Cord blood lipid correlation network profiles are associated with subsequent attention-deficit/hyperactivity disorder and autism spectrum disorder symptoms at 2 years: a prospective birth cohort study.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are neurodevelopmental conditions with early life origins. Alterations in blood lipids have been linked to ADHD and ASD; however, prospective early life data are limited. This study examined (i) associations between the cord blood lipidome and ADHD/ASD symptoms at 2 years of age, (ii) associations between prenatal and perinatal predictors of ADHD/ASD symptoms and cord blood lipidome, and (iii) mediation by the cord blood lipidome. METHODS: From the Barwon Infant Study cohort (1074 mother-child pairs, 52.3% male children), child circulating lipid levels at birth were analysed using ultra-high-performance liquid chromatography-tandem mass spectrometry. These were clustered into lipid network modules via Weighted Gene Correlation Network Analysis. Associations between lipid modules and ADHD/ASD symptoms at 2 years, assessed with the Child Behavior Checklist, were explored via linear regression analyses. Mediation analysis identified indirect effects of prenatal and perinatal risk factors on ADHD/ASD symptoms through lipid modules. FINDINGS: The acylcarnitine lipid module is associated with both ADHD and ASD symptoms at 2 years of age. Risk factors of these outcomes such as low income, Apgar score, and maternal inflammation were partly mediated by higher birth acylcarnitine levels. Other cord blood lipid profiles were also associated with ADHD and ASD symptoms. INTERPRETATION: This study highlights that elevated cord blood birth acylcarnitine levels, either directly or as a possible marker of disrupted cell energy metabolism, are on the causal pathway of prenatal and perinatal risk factors for ADHD and ASD symptoms in early life. FUNDING: The foundational work and infrastructure for the BIS was sponsored by the Murdoch Children's Research Institute, Deakin University, and Barwon Health. Subsequent funding was secured from the Minderoo Foundation, the European Union's Horizon 2020 research and innovation programme (ENDpoiNTs: No 825759), National Health and Medical Research Council of Australia (NHMRC) and Agency for Science, Technology and Research Singapore [APP1149047], The William and Vera Ellen Houston Memorial Trust Fund (via HOMER Hack), The Shepherd Foundation, The Jack Brockhoff Foundation, the Scobie & Claire McKinnon Trust, the Shane O'Brien Memorial Asthma Foundation, the Our Women Our Children's Fund Raising Committee Barwon Health, the Rotary Club of Geelong, the Ilhan Food Allergy Foundation, Geelong Medical and Hospital Benefits Association, Vanguard Investments Australia Ltd, the Percy Baxter Charitable Trust, and Perpetual Trustees.

Mapping Motor Neuron Vulnerability in the Neuraxis of Male SOD1G93A Mice Reveals Widespread Loss of Androgen Receptor Occurring Early in Spinal Motor Neurons.

Sex steroid hormones have been implicated as disease modifiers in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Androgens, signalling via the androgen receptor (AR), predominate in males, and have widespread actions in the periphery and the central nervous system (CNS). AR translocates to the cell nucleus when activated upon binding androgens, whereby it regulates transcription of target genes via the classical genomic signalling pathway. We previously reported that AR protein is decreased in the lumbar spinal cord tissue of symptomatic male SOD1G93A mice. Here, we further explored the changes in AR within motor neurons (MN) of the CNS, assessing their nuclear AR content and propensity to degenerate by endstage disease in male SOD1G93A mice. We observed that almost all motor neuron populations had undergone significant loss in nuclear AR in SOD1G93A mice. Interestingly, loss of nuclear AR was evident in lumbar spinal MNs as early as the pre-symptomatic age of 60 days. Several MN populations with high AR content were identified which did not degenerate in SOD1G93A mice. These included the brainstem ambiguus and vagus nuclei, and the sexually dimorphic spinal MNs: cremaster, dorsolateral nucleus (DLN) and spinal nucleus of bulbocavernosus (SNB). In conclusion, we demonstrate that AR loss directly associates with MN vulnerability and disease progression in the SOD1G93A mouse model of ALS.

Fatty acids and beyond: Age and Alzheimer's disease related changes in lipids reveal the neuro-nutraceutical potential of lipids in cognition.

Lipids are essential in maintaining brain function, and lipid profiles have been reported to be altered in aged and Alzheimer's disease (AD) brains as compared to healthy mature brains. Both age and AD share common metabolic hallmarks such as increased oxidative stress and perturbed metabolic function, and age remains the most strongly correlated risk factor for AD, a neurodegenerative disease. A major accompanying pathological symptom of these conditions is cognitive impairment, which is linked with changes in lipid metabolism. Thus, nutraceuticals that affect brain lipid metabolism or lipid levels as a whole have the potential to ameliorate cognitive decline. Lipid analyses and lipidomic studies reveal changes in specific lipid types with aging and AD, which can identify potential lipid-based nutraceuticals to restore the brain to a healthy lipid phenotype. The brain lipid profile can be influenced directly with dietary administration of lipids themselves, although because of synergistic effects of nutrients it may be more useful to consider a multi-component diet rather than single nutrient supplementation. Gut microbiota also serve as a source of beneficial lipids, and the value of treatments that manipulate the composition of gut microbiome should not be ignored. Lastly, instead of direct supplementation, compounds that affect pathways involved with lipid metabolism should also be considered as a way of manipulating lipid levels to improve cognition. In this review, we briefly discuss the role of lipids in the brain, the changing lipid profile in AD, current research on lipid-based nutraceuticals and their therapeutic potential to combat cognitive impairment.

Impact of Estrogens on the Regulation of White, Beige, and Brown Adipose Tissue Depots.

As adipose tissue depots are active endocrine organs, they secrete a variety of hormones (including estrogens from white adipose) and inflammatory mediators, which have important implications in numerous obesity-associated diseases. Adipose tissues are broadly characterized as consisting of white, beige, and brown depot types. The endocrine, metabolic, and inflammatory profiles of adipose are depot dependent and influenced by the estrogenic and androgenic status of the adipose tissue. Estrogen receptors mediate both the genomic and nongenomic actions of estrogens and are expressed in the brain, heart, and other peripheral tissues. All three known estrogen receptor α (ERα) and estrogen receptor ß (ERß), and the G-protein coupled estrogen receptor (GPER/GPR30) are expressed in white adipose and can modulate adipose mass. Expression of each receptor is dependent on depot location, adipose cell type, and estrogen levels. Estrogen receptor expression profiles in beige and brown adipocytes are less well established. This review will discuss the effects of estrogens on the differential deposition of the major adipose tissues and the impact of estrogens within white adipose depots. © 2019 American Physiological Society. Compr Physiol 9:457-475, 2019.

Inflammation in Traumatic Brain Injury: Roles for Toxic A1 Astrocytes and Microglial-Astrocytic Crosstalk.

Traumatic brain injury triggers neuroinflammation that may contribute to progressive neurodegeneration. We investigated patterns of recruitment of astrocytes and microglia to inflammation after brain trauma by firstly characterising expression profiles over time of marker genes following TBI, and secondly by monitoring glial morphologies reflecting inflammatory responses in a rat model of traumatic brain injury (i.e. the lateral fluid percussion injury). Gene expression profiles revealed early elevation of expression of astrocytic marker glial fibrillary acidic protein relative to microglial marker allograft inflammatory factor 1 (also known as ionized calcium-binding adapter molecule 1). Adult rat brains collected at day 7 after injury were processed for immunohistochemistry with allograft inflammatory factor 1, glial fibrillary acidic protein and complement C3 (marker of bad/disruptive astrocytic A1 phenotype). Astrocytes positive for glial fibrillary acidic protein and complement C3 were significant increased in the injured cortex and displayed more complex patterns of arbourisation with significantly increased bifurcations. Our observations suggested that traumatic brain injury changed the phenotype of microglia from a ramified appearance with long, thin, highly branched processes to a swollen amoeboid shape in the injured cortex. These findings suggest differential glial activation with astrocytes likely undergoing strategic changes in morphology and function. Whilst a detailed analysis is needed of temporal patterns of glial activation, ours is the first evidence of a role for the bad/disruptive astrocytic A1 phenotype in an open head model of traumatic brain injury.

Pericardial adipose and aromatase: A new translational target for aging, obesity and arrhythmogenesis?

A correlation exists between the extent of pericardial adipose and atrial fibrillation (AF) risk, though the underlying mechanisms remain unclear. Selected adipose depots express high levels of aromatase, capable of converting androgens to estrogens - no studies have investigated aromatase occurrence/expression regulation in pericardial adipose. The Women's Health Initiative reported that estrogen-only therapy in women elevated AF incidence, indicating augmented estrogenic influence may exacerbate cardiac vulnerability. The aim of this study was to identify the occurrence of pericardial adipose aromatase, evaluate the age- and sex-dependency of local cardiac steroid synthesis capacity and seek preliminary experimental evidence of a link between pericardial adipose aromatase capacity and arrhythmogenic vulnerability. Both human atrial appendage and epicardial adipose exhibited immunoblot aromatase expression. In rodents, myocardium and pericardial adipose aromatase expression increased >20-fold relative to young controls. Comparing young, aged and aged-high fat diet animals, a significant positive correlation was determined between the total aromatase content of pericardial adipose and the occurrence/duration of triggered atrial arrhythmias. Incidence and duration of arrhythmias were increased in hearts perfused with 17ß-estradiol. This study provides novel report of pericardial adipose aromatase expression. We show that aromatase expression is remarkably upregulated with aging, and aromatase estrogen conversion capacity significantly elevated with obesity-related cardiac adiposity. Our studies suggest an association between adiposity, aromatase estrogenic capacity and atrial arrhythmogenicity - additional investigation is required to establish causality. The potential impact of these findings may be considerable, and suggests that focus on local cardiac steroid conversion (rather than systemic levels) may yield translational outcomes.

SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24+ CD49fhi Mammary Stem Cell-Enriched Compartment.

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

Sex-dependent changes in neuronal morphology and psychosocial behaviors after pediatric brain injury.

Chronic social behavior problems after pediatric traumatic brain injury (TBI) significantly contribute to poor quality of life for survivors. Using a well-characterized mouse model of early childhood TBI, we have previously demonstrated that young brain-injured mice develop social deficits by adulthood. As biological sex may influence both normal and aberrant social development, we here evaluated potential sex differences in post-TBI psychosocial deficits by comparing the behavior of male and female mice at adulthood (8 weeks post-injury). Secondly, we hypothesized that pediatric TBI would influence neuronal morphology identified by Golgi-Cox staining in the hippocampus and prefrontal cortex, regions involved in social cognition and behavior, before the onset of social problems (3 weeks post-injury). Morphological analysis of pyramidal neurons in the ipsilateral prefrontal cortex and granule cells of the hippocampal dentate gyrus revealed a reduction in dendritic complexity after pediatric TBI. This was most apparent in TBI males, whereas neurons from females were less affected. At adulthood, consistent with previous studies, TBI males showed deficits in sociability and social recognition. TBI females also showed a reduction in sociability, but intact social recognition and increased sociosexual avoidance. Together, these findings indicate that sex is a determinant of regional neuroplasticity and social outcomes after pediatric TBI. Reduced neuronal complexity in the prefrontal cortex and hippocampus, several weeks after injury in male mice, appears to precede the subsequent emergence of social deficits. Sex-specific alterations in the social brain network are thus implicated as an underlying mechanism of social dysfunction after pediatric TBI.

Sexual dimorphism in the glucose homeostasis phenotype of the Aromatase Knockout (ArKO) mice.

We investigated the effects of estrogens on glucose homeostasis using the Aromatase Knockout (ArKO) mouse, which is unable to convert androgens into estrogens. The ArKO mouse is a model of total estrogen ablation which develops symptoms of metabolic syndrome. To determine the development and progression of whole body state of insulin resistance of ArKO mice, comprehensive whole body tolerance tests were performed on WT, ArKO and estrogen administrated mice at 3 and 12 months of age. The absence of estrogens in the male ArKO mice leads to hepatic insulin resistance, glucose and pyruvate intolerance from 3 to 12 months with consistent improvement upon estrogen treatment. Estrogen absence in the female ArKO mice leads to glucose intolerance without pyruvate intolerance or insulin resistance. The replacement of estrogens in the female WT and ArKO mice exhibited both insulin sensitizing and resistance effects depending on age and dosage. In conclusion, this study presents information on the sexually dimorphic roles of estrogens on glucose homeostasis regulation.

Estrogens do not protect, but androgens exacerbate, collagen accumulation in the female mouse kidney after ureteric obstruction.

AIMS: Controversy surrounds the gender basis of progression in chronic kidney disease. Unfortunately, most experimental studies addressing this question do not distinguish between direct effects of estrogen and indirect activation of estrogen receptors through conversion of testosterone to 17ß-estradiol by aromatase. We examined the pathogenesis of renal fibrosis in female aromatase knockout (ArKO) mice, which lack circulating and stored estrogens, while having normal levels of testosterone. MAIN METHODS: ArKO mice and their wild-type (ArWT) counterparts were subjected to unilateral ureteric obstruction (UUO), with kidney tissue collected at day(D) 0, 3 and 9 post-UUO. Effects of 5α-dihydrotestosterone (DHT) administration on each genotype were also studied. Tissue was assessed biochemically and histochemically for fibrosis. Western blot analysis was used to measure α-smooth muscle actin (α-SMA) expression and TGF-ß1 signalling. Matrix metalloproteinase-2 (MMP-2) activity was measured by zymography. KEY FINDINGS: UUO increased collagen content over time (p<0.05 (D3) and p<0.01 (D9) vs day 0), with no difference between genotypes in qualitative (collagen IV staining) and quantitative (hydroxyproline concentration) analyses. Systemic administration of non-aromatizable DHT increased collagen content after 3days of UUO in both genotypes. This was not paralleled by any change in α-SMA (myofibroblast burden) or TGF-ß1 signalling but was commensurate with DHT reducing MMP2 activity in both genotypes (p<0.05 vs genotype controls). SIGNIFICANCE: Physiological concentrations of estrogens do not protect the injured kidney from fibrosis progression. Androgens rather than estrogens are the relevant factor involved in regulating disease-related renal scarring in this model.

Fatty acids and their therapeutic potential in neurological disorders.

There is little doubt that we are what we eat. Fatty acid supplementation and diets rich in fatty acids are being promoted as ways to a healthier brain. Short chain fatty acids are a product of intestinal microbiota metabolism of dietary fibre; and their derivatives are used as an anti-convulstant. They demonstrated therapeutic potential in neurodegenerative conditions as HDAC inhibitors; and while the mechanism is not well understood, have been shown to lower amyloid ß in Alzheimer's Disease in preclinical studies. Medium chain fatty acids consumed as a mixture in dietary oils can induce ketogenesis without the need for a ketogentic diet. Hence, this has the potential to provide an alternative energy source to prevent neuronal cell death due to lack of glucose. Long chain fatty acids are commonly found in the diet as omega fatty acids. They act as an anti-oxidant protecting neuronal cell membranes from oxidative damage and as an anti-inflammatory mediator in the brain. We review which agents, from each fatty acid class, have the most therapeutic potential for neurological disorders (primarily Alzheimer's disease, Parkinson's disease, Autism Spectrum Disorder as well as possible applications to traumatic brain injury), by discussing what is known about their biological mechanisms from preclinical studies.

Effects of Estrogens on Adipokines and Glucose Homeostasis in Female Aromatase Knockout Mice.

The maintenance of glucose homeostasis within the body is crucial for constant and precise performance of energy balance and is sustained by a number of peripheral organs. Estrogens are known to play a role in the maintenance of glucose homeostasis. Aromatase knockout (ArKO) mice are estrogen-deficient and display symptoms of dysregulated glucose metabolism. We aim to investigate the effects of estrogen ablation and exogenous estrogen administration on glucose homeostasis regulation. Six month-old female wildtype, ArKO, and 17ß-estradiol (E2) treated ArKO mice were subjected to whole body tolerance tests, serum examination of estrogen, glucose and insulin, ex-vivo muscle glucose uptake, and insulin signaling pathway analyses. Female ArKO mice display increased body weight, gonadal (omental) adiposity, hyperinsulinemia, and liver triglycerides, which were ameliorated upon estrogen treatment. Tolerance tests revealed that estrogen-deficient ArKO mice were pyruvate intolerant hence reflecting dysregulated hepatic gluconeogenesis. Analyses of skeletal muscle, liver, and adipose tissues supported a hepatic-based glucose dysregulation, with a down-regulation of Akt phosphorylation (a key insulin signaling pathway molecule) in the ArKO liver, which was improved with E2 treatment. Concurrently, estrogen treatment lowered ArKO serum leptin and adiponectin levels and increased inflammatory adipokines such as tumour necrosis factor alpha (TNFα) and interleukin 6 (IL6). Furthermore, estrogen deficiency resulted in the infiltration of CD45 macrophages into gonadal adipose tissues, which cannot be reversed by E2 treatment. This study describes the effects of estrogens on glucose homeostasis in female ArKO mice and highlights a primary phenotype of hepatic glucose dysregulation and a parallel estrogen modified adipokine profile.

Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression.

We derived adult neural stem/progenitor cells (NSPCs) from the sub-ventricular zone of male and female mice to examine direct responses to principal sex hormones. In the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) NSPCs of both sexes expressed nestin and sox2, and could be maintained as neurospheres without addition of any sex hormones. The reverse was not observed; neither testosterone (T), 17ß-estradiol (E2) nor progesterone (P4) was able to support neurosphere growth in the absence of EGF and FGF2. Ten nanomolar T, E2 or P4 induced nestin(+) cell proliferation within 20 min and enhanced neurosphere growth over 7 days irrespective of sex, which was abolished by Erk inhibition with 20 µM U0126. Maintaining neurospheres with each sex hormone did not affect subsequent neuronal differentiation. However, 10 nM T, E2 or P4 added during differentiation increased ßIII tubulin(+) neuron production with E2 being more potent compared to T and P4 in both sexes. Androgen receptor (AR) inhibition with 20 µM flutamide but not aromatase inhibition with 10 µM letrozole reduced basal and T-induced neurosphere growth in females, while only concurrent inhibition of AR and aromatase produced the same effect in males. This sex-specific effect was supported by higher aromatase expression in male neurospheres compared to females measured by Western blot and green fluorescent protein (GFP) reporter. Ten micromolar menadione induced oxidative stress, impaired neurosphere growth and up-regulated aromatase expression in both sexes. However, under oxidative stress letrozole significantly exacerbated impaired neurosphere growth in males only. While both E2 and T could prevent oxidative stress-induced growth reduction in both sexes, the effects of T were dependent on innate aromatase activity. We show for the first time that intrinsic androgen and estrogen signaling may impact the capacity of NSPCs to produce neural progenitors under pathological conditions of oxidative stress.

Myocardial and cardiomyocyte stress resilience is enhanced in aromatase-deficient female mouse hearts through CaMKIIδ activation.

The role of sex steroids in cardioprotection is contentious, with large clinical trials investigating hormone supplementation failing to deliver outcomes expected from observational studies. Mechanistic understanding of androgen/estrogen myocardial actions is lacking. Using a genetic model of aromatase tissue deficiency (ArKO) in female mice, the goal of this investigation was to evaluate the capacity of a shift in cardiac endogenous steroid conversion to influence ischemia-reperfusion resilience by optimizing cardiomyocyte Ca2+ handling responses. In isolated normoxic cardiomyocytes, basal Ca2+ transient amplitude and extent of shortening were greater in ArKO myocytes, with preservation of diastolic Ca2+ levels. Isolated ArKO cardiomyocytes exposed to a high Ca2+ load exhibited greater Ca2+ transient and contractile amplitudes, associated with a greater postrest spontaneous sarcoplasmic reticulum Ca2+ load-release. Microarray differential gene expression analysis of normoxic ventricular tissues from ArKO vs wild-type identified a significant influence of aromatase on genes involved in cardiac Ca2+ handling and signaling [including calmodulin dependent kinase II (CaMKII)-δ], myofilament structure and function, glucose uptake and signaling, and enzymes controlling phosphorylation-specific posttranslational modification status. CaMKII expression was not changed in ventricular tissues, although CaMKIIδ activation and phosphorylation of downstream targets was enhanced in ArKO hearts subjected to ischemia-reperfusion. Overall, this investigation shows that relative withdrawal of estrogen in favor of testosterone through genetically induced tissue aromatase deficiency in females modifies the gene expression profile to effect inotropic support via optimized Ca2+ handling in response to stress, with a modest impact on basal function. Consideration of aromatase inhibition, acutely or chronically, may have a role in cardioprotection, of particular relevance to women.

Aromatase transgenic upregulation modulates basal cardiac performance and the response to ischemic stress in male mice.

Estrogen in females is conventionally considered a cardioprotective influence, but a role for estrogen in male cardioprotection has yet to be defined. Estrogen biosynthesis from testosterone is regulated by aromatase. Aromatase has recently been shown to be expressed in the adult heart, although little is known about its involvement in the regulation of myocardial function and stress responses. The goal of this study was to determine whether upregulation of tissue aromatase expression could improve ischemic resilience in male hearts. Isolated hearts from male transgenic aromatase-overexpressing (AROM(+); high estrogen, low testosterone) mice and wild-type (WT) mice (12 wk) were Langendorff perfused and subjected to ischemia-reperfusion (25 min ischemia and 60 min of reperfusion). Basal systolic function was lower in AROM(+) hearts (dP/dtmax: 4,121 ± 255 vs. 4,992 ± 283 mmHg/s, P < 0.05) and associated with augmented Akt phosphorylation, consistent with a suppressor action of estrogen on contractility. Ischemic contracture was attenuated in AROM(+) hearts (43 ± 3 vs. 55 ± 4 mmHg, P < 0.05), yet AROM(+) hearts were more arrhythmic in early reperfusion. At the end of 60 min of reperfusion, AROM(+) systolic functional recovery was lower (left ventricular developed pressure: 39 ± 6 vs. 56 ± 5 %basal, P < 0.05) and diastolic dysfunction was accentuated (36 ± 4 vs. 24 ± 2 mmHg, P < 0.05). This is the first study to show that in vivo aromatase upregulation modulates basal cardiac performance and the response to ischemic stress. These data suggest that while chronic exposure to enhanced estrogenic influence may have benefits in limiting ischemic contracture severity, acute functional recovery in reperfusion is compromised. A temporally targeted, tissue-specific intervention combining aromatase treatment with inotropic support may offer therapeutic potential for men and women.

Characterization of aromatase expression in the adult male and female mouse brain. I. Coexistence with oestrogen receptors α and ß, and androgen receptors.

Aromatase catalyses the last step of oestrogen synthesis. There is growing evidence that local oestrogens influence many brain regions to modulate brain development and behaviour. We examined, by immunohistochemistry, the expression of aromatase in the adult male and female mouse brain, using mice in which enhanced green fluorescent protein (EGFP) is transcribed following the physiological activation of the Cyp19A1 gene. EGFP-immunoreactive processes were distributed in many brain regions, including the bed nucleus of the stria terminalis, olfactory tubercle, medial amygdaloid nucleus and medial preoptic area, with the densest distributions of EGFP-positive cell bodies in the bed nucleus and medial amygdala. Differences between male and female mice were apparent, with the density of EGFP-positive cell bodies and fibres being lower in some brain regions of female mice, including the bed nucleus and medial amygdala. EGFP-positive cell bodies in the bed nucleus, lateral septum, medial amygdala and hypothalamus co-expressed oestrogen receptor (ER) α and ß, or the androgen receptor (AR), although single-labelled EGFP-positive cells were also identified. Additionally, single-labelled ERα-, ERß- or AR-positive cell bodies often appeared to be surrounded by EGFP-immunoreactive nerve fibres/terminals. The widespread distribution of EGFP-positive cell bodies and fibres suggests that aromatase signalling is common in the mouse brain, and that locally synthesised brain oestrogens could mediate biological effects by activating pre- and post-synaptic oestrogen α and ß receptors, and androgen receptors. The higher number of EGFP-positive cells in male mice may indicate that the autocrine and paracrine effects of oestrogens are more prominent in males than females.

Hepatic glucose intolerance precedes hepatic steatosis in the male aromatase knockout (ArKO) mouse.

Estrogens are known to play a role in modulating metabolic processes within the body. The Aromatase knockout (ArKO) mice have been shown to harbor factors of Metabolic syndrome with central adiposity, hyperinsulinemia and male-specific hepatic steatosis. To determine the effects of estrogen ablation and subsequent replacement in males on whole body glucose metabolism, three- and six-month-old male ArKO mice were subjected to whole body glucose, insulin and pyruvate tolerance tests and analyzed for ensuing metabolic changes in liver, adipose tissue, and skeletal muscle. Estrogen-deficient male ArKO mice showed increased gonadal adiposity which was significantly reduced upon 17ß-estradiol (E2) treatment. Concurrently, elevated ArKO serum leptin levels were significantly reduced upon E2 treatment and lowered serum adiponectin levels were restored to wild type levels. Three-month-old male ArKO mice were hyperglycemic, and both glucose and pyruvate intolerant. These phenotypes continued through to 6 months of age, highlighting a loss of glycemic control. ArKO livers displayed changes in gluconeogenic enzyme expression, and in insulin signaling pathways upon E2 treatment. Liver triglycerides were increased in the ArKO males only after 6 months of age, which could be reversed by E2 treatment. No differences were observed in insulin-stimulated ex vivo muscle glucose uptake nor changes in ArKO adipose tissue and muscle insulin signaling pathways. Therefore, we conclude that male ArKO mice develop hepatic glucose intolerance by the age of 3 months which precedes the sex-specific development of hepatic steatosis. This can be reversed upon the administration of exogenous E2.

The aromatase gene CYP19A1: several genetic and functional lines of evidence supporting a role in reading, speech and language.

Inspired by the localization, on 15q21.2 of the CYP19A1 gene in the linkage region of speech and language disorders, and a rare translocation in a dyslexic individual that was brought to our attention, we conducted a series of studies on the properties of CYP19A1 as a candidate gene for dyslexia and related conditions. The aromatase enzyme is a member of the cytochrome P450 super family, and it serves several key functions: it catalyzes the conversion of androgens into estrogens; during early mammalian development it controls the differentiation of specific brain areas (e.g. local estrogen synthesis in the hippocampus regulates synaptic plasticity and axonal growth); it is involved in sexual differentiation of the brain; and in songbirds and teleost fishes, it regulates vocalization. Our results suggest that variations in CYP19A1 are associated with dyslexia as a categorical trait and with quantitative measures of language and speech, such as reading, vocabulary, phonological processing and oral motor skills. Variations near the vicinity of its brain promoter region altered transcription factor binding, suggesting a regulatory role in CYP19A1 expression. CYP19A1 expression in human brain correlated with the expression of dyslexia susceptibility genes such as DYX1C1 and ROBO1. Aromatase-deficient mice displayed increased cortical neuronal density and occasional cortical heterotopias, also observed in Robo1-/- mice and human dyslexic brains, respectively. An aromatase inhibitor reduced dendritic growth in cultured rat neurons. From this broad set of evidence, we propose CYP19A1 as a candidate gene for human cognitive functions implicated in reading, speech and language.

A doxycycline-inducible, tissue-specific aromatase-expressing transgenic mouse.

Aromatase converts androgens to estrogens and it is expressed in gonads and non-reproductive tissues (e.g. brain and adipose tissues). As circulating levels of estrogens in males are low, we hypothesize that local estrogen production is important for the regulation of physiological functions (e.g. metabolism) and pathological development (e.g. breast and prostate cancers) by acting in a paracrine and/or intracrine manner. We generated a tissue-specific doxycycline-inducible, aromatase transgenic mouse to test this hypothesis. The transgene construct (pTetOAROM) consists of a full-length human aromatase cDNA (hAROM) and a luciferase gene under the control of a bi-directional tetracycline-responsive promoter (pTetO), which is regulated by transactivators (rtTA or tTA) and doxycycline. Our in vitro studies using MBA-MB-231tet cells stably expressing rtTA, showed that doxycycline treatment induced transgene expression of hAROM transcripts by 17-fold (P = 0.01), aromatase activity by 26-fold, (P = 0.0008) and luciferase activity by 9.6-fold (P = 0.0006). Pronuclear microinjection of the transgene generated four pTetOAROM founder mice. A male founder was bred with a female mammary gland-specific rtTA mouse (MMTVrtTA) to produce MMTVrtTA-pTetOAROM double-transgenic mice. Upon doxycycline treatment via drinking water, human aromatase expression was detected by RT-PCR, specifically in mammary glands, salivary glands and seminal vesicles of double-stransgenic mice. Luciferase expression and activity was detected in these tissues by in vivo bioluminescence imaging, in vitro luciferase assay and RT-PCR. In summary, we generated a transgenic mouse model that expresses the human aromatase transgene in a temporal- and spatial-specific manner, which will be a useful model to study the physiological importance of local estrogen production.

Relaxin and castration in male mice protect from, but testosterone exacerbates, age-related cardiac and renal fibrosis, whereas estrogens are an independent determinant of organ size.

This study determined the effects of castration and hormone replacement therapy on the age-related cardiac and renal pathology of male relaxin gene-knockout (RlnKO) and age-matched wild-type (RlnWT) mice and that of aged male aromatase knockout (ArKO) mice, which lack estrogens and have 5-10 times the androgen levels of male wild-type mice. One-month-old RlnWT and RlnKO mice were bilaterally gonadectomized or sham operated and maintained until 12 months. Subgroups of castrated animals received testosterone or 17ß-estradiol treatment from 9 to 12 months. Male ArKO mice and aromatase wild-type mice were aged to 12 months. Collected heart and kidney tissues were assessed for changes in organ size and fibrosis. Castration reduced body, heart, left ventricle, and kidney weights in both RlnKO and RlnWT mice, and the cardiac/renal fibrosis that was seen in sham RlnKO animals (all P < 0.05 vs. respective sham). Testosterone normalized organ weights and organ weight to body weight ratio of castrated animals and increased cardiac/renal collagen concentration to levels measured in or beyond that of sham RlnKO mice (all P < 0.05 vs. respective castrated mice). Furthermore, expression of TGF-ß1, mothers against decapentaplegic homolog 2 (Smad2), and myofibroblast differentiation paralleled the above changes (all P < 0.05 vs. respective castrated mice), whereas matrix metalloproteinase-13 was decreased in testosterone-treated RlnKO mice. Conversely, 17ß-estradiol only restored changes in organ size. Consistent with these findings, intact ArKO mice demonstrated increased cardiac/renal fibrosis in the absence of changes in organ size. These findings suggest that relaxin and castration protect, whereas androgens exacerbate, cardiac and renal fibrosis during ageing, whereas estrogens, in synergy with relaxin, regulates age-related changes in organ size.