ATAC-seq and RNA-seq analysis unravel the mechanism of sex differentiation and infertility in sex reversal chicken.

BACKGROUND: Sex determination and differentiation are complex and delicate processes. In female chickens, the process of sex differentiation is sensitive and prone to be affected by the administration of aromatase inhibitors, which result in chicken sex reversal and infertility. However, the molecular mechanisms underlying sex differentiation and infertility in chicken sex reversal remain unclear. Therefore, we established a sex-reversed chicken flock by injecting an aromatase inhibitor, fadrozole, and constructed relatively high-resolution profiles of the gene expression and chromatin accessibility of embryonic gonads. RESULTS: We revealed that fadrozole affected the transcriptional activities of several genes, such as DMRT1, SOX9, FOXL2, and CYP19A1, related to sex determination and differentiation, and the expression of a set of gonadal development-related genes, such as FGFR3 and TOX3, by regulating nearby open chromatin regions in sex-reversed chicken embryos. After sexual maturity, the sex-reversed chickens were confirmed to be infertile, and the possible causes of this infertility were further investigated. We found that the structure of the gonads and sperm were greatly deformed, and we identified several promising genes related to spermatogenesis and infertility, such as SPEF2, DNAI1, and TACR3, through RNA-seq. CONCLUSIONS: This study provides clear insights into the exploration of potential molecular basis underlying sex differentiation and infertility in sex-reversed chickens and lays a foundation for further research into the sex development of birds.

Structural insights into aldosterone synthase substrate specificity and targeted inhibition.

Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11ß-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents.

17beta-Estradiol levels in male zebra finch brain: combining Palkovits punch and an ultrasensitive radioimmunoassay.

Local aromatization of testosterone into 17beta-estradiol (E(2)) is often required for the physiological and behavioral actions of testosterone. In most vertebrates, aromatase is expressed in a few discrete brain regions. While many studies have measured brain aromatase mRNA or activity, very few studies have measured brain E(2) levels, particularly in discrete brain regions, because of technical challenges. Here, we used the Palkovits punch technique to isolate 13 discrete brain nuclei from adult male zebra finches. Steroids were extracted via solid phase extraction. E(2) was then measured with an ultrasensitive, specific and precise radioimmunoassay. Our protocol leads to high recovery of E(2) (84%) and effectively removes interfering brain lipids. E(2) levels were high in aromatase-rich regions such as caudal medial nidopallium and hippocampus. E(2) levels were intermediate in the medial preoptic area, ventromedial nucleus of the hypothalamus, lateral and medial magnocellular nuclei of anterior nidopallium, nucleus taeniae of the amygdala, and Area X. E(2) levels were largely non-detectable in the cerebellum, HVC, lateral nidopallium and optic lobes. Importantly, E(2) levels were significantly lower in plasma than in the caudal medial nidopallium. This protocol allows one to measure E(2) in discrete brain regions and potentially relate local E(2) concentrations to aromatase activity and behavior.

Morphological sex reversal upon short-term exposure to endocrine modulators in juvenile fathead minnow (Pimephales promelas).

Indications of effects on fish endocrine system have been noted when exposed to effluents of sewage treatment plants and subsequently in the receiving surface waters. For screening purposes, the concentration of vitellogenin (VTG) in plasma is employed to detect potential exposure of fish, to (anti-)estrogenic substances. However, little is known about the variability of VTG determinations and morphological endpoints (secondary sexual characteristics) in fish under exposure conditions employing compounds with hormonal activity other than estrogens. An in vivo test system was established to study the effects of methyltestosterone (MT, a potential model androgen) and fadrozole (F, an aromatase inhibitor) as well as the combination of MT and F on juvenile, sexually undifferentiated fathead minnows (Pimephales promelas). Fish were exposed to those compounds continuously in the (nominal) microg/l range (MT, 10, 50 and 100 microg/l; F, 25, 50, 100 microg/l; MT+F, 10 microg MT per l +50 microg F per l), for 14 days (MT+F) or 21 days (MT and F) using a flow-through system. The concentration of VTG and the expression of VTG mRNA was determined using whole body homogenates in an enzyme linked immunosorbant assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR), respectively. Exposure to MT alone led to de novo mRNA expression as well as up to a four-fold increase of VTG. F had no effect on the VTG mRNA expression and VTG protein synthesis. The combination of MT and F had no effect on VTG concentrations, however, this produced a strong masculinisation of the juvenile fish, e.g. after 13 days of exposure 100% of the fish showed typical male sex characteristics, e.g. formation of nose tubercles and pigmentation of the dorsal fin. The above findings suggest that in fish MT may be aromatised to an estrogen. F, on the other hand, inhibits testosterone aromatisation. Consequently, the combination of MT and F strongly morphologically masculinised the juvenile fathead minnows. VTG detection at the mRNA and protein level is a sensitive parameter, however, it does not provide for any information regarding the baseline "estrogenicity" of a given parent compound.

Molecular modelling of the human cytochrome P450 isoform CYP2A6 and investigations of CYP2A substrate selectivity.

(1) The generation of a homology model of CYP2A6, the major catalyst of human hepatic coumarin 7-hydroxylase activity, involves the use of the recently published substrate-bound CYP102 crystal structure as a template. (2) A substantial number of structurally diverse CYP2A6 substrates are found to dock satisfactorily within the putative active site of the enzyme, leading to the formulation of a structural template (or pharmacophore) for CYP2A6 specificity/selectivity. (3) The CYP2A6 model is consistent with available evidence from site-directed mutagenesis studies carried out on CYP2A subfamily isoforms, and enables some explanation of species differences in CYP2A-mediated metabolism of certain substrates. (4) Quantitative structure-activity relationship (QSAR) analysis of CYP2A5 (the mouse orthologue) mutants yields statistically significant correlations between various properties of amino acid residues and coumarin 7-hydroxylase activity.