Antrum formation and estradiol (E(2)) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E(2) on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E(2) or <em>androstenedione</em> (A(<em>4</em>)) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A(<em>4</em>), developmentally competent OGCs secreted more E(2) than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E(2) and A(<em>4</em>) on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for <em>4</em> days with E(2) (1 μg/ml; E(2)(+)), ii) GCs of OGCs cultured for <em>4</em> days without E(2) (E(2)(-)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E(2) (1 μg/ml; AF group). GCs of the E(2)(+) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E(2) biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E(2) impacts the gene expression profile of GCs to support the in vitro development of OGCs.

We studied a 35-year-old patient with female external genitalia, primary amenorrhea and XY karytotype. Plasma testosterone was 10 ng per deciliter, which did not change after administration of human chorionic gonadotropin, increased to 22 ng per deciliter after ACTH, and decreased to 0.9 ng per deciliter after dexamethasone. Plasma delta <em>4</em>-<em>androstenedione</em>, dehydroepiandrosterone and 17-hydroxyprogesterone were in the normal range. Plasma luteinizing hormone was high, but follicle-stimulating hormone normal (7.5 mlU per milliliter). There were two testes with epididymis and vas deferens, but no Mullerian structures. Microscopical examination showed hyalinization of tubules, which were lined by normal Sertoli cells and occasional immature germ cells. No Leydig cells were seen. After castration follicle-stimulating hormone increased to <em>4</em>3 mlU per milliliter. We conclude that this case of male pseudohermaphroditism was probably due to a Leydig-cell agenesis, that the epididymis and vas deferens can be developed in such a condition and the follicle-stimulating hormone secretion is regulated, at least in part, by a non-androgen substance secreted by Sertoli cells.

A controlled clinical study was designed to investigate the value of human chorionic gonadotrophin (HCG) challenge as a test for functional ovarian hyperandrogenism. Dexamethasone administration was followed by 5000 IU HCG and blood samples for steroid hormone assay were obtained 0, 8, 16, and 2<em>4</em> h thereafter. Study subjects were normal women (n = 13); women with functional ovarian hyperandrogenism, defined by androgen excess, amenorrhoea and an increased 17-hydroxyprogesterone response to nafarelin (n = 6); and normal men (n = <em>4</em>). The responses of 17-hydroxyprogesterone, <em>androstenedione</em> and testosterone to HCG in women with functional ovarian hyperandrogenism were significantly greater than in normal women. However, the 17-hydroxyprogesterone response to HCG in functional ovarian hyperandrogenism was significantly lower after HCG than after nafarelin. The oestradiol response was also significantly lower after HCG than nafarelin, although oestradiol concentration more than doubled in normal women as well as in women with functional ovarian hyperandrogenism. The responses to HCG confirm that functional ovarian hyperandrogenism abnormalities are luteinizing hormone (LH)-dependent. Therefore, the 17-hydroxyprogesterone response to HCG could represent a useful test for the diagnosis of ovarian hyperandrogenism. The lower 17-hydroxyprogesterone response to HCG than to nafarelin in functional ovarian hyperandrogenism suggests that a follicle-stimulating hormone (FSH)-responsive factor modulates thecal 17-hydroxyprogesterone secretion. The oestradiol response to HCG is consistent with HCG directly stimulating the oestradiol secretion by thecal cells.

Evidence has been accumulating for a role for metformin in reducing breast cancer risk in post-menopausal women. It inhibits growth of breast cancer cells via several mechanisms, primarily the AMPK/mTOR signalling pathway. Another possible protective mechanism may be the ability of metformin to inhibit aromatase activity. In the present study, we investigated the effects of metformin on the basal growth of MCF-7 cells, after oestradiol (E2) stimulation and after the inhibition of mTOR by rapamycin. Secondly, we investigated the effects of metformin on the activity of a number of steroidogenic enzymes and the mRNA expression of aromatase and steroid sulphatase (STS). High doses of metformin significantly inhibited both basal and oestrogen-stimulated cell division. Low-dose rapamycin (10-10 M) did not inhibit growth, but the addition of metformin induced a significant reduction in growth. High-dose rapamycin (10-8 M) inhibited growth, and this was further attenuated by the addition of metformin. Exposure to low (10-7 M) and high (10-<em>4</em> M) doses of metformin for 7-10 days significantly reduced the conversion of <em>androstenedione</em> (ANDRO) and testosterone (TESTO) (both requiring aromatase), but not the conversion of oestrone or oestrone sulphate (ES) via 17β-hydroxysteroid dehydrogenase/sulphatase to E2. This attenuation was via a downregulation in the expression of total aromatase mRNA and promoter II, whilst the expression of sulphatase was unaffected by metformin. In conclusion, plasma levels of metformin have a dual therapeutic action, first by directly inhibiting cell proliferation which can be augmented by rapamycin analogues, and secondly, by inhibiting aromatase activity and reducing the local conversion of androgens to E2.

OBJECTIVE

Hyperprolactinemia is a common hormonal disorder in women that may affect the phases of female sexual function (FSD). We investigated sexual function in patients with hyperprolactinemia.

METHODS

A total of 25 women with primary hyperprolactinemia and 16 age matched voluntary healthy women who served as the as control group were evaluated with a detailed medical and sexual history, including a female sexual function index (FSFI) questionnaire and the Beck Depression Inventory. Serum prolactin, dehydroepiandrosterone sulfate, free testosterone, androstenedione, 17alpha-hydroxyprogesterone, estradiol, free thyroxin and thyrotropin were measured. These variables were compared statistically between the 2 groups.

RESULTS

Except for prolactin serum hormone levels in women with hyperprolactinemia were not different from those in the control group. The median total FSFI score was 23.40 (IQR 17.70 to 27.30) in the hyperprolactinemic group, whereas healthy women had a median total FSFI score of 31.10 (IQR 27.55 to 32.88, p < 0.0001). FSD was diagnosed in 22 of 25 patients (88%), while 4 of 16 healthy women (25%) had FSD (p = 0.03). Desire (p = 0.001), arousal (p < 0.0001), lubrication (p = 0.001), orgasm (p = 0.001), satisfaction (p = 0.07) and pain (p = 0.003) domain scores were also significantly lower in women with hyperprolactinemia. Total FSFI (p = 0.009, r = -0.405), desire (p = 0.001, r = -0.512), arousal (p = 0.002, r = -0.466), orgasm (p = 0.026, r = 0.348) and satisfaction (p = 0.041, r = -0.320) scores negatively correlated with mean prolactin but not with the other hormones measured.

CONCLUSIONS

A significant percent of women with hyperprolactinemia whom we evaluated had sexual dysfunction. No hormonal changes other than prolactin and no depression was found as a cause of FSD.

The genomic mode of action is believed to represent the predominant effect of a steroid hormone. Recently, however, rapidly manifesting, non-genomic effects have also been observed. These are mediated mostly by allosteric interaction of a steroid with heterologous target structures such as membrane receptors, a prototype example being the GABAA. Here we describe our studies considering two interdependent questions: (1) do steroids also interact with opioid receptors in brain? Twenty different steroids, i.e. estrogens, androgens, glucocorticoids, mineralocorticoids, gestagens and a cardiac glycoside were tested with respect to their ability to compete for in vitro binding to rat brain membranes of 3H-ligands specific for delta, mu and kappa opioid receptors, respectively. Among all classes of steroids, only the estrogens were effective, all others were 20 to 100 times less effective or ineffective. The rank order among the estrogens was diethylstilbestrol>> 17 alpha-estradiol>> 17 alpha-ethinyl-estradiol>> estriol>> estrone>> 17 beta-estradiol. Next potent to estrogens (although far less) were--on average as a group--glucocorticoids, followed by mineralocorticoids, androgens, gestagens and digoxin. This global as well as within-group rank order, was, with rare exceptions, qualitatively equal irrespective of which radioligand was used, yet displayed the various radioligands different sensitivities with respect of being inhibited by steroids (irrespective of kind), i.e. in the order [3H]naloxone>> or = [3H]DAGO>> or = [3H]DADL>> [3H]DPDP>>) [3H]etorphine. The IC50 of diethylstilbestrol for displacing [3H]DAGO was approximately 30 microM and that of 17 beta-estradiol was approximately 200 microM. (2) What are the concentrations of the major steroid hormones in the brain's extracellular fluid? We have analyzed in 56 matched (i.e. simultaneously withdrawn) peripheral serum and cerebrospinal fluid (CSF) samples (from endocrinologically grossly normal patients) the concentrations of the unconjugated steroid hormones testosterone, <em>androstenedione</em>, dehydroepiandrosterone (DHEA), progesterone and cortisol (all being more or less lipophilic) as well as those of their hydrophilic counterparts, i.e. DHEA-sulfate, or their hydrophilic binding proteins, i.e. sex hormone binding globulin, corticosterone binding globulin, and albumin. Total (i.e. free plus protein-bound) CSF levels of all these steroids were found to be in the 0.02-2 nM range except for cortisol (approximately 20-50 nM), thus 3 to <em>4</em> orders of magnitude lower than the IC50 of estrogens for [3H]DAGO (see above). These total CSF values were quite similar to the reported and calculated free serum levels of these steroids and no difference existed between those of patients with intact or with disturbed (abnormally leaky) blood-brain barrier function.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Nonclassical 3 beta-hydroxy-delta 5-steroid dehydrogenase (3 beta-HSD) deficiency type of congenital adrenal hyperplasia has been hypothesized to occur in as many as 10-<em>4</em>0% of hirsute women, based on the adrenal steroidogenic responses to ACTH. However, diagnostic criteria for this "late-onset" 3 beta-HSD deficiency are not clearly established. Among <em>4</em>0 successive hyperandrogenic women undergoing evaluation of adrenal steroidogenic responses to ACTH, 8 had responses suggestive of 3 beta-HSD deficiency. Since 3 beta-HSD is present in both the ovary and adrenal, we attempted to document the defect in the ovary by stimulating their ovarian function with a gonadotropin-releasing hormone agonist test using nafarelin (6-D-[2-naphthyl]alanine-gonadotropin-releasing hormone). The eight hirsute women had steroid responses to ACTH suggestive of 3 beta-HSD deficiency, namely, the values of the delta 5-steroids, 17-hydroxypregnenolone and dehydroepiandrosterone, 30 and 60 min after ACTH in each hirsute woman were greater than 2 SD above the normal mean. Seven of the eight hirsute women had at least one elevated delta 5/delta <em>4</em>-steroid ratio; however, only three of the hirsute women had two abnormal ratios. Furthermore, the response of the delta <em>4</em>-steroid <em>androstenedione</em> and the ratio of <em>androstenedione</em> to cortisol after ACTH were significantly increased in the hirsute women, findings not consistent with 3 beta-HSD deficiency. After nafarelin, five and six hirsute patients had elevated values of the delta <em>4</em>-steroids <em>androstenedione</em> and 17-hydroxyprogesterone, respectively. No patient had an elevated delta 5/delta <em>4</em>-steroid ratio after nafarelin. Thus, ovarian steroidogenic responses to nafarelin did not support the diagnosis of 3 beta-HSD deficiency. Rather, they are consistent in most cases with polycystic ovary syndrome due to dysregulation of 17-hydroxylase and 17,20-lyase activities. We propose that increased activity of the enzyme P<em>4</em>50c17 alpha in the adrenal cortex is responsible for most of what is often termed late-onset 3 beta-HSD deficiency.

A simplified, rapid, and highly reproducible technique is described for measuring 5 alpha-reductase activity (5 alpha RA) in small skin biopsies. Human genital skin was obtained from 23 nonhirsute and 20 hirsute premenopausal women (HW) and 5 normal men. Skin samples were minced at <em>4</em> C and incubated with RPMI-16<em>4</em>0 in the presence of 95% O2-5% CO2 and <em>4</em>.15 nmol [1<em>4</em>C]testosterone ([1<em>4</em>C]T) for 2 h at 37 C. Steroids were extracted with diethyl ether and separated by Celite and paper chromatography. Radioactivity in specific eluates was quantified, and the mass of each steroid was measured by RIA. The separate formation of 5 alpha-androstane-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha, 17B diol (3 alpha diol), <em>androstenedione</em>, and androsterone from [1<em>4</em>C]T was measured. In separate experiments it was demonstrated that an incubation time of 2 h was optimum and that the addition of cofactors was unnecessary. Radiochemical purity was confirmed after chromatography. The mean +/- SE conversion ratio (CR) of T to DHT (in 2 h) in HW was higher than that in normal women (16.80 +/- 1.62% vs. <em>4</em>.<em>4</em>8 +/- 0.36%; P less than 0.01). In men, the CR of T to DHT averaged 31.60 +/- 3.96%. Individual values for the CR of T to DHT in HW and normal women did not overlap. The CR of T to 3 alpha diol was significantly higher in HW (9.66 +/- 0.86%) and men (15.98 +/- 2.0%) compared to that in normal women (2.96 +/- 0.32%; P less than 0.05). The CR of T to <em>androstenedione</em> was significantly greater in HW and men (6.18 +/- 0.<em>4</em>2 and 7.28 +/- 1.92%) compared to that in normal women (2.6<em>4</em> +/- 0.6<em>4</em>%; P less than 0.05). The CR of T to androsterone was very low and was similar in the three groups. The production of DHT in HW (<em>4</em>.50 +/- 1.0 pmol/mg X 2 h) was significantly greater than that in normal women (0.<em>4</em>8 +/- 0.08; P less than 0.01) and was similar to the production in men (6.18 +/- 1.9<em>4</em> pmol/mg X 2 h). There was a significant correlation between the CR of T to DHT and DHT production, and the CR of T to 3 alpha diol and 3 alpha diol production as well as between the CRs of T to DHT and T to 3 alpha diol. These data suggest that measurements of DHT formation are best suited for the assessment of 5 alpha RA and that the measurement of 5 alpha RA in vitro from small skin biopsies is suitable for the clinical evaluation of hirsutism.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is a potent disrupter of vertebrate endocrine systems. It was shown previously that in utero and lactational (IUL) exposure to TCDD resulted in a reduction in serum estradiol concentrations; however, the mechanism for this remains unknown. In the current study, the effects of perinatal exposure to TCDD on the pituitary-ovarian axis were examined. Pregnant rats were given a single oral dose of 1 microg TCDD/kg or vehicle as control on gestation Day 15, and female pups were killed on postnatal Day 21. Pituitaries were assayed for gonadotropin beta-subunit mRNA; additional pituitaries were cultured for <em>4</em> h and the media were assayed for FSH. Gonadotropin receptor mRNAs from vehicle- and TCDD-exposed animals were compared, with some ovaries cultured and the media assayed for estrogen secretion. LH, FSH, progesterone, and <em>androstenedione</em> concentrations were determined in serum. IUL exposure to TCDD resulted in a significant reduction of pituitary FSHbeta mRNA. Although estrogen output was shown to be reduced, neither serum FSH nor LH concentration was increased significantly, and FSH secretion in vitro was not altered. Similarly, serum progesterone and <em>androstenedione</em> were not altered by TCDD exposure, while in vitro estrogen secretion was significantly reduced. These data suggest that TCDD did not act on serum gonadotropin concentrations. The reduction in the concentration of serum estrogen appears to result from direct or indirect actions on the ovary at some point following <em>androstenedione</em> production.

Objectives of this study were to determine if concentrations of steroids, insulin-like growth factor -I (IGF-I), and IGF binding proteins (IGFBP) in follicular fluid and numbers of LH and IGF-I receptors change during growth of the dominant follicle. Ovarian follicular development was monitored daily via ultrasound in lactating Holstein cows. Animals underwent bilateral ovariectomy when the dominant follicle was first identified (days <em>4</em>-6; estrus = day 0; early; n = 5) or when it stopped growing (days 8-12; late; n = 8). All follicles were classified as dominant (DF), large (LG;>> = 6 mm in diameter, excluding DF) or small (SM; < 6 mm), follicular fluid was aspirated, and theca and granulosa cells were collected. Levels of IGFBP-2, assessed via ligand blotting, were greater (P < 0.05) in LG and SM follicles compared with DF in early cows. Levels of IGFBP-3 in follicular fluid were unaffected by follicle class. Numbers of specific 125I-hCG/LH binding sites in thecal cells were greater (P < 0.01) in DF compared with LG and SM follicles of both early and late cows. Numbers of specific 125I-hCG/LH binding sites in granulosa cells were similar for follicle sizes in early cows, but, in late cows, were greater (P < 0.01) in DF compared with SM follicles and were severalfold greater (P < 0.01) in late DF compared with early DF. Numbers of receptors for IGF-I in thecal cells were 2-fold greater (P < 0.05) in DP and LG compared with SM in late cows. Numbers of IGF-I receptors in granulosa cells were unaffected by size or growth of follicles, but were severalfold greater than in theca cells. Concentrations of estradiol were severalfold greater (P < 0.01) in DF compared with LG and SM in both early and late cows. Concentrations of <em>androstenedione</em> in early cows were greater (P < 0.05) in DF and SM compared with LG follicles. Concentrations of progesterone and IGF-I did not differ (P>> 0.10) among follicle classes, but both were greater (P < 0.10) in late LG compared with early LG follicles. Concentrations of IGF-II in follicular fluid did not differ (P>> 0.10) between early and late cows but were greater (P < 0.10) in SM than DF or LG follicles. We conclude that low amounts of IGFBP-2 and increased thecal binding sites for hCG/LH appear to be related to establishment of the dominant follicle during the first follicular wave in cattle exhibiting regular estrous cycles during late lactation.

The circadian rhythm of hormones (N = 10) and mental performance (N = 18) was investigated in male cadets during a 5-day military training course with continuous heavy physical activities corresponding to 35% of the maximal oxygen uptake, with almost total lack of food and sleep. The 2<em>4</em>-h means for <em>androstenedione</em>, dihydroepiandrosterone (DHEA), 17 alpha-hydroxyprogesterone, testosterone and thyroid-stimulating hormone decreased strongly during the course, and the circadian rhythm was extinguished below the minimum levels measured during the control experiment. The 2<em>4</em>-h means for cortisol, dihydroepiandrosterone sulfate (DHEA-S) and progesterone increased during the course, and the circadian rhythm was abolished above the maximum levels of the control experiment. A gradual increase was found in thyroxine, free thyroxine and triiodothyronine during the first 12 h of activities, followed by a constant decrease for the rest of the course. Mental performance decreased during the course and the amplitude of its circadian rhythm increased from +/- 10% to +/- 30% of the 2<em>4</em>-h mean. The circadian rhythms investigated were almost normalized after <em>4</em>-5 days of rest. However, the nocturnal rise for cortisol, <em>androstenedione</em> and DHEA appeared earlier, and the plasma levels of thyroid hormones, estradiol and DHEA-S were lower during the recovery experiment than in the control experiment. The responses to stress of the circadian rhythm for mental performance and steroid hormones during the course indicate a differential regulation.

Cytochrome P<em>4</em>50 2G1 (2G1), which is uniquely expressed in the olfactory mucosa in mammals, may have important physiological functions. In the present study, we have examined the catalytic activity of rabbit 2G1 toward a number of steroid sex hormones, including <em>androstenedione</em>, estradiol, progesterone, testosterone, and 5 alpha-dihydrotestosterone; the purified cytochrome is active toward all of these compounds in a reconstituted enzyme system with turnover numbers of 1.8<em>4</em>, 0.3<em>4</em>, 1.<em>4</em>6, 1.0<em>4</em>, and 0.8<em>4</em>, respectively, at a substrate concentration of 5 microM. In the presence of cytochrome b5, the turnover numbers are 1.58, 0.66, 1.66, 2.7<em>4</em>, and 1.3<em>4</em>, respectively. Estradiol is converted to the 2-hydroxy compound (major product) and <em>4</em>-hydroxy compound (minor product) by 2G1, and progesterone is converted to the 16 alpha-hydroxy derivative as well as the corresponding keto compound as a secondary product. The same products are formed in olfactory microsomal suspensions as major metabolites of progesterone, and the reactions are inhibited strongly by anti-2G1 IgG. In a reconstituted system, 2G1 has an apparent Km of 2.0 microM and a Vmax of 1.8 nmol/min/nmol P<em>4</em>50 for the formation of the 16 alpha-hydroxyprogesterone. Of particular interest, 2G1-catalyzed progesterone metabolism is effectively inhibited by the boar pheromones, 5 alpha-androst-16-en-3-one and 5 alpha-androst-16-en-3 alpha-ol, and to a lesser extent by a variety of odorant compounds as well as by known P<em>4</em>50 inhibitors, including ketoconazole and alpha-naphthoflavone. The broad substrate specificity and relatively high catalytic efficiency of 2G1 in sex steroid metabolism suggest a role for this unique P<em>4</em>50 isozyme in the maintenance of steroid hormone homeostasis in the olfactory mucosa.

A quantitative cytochemical method for the demonstration of 20 alpha-hydroxysteroid dehydrogenase activity (20 alpha-HSD) in the regressing corpora lutea of the adult rat ovary is described. The method employs unfixed tissue sections and relies upon the oxidation of 20 alpha-hydroxy-<em>4</em>-pregnen-3-one (20 alpha-OH-P) with nitro blue tetrazolium as the hydrogen acceptor. The enzyme was dependent upon NADP+ for its activity and was inactive when 20 beta-hydroxy-<em>4</em>-pregnen-3-one (20 beta-OH-P) was used as a substrate. The apparent Km values for 20 alpha-OH-P and NADP+ were 3 x 10(-<em>4</em>) M and 2.5 x 10(-5) M respectively. Inhibition of 20 alpha-HSD activity by steroids was demonstrable at pH 8. <em>Androstenedione</em> was by far the most potent inhibitor, followed by progesterone (the product of the enzyme activity) 17 alpha-hydroxyprogesterone, Compound S and 20 beta-OH-P. At pH 6.8, a pH more favourable to the progesterone leads to 20 alpha-OH-P reaction, only progesterone and 17 alpha-hydroxyprogesterone were inhibitory. Testosterone was without demonstrable effect at either pH.

Although luteinizing hormone (LH) affects <em>androstenedione</em> (A<em>4</em>) and progesterone (P<em>4</em>) production in theca cells, it is still unknown how LH influences molecular mechanism of A<em>4</em> and P<em>4</em> production. To examine the relationship between LH and transcription factors involved in A<em>4</em> and P<em>4</em> production, ovarian theca cells were cultured in the presence or absence of high concentrations of LH for 2<em>4</em> h (pre-treatment with high concentration of LH) and then cultured in the presence or absence of low concentration of LH for <em>4</em>8 h. Low LH enhanced production of A<em>4</em> and P<em>4</em>, and expressions of CYP17 and StAR mRNA in theca cells without pre-treatment with high LH. In addition, low LH stimulated the expression of SF-1 protein in nuclear fractions from theca cells with or without pre-treatment with high LH. The binding of SF-1 to the CYP17 and StAR promoter regions increased in theca cells treated with low LH. Although GATA-<em>4</em> and GATA-6 are both found in the nuclear fraction but not in the cytosol of theca cells, low LH enhanced the binding of GATA-6, but not of GATA-<em>4</em>, to the CYP17 promoter region without pre-treatment with high LH. Acetylation histone H3 in StAR and CYP17 promoter regions were changed by different LH-dosage. Overall, we showed that LH regulates the production of A<em>4</em> and P<em>4</em> by affecting the nuclear localization and switching of transcription factors in theca cells and that target transcription factors involved in steroid production in theca cells are changed by different LH concentration.

The role of granulosa cells on porcine follicular androgen synthesis was studied. Granulosa cells were cultured for 2<em>4</em> h either in Eagle's minimum essential medium (MEM) or in MEM plus follicle-stimulating hormone (FSH; 1 microgram/mL). Thecal preparations were cultured with or without luteinizing hormone (LH), either in MEM, 50% "spent media" from granulosa cells control (MA) or 50% "spent media" from granulosa cells incubated with FSH (MB). In the absence of LH, MB stimulated accumulation of both delta <em>4</em>-<em>androstenedione</em> (twofold) and testosterone (threefold). MB was found to contain high levels of C21-steroids, including progesterone and material which behaves chromatographically and immunologically like pregnenolone. These amounts of C21-steroids were able to stimulate thecal androgen production. The results suggest that granulosa cells contribute to thecal androgen production by providing steroid substrate. This would provide a means for granulosa cells to control the availability of aromatizable androgens.

The variation with pH of kinetic parameters was examined for 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina. The Vmax/Km profile for <em>4</em>-<em>androstenedione</em> indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.<em>4</em> and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the inactivated enzyme revealed that the inactivation arises from modification of a histidine residue. Studies with [1<em>4</em>C]diethylpyrocarbonate provided support for the idea that the 1-2 essential histidine residues are essential for the catalytic activity of the enzyme. Dye-sensitized photooxidation led to 50% inactivation of the enzyme with the decomposition of two histidine residues. This inactivation was also prevented by androstadienedione. Dancyl chloride caused a loss of the enzyme activity. Modifiers of glutamic acid, aspartic acid, cysteine, and lysine did not affect the enzyme activity. Butanedione and phenylglyoxal in the presence of borate rapidly inactivated the enzyme, indicating that arginine residues also have a crucial function in the active site. The data described support the previously proposed mechanism of beta-oxidation of 3-ketosteroid.

To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in <em>4</em> ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (<em>4</em>38.9 +/- <em>4</em>5.5 ng/ml; 875.<em>4</em> +/- 68.2 pg E2/follicle) compared to subordinate follicles (<em>4</em>0.6 +/- 69.<em>4</em> ng/ml; 99.<em>4</em> +/- 10<em>4</em>.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (11<em>4</em>7.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (<em>4</em>50.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and <em>androstenedione</em> in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 1<em>4</em>.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 1<em>4</em>.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.<em>4</em> to 32.5 +/- 1.<em>4</em> ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-<em>4</em> mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Adult hypophysectomized FSH-primed mice were used to study ovulation, fertilization, and preimplantation embryo development. Twelve days after hypophysectomy, animals were injected sc with oFSH (<em>4</em> micrograms/day) twice a day for 3 days. This resulted in large preovulatory follicles that did not secrete estrogen. Concurrent with the last FSH injection, either hCG (5 IU) or human recombinant FSH (10 IU) was injected sc to induce ovulation. Animals were mated or not and killed 1-<em>4</em> days later. The ovulation rate was similar for both the hCG-induced group (FH) and the FSH-induced group (FF), 97% and 90%, respectively. About <em>4</em>5% of the FH mice mated successfully with 56% of the eggs fertilized compared to only 25% of the FF mice with <em>4</em>5% of the eggs fertilized. However, only 5% of ovulated eggs developed to four-cell stages in vivo by day 3 for the FH animals and none in the FF group. To determine the reasons for the in vivo retarded embryo development, embryos at the one- or two-cell stage were collected on day 2 from the FH group. After 96 h of culture, 22% of two-cell embryos were converted to blastocysts, and 11% of one-cell eggs divided to the four-cell stage. In contrast, 80% of two-cell embryos from normal mice develop into blastocysts by 72 h of culture. The ovarian incubation medium and serum were used to measure progesterone (P<em>4</em>), <em>androstenedione</em> (A), and estradiol (E2). The patterns of serum and in vitro production of steroids were parallel. In FH mice, P<em>4</em> increased immediately on the day after hCG injection (day 1) and decreased progressively on days 2 and 3; A and E2 levels increased on day 2, A decreased on day 3, and E2 decreased on day <em>4</em>. When human recombinant FSH was used to induce ovulation, there were no significant changes in serum P<em>4</em> and A; E2 levels were about <em>4</em> times higher on day 1 than in the FSH-primed control, then dropped to baseline levels on days 2 and 3. However, on day 3 in both the FH and FF groups, FSH receptors were still present on the granulosa cells of antral follicles, and LH/hCG receptors were present on the granulosa cells of large antral follicles and newly formed corpora lutea.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

The inner mitochondrial membrane protein 3β-hydroxysteroid dehydrogenase 2 (3βHSD2) synthesizes progesterone and <em>androstenedione</em> through its dehydrogenase and isomerase activities. This bifunctionality requires 3βHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3βHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.<em>4</em> and <em>4</em>.5, 3βHSD2 retained its primarily α-helical character with a decrease in α-helical content at lower pH values, whereas the β-sheet content remained unchanged throughout. Titrating the pH back to 7.<em>4</em> restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3βHSD2 activity at pH <em>4</em>.5 with ~2-fold more progesterone synthesized at pH <em>4</em>.5 than at pH 3.5 and 7.<em>4</em>. Increasing the 3βHSD2 concentration from 1 to <em>4</em>0 μg resulted in a 7-fold increase in progesterone at pH <em>4</em>.5, but no change at pH 7.<em>4</em>. Incubation with guanidinum hydrochloride (GdmHCl) showed a three-step cooperative unfolding of 3βHSD2 from pH 7.<em>4</em> to <em>4</em>.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GdmHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH <em>4</em> and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH <em>4</em>, where the protein is in a pseudostable state. Based on our data, we conclude that at pH <em>4</em>-5, 3βHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.

Use of steroid biosynthesis inhibitors to suppress estrogen production is a logical strategy in the treatment of women with hormone-dependent breast cancer. The clinical availability of aminoglutethimide as an inhibitor of cytochrome P-<em>4</em>50-mediated steroid hydroxylations prompted study of the precise pharmacological and biochemical effects of this drug. Pharmacokinetic studies revealed that aminoglutethimide alters its own metabolic clearance rate as well as that of dexamethasone, a synthetic glucocorticoid. The metabolic clearance rates of other steroids such as hydrocortisone, medroxyprogesterone acetate, and <em>androstenedione</em>, and estrone are not altered by aminoglutethimide. These findings led to development of a practical regimen of escalating aminoglutethimide dosage in combination with hydrocortisone for treatment of patients with breast carcinoma. Further studies focused upon the biochemical mechanism of estrogen suppression with aminoglutethimide. In vivo, isotopic kinetic data demonstrated that aminoglutethimide inhibits peripheral aromatase by 95 to 98% in postmenopausal women. In vitro experiments indicated that aminoglutethimide can effectively block aromatase directly in human breast tumors as well. With respect to relative potency, aminoglutethimide is a 10-fold more potent aromatase inhibitor than is testololactone but is less potent than are <em>4</em>-hydroxy<em>androstenedione</em> and several brominated <em>androstenedione</em> derivatives. Taken together, these studies suggest that aminoglutethimide blocks estrogen production at three sites in women with breast carcinoma: the adrenal cortex, extraglandular peripheral tissues containing aromatase, and breast carcinoma tissue itself.

OBJECTIVE

The efficacy of acute and sustained pituitary gonadotropin down-regulation by the Nal-Glu GnRH antagonist (Nal-Glu) was evaluated in the treatment of uterine leiomyomas.

METHODS

Prospective, open clinical trial.

METHODS

Seven normally cycling women with symptomatic leiomyomas.

METHODS

Nal-Glu (50 micrograms/kg per day) was administered subcutaneously for 3 months.

METHODS

Baseline ultrasound examinations were obtained and repeated monthly throughout treatment. Each leiomyoma was mapped and measured in three dimensions. Blood samples were drawn daily for 7 days, weekly for <em>4</em> weeks, and monthly for the remaining 2 months.

RESULTS

Mean leiomyoma size decreased 52.8 +/- 7.3% (means +/- SD) after 1 month of therapy and remained unchanged for the remainder of the study. Serum levels of E2 (35.9 +/- 11.8 to 9.3 +/- 0.8 pg/mL, 131.7 +/- <em>4</em>3.3 to 3<em>4</em>.0 +/- 1.<em>4</em> pmol/L), estrone (37.3 +/- 7.5 to 13.0 +/- 2.5 pg/mL, 138.1 +/- 27.7 to <em>4</em>8.1 +/- 9.1 pmol/L), and P (1.6 +/- 1.1 to 0.3 +/- 0.01 ng/mL, 5.0 +/- 3.6 to 0.9 +/- 0.0<em>4</em> nmol/L) declined rapidly (within <em>4</em>8 hours) and remained suppressed throughout treatment. Serum LH, FSH, androstenedione, T, and DHEA levels did not change significantly. In two subjects who did not have surgical removal, leiomyomas grew to original size within the 1st month off drug. Six patients remained amenorrheic and the other subject spotted during the last 2 months of therapy.

CONCLUSIONS

Continuous treatment with Nal-Glu induces immediate and sustained pituitary-gonadal down-regulation that results in regression in leiomyoma size. By circumventing GnRH agonist-induced pituitary-ovarian up-regulation, GnRH antagonists may prove to be superior tools in the medical management of leiomyomas.

The present cross-sectional study was carried out to determine the influence of oophorectomy (OPX) on serum levels of sex steroids and bone metabolism, as well as bone mineral density (BMD), in OPX subjects in comparison with age- and body size-matched controls. Quantitative computed tomography (QCT) and dual-photon absorptiometry (DPA) demonstrated a remarkable reduction in BMD in OPX subjects. In particular, the QCT of the centrum of vertebral bone (QCT-C) in these subjects was no more than 69.33 +/- 3.<em>4</em>0% (X +/- SEM) of the control value, and this parameter was much lower than the QCT integral (QCT-I) value of total lumbar vertebrae. This means that BMD decreases specifically in spongy portions after OPX. The serum level of estrone (E1) was significantly lower in OPX subjects than in controls. The hormonal action of E1 on target organs has been thought to be only one-third of that of estradiol (E2), but the marked reduction in serum E1 level seemed to be a significant cause of the reduction in BMD. The serum level of <em>androstenedione</em> (delta <em>4</em>) significantly decreased in OPX subjects and appeared to affect bone metabolism negatively. Both bone formation and bone resorption were found to be stimulated following OPX, but the rate of bone resorption was found to be higher than that of bone formation: there was an imbalance between bone formation and bone resorption in OPX subjects. However, it was not possible to prove a relationship between Ca regulating hormone and this phenomenon. In conclusion, the QCT-C value reflects the changes in spongy vertebral BMD more sensitively than the QCT-I value or DPA.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was conducted to evaluate changes in follicular fluid (FF) insulin-like growth factor binding protein (IGFBP) proteolytic activity and levels of steroids and IGFBP during follicular development in cattle. Estrous cycles of cows were synchronized with two injections of prostaglandin F2alpha (PGF) 11 d apart and follicular growth monitored via daily rectal ultrasonography in order to identify the dominant follicle. All cows were ovariectomized <em>4</em>8 hr after the second injection of PGF. Follicular fluid was collected individually for all follicles>> 5 mm and pooled for small (1 to 5 mm) follicles. Follicular fluid estradiol and <em>androstenedione</em> levels were greater (P < 0.05) and progesterone and IGFBP-3 levels not different (P>> 0.10) in large dominant than in small (1 to 5 mm) or large (>5 mm) subordinate follicles, whereas IGFBP-2, -<em>4</em> and -5 levels were less (P < 0.05) in large dominant than in small or large subordinate follicles. To evaluate proteolysis of IGFBPs, FF was incubated with recombinant human (125) I-labeled IGFBP-2, -3, -<em>4</em>, and -5 and proteins separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of (125)I-lableled IGFBP-2 or -3. However, cleavage of (125)I-labeled IGFBP-<em>4</em> and -5 by FF from large dominant follicles was greater (P < 0.05) than by FF from small or large subordinate follicles indicating that a protease to IGFBP-<em>4</em> and -5 exists in estrogen dominant follicles. We conclude that lower levels of IGFBP-2 in estrogen dominant follicles of cattle are not due to increased proteolysis, whereas decreases in IGFBP-<em>4</em> and -5 levels are likely due, in part, to increased protease activity. Changes in IGFBP may alter levels of bioavailable IGFs that stimulate steroidogenesis and mitogenesis in developing bovine follicles.

A study of the aromatase inhibitor <em>4</em>-hydroxy-<em>androstenedione</em> (<em>4</em>-OHA) was conducted in normal healthy men to compare the oral administration of two preparations of the drug: an unformulated, micronized powder and a formulated microcrystalline material (CGP 323<em>4</em>9). The formulated material achieved a significantly higher mean peak concentration (88% greater than that obtained using the unformulated powder) and a higher mean AUC (not significant). The median time to peak was 1.5 h for both preparations and the elimination rate constants were similar (0.31 for micronized <em>4</em>-OHA and 0.36 h-1 for formulated <em>4</em>-OHA). Plasma concentrations of <em>4</em>-OHA in this group were markedly lower than those previously observed in postmenopausal breast cancer patients. Significant biological activity was demonstrated with the formulated material in its suppression of plasma oestradiol levels, whereas no significant suppression was obtained using the micronized powder. An increase in androgen levels was observed that may have been due to competitive inhibition of enzymes involved in metabolic clearance of androgens and/or to decreased feedback inhibition of gonadotrophin secretion by oestradiol.