Steroids are secreted by the gonads and adrenal glands into the blood to modulate neurophysiology and behaviour. In addition, the brain can metabolise circulating steroids and synthesise steroids de novo. Songbirds show high levels of neurosteroid synthesis. In the present study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the measurement of 10 steroids in whole blood, plasma and microdissected brain tissue (1-2 mg) of song sparrows. Our assay is highly accurate, precise, specific and sensitive. Moreover, the liquid-liquid extraction is fast, simple and effective. We quantified steroids in the blood and brain of wild male song sparrows in both breeding and non-breeding seasons. As expected, systemic androgen levels were higher in the breeding season than in the non-breeding season. Brain androgens were detectable only in the breeding season; androstenedione and 5α-dihydrotestosterone levels were up to 20-fold higher in specific brain regions than in blood. Oestrogens were not detectable in blood in both seasons. Oestrone and 17β-oestradiol were detectable in brain in the breeding season only (up to 1.4 ng g^-1 combined). Progesterone levels in several regions were higher in the non-breeding season than the breeding season, despite the lack of seasonal changes in systemic progesterone. Corticosterone levels in the blood were higher in the breeding season than in the non-breeding season but showed few seasonal differences in the brain. In general, the steroid levels presented here are lower than those in previous reports using immunoassays, because of the higher specificity of mass spectrometry. We conclude that (i) brain steroid levels can differ greatly from circulating steroid levels and (ii) brain steroid levels show region-specific seasonal patterns that are not a simple reflection of circulating steroid levels. This approach using ultrasensitive LC-MS/MS is broadly applicable to other species and allows steroid profiling in microdissected brain regions.

Keywords: aggression; aromatase; dehydroepiandrosterone; social behaviour network; stress; testosterone.

The aim was to study the effects of flutamide on cell proliferation, in vivo tumour growth and steroid production in canine and human IBC cell lines. IPC-366 and SUM149 cell cultures were exposed to flutamide concentrations for 72 hours. Additionally, IPC-366 and SUM149 xenotransplanted mice were treated subcutaneously with flutamide 3 times a week for 2 weeks. Steroid hormones determination in culture media, serum and tumour homogenates (pregnenolone, progesterone, androstenedione, testosterone, dihydrotestosterone, 17β-oestradiol and oestrone sulphate) were assayed by EIA. in vitro cell proliferation percentages showed a decrease in all flutamide dosages in IPC-366 and SUM149. in vivo flutamide reduced tumour size by 55% to 65%, and metastasis rates decreased. In treated groups, androgen levels in culture media, serum and tumour homogenates were increased as oestrogen levels decreased. These results suggest that flutamide treatment inhibits cell proliferation and promotes tumour reduction by increasing androgen levels and also support future therapy approaches.

The gonads of the fetal horse were found to be relatively devoid of 3 beta-hydroxysteroid dehydrogenase and other enzymes which metabolize dehydroepiandrosterone (DHA). In short-term in-vitro incubation experiments fetal liver converted DHA to the potential equilin precursor, 7 alpha-hydroxy DHA. DHA was converted to oestrone when incubated with extracts of horse placenta but 7 alpha-hydroxy DHA was not converted to equilin. Levels of DHA measured in peripheral blood of mares throughout pregnancy paralleled those of equilin and oestrone, and DHA concentrations fell rapidly after fetal gonadectomy, as did those of equilin and oestrone. Administration of [4-(14)C]-DHA and [7-3H]DHA to the fetus resulted in the incorporation of both 14C and 3H into maternal urinary oestrone but neither isotope was present in urinary equilin. These findings confirm the role of fetal DHA as a precursor of oestrone, but do not support the previously suggested role of the fetal liver in the synthesis of equilin. They are, however, compatible with the hypothesis that equilin is formed via a pathway which diverges from the terpenoid steroid synthetic route before DHA.

1. The sulphation of polyethyleneglycol 200 by the isolated perfused guinea pig liver is inhibited to about 60% by 10 mM ClO3- in the plasma of the perfusate when the concentration of SO4(2-) therein is 1.18 mM. 2. The inhibition is almost complete when the concentration of SO4(2-) is about 0.1 mM, a level which can be achieved by using a modified Ringer-bicarbonate solution, devoid of sulphate, to prepare the perfusate. 3. Chlorate, presumably through its action on ATP-sulphurylase, may therefore be a useful inhibitor of sulphation in the isolated perfused liver when the activity of the sulphurylase is rate-limiting. 4. The rate of bile production in the presence of chlorate is no different from that in its absence showing that, in the time scale of the perfusion, chlorate is not a general liver poison. 5. When the synthesis of PAPS is not rate-limiting, as in the sulphation of oestrone metabolites by rat liver, chlorate has no effect on the rate of sulphation.

Prepubertally castrated boars received subcutaneous injections twice weekly, from 13 to 35 weeks of age, of dehydroepiandrosterone (2 mg/5 kg) or oestrone (1 mg/5 kg). Dehydroepiandrosterone did not support the growth and secretory activity of the accessory organs, or induce copulatory behaviour. However, oestrone caused hypertrophy of the prostate, seminal vesicles and bulbourethral glands which was due to an increase in fibrous stromal tissue and not to the secretory epithelium. Oestrone also induced some male mating behaviour patterns in the presence of an oestrous gilt, although penile extrusion and ejaculation did not occur. The morphological and behavioural effects of the steroid treatments were supported by steroid profiles in blood plasma as seen in comparison with androgen and oestrogen values of intact and untreated castrated boars. It is concluded that oestrogen in the intact boar might enhance the secretion of the accessory organs by affecting the neural control of the secretory processes rather than by increasing the amount of secretory epithelium in the glands.

OBJECTIVE

To investigate possible associations between smoking habits and other coronary risk factors in postmenopausal women with known coronary heart disease (CHD).

METHODS

The study was conducted at a university clinic.

METHODS

A total of 118 postmenopausal women with CHD verified with angiography, consecutively recruited.

METHODS

Conventional treatment for CHD. The women were randomized to hormone replacement therapy (HRT) with transdermal 17-beta oestradiol and medroxyprogesterone acetate, or to a control group.

RESULTS

Smokers were younger (P = 0.005), had lower body mass index (P = 0.04) and lipoprotein Lp(a) levels (P = 0.02) compared with nonsmokers. Smokers had reduced beta-cell function (homeostasis model assessment, P = 0.006), whereas whole blood viscosity (WBV) was higher at all shear rates. WBV was not affected by HRT over a 12-month period. Oestrone levels were higher in smokers.

CONCLUSIONS

Smoking adversely affects insulin secretion (beta-cell function) and WBV in postmenopausal women with established CHD, which could be of importance as a mechanism for the increased risk of CHD in smokers. The importance of smoking as a risk factor, overrides the effect of Lp(a), which is lower in smokers compared with nonsmokers.

Four patients with untreated congenital virilizing adrenal hyperplasia (partial 21-hydroxylase deficiency) were studied by bilateral adrenal vein catheterization. Simultaneous right and left adrenal and peripheral blood samples were collected for determination of oestrone (E1) and oestradiol (E2). The concentrations of both were higher in the adrenal effluents than in the peripheral blood samples, indicating their secretion by the adrenals. All patients were also studied during a sequential test of suppression (0.5 h after i.v. administration of 4 mg dexamethasone) and stimulation (5 min after i.v. administration of 250 microgram ACTH 1-24; Synacthen). Mean peripheral E2 concentrations did not change significantly whereas E1 increased above control levels after stimulation. In contrast, suppression of adrenal venous blood concentrations with dexamethasone, and stimulation with ACTH, was demonstrated for every patient. The results indicate that in congenital adrenal hyperplasia the adrenal glands secrete significant amounts of E1 and E2.

OBJECTIVE

To compare the effects of two doses of piperazine oestrone sulphate combined with interrupted norethisterone, with that of oestradiol continuously combined with norethisterone acetate, and with placebo, in postmenopausal women.

METHODS

A prospective randomised trial.

METHODS

Two hundred postmenopausal women.

METHODS

Monocentre study with expertise in osteoporosis.

METHODS

The participants were randomly assigned to two years of treatment with alternating three-day cycles of 1.5 mg of piperazine oestrone sulphate plus 0.7 mg of norethisterone (highEP), or alternating three-day cycles of 0.75 mg of piperaine oestrone sulphate plus 0.35 mg of norethisterone (lowEP), or 2 mg of 17beta-oestradiol continuously combined with 1 mg of norethisterone acetate (E2+NETA), or placebo.

METHODS

Change in bone mineral density, lipoprotein metabolism, climacteric symptoms, and adverse effects.

RESULTS

One hundred and twenty-one women completed the study. Spinal bone mineral density was increased about 9% over two years by E2+NETA, about 6% by highEP, 4% by lowEP, but remained unchanged in the placebo group. The same pattern was seen in the hip and forearm. All hormone regimens decreased markers of bone turnover and alleviated climacteric symptoms. Serum lipoproteins decreased by about 10% in all hormone groups.

CONCLUSIONS

All hormone regimens studied prevented bone loss completely and lowered serum lipids.

The plasma oestrone and oestradiol levels of 34 postmenopausal women were studied and related to various of their other characteristics. The plasma oestrone and oestradiol-17beta levels (Mean +/- SE) were, respectively, 32.09 +/- 4.6 pg/ml and 13.9 +/- 3.1 pg/ml. There was a highly significant negative linear association between the years elapsed since the menopause and oestrone levels (P less than 0.001) and oestradiol levels (P less than 0.02). Both plasma oestrone and oestradiol levels were directly related to weight, but the relation was only significant for oestradiol. A significant positive correlation was found between vaginal bleeding and excess weight. The degree of oestrogenicity of the vaginal smear was directly related to the levels of oestrone (P less than 0.08) and oestradiol (P less than 0.005) and to the degree of endometrial hyperplasia (P less than 0.02).

Urinary incontinence was treated conservatively in 100 patients. The follow-up period was 12-24 mth. For post-menopausal women, the oestrogen therapy consisted of oral oestradiol valerate or vaginal oestrone sulphate combined with emepronium bromide. In post-menopausal patients the best results were noted when incontinence had begun at the menopause and when the duration of the complaint was not more than 3 yr. For pre-menopausal patients, the treatment given was generally emepronium bromide. During the follow-up period 15 of the patients, 11 of whom were post-menopausal, became symptomless and 77 improved; that is, the incontinence was only slight and occasional. The treatment was without any effect in 8 of the patients. Oestrogen therapy was successful in most post-menopausal women. In these patients, the best results were obtained when the duration of the incontinence was not more than 3 yr.

Serum concentrations of endogenously produced oestrogens were studied in 128 postmenopausal women with endometrial carcinoma. Radioimmunoassays of serum oestrone (E1) and oestradiol (E2) were performed on admission to hospital. Results showed a wide variation in serum concentrations of E1 and E2 in patients with endometrial carcinoma. Some patients had high E1 values and low E2, while none of the patients had low E1 and high E2 values. Hormonal levels were correlated with increasing BMI (E1; p less than 0.000001; E2; p less than 0.000001). Uterine cavity depth was also related to both E1 and E2 (E1; p less than 0.00001; E2; p less than 0.0003). Concentrations of E1 and E2 were higher in diabetic women than in the non-diabetic. (E1; p less than 0.002; E2; p less than 0.01). E2 concentration was higher in hypertensive patients than in the non-hypertensive patients (p less than 0.04). Parameters such as age, menstrual history, clinical stage and histopathology showed no significant correlation with hormone concentrations.

The plasma concentrations for unconjugated and conjugated oestrone, oestradiol-17 beta and oestriol, were measured in 25 male patients with chronic alcoholism and 25 healthy male blood donors of the same age. All persons had normal weight and were free of medication. The patients with chronic alcoholism consisted of 15 persons with and 10 without hepatomegaly. Liver biopsy was performed in the persons with hepatomegaly. Unconjugated oestrogens were separated on LH-20 micro columns after extraction with diethyl ether. Oestrogen sulphates were extracted and separated after hydrolysis with sulphatase, and glucuronides were extracted and separated after hydrolysis with glucuronidase. In the patients with hepatomegaly the plasma concentrations of unconjugated oestrone, oestradiol-17 beta and oestriol were significantly increased, and the plasma levels for the same oestrogen sulphates were significantly decreased, whereas a moderate increase was observed for oestriol glucuronide. The patients without hepatomegaly revealed a significantly lower plasma concentration for oestrone sulphate, whereas no difference was seen for the remaining oestrogen components. The ratio oestrone sulphate to oestrone was significantly reduced in both groups of patients. A significant decrease for the ratio oestradiol sulphate to oestradiol and oestriol sulphate to oestriol was seen only in the patients with hepatomegaly.

Ovaries were obtained from tammar wallabies at various stages of the reproductive cycle to examine the occurrence of oestrogens in corpora lutea, and the synthesis and metabolism of steroids in the corpus luteum and ovarian cortical and interstitial tissues. Corpora lutea contained oestradiol-17 beta and oestrone during embryonic diapause and at all stages of pregnancy studied after blastocyst activation. Aryl sulphatase, 3 beta-hydroxysteroid dehydrogenase and 17 beta-oxidoreductase were shown to be present in luteal and other ovarian tissues by incubation in vitro with labelled substrates. Aromatase was undetectable in corpora lutea or in interstitial tissue, but was present in the ovarian tissues (including follicles) which remained after removal of corpora lutea. The probable source of the oestrogens detected in the corpus luteum is discussed in relation to their role in the inhibition of follicular development during embyronic diapause.

It is controversial whether ovarian epithelial carcinoma possesses steroidogenic enzymes. We investigated aromatase expression in ovarian epithelial carcinoma, and compared it with the normal ovary and placenta. Samples were obtained from an ovarian carcinoma cell line SK-OV-3, ovarian tumour tissues from four patients with epithelial carcinoma and one patient with dysgerminoma. Aromatase enzymatic activity was measured in microsome fractions by quantitating 3H2O released from [1-3H] androstenedione and [3H]oestrone converted from [1,2,6,7-3H] androstenedione. Aromatase messenger ribonucleic acid (mRNA) was determined by reverse transcription-polymerase chain reaction (RT-PCR) using oligonucleotide primers synthesized according to the published human aromatase gene sequence. No aromatase activity was detected in either of two mucinous cystadenocarcinoma specimens or in SK-OV-3 cells, while aromatization proceeded with apparent Michaelis-Menten kinetics in the normal ovaries and placentas. The apparent Km value was 200 nmol/L for the ovary. Aromatase mRNA was detected in dysgerminoma, and the normal ovary and placenta, but not in any of three mucinous cystadenocarcinoma specimens, one serous cystadenocarcinoma specimen and SK-OV-3 cells. These results for both enzyme activity and gene expression suggest that the human ovarian epithelial carcinoma lacks aromatase. The demonstration of absence of aromatase gene expression raises the possibility that aromatase activity in ovaries bearing epithelial carcinoma may be associated with hyperplastic stromal rather than tumour cells.

OBJECTIVE

The established link between oestrogen and breast cancer occurs via both oestrogen receptor (ER)-mediated and non ER-mediated mechanisms. The term genotoxic estrogens describes mutagenic metabolites, including oestrogen catechols and quinones, which have been linked to breast carcinogenesis in post-menopausal women. We aimed to assess whether the route of administration of 17β oestradiol (E2 ) affects the accumulation of genotoxic oestrogen metabolites in a model of ovarian failure in young girls with Turner syndrome.

METHODS

Stored plasma samples obtained at 0 and 12 months were used from 40 adolescents with Turner syndrome who participated in a 12 months randomized controlled trial of the metabolic impact of E2 orally (2 mg/d) vs transdermally (100 µg/d); dose escalation allowed matching of unconjugated E2 levels in the parent study. We measured 12 oestrogen metabolites (total concentrations = conjugated and unconjugated) using a highly sensitive LCMSMS assay. Results from 48 normally menstruating adolescents were used for comparison.

RESULTS

After treatment, least square mean (SE) total E2 concentrations were higher in the oral vs transdermal group (6784 pmol/L vs 1123 [1614], P < 0.0001), as was oestrone (E1 ) (91 060 pmol/L vs 19 278 [16 534], P < 0.0001). Also, higher after oral treatment were catechol-oestrogens 4-hydroxy-E2 (149 vs 28 [±49] pmol/L), 2-hydroxy-E2 (300 vs 76 [±52]), 4-hydroxy-E1 (450 vs 105 [±113]), 2-hydroxy-E1 (3094 vs 740 [±684]) and 16α-hydroxy-E1 (3,007 vs 157 [±534]) (<0.001 between groups). Levels were much closer to controls in the transdermal group.

CONCLUSIONS

Common feminizing doses of oral oestradiol for 12 months result in substantial accumulation of unphysiologic, genotoxic oestrogens compared to transdermal oestradiol, expanding concerns about oral oestrogens' first hepatic passage. Further studies assessing long-term risks of these metabolites in women taking different forms of oestrogen are needed.

In the catfish Heteropneustes fossilis and Clarias batrachus, ovarian oestrogen-2-hydroxylase (OE-2-H) activity increased significantly at 8 h after the injection of an ovulatory dose (0.15 microg/g body weight) of a mammalian GnRH analogue ([d -Ala(6)-Pro(9)]-LHRH ethylamide) and was restored to the 0 h (control) level after egg-stripping at 16 h. On the other hand, ovarian oestradiol-17beta (OE2) level and catechol-O-methyltransferase (COMT) activity decreased significantly at 8 h. While the OE2 level was restored to the 0 h level, COMT activity increased significantly at 16 h. Changes in ovarian OE2 level and enzymes indicate higher synthesis of 2-hydroxylated catecholoestrogens and their degradation during the periovulatory period. Under in vitro conditions, the synthetic catecholoestrogens (CEs, 2- and 4-hydroxylated oestradiol17beta and oestrone (OE1)) induced germinal vesicle break down (GVBD) in a dose- (0.01-10 microg/ml) and duration- (1-36 h) dependent manner, the mean values of the responses being in the order 2-OH OE2>4-OH OE2> 2-OH OE1>4-OH OE1. The CE-induced GVBD response (8 h induction) was not blocked by prior and subsequent incubations with steroid synthesis inhibitors (cyanoketone, epostane and aminoglutethimide) up to 36 h, suggesting that de novo steroidogenesis is not essential for the response. The percentage of GVBD response to 2-h induction by CEs was significantly inhibited by actinomycin D (a transcriptional inhibitor) and cycloheximide (a translational inhibitor), indicating the involvement of both RNA and protein synthesis. The CE-induced 8-h stimulation of GVBD was mildly blocked by propranolol, the beta-adrenergic inhibitor, suggesting the response was partly mediated through a beta-adrenergic receptor mechanism. Incubations with phentolamine, an alpha-adrenergic inhibitor, did not interfere with the CE-induced GVBD response. The results demonstrate CE-related enzymatic changes in teleost (catfish) ovaries and maturation-inducing substance activity of CEs.

We have examined the effect of the catechol oestrogens 2-hydroxyoestradiol (2-OHE2), 4-hydroxyoestradiol (4-OHE2) and 2-hydroxyoestrone (2-OHE1) and their corresponding primary oestrogens on secretion of LH and FSH by enzymatically dispersed rat anterior pituitary cells in monolayer culture. Basal LH levels in the medium were significantly higher than in control wells when cells were exposed to 10(-8) M-oestradiol-17 beta for 40 h: oestrone and all three catechol oestrogens (in the same doses) also stimulated basal LH concentrations to levels quantitatively similar to those seem after oestradiol treatment. The same effects were observed when steroids were given at 10(-9) mol/l. Oestradiol, 2-OHE2, and 4-OHE2 but not 2-OHE1 increased pituitary responsiveness to LH releasing hormone (LH-RH) (given in a range of doses from 10(-11) to 10(-6) mol/l). The responses of cells treated with 2-OHE2 and 4-OHE2 were similar, though less than the response seen after treatment with oestradiol. This contrasts with the very different oestrogenic effects of 2- and 4-OHE2 previously observed in vivo. Neither oestradiol nor the catechol oestrogens had any effect on basal or LH-RH-stimulated FSH release.

Metabolic disorder is a major health problem and is associated with a number of metabolic diseases. Due to native hyperglycaemia and resistance to exogenous insulin, chickens as a model had used in the studies of adipose tissue biology, metabolism and obesity. But no detailed information is available about the comprehensive changes of serum metabolites at different stages of chicken embryonic development. This study employed LC/MS-QTOF to determine the changes of major functional metabolites at incubation day 14 (E14d), 19 (E19d) and hatching day 1 (H1d), and the associated pathways of differential metabolites during chicken embryonic development were analysed using Metabolite Set Enrichment Analysis method. Results showed that 39 metabolites were significantly changed from E14d to E19d and 68 metabolites were significantly altered from E19d to H1d in chicken embryos. Protein synthesis was promoted by increasing the concentrations of L-glutamine and threonine, and gonadal development was promoted through increasing oestrone content from E14d to E19d in chicken embryos, which indicated that serum glutamine, threonine and oestrone contents may be considered as the candidate indicators for assessment of early embryonic development. 2-oxoglutaric acid mainly contributed to enhancing the citric cycle, and it plays an important role in improving the growth of chicken embryos at the late development; the decreasing of L-glutamine, L-isoleucine and L-leucine contents from E19d to H1d in chicken embryonic development implied their possible functions as the feed additive during early posthatch period of broiler chickens to satisfy the growth. These results provided insights into understand the roles of serum metabolites at different developmental stages of chicken embryos, it also provides available information for chicken as a model to study metabolic disease or human obesity.

This paper reviews the equine granulosa cell tumour (GCT) and describes the clinicopathological features, treatment and outcome in seven cases of GCT in mares. Mares were presented with unilateral ovarian enlargement during the 2007 to 2010 breeding seasons. The mean (sd) age of the mares was 11.7 (5.96) years. Three mares were multiparous barren, three were nulliparous and one was primigravida. Behaviour at presentation was 57 per cent anoestrus, 28 per cent with stallion-like behaviour and 14 per cent with persistent oestrus. All mares had unilateral ovarian enlargement. Six non-pregnant mares had a small and inactive contralateral ovary; the pregnant mare had a single small corpus luteum on the contralateral ovary and was at three-and-a-half months' gestation. Enlarged ovaries measured 7 cm to an estimated 30 cm in diameter. 28 per cent had a multicystic ultrasound appearance, 57 per cent were dense structures and 14 per cent were of mixed appearance. Mean concentrations of progesterone were <1 ng/ml, oestrone sulphate 3.06 (2.32) ng/ml and testosterone 0.58 (0.64) nmol/l in non-pregnant mares. Inhibin was elevated in all non-pregnant cases at 7.6 (12.45) ng/ml. The pregnant mare had concentrations of progesterone 2.5 ng/ml, oestrone sulphate 81.0 ng/ml, testosterone 1.9 nmol/l and inhibin 1.31 ng/ml. Mares demonstrating stallion-like behaviour had a significantly higher (P<0.001) testosterone concentration (1.85 [0.07] nmol/l) than those that did not (0.34 [0.26] nmol/l). Three mares underwent unilateral ovariectomy and resumed cyclic ovarian activity within nine months of surgery.