Structural basis for FGF hormone signalling.

α/ßKlotho coreceptors simultaneously engage fibroblast growth factor (FGF) hormones (FGF19, FGF21 and FGF23)1,2 and their cognate cell-surface FGF receptors (FGFR1-4) thereby stabilizing the endocrine FGF-FGFR complex3-6. However, these hormones still require heparan sulfate (HS) proteoglycan as an additional coreceptor to induce FGFR dimerization/activation and hence elicit their essential metabolic activities6. To reveal the molecular mechanism underpinning the coreceptor role of HS, we solved cryo-electron microscopy structures of three distinct 1:2:1:1 FGF23-FGFR-αKlotho-HS quaternary complexes featuring the 'c' splice isoforms of FGFR1 (FGFR1c), FGFR3 (FGFR3c) or FGFR4 as the receptor component. These structures, supported by cell-based receptor complementation and heterodimerization experiments, reveal that a single HS chain enables FGF23 and its primary FGFR within a 1:1:1 FGF23-FGFR-αKlotho ternary complex to jointly recruit a lone secondary FGFR molecule leading to asymmetric receptor dimerization and activation. However, αKlotho does not directly participate in recruiting the secondary receptor/dimerization. We also show that the asymmetric mode of receptor dimerization is applicable to paracrine FGFs that signal solely in an HS-dependent fashion. Our structural and biochemical data overturn the current symmetric FGFR dimerization paradigm and provide blueprints for rational discovery of modulators of FGF signalling2 as therapeutics for human metabolic diseases and cancer.

Development of Fe3O4/reduced graphene oxide nanocomposite-based dispersive solid-phase extraction for the quantification of five steroid hormones.

Aim: An accurate and sensitive analytical method was proposed to detect some steroid hormones in biological samples. Materials & methods: An Fe3O4/reduced graphene oxide nanocomposite-based dispersive solid-phase extraction was developed for the effective and simple preconcentration of steroid hormones from human serum samples. Results & conclusion: The nanocomposite was firstly used as adsorbent to simultaneously extract the selected hormones. Limit of detection values for the selected hormones were calculated between 5.5 and 39.2 ng/kg (mass based). An artificial serum sample was used to test the applicability and accuracy of the developed method; percentage recovery results obtained from two different spiked concentrations were found to be in the range of 80.5-99.9%.

Gestational Diabetes Mellitus Is Associated with Differences in Human Milk Hormone and Cytokine Concentrations in a Fully Breastfeeding United States Cohort.

It is unclear whether gestational diabetes mellitus (GDM) alters breast milk composition. We prospectively examined associations of GDM status with concentrations of six potentially bioactive elements (glucose, insulin, C-reactive protein (CRP), interleukin-6 (IL-6), leptin, and adiponectin) in human milk. These were measured at both 1 and 3 months postpartum in 189 fully breastfeeding women. Mixed-effects linear regression assessed GDM status-related differences in these milk bioactives, adjusting for demographics, maternal factors, and diet. At 1 and 3 months postpartum, milk CRP was higher (1.46 ± 0.31 ng/mL; p < 0.001 and 1.69 ± 0.31 ng/mL; p < 0.001) in women with GDM than in women without GDM, whereas milk glucose (−5.23 ± 2.22 mg/dL; p = 0.02 and −5.70 ± 2.22; p = 0.01) and milk insulin (−0.38 ± 0.17 µIU/mL; p = 0.03 and −0.53 ± 0.17; p = 0.003) were lower in women with GDM. These significant associations remained similar after additional adjustment for maternal weight status and its changes. No difference was found for milk IL-6, leptin, and adiponectin. There was no evidence of association between these milk bioactive compounds and 1 h non-fasting oral glucose challenge serum glucose in the women without GDM. This prospective study provides evidence that potentially bioactive elements of human milk composition are altered in women with GDM.

Homogeneous liquid-liquid extraction as an alternative sample preparation technique for biomedical analysis.

Liquid-liquid extraction is a widely used technique of sample preparation in biomedical analysis. In spite of the high pre-concentration capacities of liquid-liquid extraction, it suffers from a number of limitations including time and effort consumption, large organic solvent utilization, and poor performance in highly polar analytes. Homogeneous liquid-liquid extraction is an alternative sample preparation technique that overcomes some drawbacks of conventional liquid-liquid extraction, and allows employing greener organic solvents in sample treatment. In homogeneous liquid-liquid extraction, a homogeneous phase is formed between the aqueous sample and the water-miscible extractant, followed by chemically or physically induced phase separation. To form the homogeneous phase, aqueous samples are mixed with water-miscible organic solvents, water-immiscible solvents/cosolvents, surfactants, or smart polymers. Then, phase separation is induced chemically (adding salt, sugar, or buffer) or physically (changing temperature or pH). This mode is rapid, sustainable, and cost-effective in comparison with other sample preparation techniques. Moreover, homogeneous liquid-liquid extraction is more suitable for the extraction of delicate macromolecules such as enzymes, hormones, and proteins and it is more compatible with liquid chromatography with tandem mass spectrometry, which is a vital technique in metabolomics and proteomics. In this review, the principle, types, applications, automation, and technical aspects of homogeneous liquid-liquid extraction are discussed.

A Photohormone for Light-Dependent Control of PPARα in Live Cells.

Photopharmacology enables the optical control of several biochemical processes using small-molecule photoswitches that exhibit different bioactivities in their cis- and trans-conformations. Such tool compounds allow for high spatiotemporal control of biological signaling, and the approach also holds promise for the development of drug molecules that can be locally activated to reduce target-mediated adverse effects. Herein, we present the expansion of the photopharmacological arsenal to two new members of the peroxisome proliferator-activated receptor (PPAR) family, PPARα and PPARδ. We have developed a set of highly potent PPARα and PPARδ targeting photohormones derived from the weak pan-PPAR agonist GL479 that can be deactivated by light. The photohormone 6 selectively activated PPARα in its trans-conformation with high selectivity over the related PPAR subtypes and was used in live cells to switch PPARα activity on and off in a light- and time-dependent fashion.

Molecular basis for control of antibiotic production by a bacterial hormone.

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.

Leucokinins: Multifunctional Neuropeptides and Hormones in Insects and Other Invertebrates.

Leucokinins (LKs) constitute a neuropeptide family first discovered in a cockroach and later identified in numerous insects and several other invertebrates. The LK receptors are only distantly related to other known receptors. Among insects, there are many examples of species where genes encoding LKs and their receptors are absent. Furthermore, genomics has revealed that LK signaling is lacking in several of the invertebrate phyla and in vertebrates. In insects, the number and complexity of LK-expressing neurons vary, from the simple pattern in the Drosophila larva where the entire CNS has 20 neurons of 3 main types, to cockroaches with about 250 neurons of many different types. Common to all studied insects is the presence or 1-3 pairs of LK-expressing neurosecretory cells in each abdominal neuromere of the ventral nerve cord, that, at least in some insects, regulate secretion in Malpighian tubules. This review summarizes the diverse functional roles of LK signaling in insects, as well as other arthropods and mollusks. These functions include regulation of ion and water homeostasis, feeding, sleep-metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. Other functions are implied by the neuronal distribution of LK, but remain to be investigated.

Noncanonical Sequences Involving NHERF1 Interaction with NPT2A Govern Hormone-Regulated Phosphate Transport: Binding Outside the Box.

Na+/H+ exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser290. The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located "outside the box" explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

Human Family 1-4 cytochrome P450 enzymes involved in the metabolic activation of xenobiotic and physiological chemicals: an update.

This is an overview of the metabolic activation of drugs, natural products, physiological compounds, and general chemicals by the catalytic activity of cytochrome P450 enzymes belonging to Families 1-4. The data were collected from > 5152 references. The total number of data entries of reactions catalyzed by P450s Families 1-4 was 7696 of which 1121 (~ 15%) were defined as bioactivation reactions of different degrees. The data were divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds, presented in tabular form. The metabolism and bioactivation of selected examples of each group are discussed. In most of the cases, the metabolites are directly toxic chemicals reacting with cell macromolecules, but in some cases the metabolites formed are not direct toxicants but participate as substrates in succeeding metabolic reactions (e.g., conjugation reactions), the products of which are final toxicants. We identified a high level of activation for three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the generally low participation of Physiological Compounds in bioactivation reactions. In the group of General Chemicals, P450 enzymes 1A1, 1A2, and 1B1 dominate in the formation of activated metabolites. Drugs are mostly activated by the enzyme P450 3A4, and Natural Products by P450s 1A2, 2E1, and 3A4. Physiological Compounds showed no clearly dominant enzyme, but the highest numbers of activations are attributed to P450 1A, 1B1, and 3A enzymes. The results thus show, perhaps not surprisingly, that Physiological Compounds are infrequent substrates in bioactivation reactions catalyzed by P450 enzyme Families 1-4, with the exception of estrogens and arachidonic acid. The results thus provide information on the enzymes that activate specific groups of chemicals to toxic metabolites.

Protein hormone fragmentation in intercellular signaling: hormones as nested information systems.

This study explores the hypothesis that protein hormones are nested information systems in which initial products of gene transcription, and their subsequent protein fragments, before and after secretion and initial target cell action, play additional physiological regulatory roles. The study produced four tools and key results: (1) a problem approach that proceeds, with examples and suggestions for in vivo organismal functional tests for peptide-protein interactions, from proteolytic breakdown prediction to models of hormone fragment modulation of protein-protein binding motifs in unrelated proteins; (2) a catalog of 461 known soluble human protein hormones and their predicted fragmentation patterns; (3) an analysis of the predicted proteolytic patterns of the canonical protein hormone transcripts demonstrating near-universal persistence of 9 ± 7 peptides of 8 ± 8 amino acids even after cleavage with 24 proteases from four protease classes; and (4) a coincidence analysis of the predicted proteolysis locations and the 1939 exon junctions within the transcripts that shows an excess (P < 0.001) of predicted proteolysis within 10 residues, especially at the exonal junction (P < 0.01). It appears all protein hormone transcripts generate multiple fragments the size of peptide hormones or protein-protein binding domains that may alter intracellular or extracellular functions by acting as modulators of metabolic enzymes, transduction factors, protein binding proteins, or hormone receptors. High proteolytic frequency at exonal junctions suggests proteolysis has evolved, as a complement to gene exon fusion, to extract structures or functions within single exons or protein segments to simplify the genome by discarding archaic one-exon genes.

Beta-Cyclodextrin-Decorated Magnetic Activated Carbon as a Sorbent for Extraction and Enrichment of Steroid Hormones (Estrone, ß-Estradiol, Hydrocortisone and Progesterone) for Liquid Chromatographic Analysis.

A ß-cyclodextrin-decorated magnetic activated carbon adsorbent was prepared and characterized using various analytical techniques (X-ray diffraction (XRD), scanning electron microscopy-electron diffraction spectroscopy (SEM-EDS) and transmission electron microscopy (TEM)), and the adsorbent was used in the development of a magnetic solid-phase microextraction (MSPE) method for the preconcentration of estrone, ß-estradiol, hydrocortisone and progesterone in wastewater and river water samples. This method was optimized using the central composite design in order to determine the experimental parameters affecting the extraction procedure. The quantification of hormones was achieved using high-performance liquid chromatography equipped with a photodiode array detector (HPLC-DAD). Under optimum conditions, the linearity ranged from 0.04 to 300 µg L-1 with a correlation of determinations of 0.9969-0.9991. The limits of detection and quantification were between 0.01-0.03 and 0.033-0.1 µg L-1, with intraday and interday precisions at 1.1-3.4 and 3.2-4.2. The equilibrium data were best described by the Langmuir isotherm model, and high adsorption capacities (217-294 mg g-1) were obtained. The developed procedure demonstrated high potential as an effective technique for use in wastewater samples without significant interferences, and the adsorbent could be reused up to eight times.

Quantitative MALDI-MS assay of steroid hormones in plasma based on hydroxylamine derivatization.

Measuring the concentrations of steroid hormones in plasma is critical for understanding their role in various vital physiological processes. The detection of underivatized steroid hormones in biofluids through mass spectrometry (MS) is typically hindered by low ionization efficiency. We described a novel matrix-assisted laser desorption/ionization-MS (MALDI-MS) approach based on hydroxylamine derivatization (HA-D) to analyze low-concentration steroid hormones in plasma. The ketonic carbonyl group containing steroid hormones could be derivatized using HA to form oxime derivatives, which considerably enhanced the MS sensitivity for detecting steroid hormones. By using the optimized conditions, estrone (E1), testosterone (T), and progesterone (Prog), could be simultaneously quantified in plasma with a limit of detection (LOD) from 0.019 to 0.031 nM, recoveries from 86% to 108%, and coefficient of variation (CV%) from 4.59% to 11.90%. HA-D/MALDI-MS exhibited higher sensitivity than those using Girard T (GT). To establish potential utility of our method, we characterized fatty liver patient plasmas to demonstrate that the HA-D/MALDI-MS procedure could generate quantitative results comparable to the current clinical liquid chromatography-electrospray ionization tandem MS (LC-ESI MS/MS) method. This approach facilitates the rapid and accurate characterization of plasma hormones, and renders the MALDI-MS approach for steroid hormones more adaptable for clinical research and use.

Rapid Induction of the Unfolded Protein Response and Apoptosis by Estrogen Mimic TTC-352 for the Treatment of Endocrine-Resistant Breast Cancer.

Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E4), which are proposed as safer estrogenic agents compared with 17ß-estradiol (E2), for the treatment of endocrine-resistant breast cancer. TTC-352 and E4 are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, ERE DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E2, less potent full agonist E4, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators' binding profiles with SEMs and E4 We describe TTC-352's pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352's benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E2-enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk.

Estrous cyclicity and reproductive success are unaffected by translocation for the formation of new reproductive pairs in captive red wolves (Canis rufus).

This study investigated possible female-related causes for inconsistent success among reproductive pairs in the zoo-based red wolf (Canis rufus) population. Females (n = 13) at seven institutions were assessed for evidence of ovulation and normal reproductive cycles through the measurement of estradiol and progesterone metabolite excretion in feces. Fecal cortisol metabolites (FCM) were also measured. Factors potentially affecting FCM and/or estrous cyclicity were recorded, including exhibit status (on vs. off), reproductive history (proven vs. unproven), copulatory behaviors (ties observed: yes or no), pregnancy/parturition (pups or no pups produced), and translocation before the observed breeding season (yes or no). No differences were observed in baseline FCM between females housed on versus off-exhibit (p = .46) or between females producing pups and those who did not (p = .19). Baseline FCM were significantly lower among females observed in copulatory ties compared to females never observed in a tie (p = .04), and tended to be higher in females translocated before the breeding season compared to females in existing reproductive pairs (p = .11), and among historically unproven females compared to proven females (p = .11). All females evaluated had an endocrine profile indicative of ovulation and among the four females translocated to be paired with a new male before the breeding season, two had successful pregnancies, producing litters. Therefore, despite observed differences in baseline FCM among factors, estrous cyclicity and reproductive success are unaffected by translocation for the formation of new reproductive pairs in the zoo-based red wolf population.

Simultaneous Determination of Prostaglandin and Hormones in Excreta of Trogopterus xanthipes.

The excreta of Trogopterus xanthipes (also called Wulingzhi in Chinese, WLZ) is a well-known traditional Chinese medicine used for the treatment of irregular menstruation in clinic. Few reports are available on the chemical profiling of WLZ. In this work, qualitative and quantitative analyses of endogenous prostaglandin and hormones in WLZ were performed using UHPLC-orbitrap-MSn. In total, 48 compounds were identified in urine of T. xanthipes. Furthermore, the contents of four target compounds were simultaneously quantitated in 20 batches of samples by UPLC-MS/MS. The quantitative method showed a good linear correlation (R > 0.995) in a wide range for each compound. The method had a high sensitivity with LOD (0.5-1.0 ng/mL) and LOQ (1.0-2.5 ng/mL). The intra- and inter-day precisions were < 9.17 (RSD %), and repeatability and stability were < 6.14 (RSD %). The recovery of the analytes varied between 85.8% and 97.3% at three different concentrations. The present integrated qualitative and quantitative assessment of WLZ provides an evaluation strategy to assess the constituent in traditional Chinese medicine.

Peptide KE in Human Proteome.

Peptide KE exhibits immunoprotective, geroprotective, and oncostatic activities and stimulates functional activity of fibroblasts. The KE motif is present in amino acid sequences of some cytokines and peptide hormones functionally similar to KE peptide. However, the relationship between the presence of KE motif and protein functions on the scale of known human proteome has not yet received sufficient attention. The incidence of bioregulatory peptide KE in proteins of various functional groups constituting human proteome is studied. The study is carried out with the use of the available data on the human proteome (UniProt portal) comprising 20,417 proteins. The levels of KE motifs were maximum in cytoplasmic and nuclear proteins, while the presence of KE in the membrane and all other proteins was the minimum. KE peptide molecules released from nuclear proteins during limited proteolysis can bind to DNA and regulate gene expression.

Metabolic profiling by reversed-phase/ion-exchange mass spectrometry.

Metabolic profiling is commonly achieved by mass spectrometry (MS) following reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) either performed independently, leading to overlapping datasets, or in a coupled configuration, requiring multiple liquid chromatography (LC) systems. To overcome these limitations, we developed a single, 20-minute chromatographic method using an in-line RP-ion-exchange (IEX) column arrangement and a single LC system. This configuration separates clinically significant polar and non-polar compounds without derivatization or ion-pairing reagents, allowing ionization in both polarities. An in-house library was created with 397 authentic standards, including acylcarnitines, amino acids, bile acids, nucleosides, organic acids, steroid hormones, and vitamins. Analysis of pooled plasma and urine samples revealed 5445 and 4111 ion features, leading to 88 and 82 confirmed metabolite identifications, respectively. Metabolites were detected at clinically relevant concentrations with good precision, and good chromatographic separation was demonstrated for clinically significant isomers including methylmalonic acid and succinic acid, as well as alloisoleucine and isoleucine/leucine. Evaluation of the samples by unsupervised principal component analysis showed excellent analytical quality.

A Multiplexed Microfluidic Platform toward Interrogating Endocrine Function: Simultaneous Sensing of Extracellular Ca2+ and Hormone.

Extracellular Ca2+ ([Ca2+]ex) is an important regulator of various physiological and pathological functions, including intercellular communication for synchronized cellular activities (e.g., coordinated hormone secretion from endocrine tissues). Yet it is rarely possible to concurrently quantify the dynamic changes of [Ca2+]ex and related bioactive molecules with high accuracy and temporal resolution. This work aims to develop a multiplexed microfluidic platform to enable monitoring oscillatory [Ca2+]ex and hormone(s) in a biomimetic environment. To this end, a low-affinity fluorescent indicator, Rhod-5N, is identified as a suitable sensor for a range of [Ca2+]ex based on its demonstrated high sensitivity and selectivity to Ca2+ in biomedical samples, including human serum and cell culture medium. A microfluidic chip is devised to allow for the immobilization of microscale subjects (analogous to biological tissues), precise control of the perfusion gradient at sites of interest, and integration of modalities for fluorescence measurement and enzyme-linked immunosorbent assay. As this analytical system is demonstrated to be viable to quantify the dynamic changes of Ca2+ (0.2-2 mM) and insulin (15-150 mU L-1) concurrently, with high temporal resolution, it has the potential to provide key insights into the essential roles of [Ca2+]ex in the secretory function of endocrine tissues and to identify novel therapeutic targets for human diseases.

Structure-dependent retention of steroid hormones by common laboratory materials.

The tendency of steroid molecules to adsorb to various materials, particularly plastics, has been known of for decades but has not received widespread attention in the scientific community, and a modern, systematic study is lacking. This adsorption is an important consideration for researchers working with steroid hormones as it could skew the results of various experiments. Here we show that steroids adsorb to various vessels used in experiments, including microcentrifuge tubes, glass vials, and cell culture plates, in a manner that depends on the steroid's molecular structure and on the type of vessel. The lipophilicity of steroids is a strong predictor of the degree of adsorption, with nearly 50 % of the most lipophilic steroid tested, pregnenolone, retained in a high-adsorbing microcentrifuge tube after one hour incubation of an aqueous pregnenolone solution followed by removal of the aqueous solvent. We also show the effects of other factors such as incubation time, centrifugation, and temperature on adsorption, and show that adsorption can be mostly prevented by the presence of serum proteins in steroid solutions and/or by the use of low-adsorbing tubes.

The influence of physiological status on the reproductive behaviour of humpback whales (Megaptera novaeangliae).

For most cetacean species, there is little known about how an individual's physiology influences its behaviour. Humpback whales (Megaptera novaeangliae) are a good candidate to examine such links as they have a well-described distribution and behaviour, can be consistently sampled using remote biopsy systems, and have been the subject of several previous endocrine studies. The objective here was to examine whether a female humpback whale's social state (i.e. escorted by a male or not) is related to her endocrine condition, and whether male dominance ranking is related to testosterone levels. Skin and blubber biopsies were collected from the east and west Australian humpback whale populations in 2010-2016 (n = 252) at multiple times throughout the winter-spring breeding season. Steroid hormones were extracted from blubber and concentrations of progesterone (a marker for pregnancy), testosterone (a marker of male testicular activity) and oestradiol (a potential marker of ovarian activity) measured using enzyme-immunoassays. Principal escorts-the dominant males in mixed sex groups-had significantly higher blubber testosterone levels (mean ±â€¯SE; 1.43 ±â€¯0.20 ng/g wet weight) than subordinate, secondary escorts (0.69 ±â€¯0.06 ng/g wet weight). Females that were escorted by males typically possessed elevated blubber oestradiol levels (1.96 ±â€¯0.25 ng/g wet weight; p = 0.014); few were considered to be pregnant (p = 0.083). 'Unescorted' females displayed characteristically lower blubber oestradiol levels (0.56 ±â€¯0.06 ng/g wet weight). Together, these results are consistent with 'challenge hypothesis' theory and suggest the existence of associated reproductive patterns in humpback whales.