Major Steroid Research Articles

Determination of estradiol in human serum using magnetic particles-based chemiluminescence immunoassay

A magnetic particles (MPs)-based chemiluminescence immunoassay (CLIA) with high sensitivity, specificity, and reproducibility was proposed for the evaluation of estradiol (E 2) in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and the horseradish peroxidase (HRP)–luminol–H 2O 2 chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E 2, without cross-reaction for the major steroids, including estrone (E 1), estriol (E 3), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E 2 in human serum without extraction. Besides, the effects of several physicochemical parameters, including the dilution ratios of E 2–6-HRP conjugate and anti-E 2 polyclonal antibody, immunoreaction time, chemiluminescent (CL) substrate volume, volume of MPs, and CL reaction time, were studied and optimized. The proposed method had a detection limit of 2.51 pg mL −1 with a larger working range of 15–1000 pg mL −1. The inter-assay and intra-assay coefficient of variation (CV) were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3, 106 and 101%, respectively. The method has been successfully applied to the determination of E 2 in 105 human sera and showed a good correlation compared with the commercial radioimmunoassay (RIA) kit with a correlative coefficient of 0.9892. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E 2 in human serum.

Comparative studies on the interplay of testosterone, estrogen and progesterone in collagen induced arthritis in rats

Rheumatoid arthritis (RA) is a sexually dimorphic autoimmune disorder exhibiting a higher disease prevalence and severity among females. This study was carried out to understand the role of the major sex hormones viz., testosterone, estrogen and progesterone on the severity in arthritis. The interplay of the sex hormones was studied in a rat model of collagen induced arthritis (CIA). The parameters used for analyzing the disease severity included paw volume, radiology, histopathology of joint, markers for bone turnover, cytokine profile, levels of pain mediator (prostaglandin E 2) and immune response to type II collagen. Arthritis induction to castrated and ovariectomised rats resulted in enhanced inflammation thereby indicating the importance of sex hormones. Treatment with physiological doses of dihydrotestosterone and estrogen attenuated the inflammation, with estrogen exhibiting higher potency. Progesterone was not shown to have any significance in disease modification; however, when progesterone was administered in combination with estrogen, the protective effects of estrogen were noticed to decrease.

Juvenile male rats display lower cortical metabolic capacity than females

The juvenile brain undergoes marked maturational changes accompanied by major sex hormone changes. In particular, sex differences in neural substrates could underlie male-specific dysfunction in behavioral responses related to the prefrontal cortex. Sex differences in regional metabolic capacity of the cerebral cortex were investigated in juvenile Sprague-Dawley rats. At 6 weeks of age the brains were processed for quantitative histochemistry of cytochrome oxidase, a rate-limiting enzyme in cellular respiration, which is an index of brain metabolic capacity. Quantitative image analysis revealed a main effect of sex with males displaying lower regional metabolic capacity than females in the dorsolateral and orbital prefrontal cortex and in the posterior parietal cortex. In addition, males separated for 6 h/day from their mothers as pups showed greater ambulatory behavior in the novel open field and higher metabolism in the posterior parietal cortex relative to males separated for 15 min/day. This is the first study to show sex differences in brain metabolic capacity in regions such as the prefrontal cortex that may be hypometabolic in juvenile males relative to females.

Naturally occurring progesterone in loblolly pine (Pinus taeda L.): A major steroid precursor of environmental androgens

AbstractProgesterone, androstenedione, and androstadienedione were previously identified in the water and sediment of the Fenholloway River (Taylor County, FL, USA), a river that contains populations of masculinized female mosquitofish downstream of a paper mill, at levels higher than those in the nearby Spring Creek. Plant sterols, such as β‐sitosterol in mill effluent derived from pine tree pulp, were suggested to be metabolized by bacteria to progesterone and androgens to account for the masculinization phenomenon. The current study made use of standard solid‐phase methanol extraction procedures, high‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and a cell‐based, androgen‐receptor transcription assay to determine naturally occurring progesterone levels in mature pine trees. Progesterone concentrations in the loblolly pine (Pinus taeda L.) were 49.34 ± 4.1 nmol/g dry mature wood (15.5 ± 1.29 μg/g), 12.26 ± 1.78 nmol/g pine needles (3.85 ± 0.56 μg/g), and 3.81 ± 0.36 nmol/g pine bark (1.19 ± 0.11 μg/g). The results suggest that naturally occurring progesterone from pine wood pulp contributes to increased progesterone levels downstream of paper mill effluent discharges and may serve as the natural steroid precursor for environmental androgen production that causes masculinization of female mosquitofish.

Financial Incentives and League Policy: The Example of Major League Baseball's Steroid Policy

This paper illustrates possible changes in revenue from league drug policies. We argue that there were perceived financial incentives for Major League Baseball (MLB) to not have a steroid policy. MLB did not begin testing players for steroids in earnest until the 2005 season although the league office acknowledged use a decade and a half earlier. Only after outside pressure did the league implement a policy with specific, enforceable penalties. This paper uses regression analysis to assess whether the absence of a true steroid policy prior to 2005 affected scoring, then quantifies the amount of revenue baseball owners lost as a consequence of the updated steroid policy.

Effect of dehydroepiandrosterone (DHEA) on monoamine oxidase activity, lipid peroxidation and lipofuscin accumulation in aging rat brain regions

Dehydroepiandrosterone (DHEA), one of the major steroid hormones, and its ester have recently received attention with regard to aging and age-related diseases like Alzheimer and others. DHEA is synthesized de novo in the brain and its substantial fall with age has been shown to be associated with neuronal vulnerability to neurotoxicity processes. Thus, DHEA is considered to be a neuroactive pharmacological substance with potential antiaging properties. A prominent feature that accompanies aging is an increase in monoamine oxidase (MAO). Increased MAO activity with correlated increase in lipid peroxidation in the aging rat brain supports the hypothesis that catecholamine oxidation is an important source of oxidative stress. The progressive accumulation of lipofuscin in neuronal cells is one of the most characteristic age related changes, an increase in body weight was also observed at 24 months. The objective of this study was to observe the changes in monoamine oxidase activity, lipid peroxidation levels and lipofuscin accumulation occurring in aging rat brain regions, and to see whether these changes are restored to normal levels after exogenous administration of DHEA (30 mg/kg/day for 1 month). The results obtained in the present work revealed that normal aging was associated with significant increases in the activity of monoamine oxidase, lipid peroxidation levels and lipofuscin accumulation in brain regions of 4, 14 and 24 months age group male rats. The present study showed that DHEA treatment significantly decreased monoamine oxidase activity, lipid peroxidation and lipofuscin accumulation in brain regions of aging rats, the increased body weight at 24 months also decreased more than the age matched controls. It can therefore be suggested that DHEA's beneficial effects seemed to arise from its antioxidant, antiobesity, antilipofuscin, antilipidperoxidative and thereby anti-aging actions. The results of this study will be useful for pharmacological modification of the aging process and development of new drugs for age related disorders.

Normal Bone Growth Requires Optimal Estrogen Levels: Negative Effects of Both High and Low Dose Estrogen on the Number of Growth Plate Chondrocytes

Endochondral bone formation at epiphyseal growth plate consists of the synchronized processes of chondrogenesis and cartilage ossification. Estrogen, the major female sex hormone, plays an important role in this process, particularly during the pubertal growth spurt. However, its effects on the growth plate are not completely understood. The aims of this study were to clarify the effects of estrogen on the kinetics of chondrocytes in the growth plates of 10- to 25-week-old female rabbits by studying the effects of ovariectomy or high-dose administration of estrogen on the balance between cell proliferation and death. Forty-eight Japanese white rabbits were divided into three groups: sham operated, ovariectomized, or ovariectomized with subsequent weekly injection of high dose estrogen from 10 weeks. The chondrocyte kinetics was investigated by histomorphometry and immunohistochemistry, using antibodies for caspase-3, a marker of apoptosis, and for proliferating cell nuclear antigen. Both ovariectomized and estrogen-injected rabbits showed a declination of the chondrocyte number although the latter animals indicated a more dramatic effect. Estrogen-injected rabbits showed a decrease in the cell proliferating ability together with an increase in chondrocytes undergoing apoptosis while ovariectomy mainly reduced the cell proliferating ability. Given the known importance of estrogen for bone growth, one would expect that ovariectomy and high-dose administration of estrogen would have opposite effects. However, the present study indicated that both low and high concentration had a similar effect: a decrease in the chondrocyte number compared with control, suggesting that estrogen has to be maintained within a narrow range for optimal bone growth.

Chronic administration of dehydroepiandrosterone (DHEA) to female monkey and rat has no effect on mammary gland histology

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, has been shown to exert chemopreventive effect on the development of carcinogen-induced rat mammary tumors. Since little is known on the effect of DHEA administration on mammary gland physiology and histology, we have studied the effect of long-term administration of DHEA to normal female monkey and rat on mammary gland histology as well as on serum DHEA, DHEA sulphate (DHEA-S), testosterone and estradiol levels. In monkeys, DHEA treatment (2 or 10 mg/(kg b.w. day)) induced a dose-related increase in serum DHEA and DHEA-S (above 20-fold) levels. At the highest dose of DHEA, serum testosterone levels were significantly increased (three- to fourfold), while serum estradiol concentration was not modified. DHEA treatment did not modify the histological characteristics of monkey mammary glands. In the rat, following DHEA administration (10 or 100 mg/(kg b.w. day)), a dose-related marked increase in serum DHEA and DHEA-S was observed. Serum testosterone was also increased in DHEA-treated animals, while no significant changes in serum estradiol levels were detected. As in the monkey, the histology of the female rat mammary gland remained unchanged following long-term treatment with any of the two doses of DHEA.

Nutritional Quality of Dried Pig Placenta

Nutrients and hormone levels of dried pig placenta were studied. Placentas were freeze-dried (FD), oven-dried at 60 (OD-60), and 90℃ (OD-90) and then crushed by a blender into small pieces. FD and OD-60 pig placenta had a higher moisture content than did OD-90, with no difference between FD and OD-60. There were no large differences in compositions of crude protein, crude fat, and crude ash of dried placenta among the treatments and the contents of K, Fe, and α-tocopherol were highest in FD (p＜0.05). Glutamine and glycine were the most abundant amino acids in all dried placenta and tyrosine was highly retained in FD placenta, compared with OD (p＜0.05). Estradiol was the major sex hormone, followed by progesterone and testosterone in all dried placentas. Antibiotics including amoxicillin, sulfamethazine, tylosin, and chlorotetracyclin were not detected from the pig placentas tested. These results demonstrate that placenta is a good biomaterial with high nutritional quality, and that freeze drying is superior to oven drying for processing pig placenta.

Novel approaches to the analysis of steroid estrogens in river sediments

A wide range of estrogenic contaminants has been detected in the aquatic environment. Among these, natural and synthetic steroid estrogens, typically present in municipal sewage-treatment plant (STP) effluents, are the most potent. In this study a new GC-MS method has been developed for direct analysis of five major steroid estrogens (estrone, 17beta-estradiol, 17alpha-ethinylestradiol, dienestrol, and diethylstilbestrol) in river sediments. Four GC-MS systems used for analysis of underivatized analytes in purified extracts were compared. Relatively low detection limits (1.5-5 ng g(-1) dried sediment) and good repeatability of GC splitless injection (RSD 1-2%) were achieved by use of a system combining low-pressure gas chromatography with a single-quadrupole mass analyzer (LP-GC-MS). Use of orthogonal gas chromatography (GCxGC) hyphenated with high-speed time-of-flight mass spectrometry (HSTOF-MS) enabled not only significantly better resolution of target analytes, and their unequivocal identification, but also further improvement (decrease) of their detection limits. In addition to these outcomes, use of this unique GCxGC-TOF-MS system enabled identification of several other non-target chemicals, including pharmaceutical steroids, present in purified sediment extracts.

Screening of mycelial fungi for 7α- and 7β-hydroxylase activity towards dehydroepiandrosterone

In total, 481 fungal strains were screened for the ability to carry out 7(α/β)-hydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androsten-17-one). Representatives of 31 genera of 15 families and nine orders of ascomycetes, 17 genera of nine families and two orders of zygomycetes, two genera of two families and two orders of basidiomycetes, and 14 genera of mitosporic fungi expressed 7(α/β)-hydroxylase activity. The majority of strains were able to introduce a hydroxyl group to position 7α. Active strains selectively producing 3β,7α-dihydroxy-5-androsten-17-one were found among Actinomucor, Backusella, Benjaminiella, Epicoccum, Fusarium, Phycomyces and Trichothecium, with the highest yield of 1.25 and 1.9 g L−1 from 2 and 5 g L−1 DHEA, respectively, reached with F. oxysporum. Representatives of Acremonium, Bipolaris, Conidiobolus and Curvularia formed 3β,7β-dihydroxy-5-androsten-17-one as a major product from DHEA. The structures of the major steroid products were confirmed by TLC, gas chromatography (GC), mass spectra (MS), and 1H-NMR analyses.

Circadian rhythm of urinary steroid metabolites

Samples submitted for urinary steroid profile analysis are often untimed, but influence of collection time on interpretation is unknown. We report circadian rhythms of the major steroid metabolites and derived ratios in urine collected at 3-h intervals over 24 h, after first establishing that disturbance of sleep associated with collection does not alter rhythms on the succeeding day. Assay of steroid metabolites (gas chromatography) and creatinine in urine collections made by 10 men and 10 women every 3 h starting at 2,100 for 24 h. Data were subjected to cosinor analysis. Summed cortisol and androgen metabolites exhibited significant circadian rhythms, as expected, but with a surprisingly long time-lag (maxima at around 1,400). Amplitudes were different, so that the ratio cortisol/androgen metabolites also showed a significant rhythm. The ratios 5alpha/5beta tetrahydrocortisol and 20-hydroxy/20-oxo cortisol metabolites showed significant rhythms which were not in phase with total cortisol metabolites, while 11-hydroxy/11-oxo cortisol metabolites showed no rhythm. There were no gender differences in time of maxima. Previously established gender differences in metabolite levels were confirmed. Creatinine levels showed no circadian rhythm. Circadian variation should be considered when interpreting results from urine steroid analysis. Calculation of steroid/steroid or steroid/creatinine ratios is not informative in untimed collections.

Ovarian steroids and the human breast: Regulation of stem cells and cell proliferation

Ovarian steroidal control of mammary gland proliferation and differentiation is not well defined in the human. We therefore developed the athymic nude mouse model in which intact normal human breast tissue is xenografted subcutaneously and treated with human physiological serum levels of oestrogen (E) and/or progesterone (P). We showed that: (i) E, and not P, is the major steroid hormone inducing proliferation of epithelial cells in the adult non-pregnant, non-lactating breast; (ii) E induces progesterone receptor (PR) expression; and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E was required to induce proliferation. Using double label immuno-fluorescence, we demonstrated that cells expressing the oestrogen receptor-α (ERα) invariably contained the PR but that steroid receptor expression and cell proliferation (Ki67 antigen) were dissociated. Recently, we have demonstrated that some ERα/PR-positive epithelial cells are quiescent breast stem cells suggesting that they act as “steroid hormone sensors” that secrete paracrine factors to regulate the proliferative activity of adjacent ERα/PR-negative epithelial cells. The dissociation between steroid receptor expression and cell proliferation in normal epithelium was lost at an early stage in ERα/PR-positive breast tumour formation perhaps indicating that they arise from deregulation of the normally quiescent breast stem cells.

Ketosteroids and hydroxyketosteroids, minor metabolites of sugarcane wax

Besides β-sitosterol and stigmasterol, the major steroids of sugarcane, the following minor steroids have been isolated and identified from sugarcane wax: 3,6-diketosteroids, Δ 4-3-keto steroids, and Δ 4-6-hydroxy-3-keto steroids. Their structures were established by spectroscopic techniques and chemical correlations.

Estrogen in men: effects on bone accrual, maintenance and prevention of bone loss

Sex steroid hormones play an important role in the maintenance of bone mass in males as well as in females. Even though androgens represent the major sex steroid class in men, their primacy in regulating male skeletal remodeling has been questioned increasingly as direct and indirect evidence has emerged suggesting that estrogens also play a major role in male skeletal health. This review summarizes clinical and experimental evidence that estrogens are essential for bone accrual in the growing skeleton, maintenance of bone mass and prevention of bone loss in men.

Development of a Panel of Monoclonal Antibodies against the Mineralocorticoid Receptor

Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids. They are expressed in multiple tissues and mediate diverse functions. Less is known about MR regulation and function compared with other major steroid receptors, although its importance has become increasingly apparent. A significant obstacle to such studies has been the dearth of specific high-affinity MR antibodies. We have produced monoclonal antibodies against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain), with the anticipation that their individual affinities for the MR would differ depending upon its conformation, which in turn, is dependent upon the location of the receptor within the cell and the proteins associated with it. Hybridoma clones with high titers to the cognate peptide ELISA were analyzed by Western blots using protein from Chinese hamster ovary cells transfected with enhanced green fluorescent protein-rat MR cDNA and from hippocampal cytosol from adrenalectomized rats. Immunohistochemistry was done on kidney, heart, colon, and brain. Antibodies that proved to be most useful for Western blot analysis and immunohistochemistry include those raised against peptides comprising amino acids 1-18, 64-82, 79-97, and 365-381. The intensity of immunoreactivity in the cytosol compared with nucleus in the same cells differed between antibodies, suggesting that certain receptor epitopes were more or less exposed depending on the location of the receptor within the cell. In summary, several antibodies are described that recognize different parts of the MR that should facilitate the study of this important mediator of two classes of steroid hormone action.

Just the Right Size

Two parameters control the eventual size of insects: their growth rate and the length of their growth period. Ecdysone, a major steroid hormone, functions as a developmental timer that controls the length of the growth period. Colombani et al. (see the Perspective by King-Jones and Thummel) now show that ecdysone from the prothoracic gland of Drosophila also regulates the speed at which the animals grow by inhibiting insulin/insulin-like growth factor signaling. This work provides a conceptual framework for understanding how the final size of an organism is determined and establishes a link between steroid hormone and insulin signaling. J. Colombani, L. Bianchini, S. Layalle, E. Pondeville, C. Dauphin-Villemant, C. Antoniewski, C. Carré, S. Noselli, P. Léopold, Antagonistic actions of ecdysone and insulins determine final size in Drosophila . Science 310 , 667-670 (2005). [Abstract] [Full Text] K. King-Jones, C. S. Thummel, Less steroids make bigger flies. Science 310 , 630-631 (2005). [Summary] [Full Text]

Changes in steroidogenic enzyme and steroidogenic acute regulatory protein messenger RNAs in ovarian follicles during ovarian development of rainbow trout ( Oncorhynchus mykiss)

In salmonid fishes, estradiol-17β (E2) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) are the major steroid hormones controlling oocyte growth (vitellogenesis) and final maturation (resumption of meiosis). The aim of this study was to determine changes in mRNAs encoding ovarian steroidogenic enzymes and steroidogenic acute regulatory protein (StAR) during ovarian development in female rainbow trout. We analyzed the levels of mRNAs encoding the enzymes P450 side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase/C17–C20 lyase (P450c17), aromatase (P450arom), and carbonyl reductase-like 20β-hydroxysteroid dehydrogenase (20β-HSD), and StAR in developing ovarian follicles of rainbow trout by Northern blot, in relation to the pattern of serum E2 and 17,20β-P levels. Serum E2 levels were elevated during vitellogenesis and decreased prior to an ovulatory increase in 17,20β-P. Transcripts for P450scc and P450c17 increased in late vitellogenic follicles, then decreased in post-ovulatory follicles. In contrast, P450arom transcripts were abundant during vitellogenesis and then declined as vitellogenesis was completed and were barely detectable in post-ovulatory follicles. 3β-HSD mRNA levels increased in late vitellogenic follicles and were maintained at high levels in post-ovulatory follicles. 20β-HSD and StAR mRNA levels were very low during vitellogenesis, and then strongly increased during late vitellogenesis to a peak in post-ovulatory follicles. These results indicate that the expression of genes encoding steroidogenic enzymes and StAR change dynamically, dependent on the developmental stages of rainbow trout follicles. The acquisition of the ability of later stage follicles to rapidly produce large quantities of 17,20β-P appears to be supported by a preparatory increase in mRNAs encoding StAR and other steroidogenic enzymes.

Is There a Receptor for Dehydroepiandrosterone or Dehydroepiandrosterone Sulfate?

It remains unknown whether dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) have a physiological role other than serving as metabolic intermediates in androgen synthesis. Apart from intracellular metabolism, there is no convincing cellular mechanism of action for physiological concentrations of DHEA(S). Unlike other major steroids, a receptor for DHEA(S) has not been definitively isolated. This article will review the evidence supporting a receptor-dependent basis for the direct physiological effects of DHEA(S). The data supporting an intracellular receptor for DHEA(S) are relatively weak and do not allow us to determine whether DHEA(S) directly, or a metabolite of DHEA(S), acts as a direct receptor ligand. Recent data strongly support a plasma membrane receptor for DHEA, but this potential receptor is yet to be isolated. Definitive characterization of the molecular mechanism (receptor or otherwise) of DHEA(S) action is necessary before we can determine whether DHEA(S) has a biological role other than as an androgen precursor.

Dehydroepiandrosterone and ageing.

Dehydroepiandrosterone (DHEA) is the major steroid produced by the adrenal zona reticularis and, in contrast to cortisol and aldosterone, its secretion declines with ageing. This has generated major interest in its putative role as an 'anti-ageing' hormone. However, it is not clear that the age-associated, physiological decline in DHEA secretion represents a harmful deficiency. DHEA exhibits its action mainly by conversion to sex steroids. In addition, DHEA has neurosteroidal properties and may exhibit direct action via specific binding sites on endothelial cells. There is convincing evidence for beneficial effects of DHEA in patients with adrenal insufficiency and future research will hopefully elucidate its role in patients receiving pharmacological glucocorticoid treatment. However, in healthy elderly subjects, current evidence from randomised, controlled trials does not justify the use of DHEA, with no major beneficial effects reported and, in addition, potentially adverse effects on sex steroid-dependent tumour growth need to be considered.