Testicular induced corticosterone synthesis in male rats under fasting stress.

Testicular steroidogenesis is depressed by adrenal-secreted corticosterone (CORT) under stress. However, the mechanisms are not well understood. This study investigated the details of testicular steroidogenesis depression during fasting. Blood levels of adrenocorticotropic hormone secreted from the pituitary glands increased, but blood CORT was not changed in rats that fasted for 96 h, in spite of the rats being severely stressed. CORT in fasting adult male rats increased more than three times in the testis, but reduced testicular testosterone (T) and blood T levels to 5% and 2% of the control, respectively, was observed. The contents of T precursor (except PGN) were drastically reduced in the fasted-rat testes. Testicular CORT levels were elevated, but the enzymatic activity of cytochrome P45011ß, which produces CORT, remained unchanged. The enzymatic activities of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), mediating the conversion of pregnenolone to progesterone, decreased in the fasted-rat testes. Thus, fasting suppressed testicular steroidogenesis by affecting the enzyme activity of 3ß-HSD in the testes and drastically reduced T and increased CORT synthesis. It can be considered that T synthesis involved in cell proliferation is suppressed due to lack of energy during fasting. Conversely, 11ß-hydroxylase enzyme activity was induced and CORT synthesis is increased to cope with the fasting stress. Hence, it can be concluded that CORT synthesis in the testes plays a role in the local defense response.

Electro-acupuncture regulates glucose metabolism in chronic stress model rats.

Studies have shown that acupuncture is very effective in treating chronic stress depression. However, little is known about the therapeutic mechanism of electro-acupuncture. Metabolomics, on the other hand, is a technology that determines the metabolic changes of organisms caused by various interventions as a whole and is related to the overall effect of electro-acupuncture (EA). 1HNMR, serum sample analysis, and histopathology and molecular biology analysis were used to evaluate the effects of EA. The results show that electro-acupuncture points can regulate the heat pain threshold of chronic stress model rats and change the morphology of adrenal cortex cells Structure, and regulate the contents of corticotropin-releasing hormone, Corticosterone (CORT), glucose, alanine and valine in the samples. These findings help to clarify the therapeutic mechanism of electro-acupuncture on heterologous chronic stress model rats. The effect of electro-acupuncture on improving chronic stress is likely to be achieved by regulating glucose metabolism, which can provide a reference for clinical acupuncture treatment of chronic stress depression.

Insights into the Therapeutic Potential of Glucocorticoid Receptor Modulators for Neurodegenerative Diseases.

Glucocorticoids are crucial for stress-coping, resilience, and adaptation. However, if the stress hormones become dysregulated, the vulnerability to stress-related diseases is enhanced. In this brief review, we discuss the role of glucocorticoids in the pathogenesis of neurodegenerative disorders in both human and animal models, and focus in particular on amyotrophic lateral sclerosis (ALS). For this purpose, we used the Wobbler animal model, which mimics much of the pathology of ALS including a dysfunctional hypothalamic-pituitary-adrenal axis. We discuss recent studies that demonstrated that the pathological cascade characteristic for motoneuron degeneration of ALS is mimicked in the genetically selected Wobbler mouse and can be attenuated by treatment with the selective glucocorticoid receptor antagonist (GRA) CORT113176. In long-term treatment (3 weeks) GRA attenuated progression of the behavioral, inflammatory, excitatory, and cell-death-signaling pathways while increasing the survival signal of serine-threonine kinase (pAkt). The action mechanism of the GRA may be either by interfering with GR deactivation or by restoring the balance between pro- and anti-inflammatory signaling pathways driven by the complementary mineralocorticoid receptor (MR)- and GR-mediated actions of corticosterone. Accordingly, GR antagonism may have clinical relevance for the treatment of neurodegenerative diseases.

Quantitative analysis of 11-dehydrocorticosterone and corticosterone for preclinical studies by liquid chromatography/triple quadrupole mass spectrometry.

RATIONALE: The activity of the glucocorticoid activating enzyme 11ß-hydroxysteroid dehydrogenase type-1 (11ßHSD1) is altered in diseases such as obesity, inflammation and psychiatric disorders. In rodents 11ßHSD1 converts inert 11-dehydrocorticosterone (11-DHC) into the active form, corticosterone (CORT). A sensitive, specific liquid chromatography/tandem mass spectrometry method was sought to simultaneously quantify total 11-DHC and total and free CORT in murine plasma for simple assessment of 11ßHSD1 activity in murine models. METHODS: Mass spectrometry parameters were optimised and a method for the chromatographic separation of CORT and 11-DHC was developed. Murine plasma was prepared by 10:1 chloroform liquid-liquid extraction (LLE) for analysis. Limits of quantitation (LOQs), linearity and other method criteria were assessed, according to bioanalytical method validation guidelines. RESULTS: Reliable separation of 11-DHC and CORT was achieved using an ACE Excel 2 C18-AR (2.1 × 150 mm; 2 µm) fused core column at 25°C, with an acidified water/acetonitrile gradient over 10 min. Analytes were detected by multiple reaction monitoring after positive electrospray ionisation (m/z 345.1.1 ➔ 121.2, m/z 347.1 ➔ 121.1 for 11-DHC and CORT, respectively). The LOQs were 0.25 and 0.20 ng/mL for 11-DHC and CORT, respectively. CONCLUSIONS: This LC/MS method is suitable for the reliable analysis of 11-DHC and CORT following simple LLE of murine plasma, bringing preclinical analysis in line with recommendations for clinical endocrinology and biochemistry.

Corticosterone in Lizard Egg Yolk Is Reduced by Maternal Diet Restriction but Unaltered by Maternal Exercise.

When females face adverse environmental conditions, physiological changes, such as elevated corticosterone levels, to cope with the stressors may also impact their offspring. Such maternal effects are often considered adaptive and may "prime" the offspring for the same adverse environment, but maternal corticosterone levels do not always match that of the eggs produced. We examined how diet restriction and increased locomotor activity, via exercise training, affected steroid hormone levels of female green anole lizards, as well as the hormone levels in the yolk of their eggs. Diet restriction did not affect female hormone levels, but training increased corticosterone levels. Despite this, training did not affect yolk steroid levels, but eggs from females with diet restriction had lower corticosterone levels in yolk. This suggests that two common stressors, food shortage and increased locomotor activity, impact female physiology in a way that is not translated to her offspring.

Posthatching Parental Care and Offspring Growth Vary with Maternal Corticosterone Level in a Wild Bird Population.

Corticosterone is the primary metabolic steroid in birds and is vital for maintaining homeostasis. However, the relationship between baseline corticosterone and reproduction is unclear, and we lack an understanding of how differences in baseline corticosterone at one stage of the breeding cycle influence reproductive effort at later stages. In a wild population of house wrens, we quantified the concentration of corticosterone in yolks of freshly laid eggs as an integrated measure of maternal physiology and related this to a behavioral measure of stress reactivity made during the nestling period, namely, the latency with which females resumed parental activities following a standardized disturbance at their nest (setting up a camera to record provisioning). Females that recently produced eggs containing higher corticosterone concentrations, which were significantly repeatable within females, took longer to resume activity related to parental care (i.e., feeding and brooding young) following the disturbance. Moreover, a female's latency to resume parental activities negatively predicted her provisioning of nestlings with food and the condition of these young at fledging but did not predict the number fledged. We cross-fostered offspring prior to hatching so these effects on maternal behavior are independent of any prenatal maternal effects on nestlings via the egg. These results are consistent with earlier findings, suggesting that females with higher baseline corticosterone during egg laying or early incubation tend to prioritize self-maintenance over reproduction compared with females with lower baseline corticosterone and suggest that a female's latency to return to her nest and resume parental care following a disturbance might represent a simple, functional measure of maternal stress reactivity.

Measurement of 11-dehydrocorticosterone in mice, rats and songbirds: Effects of age, sex and stress.

Glucocorticoids (GCs) are secreted into the blood by the adrenal glands and are also locally-produced by organs such as the lymphoid organs (bone marrow, thymus, and spleen). Corticosterone is the primary circulating GC in many species, including mice, rats and birds. Within lymphoid organs, corticosterone can be locally produced from the inactive metabolite, 11-dehydrocorticosterone (DHC). However, very little is known about endogenous DHC levels, and no immunoassays are currently available to measure DHC. Here, we developed an easy-to-use and inexpensive immunoassay to measure DHC that is accurate, precise, sensitive, and specific. The DHC immunoassay was validated in multiple ways, including comparison with a mass spectrometry assay. After assay validations, we demonstrated the usefulness of this immunoassay by measuring DHC (and corticosterone) in mice, rats and song sparrows. Overall, corticosterone levels were higher than DHC levels across species. In Study 1, using mice, we measured steroids in whole blood and lymphoid organs at postnatal day (PND) 5, PND23, and PND90. Corticosterone and DHC showed distinct tissue-specific patterns across development. In Studies 2 and 3, we measured circulating corticosterone and DHC in adult rats and song sparrows, before and after restraint stress. In rats and song sparrows, restraint stress rapidly increased circulating levels of both steroids. This novel DHC immunoassay revealed major changes in DHC concentrations during development and in response to stress, which have important implications for understanding GC physiology, effects of stress on immune function, and regulation of local GC levels.

Clinical, Physiologic, and Behavioral Evaluation of Permanently Catheterized NMRI Mice.

Vascular catheterization is becoming a popular technique in laboratory rodents, facilitating repetitive blood sampling and infusion in individual animals. In mice, catheterization is complicated by their small body size, which may increase the risk of postoperative complications that may both threaten catheter longevity and animal welfare. Less obvious complications to a permanent catheter may include subclinical infection, visceral tissue damage from disseminating microthrombi released from the catheter, and distress from being isolated from conspecifics and other experimental stressors. Such complications may go unnoticed and may affect animal welfare as well as confound research outcomes. This study investigated the implications of long-term arterial catheterization in NMRI mice by evaluating clinical, physiologic and behavioral parameters. Body weight and food and water consumptions were monitored during the study period. Fecal corticosterone metabolites were quantified as biomarkers of stress, and nucleic acid metabolites (8-oxo-7,8-dihydro-2'-deoxyguanisine and 8-oxo-7,8-dihydroguanosine) as biomarkers of oxidative damage. Behavioral dysfunction was studied by scoring animal welfare and nest building. Catheters were placed the right common carotid artery of mice; catheterized mice were compared with sham-operated and nonsurgical control mice. Except for an increase in the body weight of catheterized mice during the experimental period, clinical parameters (body weight and food and water consumptions) did not differ between groups. Physiologic parameters (oxidized nucleic acid metabolites and fecal corticosterone metabolites) were higher in control mice during the first week of experimentation compared with the end of study but did not differ between groups. Likewise, catheterization had no effect on behavioral parameters (nest building and animal welfare assessment). Long-term arterial catheterization of mice had no detectable implications on animal welfare in this study.

Preventing ectoparasite infestations reduces glucocorticoid concentrations in the hair of cows - short communication.

The aim of this study was to compare hair corticosteroid concentrations in ectoparasiticide-treated (n = 10) and non-treated (n = 12) Holstein cows. Animals in the treated group received cyfluthrin three times, on days 0, 28 and 56 of the experiment. Hair samples were collected from all cows on days 0, 21, 42, 63, and 84 of the experiment for the determination of cortisol and corticosterone concentrations using immunoassays. The respective hair cortisol concentrations in the treated group averaged 209.78, 165.10, 260.78, 177.44 and 183.11 ng/g, while in the non-treated group hair cortisol concentrations averaged 172.58, 243.58, 309.73, 199.75 and 207.09 ng/g. These results indicate that the control of ectoparasites reduced hair cortisol levels in dairy cows. The respective hair corticosterone concentrations in the treated group averaged 19.06, 22.95, 21.95, 21.60 and 24.84 ng/g and in the non-treated group the hair corticosterone concentrations averaged 17.28, 21.94, 34.05, 26.27 and 26.91 ng/g. The results suggest that longterm stress can be estimated better by the determination of hair cortisol rather than corticosterone concentrations.

Bioconversion of Corticosterone into Corticosterone-Glucoside by Glucosyltransferase.

Glucosylation of the 21-hydroxyl group of glucocorticoid changes its solubility into hydrophilicity from hydrophobicity and, as with glucocorticoid glucuronides as a moving object in vivo, it is conceivable that it exhibits the same behavior. Therefore, glucosylation to the 21-hydroxyl group while maintaining the 11ß-hydroxyl group is particularly important, and glucosylation of corticosterone was confirmed by high-resolution mass spectrometry and 1D (¹H and 13C) and 2D (COSY, ROESY, HSQC-DEPT and HMBC) NMR. Moreover, the difference in bioactivity between corticosterone and corticosterone 21-glucoside was investigated in vitro. Corticosterone 21-glucoside showed greater neuroprotective effects against H2O2-induced cell death and reactive oxygen species (ROS) compared with corticosterone. These results for the first time demonstrate that bioconversion of corticosterone through the region-selective glucosylation of a novel compound can present structural potential for developing new neuroprotective agents.

Effects of Nesting Material on the Toxicologic Assessment of Cyclophosphamide in Crl:CD1(ICR) Mice.

The provision of nesting material benefits mice by reducing cold stress, improving feed conversion, increasing litter size, and improving adaptive immunity. The effects of toxins are sensitive to environmental changes, and the introduction of novel items can alter results in some toxicologic studies. We hypothesized that nesting material would reduce stress and positively alter immunologic parameters in Crl:CD1(ICR) mice, thus changing typical results from a well-studied immunomodulating drug, cyclophosphamide. A 13-wk study assessed the following treatments in a factorial design (n = 4; 32 cages total): nesting (0 or 10 g) and drug (50 mg/kg cyclophosphamide or 10 mL/kg saline; IP weekly). Detailed examinations and body weights were recorded weekly, and nests were scored twice weekly. Fecal pellets were collected at 0, 4, 6, and 12 wk for analysis of corticosterone metabolites. At study termination, clinical pathology and immune parameters were collected, a necropsy performed, and lymphoid organs and adrenal glands were submitted for histopathology. All expected results due to cyclophosphamide were observed. Nesting reduced the proportion of mice with piloerection, and body weights were highest in saline-nested male mice. No differences in hematology, clinical chemistry, or absolute lymphocyte counts were observed. Corticosterone metabolites in all nested groups were not different from baseline levels but all nonnested groups had higher levels than baseline. Nested cyclophosphamide-treated groups had significantly lower corticosterone levels than nonnested cyclophosphamide-treated groups. This study illustrates that nesting material does not alter the results of a standard toxicology study of cyclophosphamide but alleviates study-related stress and improves mouse welfare.

Tracking Down a New Steroid-Hydroxylating Promiscuous Cytochrome†P450: CYP154C8 from Streptomyces sp. W2233-SM.

CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11-ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α-hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16-hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α-dihydroxyprogesterone and 6ß,16α-dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications.

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species.

Repeated social defeat and the rewarding effects of cocaine in adult and adolescent mice: dopamine transcription factors, proBDNF signaling pathways, and the TrkB receptor in the mesolimbic system.

RATIONALE: Repeated social defeat (RSD) increases the rewarding effects of cocaine in adolescent and adult rodents. OBJECTIVE: The aim of the present study was to compare the long-term effects of RSD on the conditioned rewarding effects of cocaine and levels of the transcription factors Pitx3 and Nurr1 in the ventral tegmental area (VTA), the dopamine transporter (DAT), the D2 dopamine receptor (D2DR) and precursor of brain-derived neurotrophic factor (proBDNF) signaling pathways, and the tropomyosin-related kinase B (TrkB) receptor in the nucleus accumbens (NAc) in adult and adolescent mice. METHODS: Male adolescent and young adult OF1 mice were exposed to four episodes of social defeat and were conditioned 3 weeks later with 1 mg/kg of cocaine. In a second set of mice, the expressions of the abovementioned dopaminergic and proBDNF and TrkB receptor were measured in VTA and NAc, respectively. RESULTS: Adolescent mice experienced social defeats less intensely than their adult counterparts and produced lower levels of corticosterone. However, both adult and adolescent defeated mice developed conditioned place preference for the compartment associated with this low dose of cocaine. Furthermore, only adolescent defeated mice displayed diminished levels of the transcription factors Pitx3 in the VTA, without changes in the expression of DAT and D2DR in the NAc. In addition, stressed adult mice showed a decreased expression of proBDNF and the TrkB receptor, while stressed adolescent mice exhibited increased expression of latter without changes in the former. CONCLUSION: Our findings suggest that dopaminergic pathways and proBDNF signaling and TrkB receptors play different roles in social defeat-stressed mice exposed to cocaine.

The impact of the clinical CYP11B2 mutation V386A strongly depends on the enzyme's genetic background.

Human cytochrome P450 11B2 (CYP11B2) is an essential enzyme in the steroid hormone biosynthesis, which catalyzes the last three reaction steps of the aldosterone synthesis. These reactions comprise a hydroxylation at position C11 of the steroid intermediate deoxycorticosterone yielding corticosterone, followed by a hydroxylation at position C18 yielding 18-hydroxy-corticosterone and a subsequent oxidation of the hydroxyl group at C18, which results in the formation of aldosterone. Alterations in the amino acid sequence of CYP11B2 often cause severe disease patterns. We previously described a procedure for expression and purification of human CYP11B2 employing recombinant E. coli, which allows the rapid characterization of CYP11B2 mutants on a molecular level. This system was now utilized for the examination of the influence of the polymorphism at position 173 in combination with the mutation V386A on the activity of CYP11B2. Our in vitro findings show that the combination of the V386A mutation with the variant CYP11B2 173Arg only slightly reduces the 18-hydroxylase and 18-oxidase activity, whereas the V386A mutation with the CYP11B2 173Lys variant almost abolishes the 18-hydroxylation and 18-oxidation. In both cases the 11-hydroxylase activity is not affected. These findings highlight the importance of the genetic background of an enzyme when regarding the effect of clinical mutations.

Steroid 17-hydroxylase and 17,20-lyase deficiencies, genetic and pharmacologic.

Steroid 17-hydroxylase 17,20-lyase (cytochrome P450c17, P450 17A1, CYP17A1) catalyzes two major reactions: steroid 17-hydroxylation followed by the 17,20-lyase reactions. The most severe mutations in the cognate CYP17A1 gene abrogate all activities and cause combined 17-hydroxylase/17,20-lyase deficiency (17OHD), a biochemical phenotype that is replicated by treatment with the potent CYP17A1 inhibitor abiraterone acetate. The adrenals of patients with 17OHD synthesize 11-deoxycorticosterone (DOC) and corticosterone but no 19-carbon steroids, similar to the rodent adrenal, and DOC causes hypertension and hypokalemia. Loss of 17,20-lyase activity precludes sex steroid synthesis and leads to sexual infantilism. Rare missense CYP17A1 mutations minimally disrupt 17-hydroxylase activity but cause isolated 17,20-lyase deficiency (ILD), Mutations in the POR gene encoding the required cofactor protein cytochrome P450-oxidoreductase causes a spectrum of disease from ILD to 17OHD combined with 21-hydroxylase and aromatase deficiencies, sometimes including skeletal malformations. Mutations in the CYB5A gene encoding a second cofactor protein cytochrome b5 also selectively disrupt 17,20-lyase activity and cause the purest form of ILD. The clinical manifestations of these conditions are best understood in the context of the biochemistry of CYP17A1.

An experimental examination of interindividual variation in feather corticosterone content in the house sparrow, Passer domesticus in southeast Australia.

Non-invasive techniques for measuring glucocorticoids (GCs) have become more prevalent, due to the advantage of eliminating the effects of animal disturbance on GC levels and their potential to provide an integrated, historic estimate of circulating GC levels. In the case of birds, corticosterone (CORT) is deposited in feathers, and may reflect a bird's GC status over the period of feather synthesis. This technique thus permits a retrospective view of the average circulating GC levels during the moult period. While it is generally assumed that differences in feather CORT content (CORTf) between individuals reflects their different stress histories during either natural or induced moult, it is not clear how much of this variation is due to extrinsic versus intrinsic factors. We examined this question by determining CORTf in free-living house sparrows (Passer domesticus) from two populations, one urban and the other rural, that were plucked before and after exposure to different plasma CORT levels while held captive. We experimentally manipulated plasma CORT by implanting birds with either a corticosterone-filled, metyrapone-filled, or empty ('sham') silastic capsule as replacement feathers first emerged. The pattern of post-treatment CORTf was consistent with our expectations, based on plasma CORT levels of an experimentally implanted reference group. However, there was no statistically significant difference in CORTf between these treatment groups unless sex, population origin, and CORTf of original feathers for each individual were included in a model. Thus, birds with higher CORTf in feathers removed for this experiment tended to have higher CORTf in post-treatment replacement feathers, irrespective of treatment. In addition, we found that feather fault bar scores were significantly higher in CORT-treated birds than in the other two treatment groups, but did not vary directly with CORTf level. Our study therefore broadly confirms the use of feathers as a non-invasive tool to estimate plasma CORT during moult in birds, but importantly demonstrates the potential for intrinsic differences in stress characteristics between populations and individuals to obscure the effects extrinsic stressors might have on CORTf.

Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium.

Cytochrome P450 monooxygenases (P450s) are attractive enzymes for the pharmaceutical industry, in particular, for applications in steroidal drug synthesis. Here, we report a comprehensive functional and structural characterization of CYP109E1, a novel steroid-converting cytochrome P450 enzyme identified from the genome of Bacillus megaterium DSM319. In vitro and whole-cell in vivo turnover experiments, combined with binding assays, revealed that CYP109E1 is able to hydroxylate testosterone at position 16ß. Related steroids with bulky substituents at carbon C17, like corticosterone, bind to the enzyme without being converted. High-resolution X-ray structures were solved of a steroid-free form of CYP109E1 and of complexes with testosterone and corticosterone. The structural analysis revealed a highly dynamic active site at the distal side of the heme, which is wide open in the absence of steroids, can bind four ordered corticosterone molecules simultaneously, and undergoes substantial narrowing upon binding of single steroid molecules. In the crystal structures, the single bound steroids adopt unproductive binding modes coordinating the heme-iron with their C3-keto oxygen. Molecular dynamics (MD) simulations suggest that the steroids may also bind in ~180° reversed orientations with the C16 carbon and C17-substituents pointing toward the heme, leading to productive binding of testosterone explaining the observed regio- and stereoselectivity. The X-ray structures and MD simulations further identify several residues with important roles in steroid binding and conversion, which could be confirmed by site-directed mutagenesis. Taken together, our results provide unique insights into the CYP109E1 activity, substrate specificity, and regio/stereoselectivity. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5L90 (steroid-free CYP109E1), 5L91 (CYP109E1-COR4), 5L94 (CYP109E1-TES), and 5L92 (CYP109E1-COR). ENZYMES: Cytochrome P450 monooxygenase CYP109E1, EC 1.14.14.1, UniProt ID: D5DKI8, Adrenodoxin reductase EC 1.18.1.6.

Relationship of glucocorticoids and hematological measures with feed intake, growth, and efficiency of finishing beef cattle.

The objective of this experiment was to determine the association of glucocorticoids and markers for immune status in finishing beef steers and heifers with DMI, growth, and efficiency. Steers ( = 127) and heifers ( = 109) were individually fed a finishing ration for 84 d with BW measured every 21 d. Blood samples were collected via jugular venipuncture for metabolite (glucose and lactate) and cortisol analysis and rectal grab samples of feces were collected for corticosterone analysis on d 83 of the experiment. Plasma cortisol was not correlated to DMI ( = -0.08, > 0.05) or fractional DMI (g DMI/kg BW; = -0.03, > 0.05) but was negatively correlated with ADG ( = -0.17, < 0.01) and G:F ( = -0.20, < 0.01) and positively correlated to residual feed intake (RFI; = 0.14, < 0.05). Fecal corticosterone was positively correlated to fractional DMI ( = 0.15, < 0.05) and RFI ( = 0.23, < 0.01) and negatively correlated to G:F ( = -0.18, < 0.01). Using a mixed model analysis, none of the metabolites or hormones were associated with DMI ( > 0.05) but fecal corticosterone was positively associated with fractional DMI only in heifers ( = 0.04). Plasma lactate ( < 0.01) was and plasma cortisol ( < 0.10) tended to be negatively associated with ADG. Plasma cortisol ( < 0.05) and fecal corticosterone tended ( < 0.10) to be negatively associated with G:F. Fecal corticosterone was positively associated with RFI in heifers ( < 0.04). In a mixed model analysis, total leukocyte count was positively associated with ADG ( < 0.04) and tended to be positively associated with G:F ( < 0.06). Among leukocyte subtypes, neutrophil count was positively associated with ADG in steers ( < 0.02) and monocytes were positively associated with ADG in heifers ( < 0.03). Lymphocyte counts (LY) in steers were negatively associated with DMI ( = 0.03) and fractional DMI ( < 0.03). In heifers, LY tended to be positively associated with DMI ( < 0.09) and fractional DMI ( < 0.06). Lymphocyte count was also positively associated with ADG ( < 0.01) and G:F ( = 0.05) in heifers. The association of production traits with immune status seems to be different between steers and heifers. There was a stronger relationship of cortisol than fecal corticosterone to feed efficiency measures, suggesting that cortisol concentrations could be a better marker for feed efficiency traits than fecal corticosterone concentrations.

Effects of diet, time since defecation, and drying process of the droppings on corticosterone metabolite measurements in Japanese quail.

The use of noninvasive methods for measuring fecal glucocorticoid metabolites is a useful tool for endocrine assessment particularly in studies where animals cannot be captured, when they should be sampled without disturbing their activities, and/or when welfare needs to be maximized. However, still no complete standardization exists for the methodology, and some confounding variables may play an important role affecting measurements and interpretation of results. The present study focused on whether two different diets (laying feed or seed mixture), the time since defecation (0, 4, 24, or 48 h) and the drying method of those samples (oven-dried or naturally nonoven-dried) may affect concentrations of corticosterone metabolites (CM) measured in male Japanese quail. Half of the birds were provided with plain water (control) and the other half received a corticosterone solution. Birds fed with a seed mixture exhibited higher values of CM (nanogram/gram) in droppings than quail that received a laying feed diet suggesting that diet should be carefully considered as a potential source of variation. As expected both groups exhibited higher CM concentration after corticosterone treatment. While CM concentrations increased significantly in nonoven-dried samples over time (0 < 4 < 24 = 48 h), oven-dried samples exhibited similar high CM values. At 24 and 48 h postdefecation, nonoven-dried samples had similar CM concentrations as all oven-dried samples. Drying of samples may be considered a reliable method to reduce variations due to water loss over time, facilitating comparisons up to 48 h postdefecation. This finding would allow to enhance the range of application of this noninvasive and welfare friendly method to situations where samples cannot be collected or frozen shortly after defecation.