrone, estrone sulfate, dehydroepiandrosterone, and follicle stimulating hormone. There was a significant decline in the concentrations of estradiol, dihydrotestosterone, dehydroepiandrosterone sulfate, and luteinizing hormone. There was a rise in the levels of androstenedione and testosterone.

CONCLUSIONS

The changes in the concentration of sex hormone-binding globulin could be fitted to a quadratic equation with an initial rise in the concentration followed by a decline. Thus, the trend in hormone concentrations shows no set pattern with aging in the postmenopausal years.

This study examined the effects of acute dehydroepiandrosterone (DHEA) ingestion on serum steroid hormones and the effect of chronic DHEA intake on the adaptations to resistance training. In 10 young men (23 +/- <em>4</em> yr old), ingestion of 50 mg of DHEA increased serum <em>androstenedione</em> concentrations 150% within 60 min (P < 0.05) but did not affect serum testosterone and estrogen concentrations. An additional 19 men (23 +/- 1 yr old) participated in an 8-wk whole body resistance-training program and ingested DHEA (150 mg/day, n = 9) or placebo (n = 10) during weeks 1, 2, <em>4</em>, 5, 7, and 8. Serum <em>androstenedione</em> concentrations were significantly (P < 0.05) increased in the DHEA-treated group after 2 and 5 wk. Serum concentrations of free and total testosterone, estrone, estradiol, estriol, lipids, and liver transaminases were unaffected by supplementation and training, while strength and lean body mass increased significantly and similarly (P < 0.05) in the men treated with placebo and DHEA. These results suggest that DHEA ingestion does not enhance serum testosterone concentrations or adaptations associated with resistance training in young men.

Dihydrotestosterone metabolism was studied with a constant infusion technique in three men, three women, five hirsute women, and four estrogen-treated hirsute women. The mean dihydrotestosterone metabolic clearance rate was higher in men (336 liters/2<em>4</em> hr per m(2) [range, 239-<em>4</em><em>4</em>8]) than in women (153 liters/2<em>4</em> hr per m(2) [range, 108-18<em>4</em>]). The metabolic clearance rates in hirsute patients were intermediate between those men and women and were decreased by estrogen treatment. These observations demonstrate similarities in the metabolic rates of testosterone and dihydrotestosterone. The conversion of plasma testosterone and <em>androstenedione</em> to dihydrotestosterone was studied in men and hirsute women. Approximately <em>4</em> and 2% of plasma testosterone and <em>androstenedione</em>, respectively, were converted to plasma dihydrotestosterone in both groups. From these observations it was determined that a major fraction of plasma dihydrotestosterone was derived from these plasma precursors rather than from glandular secretion. Both 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstan-3beta,17beta-diol (3beta-diol) were identified in plasma during dihydrotestosterone and testosterone infusions. The conversion ratio of dihydrotestosterone to 3alpha-diol (C(BB) (DHT-3alpha)) was greater than the conversion ratio to the 3beta-isomer (C(BB) (DTH-3beta)) in all the patients studied. Both C(BB) (DHT-3alpha) and C(BB) (DHT-3beta) were higher in men (mean values of 0.151 [range, 0.110-0.222] and 0..031 [range, 0.022-0.0<em>4</em>2]) than in women (means of 0.0<em>4</em><em>4</em> [range, 0.037-0.0<em>4</em>8] and 0.012 [range 0.010-0.013]). A smaller fraction of testosterone was converted to 3alpha-diol and 3beta-diol.

The FecB (Booroola) mutation, which leads to increased ovulation rates and multiple births in sheep, is now known to occur in the signaling domain of the bone morphogenic protein (BMP)-1B receptor. We examined the effect of the mutation on the responsiveness of granulosa (GC) and theca cells (TC) to BMPs and other local regulators using tissue from animals with (Fec(B/B)) and without (Fec(+/+)) the FecB mutation. Experiments examined the effect of BMP-2, -<em>4</em>, and -6 (0.005-50 ng/ml), and their interaction with IGF-I (0.1-10 ng/ml LR3 analog) and gonadotropins, on the proliferation and differentiation of GCs and TCs isolated from small (<2 mm) antral follicles and maintained in serum-free culture for up to 8 d. Dose-finding studies using ovaries from wild-type sheep obtained from the abbattoir showed no difference among the different BMPs in stimulating (P < 0.001) estradiol (E2) production by GCs cultured with FSH (10 ng/ml), but there was a clear interaction (P < 0.001) with IGF-I. BMPs had no effect on GC proliferation or the sensitivity of GCs to FSH. In contrast, higher doses of BMPs (5-50 ng/ml) inhibited LH-stimulated <em>androstenedione</em> production by TCs, whereas lower doses (0.005-0.05 ng/ml) stimulated TC proliferation (P < 0.01). Regardless of dose of IGF-I, at the end of culture (96-192 h) hormone production by GCs (E2, inhibin A) and TCs (<em>androstenedione</em>) was <em>4</em>- to 5-fold greater (P < 0.001) by cells from Fec(B/B), compared with Fec(+/+) ewes exposed to the same dose of gonadotropin. In the presence of low concentrations of IGF-I (0.1 ng/ml), the maximum increase in the production of E2 and inhibin A by GCs from FF ewes in response to BMPs was observed at doses that were 3- to 10-fold lower (3-10 ng/ml) than ++ (30 ng/ml; P < 0.001). Low doses of BMPs stimulated proliferation of TCs from ++ (P < 0.01) but not FF ewes. Immunohistochemistry confirmed BMP-6 protein expression in the oocyte, granulosa, and thecal layers of antral follicles from both genotypes. These results confirm a major role for BMPs in controlling ovarian somatic cell function in sheep and provide evidence to support the hypothesis that the FecB mutation increases the BMP response of somatic cells when stimulated to differentiate by gonadotropins.

Aromatase cytochrome P-<em>4</em>50 has been purified from human placenta to homogeneity, as demonstrated by electrophoresis on polyacrylamide gels with SDS, and by double diffusion against an antibody raised in rabbits. The enzyme converts <em>androstenedione</em> to estrone (Vmax 13.3 n moles/min/n mole P-<em>4</em>50; Km 30 microM) and testosterone to estradiol. Aromatase activity requires P-<em>4</em>50, P-<em>4</em>50 reductase and NADPH. Enzyme activity is inhibited by anti-aromatase antibodies and by <em>4</em>-hydroxy<em>androstenedione</em>. The enzyme shows a molecular weight of 55,000, is extremely unstable and spontaneously forms P-<em>4</em>20 with a half-life of 2.5 days.