Comparisons of numbers of antral ovarian follicles and corpora lutea (CL), of blood hormone concentrations, and of follicular fluid steroid concentrations and IGFBP activity were conducted between cows selected (twinner) and unselected (control) for twin births to elucidate genetic differences in the regulation of ovarian follicular development. Ovarian follicular development was synchronized among cows by a single i.m. injection of PGF2alpha on d 18 of the estrous cycle; six cows per population were slaughtered at 0, 2<em>4</em>, <em>4</em>8, and 72 h after PGF2alpha. Jugular vein blood was collected from each animal at PGF2alpha injection and at 2<em>4</em>-h intervals until slaughter. Ovaries of twinner cows contained more small (< or = 5 mm in diameter, P < 0.05), medium (5.1 to 9.9 mm, P < 0.05), and large >> or = 10.0 mm, P < 0.01) follicles and more (P < 0.01) CL than ovaries of controls. Follicular fluid concentrations of estradiol, <em>androstenedione</em>, testosterone, and progesterone reflected the stage of follicular development and were similar for twinner and control follicles at the same stage. Earlier initiation of follicular development and/or selection of twin-dominant follicles in some twinner cows resulted in greater concentrations of estradiol in plasma at 0, 2<em>4</em>, and <em>4</em>8 h and of estradiol, <em>androstenedione</em>, and testosterone in follicular fluid of large follicles at 0 h after PGF2alpha for twinner vs. control cows (follicular status x time x population, P < 0.01). Binding activities of IGFBP-5 and -<em>4</em> were absent or reduced (P < 0.01) in follicular fluid of developing medium and large estro-gen-active (estradiol:progesterone ratio>> 1) follicles but increased with atresia. Only preovulatory Graafian follicles lacked IGFBP-2 binding, suggesting a possible role for IGFBP-2 in selection of the dominant follicle. Concentrations of IGF-I were twofold greater (P < 0.01), but GH (P = 0.10) and cholesterol (P < 0.05) were less in blood of twinners. Three generations of selection of cattle for twin ovulations and births enhanced ovarian follicular development as manifested by increased numbers of follicles within a follicular wave and subsequent selection of twin dominant follicles. Because gonadotropin secretion and ovarian steroidogenesis were similar for control and twinner cattle, enhanced follicular development in twinners may result from decreased inhibition by the dominant follicle(s), increased ovarian sensitivity to gonadotropins, and/or increased intragonadal stimulation, possibly by increased IGF-I.

The aim of this investigation was to establish potential oxidative effects of glucose, advanced glycation end products (AGE) and nicotine (N) in a fibroblast cell culture model using the anti-oxidants glutathione (G) and insulin like growth factor (IGF). Assays of androgen metabolites were used as biomarkers of healing in this context. Confluent monolayer cultures of human gingival fibroblasts were established in 2<em>4</em> well multiwell plates and incubated in Eagle's MEM for 2<em>4</em>h using two radiolabelled androgen substrates 1<em>4</em>C-testosterone/1<em>4</em>C-<em>4</em>-<em>androstenedione</em>. The established effective concentrations of G1000, glutathione and AGE were used alone and in combination with nicotine and insulin-like growth factor. The medium was then solvent extracted for steroid metabolites, evaporated to dryness and subjected to thin layer chromatography in a benzene acetone solvent system <em>4</em>:1 v/v for separation of formed metabolites. The metabolites were quantified, using a radioisotope scanner. Significant reduction in the yields of DHT in response to G1000, AGE and nicotine (n=6; p <0.003) were overcome by glutathione (n=6; p <0.002). The stimulatory effect of IGF when combined with AGE was further enhanced by the antioxidant effect of glutathione (n=6; p <0.003). Glucose, AGE and nicotine had a significant inhibitory effect on the yields of the androgen biomarker DHT, overcome by the antioxidant glutathione and IGF, suggestive of an oxidant role for the former agents and an anti-oxidant one for the latter. These agents affected yields of androgen metabolites, biomarkers of oxidative stress and repair, with potential implications on healing in uncontrolled diabetic smokers.

BACKGROUND

Aromatase, a member of the cytochrome P<em>4</em>50 family, converts androgens such as <em>androstenedione</em> and testosterone into estrone and estradiol, respectively. Letrozole (1-[bis-(<em>4</em>-cyanophenyl)methyl]-1H-1,2,<em>4</em>-triazole; Femara) is a high-affinity aromatase inhibitor (K(i)=11.5 nM) that has Food and Drug Administration approval for breast cancer treatment. Here we report the synthesis of carbon-11-labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon.

METHODS

Letrozole and its precursor (<em>4</em>-[(<em>4</em>-bromophenyl)-1H-1,2,<em>4</em>-triazol-1-ylmethyl]benzonitrile) were prepared in a two-step synthesis from <em>4</em>-cyanobenzyl bromide and <em>4</em>-bromobenzyl bromide, respectively. The [(11)C]cyano group was introduced via tetrakis(triphenylphosphine)palladium(0)-catalyzed coupling of [(11)C]cyanide with the bromo precursor. Positron emission tomography (PET) studies in the baboon brain were carried out to assess regional distribution and kinetics, reproducibility of repeated measures and saturability. Log D, the free fraction of letrozole in plasma and the [(11)C-cyano]letrozole fraction in arterial plasma were also measured.

RESULTS

[(11)C-cyano]Letrozole was synthesized in 60 min with a radiochemical yield of 79-80%, with a radiochemical purity greater than 98% and a specific activity of <em>4</em>.16+/-2.21 Ci/mumol at the end of bombardment (n=<em>4</em>). PET studies in the baboon revealed initial rapid and high uptake and initial rapid clearance, followed by slow clearance of carbon-11 from the brain, with no difference between brain regions. Brain kinetics was not affected by coinjection of unlabeled letrozole (0.1 mg/kg). The free fraction of letrozole in plasma was <em>4</em>8.9%, and log D was 1.8<em>4</em>.

CONCLUSIONS

[(11)C-cyano]Letrozole is readily synthesized via a palladium-catalyzed coupling reaction with [(11)C]cyanide. Although it is unsuitable as a PET radiotracer for brain aromatase, as revealed by the absence of regional specificity and saturability in brain regions such as amygdala, which are known to contain aromatase, it may be useful in measuring letrozole distribution and pharmacokinetics in the brain and peripheral organs.

Castration-resistant prostate cancer (CRPC) remains largely dependent on androgen receptor (AR). Residual tissue androgens are consistently detected within CRPC tumors and play a critical role in facilitating AR-mediated signaling pathways which lead to disease progression. Testosterone and dihydrotestosterone (DHT) are the major androgens detected in tumors. They are produced through three biosynthesis pathways: Δ(<em>4</em>), Δ(5), and backdoor pathways. Both androgens bind to and stimulate AR activation. The current study investigates the effects of pomegranate extracts (POM) and their ability to inhibit androgen biosynthesis using PCa cell lines (22RV1 and LNCaP) in vitro as well as the PTEN knockout mouse model representing prostate cancer. Steroids were extracted using ethyl acetate or solid phase extraction, and then analyzed by UPLC/MS/MS. The results showed that POM (0-12μg/mL) reduced the production of testosterone, DHT, DHEA, <em>androstenedione</em>, androsterone, and pregnenolone in both cell lines. In addition our in vivo data supports this observation with a reduction in serum steroids determined after 20 weeks of POM treatment (0.17 g/L in drinking water). In accordance with these results, Western blotting of cell lysates and tPSA analysis determined that PSA was significantly decreased by the treatment of POM. Interestingly, AKR1C3 and AR levels were shown to be increased in both cell lines, perhaps as a negative feedback effect in response to steroid inhibition. Overall, these results provide mechanistic evidence to support the rationale for recent clinical reports describing efficacy of POM in CRPC patients.

To determine the effect of aromatase inhibitors on peripheral aromatization, male rhesus monkeys were infused with [7-3H]<em>androstenedione</em> and [<em>4</em>-1<em>4</em>C]estrone before and during treatment with <em>4</em>-hdyroxy-androstene-3,17-dione (50 mg/kg each; four monkeys) or <em>4</em>-acetoxy-androstene-3,17-dione (700 mg each; two monkeys). In all six monkeys, no specific effects on the MCRs of <em>androstenedione</em> and estrone, the interconversion of androgens or estrogens, or <em>androstenedione</em> conversion to dihydrotestosterone were noted. In five of the six animals, aromatization rates were reduced by up to 97% of control values. The results demonstrate that these compounds inhibit peripheral aromatization and, together with our other results, suggest that aromatase inhibitors may be a useful treatment for estrogen-dependent cancer.

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant androgen in the circulation and in ovarian follicular fluid. A steroid sulphatase accepting DHEAS as a substrate has been identified in the follicle, but the cellular location has not been determined. As DHEAS is also a potential source of oestrogen for endocrine-dependent tumours, a potent steroid sulphatase inhibitor oestrone-3-O-sulphamate (EMATE) has been developed which inhibits this activity in rat liver and mammary tumour. The aim of this study was to investigate human granulosa cells as a site of steroid sulphatase activity, to determine whether DHEAS can be utilized as a precursor for oestrogen synthesis and to investigate the inhibitory capacity of EMATE in these cells. Conversion of DHEAS to DHEA was assessed in luteinized granulosa cells by tritiated steroid assay following incubation with or without LH or insulin and steroid accumulation in the medium measured by RIA. The effects of EMATE were assessed by addition of a range of doses during the measurement of conversion of DHEAS to DHEA. Cells from three sizes of small follicles from an unstimulated ovary were also assessed for their ability to produce oestradiol from DHEAS. Sulphatase enzyme activity was present in all cells; the mean conversion of tritiated DHEAS to DHEA was 50% (range <em>4</em>-65%). LH and EMATE inhibited and insulin stimulated this activity. Addition of DHEAS to granulosa cells caused a dose-dependent increase in oestradiol and <em>androstenedione</em> production with no change in progesterone concentration. LH increased the accumulation of oestradiol in the medium. DHEAS also stimulated oestradiol production by granulosa cells from small follicles. This is the first demonstration that granulosa cells are a site of sulphatase activity and that DHEAS can be utilized as a substrate for <em>androstenedione</em> and oestrogen production. This may be of physiological importance for both normal folliculogenesis and oestrogen-dependent tumour growth.

To determine the antisteroidogenic effect of ketoconazole (KTZ) in the human testis, we measured the plasma delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, dehydroepiandrosterone (DHEA), progesterone, 17 alpha-hydroxyprogesterone, <em>androstenedione</em> (A), and testosterone (T) concentrations in three men with previously untreated metastatic prostate cancer at various time intervals for 2<em>4</em> h before and <em>4</em>8 h after the administration of 200 mg oral KTZ every 8 h. The adrenal glands of these three patients were suppressed (as measured by the plasma cortisol levels) by the administration of 1.0 mg dexamethasone daily for 7 days before and during the study. After six doses of KTZ, bilateral orchiectomy was performed, and the intratesticular concentration of the aforementioned seven steroids and the intratesticular activities of the 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase enzymes in the delta <em>4</em>-steroidogenic pathway were determined. These seven intratesticular steroids and three intratesticular enzyme activities were compared to those in five men with previously untreated prostate cancer who underwent orchiectomy as primary treatment for their disease. Plasma A, DHEA, and T all significantly decreased during KTZ therapy. There was no significant change in the other four steroids in the plasma. In the testis, delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, and delta <em>4</em>-17 alpha-hydroxyprogesterone were all significantly elevated, whereas intratesticular DHEA, A, and T were significantly decreased in the three KTZ-treated patients compared to levels in the five non-KTZ-treated patients. Measurement of the enzyme activities demonstrated a significant reduction in both 17 alpha-hydroxylase and 17,20-desmolase, but no change in 17 beta-hydroxysteroid dehydrogenase, in the KTZ-treated patients compared to the levels in the non-KTZ-treated patients. We conclude that oral KTZ decreases testicular T production by inhibiting the 17,20-desmolase and also the 17 alpha-hydroxylase steps in both the delta <em>4</em>- and delta 5-T biosynthetic pathways.

Flutamide is rapidly metabolized by hydroxylation of the side chain to SCH 16<em>4</em>23 (alpha, alpha, alpha-trifluoro-2-methyl-<em>4</em>'-nitro-m-lactotoluidide), the major metabolic product in all species studied, which is biologically active in vivo and in vitro studies. Flutamide exhibits its antiandrogenic activity by inhibiting androgen uptake and/or inhibition of nuclear binding of the androgens in the target tissues. At daily doses from 1 to 50 mg/kg body weight, flutamide reduced seminal vesicle and ventral prostate weights of intact male rats without affecting sexual potency. In addition, flutamide reduced the rate of DNA synthesis in the prostate of rats to a greater degree than other steroidal antiandrogens. The antiandrogenic activity was corroborated by the inhibition of androgen-induced prostate hypertrophy in orchiectomized rats through the use of testosterone, testosterone propionate, dihydrotestosterone, <em>androstenedione</em>, and dehydroepiandrosterone. Flutamide, given orally, reduced prostatic size in aged dogs with benign prostate hyperplasia after six weeks and one year. The baboon prostate was also reduced in size when flutamide was administered three times a week for four weeks.

BACKGROUND

Body weight influences fertility and studies in mice have indicated that leptin is one of the mediators of this effect. Leptin is believed to centrally stimulate the hypothalamic-pituitary axis resulting in increased gonadotropin release. Moreover, leptin is present in follicular fluid and the receptor is expressed in the human ovary. The aim of this study was to evaluate the direct effect of leptin on cultured human granulosa cell steroidogenesis.

METHODS

Granulosa cells were obtained in connection with IVF procedures, and then cultured in a serum-free medium containing <em>androstenedione</em> (1 microM) for a total of <em>4</em> days. After 2 days of culture the medium was changed and the hormones under study were added. We tested the effect of leptin (1, 20, 100 ng/ml) on basal, FSH (10-100 ng/ml), and FSH (10-100 ng/ml)+IGF-I (30 ng/ml) stimulated steroidogenesis.

RESULTS

Leptin (20 ng/ml and 100 ng/ml) significantly reduced basal and FSH-stimulated estradiol secretion (p<0.05). Basal and FSH (10 and 30 ng/ml) stimulated progesterone production was significantly inhibited by leptin 20 ng/ml, whereas leptin 100 ng/ml significantly reduced basal but not FSH stimulated progesterone production. Finally, steroidogenesis stimulated by IGF-I alone and in combination with FSH was not influenced by leptin.

CONCLUSIONS

These results suggest that leptin acts directly to inhibit basal and FSH stimulated estradiol and progesterone production in cultured human granulosa cells. This raises the possibility that high circulating leptin levels as seen in obese women may compromise fertility through peripheral mechanisms.

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A5<em>4</em>9, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A5<em>4</em>9 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A5<em>4</em>9 cells. When cultured in the presence of normal FCS, A5<em>4</em>9 intact cells converted DHEA to androstenediol, <em>androstenedione</em> principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A5<em>4</em>9 RNA indicated the presence of 17beta-HSD type <em>4</em> and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, <em>androstenedione</em> was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A5<em>4</em>9 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

OBJECTIVE

To confirm the results of an earlier study showing premenopausal equol excretors to have hormone profiles associated with reduced breast cancer risk, and to investigate whether equol excretion status and plasma hormone concentrations can be influenced by consumption of probiotics.

METHODS

A randomized, single-blinded, placebo-controlled, parallel-arm trial.

METHODS

In all, 3<em>4</em> of the initially enrolled 37 subjects completed all requirements.

METHODS

All subjects were followed for two full menstrual cycles and the first seven days of a third cycle. During menstrual cycle 1, plasma concentrations of estradiol (E(2)), estrone (E(1)), estrone-sulfate (E(1)-S), testosterone (T), <em>androstenedione</em> (A), dehydroepiandrosterone-sulfate (DHEA-S), and sex-hormone-binding globulin (SHBG) were measured on cycle day 2, 3, or <em>4</em>, and urinary equol measured on day 7 after a <em>4</em>-day soy challenge. Subjects then received either probiotic capsules (containing Lactobacillus acidophilus and Bifidobacterium longum) or placebo capsules through day 7 of menstrual cycle 3, at which time both the plasma hormone concentrations and the post-soy challenge urinary equol measurements were repeated.

RESULTS

During menstrual cycle 1, equol excretors and non-excretors were not significantly different with respect to subject characteristics, diet, or hormone concentrations. Significant inverse correlations were found between E(2) and body mass index (BMI) (P=0.02), SHBG and BMI (P=0.01), DHEA-S and dietary fiber (P=0.0<em>4</em>), and A and protein:carbohydrate ratio (P=0.02). Probiotic consumption failed to significantly alter equol excretor status or hormone concentrations during menstrual cycle 3, although there were trends towards decreased concentrations of T (P=0.1<em>4</em>) and SHBG (P=0.10) in the probiotic group.

CONCLUSIONS

We were unable to verify a previously reported finding of premenopausal equol excretors having plasma hormone concentrations different from those of nonexcretors. Furthermore, a 2-month intervention with probiotic capsules did not significantly alter equol excretion or plasma hormone concentrations.

Various age-related diseases increase in incidence during perimenopause. However, our understanding of the effects of aging compared with hormonal changes of perimenopause in mediating these disease risks is incomplete, in part due to the lack of an experimental perimenopause model. We therefore aimed to determine whether manipulation of the transition to ovarian failure in rats via the use of <em>4</em>-vinylcyclohexene diepoxide (VCD) could be used to model and accelerate hormonal changes characteristic of perimenopause. We examined long-term (11 to 20 mo), dose-dependent effects of VCD on reproductive function in 1- and 3-mo-old female Sprague-Dawley rats. Twenty-five daily doses of VCD (80 or 160 mg/kg daily compared with vehicle alone) depleted ovarian follicles in a dose-dependent fashion in rats of both ages, accelerated the onset of acyclicity, and caused dose-dependent increases in follicle-stimulating hormone that exceeded those naturally occurring with age in control rats but left serum levels of 17β-estradiol unchanged, with continued ovarian production of <em>androstenedione</em>. High-dose VCD caused considerable nonovarian toxicities in 3-mo-old Sprague-Dawley rats, making this an unsuitable model. In contrast, 1-mo-old rats had more robust dose-dependent increases in follicle-stimulating hormone without evidence of systemic toxicity in response to either VCD dose. Because perimenopause is characterized by an increase in follicle-stimulating hormone with continued secretion of ovarian steroids, VCD acceleration of an analogous hormonal milieu in 1-mo-old Sprague-Dawley rats may be useful for probing the hormonal effects of perimenopause on age-related disease risk.

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

Local regulation of ovulation involves the interaction of LH and intrafollicular factors including steroids, prostaglandins, and peptides derived from endothelial cells, leukocytes, fibroblasts, and steroidogenic cells. To estimate the intrafollicular role of endothelin-1 (ET-1) and its possible interaction with LH, tumor necrosis factor alpha (TNFalpha), and interleukin-1beta (IL-1beta), a microdialysis system was implanted into the theca layer of preovulatory bovine follicles that were maintained in organ culture chambers. The effects of LH, ET-1, TNFalpha, and IL-1beta on the local release of steroids, prostaglandin E2 (PGE2), and ET-1 from the cells surrounding the implanted capillary membrane were investigated. Each preovulatory follicle (selected based on the concentrations of steroids and PGE2) was dissected from surrounding stromal tissue and implanted with <em>4</em> capillary dialysis membranes (control, LH, cytokines or ET-1, and LH+cytokine or LH+ET-1) into the theca layer. They were then incubated in organ culture chambers and perfused with Ringer's solution for 1<em>4</em> h after pre-perfusion for 2 h. The stimulation with LH (5 microg/ml) between <em>4</em> and 6 h increased the release of progesterone (P<em>4</em>), <em>androstenedione</em> (A), estradiol-17beta (E2), PGE2 (p < 0.001), and ET-1 (p < 0.05). The infusion of ET-1 (250 ng/ml) between 8 and 10 h inhibited P<em>4</em> and A release but stimulated E2 release (p < 0.05). The infusion of TNFalpha (100 ng/ml) between 8 and 10 h after LH exposure suppressed the release of A and E2 (p < 0.05). IL-1beta (10 ng/ml) between 8 and 10 h stimulated E2 release but inhibited A release (p < 0.05). Moreover, ET-1 and cytokines clearly stimulated PGE2 release (p < 0.05). ET-1 and TNFalpha induced further release of PGE2 stimulated by LH (p < 0. 05). Also, TNFalpha and IL-1beta induced further release of ET-1 stimulated by LH (p < 0.05). These results show that ET-1 is released from the theca layer of mature bovine follicles in vitro and that it affects follicular steroids and PGE2 secretion. The overall results suggest that interactions among ET-1, PGE2, and cytokines may have key roles in a local intermediatory/amplifying system of the LH-triggered ovulatory cascade in the bovine preovulatory follicle.

The authors examined the effects of the immunosuppressive drug cyclosporine (CsA) on the male reproductive system in prepubertal rats. Twenty-one-day-old rats were subcutaneously injected with either cremaphorsaline vehicle or CsA (1 and 2 mg/kg/d). The animals were treated until they were 66 days old. Cyclosporine did not affect the weights of the body or testis but decreased the weights of all sex accessory organs. Quantitative analysis of the tubules in stage VII of spermatogenesis revealed a decline in the cell counts of pachytene spermatocytes and step VII spermatids. Testicular and epididymal sperm counts and motility were decreased by 50% and fertility by 60%. Cyclosporine lowered serum testosterone despite an elevation of LH, indicating that the drug directly inhibited testosterone synthesis. Serum creatinine levels were normal in the treated animals, precluding renal failure as the cause for this impairment. Intratesticular concentrations of pregnenolone and 17-hydroxy progesterone were significantly elevated, while those of progesterone, <em>androstenedione</em>, and testosterone were markedly reduced. Determination of steroidogenic enzyme activities indicated that the administration of CsA inhibited the activity of delta 5-3B-hydroxy steroid dehydrogenase-delta 5-<em>4</em> isomerase (3 beta-HSD). These results clearly indicate that CsA in the doses used is harmful to the male reproductive function in prepubertal rats.

BACKGROUND

There is considerable controversy as to how long the beneficial effects of laparoscopic ovarian drilling (LOD) last. This follow-up study was undertaken to investigate the long-term effects of LOD.

METHODS

The study included 116 anovulatory women with polycystic ovary syndrome (PCOS) who underwent LOD between 1991 and 1999 (study group) and 3<em>4</em> anovulatory PCOS women diagnosed during the same period, who had not undergone LOD (comparison group). The hospital records were reviewed and most patients attended for a transvaginal ultrasound scan and blood sampling to measure the serum concentrations of LH, FSH, testosterone, <em>androstenedione</em> and sex hormone-binding globulin. The results before and at different intervals, short- (<1 year), medium- (1-3 years) and long-term (<em>4</em>-9 years), after LOD were compared.

RESULTS

The LH:FSH ratio, mean serum concentrations of LH and testosterone and free androgen index decreased significantly after LOD and remained low during the medium- and long-term follow-up periods. The mean ovarian volume decreased significantly (P < 0.05) from 11 ml before LOD to 8.5 ml at medium-term and remained low (8.<em>4</em> ml) at long-term follow-up.

CONCLUSIONS

The beneficial endocrinological and morphological effects of LOD appear to be sustained for up to 9 years in most patients with PCOS.

The narcotic agent etomidate and the antimycotic drug ketoconazole are known to block steroid biosynthesis in man. To study the different effects of these imidazole derivatives on human adrenal steroid biosynthesis we incubated slices of human adrenal glands with 3H-labeled precursors and increasing concentrations of etomidate or ketoconazole (0-2000 microM). After extraction the labeled metabolites were separated by thin-layer chromatography and quantified by scintillation counting. Etomidate inhibited most potently 11 beta-hydroxylase activity by suppressing the formation of corticosterone from 11-deoxycorticosterone to 1% of control [50% inhibitory concentration (IC50) 0.03 microM] while ketoconazole suppressed 11 beta-hydroxylase to only 39% of control activity (IC50 15 microM). Ketoconazole however, most potently blocked the conversion of 17 alpha-hydroxy-progesterone to <em>androstenedione</em> by C17,20-desmolase to about 15% of control activity (IC50 1 microM) while etomidate showed a much weaker effect on this enzyme with a suppression to 50% of C17,20-desmolase control activity at a concentration of 380 microM. Both imidazole drugs showed a similar strong inhibitory effect on the activity of 17 alpha-hydroxylase (IC50 6-18 microM) and 16 alpha-hydroxylase (IC50 <em>4</em>-8 microM) and did not affect 21-hydroxylase. These in vitro data indicate a predominant inhibitory effect of etomidate on corticosteroid biosynthesis by relative selective inhibition of 11 beta-hydroxylase and of ketoconazole on the adrenal androgen biosynthesis by a predominant inhibition of C17,20-desmolase. This differential inhibitory effect of etomidate and ketoconazole on human steroid biosynthesis may be of clinical importance for a possible therapeutic use of these imidazole derivatives in endocrine disorders.

OBJECTIVE

We determined hormone concentrations (estradiol [E2], estrone [E1], estrone conjugates [E1-C], androstenedione [A], testosterone [T]) before and on anastrozole therapy where we also determined plasma concentrations of anastrozole and its metabolites.

METHODS

Postmenopausal women who were to receive adjuvant anastrozole for resected early breast cancer were studied. Pretreatment, blood samples were obtained for the acquisition of DNA and for plasma hormone measurements (E2, E1, E1-C, A, and T). A second blood draw was obtained at least 4 weeks after starting anastrozole for hormone, anastrozole and metabolite measurements. For hormone assays, a validated bioanalytical method using gas chromatography negative ionization tandem mass spectrometry was used. Anastrozole and metabolite assays involved extraction of plasma followed by LC/MS/MS assays.

RESULTS

649 patients were evaluable. Pretreatment and during anastrozole, there was large inter-individual variability in E2, E1, and E1-C as well as anastrozole and anastrozole metabolite concentrations. E2 and E1 concentrations were below the lower limits of quantitation in 79% and 70%, respectively, of patients on anastrozole therapy, but those with reliable concentrations had a broad range (0.627-234.0 pg/mL, 1.562-183.2 pg/mL, respectively). Considering E2, 8.9% had the same or higher concentration relative to baseline while on anastrozole, documented by the presence of drug.

CONCLUSIONS

We demonstrated large inter-individual variability in anastrozole and anastrozole metabolite concentrations as well as E1, E2, E1-C, A, and T concentrations before and while on anastrozole. These findings suggest that the standard 1mg daily dose of anastrozole is not optimal for a substantial proportion of women with breast cancer.

Massively obese males often show symptoms of hypogonadism, but the mechanism for this is unclear. Increased endogenous opioid inhibition of the hypothalamic GnRH pulse generator resulting in insufficient stimulation of the pituitary gonadotroph has been proposed as a possible mechanism. If this hypothesis is correct, obese males should be more sensitive to the LH-elevating effects of the opiate antagonist, naloxone, than men of normal weight and gonadal status. This study investigated the etiology of obesity-related hypogonadism by examining luteinizing hormone (LH) and follicle stimulating hormone (FSH) responses to gonadotropin-releasing hormone (GnRH) and to infusions of saline or naloxone. Subjects were five obese (201 +/- 1<em>4</em>% IBW) and five normal weight (control) (97 +/- <em>4</em>% IBW) males. Before treatment, obese males had significantly (p < 0.05) lower testosterone levels than control subjects (307 +/- 72 vs. 597 +/- <em>4</em>9 ng/dl), whereas estradiol, <em>androstenedione</em>, and dehydroepiandrosterone levels were not different between the two groups. Both groups showed equivalent elevations in LH (fourfold to sixfold) in response to GnRH stimulation, but obese patients had significantly lower basal (p < 0.05) and GnRH-stimulated (p < 0.01) FSH levels. Infusions of naloxone (but not saline) led to significant (p < 0.01) increases in LH above preinfusion baseline levels (20.5 +/- 2.8% in obese and 28.6 +/- 6.3% in controls). In control subjects, integrated LH levels during naloxone infusion were not significantly elevated above those found during saline infusion, while obese subjects exhibited a <em>4</em>3% augmentation of integrated LH (31.0 +/- 5.3 ng/ml during naloxone vs. 21.7 +/- 1.8 ng/ml during saline, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

During the transition from the follicular to the luteal phase, follicular steroid secretion shifts from predominantly 17 beta-estradiol and androgen production before the LH surge to decreased androgen and estrogen and increased progesterone production after the LH surge. Our objective was to determine if changes in 17 beta-estradiol and androgen production by the preovulatory follicle are effected via changes in mRNA levels for the steroidogenic enzymes cytochrome P<em>4</em>50 17 alpha-hydroxylase/C17, C20-lyase (P<em>4</em>50-17 alpha) and cytochrome P<em>4</em>50 aromatase (P<em>4</em>50arom). Heifers were injected with prostaglandin F2 alpha (PGF2 alpha) during the luteal phase to initiate luteolysis and a follicular phase. Preovulatory follicles were obtained at two times before the LH surge, in the early follicular phase (2<em>4</em> h after PGF2 alpha injection) and in the midfollicular phase (<em>4</em>8 h after PGF2 alpha), and at one time in the late follicular phase, after the LH surge but before ovulation (20 h after the onset of estrus, about 20 h after the LH surge). Granulosa cells and theca interna were isolated from preovulatory follicles obtained during the early follicular phase and cultured with or without ovine LH (100 ng/ml; n = <em>4</em> follicles isolated 2<em>4</em> h after PGF2 alpha). Total RNA extracted from granulosa cells and theca interna, either immediately after cell isolation during the early (n = 5), mid (n = <em>4</em>)-, or late follicular phase (n = 5) or after 2<em>4</em> or 72 h of culture, was subjected to Northern analysis for P<em>4</em>50-17 alpha and P<em>4</em>50arom mRNA. Autoradiographs were scanned densitometrically, and values were adjusted for loading and transfer efficiency. P<em>4</em>50-17 alpha mRNA was highly abundant in theca interna before the LH surge, but decreased by 96% after the LH surge (P < 0.001). In contrast, P<em>4</em>50-17 alpha mRNA in the granulosa cell fraction was very low at all three times of follicle isolation and most likely was due to a slight contamination of the granulosa cell preparation with theca interna. P<em>4</em>50arom mRNA was abundant in granulosa cells isolated in the early and midfollicular phase, but decreased by 9<em>4</em>% after the LH surge (P < 0.001). In contrast, P<em>4</em>50arom mRNA was undetectable in theca interna. Levels of <em>androstenedione</em> and 17 beta-estradiol in follicular fluid were high during the early and midfollicular phase and decreased dramatically after the LH surge (P < 0.05 and P < 0.001, respectively). P<em>4</em>50-17 alpha and P<em>4</em>50arom mRNA were undetectable after 2<em>4</em> h of culture in the presence or absence of LH and thereafter.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

It is not possible to differentiate reliably between male idiopathic hypothalamic hypogonadism (HH) and severe constitutional delay of puberty (CD) on the basis of a standard GnRH bolus test (GBT) or other known endocrine or clinical parameters. Therefore, we studied the response of 17 hypogonadal men, 8 with a diagnosis of HH (age, 15.5-<em>4</em>1; bone age, 12.5-19 yr; testes, 1-<em>4</em> ml) and 9 with CD (age, 1<em>4</em>.5-20; bone age, 11-15 yr, testes, 2-10 ml) to pulsatile GnRH stimulation. Basal and peak LH and FSH levels after a single dose of GnRH greatly overlapped between the two groups. In each patient, a spontaneous nocturnal plasma profile of LH and FSH, sampled every 20 min, was followed by a pulsatile GnRH stimulation (5 micrograms iv every 90 min) via a portable minipump for 36 h. Before and after this pulsatile GnRH stimulation, a GBT (60 micrograms/m2 iv) was performed and plasma LH, FSH, testosterone, <em>androstenedione</em>, and dehydroepiandrosterone sulfate were measured. Pulse analysis revealed 0-5 spontaneous nocturnal LH peaks in the CD patients but only one in all of the HH patients. During the 36 h of pulsatile GnRH, mean LH and FSH levels were significantly higher (P less than 0.0001) than during the spontaneous nocturnal profile in all patients (except 1 from each group for LH). The GBT after pulsatile stimulation caused significantly higher (P less than 0.001) LH increments in CD than in HH patients, with no overlap between the two groups (range, <em>4</em>.1-15.6 in CD vs. 0.8-2.<em>4</em> mIU/ml in HH). Plasma testosterone rose significantly (P less than 0.01) during pulsatile GnRH from 67 to 155 ng/dl (median) in the CD men, but did not change in the HH group (21 to 22.5 ng/dl). Plasma <em>androstenedione</em> and dehydroepiandrosterone sulfate did not rise in either group. We conclude that, in contrast to other parameters investigated so far, the LH increment in the second GBT after 36 h of pulsatile GnRH allows clear-cut differentiation between CD and HH. These results indicate significantly lower pituitary LH reserve in patients with permanent HH after short term priming of the pituitary by pulsatile GnRH administration.

Changes in the serum levels of gonadotrophins and steroid hormones with increasing age were studied in <em>4</em><em>4</em>9 women aged <em>4</em>0 and over to investigate the relationships between these hormones even very late in life. The levels of oestradiol (E2) and dehydroepiandrosterone sulphate (DHEA-S) fell after age 50 and remained low thereafter. However, while serum oestrone (E1), testosterone (T), delta-<em>4</em>-<em>androstenedione</em> (A) and prolactin (PRL) concentrations also decreased initially after age 50 they subsequently rose again progressively and this increase was in fact significant in the case of E1. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) rose after age 50, but whereas FSH remained elevated, LH decreased late in life. Cortisol (F) increased significantly after age 70. There was a significant correlation between androgens and E1 as well as between E2 and LH, even after age 60. Owing to the great heterogeneity of the population studied, it is not yet possible to speculate as to the physiopathological significance of these observations. It would seem, however, that the negative feedback effect of oestrogens on LH secretion remains operational very late in life.

The effects of PCBs (mixture of 2, 3, <em>4</em>, 5-tetra; 2, 2', <em>4</em>, 5, 5'-penta; 2, 2', 3, 3', 6, 6'-hexa and 2, 2', 3, 3', <em>4</em>, <em>4</em>', 5, 5'-octa congeners) on androgen production were investigated by suspension of Leydig cells from adult rat testis. hCG-stimulated androgen production was significantly inhibited by PCBs while progesterone level was not affected. Progesterone supported testosterone production was also decreased by PCBs, while conversion of <em>androstenedione</em> to testosterone was unchanged. These results suggest that the activity of microsomal enzyme C21 side-chain cleavage P<em>4</em>50 was decreased by PCB treatment of Leydig cells in vitro.

The basal plasma levels of cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), and testosterone were studied in 20 patients with advanced prostatic cancer receiving combined treatment with an LHRH agonist and an antiandrogen [5,5-dimethyl-3-[<em>4</em>-nitro-3-(trifluoromethyl)-phenyl]-2 <em>4</em>-imidazolidinedione]. After 60 days of combined antihormonal therapy, plasma levels of testosterone decreased from 5.<em>4</em><em>4</em> +/- 0.<em>4</em><em>4</em> (SEM) to 0.136 +/- 0.052 ng/ml (2.5% of control). Somewhat unexpectedly, the plasma concentrations of the adrenal androgens DHEA and DHEA-S were reduced to <em>4</em>5 +/- 7 and 6<em>4</em> +/- <em>4</em>% of control, respectively. The maximal reduction in plasma adrenal androgen levels occurred between 2 and <em>4</em> weeks of treatment. Whereas the increase in serum cortisol, 17-hydroxypregnenolone, and 17-hydroxyprogesterone concentrations 2 1/2 h after the injection of 0.25 mg human ACTH 1-2<em>4</em> was not affected by the combined treatment, the increment of DHEA and <em>androstenedione</em> after the same stimulus was reduced from 3.1 +/- 0.98 and 0.73 +/- 0.11 to 1.<em>4</em>8 +/- 0.5 and 0.31 +/- 0.05 ng/ml, respectively. The reduced levels of serum DHEA and DHEA-S were not due to the LHRH agonist by itself, since similarly low levels of serum DHEA and DHEA-S were found in patients surgically castrated and receiving the same antiandrogen. These data suggest that treatment with an antiandrogen in castrated men inhibit the formation of adrenal androgens due to a blockade at the level of 17, 20-desmolase. The efficiency of the new combined antihormonal therapy (castration and antiandrogen) aimed at complete androgen neutralization in prostate cancer is thus further facilitated.