Aromatase Knockout Mice Research Articles

Sex‐specific impact of brain estrogen deficiency on astrocyte reactivity, neurodegeneration, and memory loss in aromatase knockout mouse models

AbstractBackgroundNearly two‐thirds of Americans with Alzheimer’s disease (AD) are women, however, the molecular mechanisms underlying this sex difference in AD vulnerability remain unclear. Prior research has reported that women with AD have lower brain estrogen. Yet, how estrogen deficiency modulates this sex difference in AD is not well understood. Aromatase, the key enzyme for estrogen biosynthesis, is expressed in both neurons and astrocytes of the brain, with high levels in the hippocampus. To explore the underlying mechanism of estrogen deficiency in sex‐specific AD vulnerability, we generated mouse models with brain‐specific aromatase knockout (bArKO) and whole‐body total aromatase knockout (tArKO).MethodWe performed a series of memory and behavior tests including spontaneous alternation, social interaction, and tail suspension tests on 6‐ and 19‐month‐old bArKO and tArKO mice with both sexes (n = 10/group). Hippocampus tissues from these mice (n = 3∼4) were collected for bulk RNA‐seq and qPCR. Brain tissues (n = 5) were fixed and stained for astrocytes and neurons by immunofluorescence. Serum and tissue estrogen were measured using liquid chromatography‐mass spectrometry.ResultAromatase deletion decreased brain estrogen levels in bArKO mice and circulating and brain estrogen levels in tArKO mice. Impairments of spatial working memory and social interaction behavior were found in 19‐month‐old female bArKO and tArKO mice. Female tArKO mice also displayed depression‐like behavior at 6‐ and 19‐month‐old. The number of neurons in the CA1 region was significantly decreased in 19‐month‐old female (but not male) tArKO mice. Bulk RNA‐seq showed abnormal expression of genes associated with astrocyte reactivity (e.g., Gfap, Ctgf, Apoe, Lcn2, Sparc, Bdnf, Col1a1, Col1a2) only in aged female bArKO mice. Given growing evidence indicating a causal relationship between neurotoxic reactive astrocytes and neurodegeneration, this finding indicates brain estrogen deficiency triggering neurotoxic astrocyte reactivity in the hippocampus of aged female bArKO mice.ConclusionOur findings provide novel mechanisms that link estrogen deficiency to sex‐specific astrocyte reactivity in the hippocampus, and neurodegeneration and memory loss in aged female mice. Future studies will focus on defining impact of estrogen signaling deficiency on AD mouse models and identifying druggable targets to inform the development of new therapeutic strategies for the prevention and treatment of AD.

P-677 Letrozole inhibits ovulation via reducing responsiveness to luteinizing hormone in in vitro-grown mouse follicles

Abstract Study question Does letrozole reduce responsiveness to luteinizing hormone (LH) in in vitro-grown mouse follicles? Summary answer We found that letrozole reduced responsiveness to LH via reducing of luteinizing hormone/chorionic gonadotropin receptor (Lhcgr) transcription in in vitro. What is known already The continuous dosing of letrozole, the third-generation aromatase inhibitor, is used for the controlled ovarian stimulation (COS) of fertility preservation cycles in women with hormone-sensitive breast cancer to prevent the transient rise of serum estrogen levels. Recently, lower oocyte maturation rates and higher abnormal fertilization rates in COS with letrozole compared to standard COS have been reported. The studies using aromatase knockout mice and estrogen receptor beta knockout mice showed that intra-follicular estrogen is essential for responsiveness to LH of granulosa cells. However, whether letrozole reduces responsiveness to LH of follicles remains unknown. Study design, size, duration We evaluated the effect of letrozole on responsiveness to LH using in vitro mouse follicle culture system. Preantral follicles (150-200 µm) with a small number of stromal cells were isolated from 3 weeks old C57BL/6jjcl mouse ovaries and cultured in the presence of letrozole. We analyzed in vitro ovulation ability, and expression levels of ovulation-related genes to clarify the effect of letrozole on responsiveness to LH of follicles. Participants/materials, setting, methods The follicles were cultured with letrozole (0.01µM, 0.1µM, or 1µM) or vehicle (0.1% DMSO) for 5 days followed by 5 IU/ml hCG and 5 ng/ml hEGF stimulation as ovulation induction. The diameter and survival rate of follicles were assessed. After16 hours after ovulation induction, we assessed the ovulation and measured the transcription of Lhcgr, Ptgs2, Runx1 and Actb (internal control) in the follicles by RT-qPCR. We conducted a collateral experiment in the presence of estradiol. Main results and the role of chance The follicle growth and survival rate did not change by letrozole in vitro. However, in vitro ovulation was reduced by the addition of letrozole in a dose-dependent manner (Cochran-Armitage trend test, P < 0.0001): 41.2% in the 0.01µM letrozole treated group (n = 23), 10.5% in the 0.1µM letrozole treated-group (n = 17), and no ovulation in the 1µM letrozole treated-group (n = 19) compared with 69.6% in the vehicle-group (n = 18). Exogenous estrogen addition restored the ovulation ability of 1µM letrozole-treated follicle to the same levels as the vehicle group. RT-qPCR revealed that 0.1 µM letrozole significantly reduce the transcription levels of Lhcgr (P = 0.0027), Runx1 (P = 0.0124), and Ptgs2 (P = 0.0067) compared with vehicle-treated follicles. Exogenous estrogen addition restored the transcription of these genes to levels observed in vehicle-treated follicles. We also observed a positive correlation between the transcription of Lhcgr and Ptgs2 (R = 0.55, P = 0.0003), Lhcgr and Runx1 (R = 0.71, P < 0.0001), and Ptgs2 and Runx1 (R = 0.77, P < 0.0001). Our results suggested that letrozole-treated follicles impaired ovulation due to dysregulation of Lhcgr transcription and its downstream cascade caused by estrogen deficiency. Limitations, reasons for caution This is an in vitro experiment using a mouse model. Further research is needed to determine whether our findings are applicable to humans. Wider implications of the findings Our findings imply that letrozole may have disadvantages in not only ovulation, but also subsequent reproductive processes affected by the effectiveness of the LH surge, such as oocyte maturation, fertilization, and preimplantation embryo development. When evaluating oocyte cryopreservation for fertility preservation, it may be necessary to consider the COS protocol. Trial registration number Not applicable

Development and Characterization of Inducible Astrocyte-Specific Aromatase Knockout Mice.

17β-estradiol (E2) is produced in the brain as a neurosteroid, in addition to being an endocrine signal in the periphery. The current animal models for studying brain-derived E2 include global and conditional non-inducible knockout mouse models. The aim of this study was to develop a tamoxifen (TMX)-inducible astrocyte-specific aromatase knockout mouse line (GFAP-ARO-iKO mice) to specifically deplete the E2 synthesis enzymes and aromatase in astrocytes after their development in adult mice. The characterization of the GFAP-ARO-iKO mice revealed a specific and robust depletion in the aromatase expressions of their astrocytes and a significant decrease in their hippocampal E2 levels after a GCI. The GFAP-ARO-iKO animals were alive and fertile and had a normal general brain anatomy, with a normal astrocyte shape, intensity, and distribution. In the hippocampus, after a GCI, the GFAP-ARO-iKO animals showed a major deficiency in their reactive astrogliosis, a dramatically increased neuronal loss, and increased microglial activation. These findings indicate that astrocyte-derived E2 (ADE2) regulates the ischemic induction of reactive astrogliosis and microglial activation and is neuroprotective in the ischemic brain. The GFAP-ARO-iKO mouse models thus provide an important new model to help elucidate the roles and functions of ADE2 in the brain.

Brain-Derived Estrogen and Neurological Disorders.

Astrocytes and neurons in the male and female brains produce the neurosteroid brain-derived 17β-estradiol (BDE2) from androgen precursors. In this review, we discuss evidence that suggest BDE2 has a role in a number of neurological conditions, such as focal and global cerebral ischemia, traumatic brain injury, excitotoxicity, epilepsy, Alzheimer's disease, and Parkinson's disease. Much of what we have learned about BDE2 in neurological disorders has come from use of aromatase inhibitors and global aromatase knockout mice. Recently, our group developed astrocyte- and neuron-specific aromatase knockout mice, which have helped to clarify the precise functions of astrocyte-derived 17β-estradiol (ADE2) and neuron-derived 17β-estradiol (NDE2) in the brain. The available evidence to date suggests a primarily beneficial role of BDE2 in facilitating neuroprotection, synaptic and cognitive preservation, regulation of reactive astrocyte and microglia activation, and anti-inflammatory effects. Most of these beneficial effects appear to be due to ADE2, which is induced in most neurological disorders, but there is also recent evidence that NDE2 exerts similar beneficial effects. Furthermore, in certain situations, BDE2 may also have deleterious effects, as recent evidence suggests its overproduction in epilepsy contributes to seizure induction. In this review, we examine the current state of this quickly developing topic, as well as possible future studies that may be required to provide continuing growth in the field.

Neuron-Derived Estrogen-A Key Neuromodulator in Synaptic Function and Memory.

In addition to being a steroid hormone, 17β-estradiol (E2) is also a neurosteroid produced in neurons in various regions of the brain of many species, including humans. Neuron-derived E2 (NDE2) is synthesized from androgen precursors via the action of the biosynthetic enzyme aromatase, which is located at synapses and in presynaptic terminals in neurons in both the male and female brain. In this review, we discuss evidence supporting a key role for NDE2 as a neuromodulator that regulates synaptic plasticity and memory. Evidence supporting an important neuromodulatory role of NDE2 in the brain has come from studies using aromatase inhibitors, aromatase overexpression in neurons, global aromatase knockout mice, and the recent development of conditional forebrain neuron-specific knockout mice. Collectively, these studies demonstrate a key role of NDE2 in the regulation of synapse and spine density, efficacy of excitatory synaptic transmission and long-term potentiation, and regulation of hippocampal-dependent recognition memory, spatial reference memory, and contextual fear memory. NDE2 is suggested to achieve these effects through estrogen receptor-mediated regulation of rapid kinase signaling and CREB-BDNF signaling pathways, which regulate actin remodeling, as well as transcription, translation, and transport of synaptic proteins critical for synaptic plasticity and function.

Astrocyte-Derived Estrogen Regulates Reactive Astrogliosis and is Neuroprotective following Ischemic Brain Injury.

Expression of the 17β-estradiol (E2) synthesis enzyme aromatase is highly upregulated in astrocytes following brain injury. However, the precise role of astrocyte-derived E2 in the injured brain remains unclear. In the current study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse model to deplete astrocyte-derived E2 in the brain and determine its roles after global cerebral ischemia (GCI) in male and female mice. GFAP-ARO-KO mice were viable and fertile, with normal gross brain structure, normal morphology, intensity and distribution of astrocytes, normal aromatase expression in neurons, and normal cognitive function basally. In contrast, after GCI, GFAP-ARO-KO mice: (1) lacked the normal elevation of astrocyte aromatase and hippocampal E2 levels; (2) had significantly attenuated reactive astrogliosis; and (3) displayed enhanced neuronal damage, microglia activation, and cognitive deficits. RNA-sequencing (RNA-seq) analysis revealed that the ischemic GFAP-ARO-KO mouse hippocampus failed to upregulate the "A2" panel of reactive astrocyte genes. In addition, the JAK-STAT3 pathway, which is critical for the induction of reactive astrogliosis, was significantly downregulated in the GFAP-ARO-KO hippocampus following GCI. Finally, exogenous E2 administration fully rescued the compromised JAK-STAT3 pathway and reactive astrogliosis, and reversed the enhanced neuronal damage and microglial activation in the GFAP-ARO-KO mice after GCI, suggesting that the defects in the KO mice are because of a loss of E2 rather than an increase in precursor androgens. In conclusion, the current study provides novel genetic evidence for a beneficial role of astrocyte-derived E2 in reactive astrogliosis, microglial activation, and neuroprotection following an ischemic injury to the brain.SIGNIFICANCE STATEMENT Following cerebral ischemia, reactive astrocytes express the enzyme aromatase and produce 17β-estradiol (E2), although the precise role of astrocyte-derived E2 is poorly understood. In this study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse to deplete astrocyte-derived E2 and elucidate its roles after global cerebral ischemia (GCI). The GFAP-ARO-KO mice exhibited significantly attenuated reactive astrogliosis, as well as enhanced microglial activation, neuronal damage, and cognitive dysfunction after GCI. Transcriptome analysis further revealed that astrocyte-derived E2 was critical for the induction of the JAK-STAT3 signaling pathway, as well as the A2 reactive astrocyte phenotype after ischemia. Collectively, these findings indicate that astrocyte-derived E2 has a key role in the regulation of reactive astrogliosis, microglial activation, and neuroprotection after cerebral ischemia.

Brain Aromatase and the Regulation of Sexual Activity in Male Mice.

The biologically active estrogen estradiol has important roles in adult brain physiology and sexual behavior. A single gene, Cyp19a1, encodes aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol in the testis and brain of male mice. Estradiol formation was shown to regulate sexual activity in various species, but the relative contributions to sexual behavior of estrogen that arises in the brain versus from the gonads remained unclear. To determine the role of brain aromatase in regulating male sexual activity, we generated a brain-specific aromatase knockout (bArKO) mouse. A newly generated whole-body total aromatase knockout mouse of the same genetic background served as a positive control. Here we demonstrate that local aromatase expression and estrogen production in the brain is partially required for male sexual behavior and sex hormone homeostasis. Male bArKO mice exhibited decreased sexual activity in the presence of strikingly elevated circulating testosterone. In castrated adult bArKO mice, administration of testosterone only partially restored sexual behavior; full sexual behavior, however, was achieved only when both estradiol and testosterone were administered together. Thus, aromatase in the brain is, in part, necessary for testosterone-dependent male sexual activity. We also found that brain aromatase is required for negative feedback regulation of circulating testosterone of testicular origin. Our findings suggest testosterone activates male sexual behavior in part via conversion to estradiol in the brain. These studies provide foundational evidence that sexual behavior may be modified through inhibition or enhancement of brain aromatase enzyme activity and/or utilization of selective estrogen receptor modulators.

OR09-03 Brain Aromatase Is Essential for Regulation of Sexual Activity in Male Mice

Introduction: The biologically active form of estrogen, estradiol (E2), has important organizational roles in brain development and activational roles in adult brain physiology and behavior. It has been proposed that E2 formation in the brain might regulate sexual activity in various species. The mechanisms that link estrogen formation in the brain and sexual behavior, however, remain unclear. Aromatase is the key enzyme that catalyzes the conversion of testosterone (T) to E2 in the testis and brain of male mice. To determine the role of brain aromatase in male sexual activity, we generated a brain-specific aromatase knockout (bArKO) mouse model. Additionally, a newly generated total aromatase knockout (tArKO) mouse model served as a positive control. Methods: We generated the floxed aromatase mice (Aromfl/fl), which flanked the transcription and translation start sites and the common splice acceptor site for the upstream brain promoter I.f of the aromatase gene. We then crossed Nestin-Cre mice with Aromfl/fl mice to generate bArKO mice. Using the same Aromfl/fl mice, we bred tArKO via crossing with ZP3-Cre mice. Circulating and tissue (brain and testis) E2 levels were measured using liquid chromatography-tandem mass spectrometry. We assessed sexual activity in 12-14 week-old bArKO, tArKO and littermate control males over two 30-minute trials. The interactions were monitored and videotaped, and the videotape was scored for the sexual activity. To investigate whether the lack of estrogen production in the brain was causative for altered sexual behavior, 20 bArKO and 20 control mice were castrated at ~nine weeks of age and supplemented with exogenous sex hormone via 60-day time release pellet implantation. Results: E2 levels are significantly decreased in the brain but not the testis of bArKO mice as compared to control mice (P < 0.05, n=6-12). As expected, E2 levels in the brain and testis are significantly lower in tArKO mice compared with their WT littermates (n=6-9). Furthermore, we demonstrate that local aromatase expression and estrogen production in the brain is required for male sexual behavior and sex hormone homeostasis. Male bArKO mice exhibited significantly decreased sexual activity in the presence of strikingly elevated circulating T (n=5). In castrated adult bArKO mice, administration of E2 together with T restored maximum sexual behavior (n=5). Thus, aromatase in the brain is necessary for T-dependent male sexual activity. We also found that brain aromatase is required for negative feedback regulation of circulating T of testicular origin. Conclusion: Our findings suggest T activates male sexual behavior in part via conversion to E2 in the brain and provide the foundation for inhibition or enhancement of brain aromatase enzyme activity and/or utilization of selective estrogen receptor modulators in modifying sexual behavior.DCB and HZ contributed equally to this work.

Changes in nucleus accumbens gene expression accompany sex-specific suppression of spontaneous physical activity in aromatase knockout mice

Aromatase catalyzes conversion of testosterone to estradiol and is expressed in a variety of tissues, including the brain. Suppression of aromatase adversely affects metabolism and physical activity behavior, but mechanisms remain uncertain. The hypothesis tested herein was that whole body aromatase deletion would cause gene expression changes in the nucleus accumbens (NAc), a brain regulating motivated behaviors such as physical activity, which is suppressed with loss of estradiol. Metabolic and behavioral assessments were performed in male and female wild-type (WT) and aromatase knockout (ArKO) mice. NAc-specific differentially expressed genes (DEGs) were identified with RNAseq, and associations between the measured phenotypic traits were determined. Female ArKO mice had greater percent body fat, reduced spontaneous physical activity (SPA), consumed less energy, and had lower relative resting energy expenditure (REE) than WT females. Such differences were not observed in ArKO males. However, in both sexes, a top DEG was Pts, a gene encoding an enzyme necessary for catecholamine (e.g., dopamine) biosynthesis. In comparing male and female WT mice, top DEGs were related to sexual development/fertility, immune regulation, obesity, dopamine signaling, and circadian regulation. SPA correlated strongly with Per3, a gene regulating circadian function, thermoregulation, and metabolism (r = −0.64, P = .002), which also correlated with adiposity (r = 0.54, P = .01). In conclusion, aromatase ablation leads to gene expression changes in NAc, which may in turn result in reduced SPA and related metabolic abnormalities. These findings may have significance to post-menopausal women and those treated with an aromatase inhibitor.

A loss of estrogen signaling in the aromatase deficient mouse penis results in mild hypospadias

Hypospadias is the abnormal opening of the urethra on the underside of the penis and occurs in approximately 1/125 live male births worldwide. The incidence rate of hypospadias has dramatically increased over the past few decades. This is now attributed, at least in part, to our exposure to endocrine-disrupting chemicals (EDCs) which alter the hormonal signals required for development of the penis. In humans androgens are the main drivers of fusion of the urethral folds to form the urethra within the shaft of the penis, a process required for termination of the urethra in its normal location at the tip of the penis. However, recent research has suggested that estrogen also plays a role in this process. To better understand how EDCs impact urethral development it is essential that we understand the normal function of hormones during development of the penis. To define the role of estrogen in urethral development we examined development of the penis in the aromatase (Cyp19a1) Knockout (ArKO) mouse strain in which endogenous estrogen production is completely ablated. We found that the ArKO penis had a mild hypospadias phenotype. The developing ArKO postnatal penis displayed an early disruption in preputial development, which likely causes the mild hypospadias observed in adults. Using qPCR, we found altered expression of keratin genes and key urethral patterning genes in response to the disrupted estrogen signaling. The hypospadias phenotype was almost identical to that reported for the estrogen receptor α (ERα) knockout confirming that ERα is the predominant receptor for mediating estrogen action during development of the mouse penis. Our results show that estrogen is required for normal prepucial development and placement of the mature urethral opening at the distal aspect of the penis. We also identified several genes which are potential downstream targets of estrogen during normal urethral closure. With this knowledge, we can now better understand how anti-estrogenic as well as estrogenic EDCs disrupt urethral closure to cause mild hypospadias in both mice and humans.

Sex‐dependency of oestrogen‐induced structural synaptic plasticity: Inhibition of aromatase versus application of estradiol in rodents

Sex-dependent differences in learning and memory formation in humans have been frequently shown. The mechanisms underlying the formation and retention of memories are assumed to involve synaptic plasticity in the hippocampus. Estradiol was shown to effect synaptic plasticity in the hippocampus of rodents. The effects after exogenous application of estradiol to animals frequently produce inconsistent results, in particular, if sex is not considered in the studies. Recently we provided evidence that locally synthesized estradiol plays an essential role on synaptic connectivity in the hippocampus of females but not of male mice. In females, inhibition of local estradiol synthesis leads to synapse loss, which results from impairment of long-term potentiation and dephosphorylation of cofilin, and thereby the destabilization of postsynaptic dendritic spines. This sex-dependency was also seen in the classical aromatase knock-out mouse. Intriguingly, no differences between sexes have been found in a conditional forebrain-specific aromatase knock-out mouse. Altogether, the findings underscore the necessity of including 'Sex as a Biological Variable' in studies of sex steroid-induced synaptic plasticity.

Higher Insulin Resistance and Adiposity in Postmenopausal Women With Breast Cancer Treated With Aromatase Inhibitors.

ContextAromatase deficiency causes obesity and insulin resistance in aromatase knockout mice and humans with rare mutations of the aromatase gene (CYP19). Aromatase inhibitors are a commonly prescribed therapy for postmenopausal breast cancer.ObjectiveWe hypothesized that aromatase inhibitors induce obesity and insulin resistance when used in treatment of breast cancer.DesignCase-control study.SettingUniversity teaching hospital.ParticipantsPatients with postmenopausal breast cancer (n = 20) treated with aromatase inhibitors and 20 age-matched control subjects.Main outcome measuresThe primary outcome measure was insulin sensitivity index – Matsuda, derived from a 75-g oral glucose tolerance test. Body composition was assessed by dual energy x-ray absorptiometry and biopsy specimens of subcutaneous adipose tissue obtained for assessment of mRNA transcript levels. Data are reported as mean ± SEM (patients receiving inhibitors vs control group, respectively).ResultsAromatase inhibitor therapy was associated with significantly lower insulin sensitivity (5.15 ± 0.45 vs 6.80 ± 0.64; P = 0.041), higher peak insulin concentration after oral glucose tolerance test (693.4 ± 78.6 vs 527.6 ± 85.5 pmol/L; P = 0.035), greater percentage of body fat (38.4% ± 1.0% vs 34.6% ± 1.3%; P = 0.026), and higher plasma leptin concentration (23.5 ± 2.8 vs 15.5 ± 2.3 ng/mL; P = 0.035).ConclusionWomen who received aromatase inhibitors for postmenopausal breast cancer had greater percentage body fat and insulin resistance compared with control subjects with no history of breast cancer.

Neuron-Derived Estrogen Regulates Synaptic Plasticity and Memory.

17β-estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but its precise functions in the brain are unclear. Here, we used a forebrain-neuron-specific aromatase knock-out (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain of mice and thereby elucidate its functions. FBN-ARO-KO mice showed a 70–80% decrease in aromatase and forebrain E2 levels compared with FLOX controls. Male and female FBN-ARO-KO mice exhibited significant deficits in forebrain spine and synaptic density, as well as hippocampal-dependent spatial reference memory, recognition memory, and contextual fear memory, but had normal locomotor function and anxiety levels. Reinstating forebrain E2 levels via exogenous in vivo E2 administration was able to rescue both the molecular and behavioral defects in FBN-ARO-KO mice. Furthermore, in vitro studies using FBN-ARO-KO hippocampal slices revealed that, whereas induction of long-term potentiation (LTP) was normal, the amplitude was significantly decreased. Intriguingly, the LTP defect could be fully rescued by acute E2 treatment in vitro. Mechanistic studies revealed that FBN-ARO-KO mice had compromised rapid kinase (AKT, ERK) and CREB-BDNF signaling in the hippocampus and cerebral cortex. In addition, acute E2 rescue of LTP in hippocampal FBN-ARO-KO slices could be blocked by administration of a MEK/ERK inhibitor, further suggesting a key role for rapid ERK signaling in neuronal E2 effects. In conclusion, the findings provide evidence of a critical role for neuron-derived E2 in regulating synaptic plasticity and cognitive function in the male and female brain.SIGNIFICANCE STATEMENT The steroid hormone 17β-estradiol (E2) is well known to be produced in the ovaries in females. Intriguingly, forebrain neurons also express aromatase, the E2 biosynthetic enzyme, but the precise functions of neuron-derived E2 is unclear. Using a novel forebrain-neuron-specific aromatase knock-out mouse model to deplete neuron-derived E2, the current study provides direct genetic evidence of a critical role for neuron-derived E2 in the regulation of rapid AKT-ERK and CREB-BDNF signaling in the mouse forebrain and demonstrates that neuron-derived E2 is essential for normal expression of LTP, synaptic plasticity, and cognitive function in both the male and female brain. These findings suggest that neuron-derived E2 functions as a novel neuromodulator in the forebrain to control synaptic plasticity and cognitive function.

Cognitive Effects of Aromatase and Possible Role in Memory Disorders.

Diverse cognitive functions in many vertebrate species are influenced by local conversion of androgens to 17β-estradiol (E2) by aromatase. This enzyme is highly expressed in various brain regions across species, with some inter-species variation in terms of regional brain expression. Since women with breast cancer and men and women with other disorders are often treated with aromatase inhibitors (AI), these populations might be especially vulnerable to cognitive deficits due to low neuroE2 synthesis, i.e., synthesis of E2 directly within the brain. Animal models have been useful in deciphering aromatase effects on cognitive functions. Consequences of AI administration at various life cycle stages have been assessed on auditory, song processing, and spatial memory in birds and various aspects of cognition in rodent models. Additionally, cognitive deficits have been described in aromatase knockout (ArKO) mice that systemically lack this gene throughout their lifespan. This review will consider evidence to date that AI treatment in male and female rodent models, birds, and humans results in cognitive impairments. How brain aromatase regulates cognitive function throughout the lifespan, and gaps in current knowledge will be considered, along with future directions to better define how aromatase might guide learning and memory from early development through the geriatric period. Better understanding the importance of E2 synthesis on neurobehavioral responses at various ages will likely aid in the discovery of therapeutic strategies to prevent potential cognitive deficits, including Alzheimer's Disease, in individuals treated with AI or those possessing CYP19 gene polymorphisms, as well as cognitive effects of normal aging that may be related to changes in brain aromatase activity.

Sexually Dimorphic Effects of Aromatase on Neurobehavioral Responses.

Aromatase is the enzyme responsible for converting testosterone to estradiol. In mammals, aromatase is expressed in the testes, ovaries, brain, and other tissues. While estrogen is traditionally associated with reproduction and sexual behavior in females, our current understanding broadens this perspective to include such biological functions as metabolism and cognition. It is now well-recognized that aromatase plays a vital lifetime role in brain development and neurobehavioral function in both sexes. Thus, ongoing investigations seek to highlight potentially vital sex differences in the role of aromatase, particularly regarding its centrally mediated effects. To characterize the role of aromatase in mediating such functions, effects of aromatase inhibitor (AI) treatments on humans and animal models have been determined. Aromatase knockout (ArKO) mice that systemically lack the enzyme have also been employed. Humans possessing mutations in the gene encoding aromatase, CYP19, have also provided critical insight into how aromatase affects brain function in a possible sex-dependent manner. A better understanding of how AIs, used to treat breast cancer and other clinical conditions, may detrimentally affect neurobehavioral responses will likely promote development of future therapies to combat these effects. Herein, we will provide a critical review of the current knowledge of sex differences in aromatase regulation of various neurobehavioral functions. Although many species have been used to better understand the functions of aromatase, this review focuses on rodent models and humans. Critical gaps in our present understanding of this area will be considered, and important future research directions will be discussed.

Impact of aromatase absence on murine intraocular pressure and retinal ganglion cells

We hypothesize that aromatase, an enzyme that regulates estrogen production, plays a significant role in the control of intraocular pressure (IOP) and retinal ganglion cells (RGCs). To begin to test our hypothesis, we examined the impact of aromatase absence, which completely eliminates estrogen synthesis, in male and female mice. Studies were performed with adult, age-matched wild type (WT) and aromatase knockout (ArKO) mice. IOP was measured in a masked fashion in both eyes of conscious mice at 12 and 24 weeks of age. Retinas were obtained and processed for RGC counting with a confocal microscope. IOP levels in both 12- and 24-week old female ArKO mice were significantly higher than those of age- and sex-matched WT controls. The mean increase in IOP was 7.9% in the 12-week-, and 19.7% in the 24-week-old mice, respectively. These changes were accompanied by significant 9% and 7% decreases in RGC numbers in the ArKO female mice, relative to controls, at 12- and 24-weeks, respectively. In contrast, aromatase deficiency did not lead to an increased IOP in male mice. There was a significant reduction in RGC counts in the 12-, but not 24-, week-old male ArKO mice, as compared to their age- and sex-matched WT controls. Overall, our findings show that aromatase inhibition in females is associated with elevated IOP and reduced RGC counts.

A Review on the Effects of Bisphenol A and Its Derivatives on Skeletal Health.

Bisphenol A (BPA) is an endocrine disruptor which can bind to the oestrogen receptor. It also possesses oestrogenic, antiandrogenic, inflammatory and oxidative properties. Since bone responds to changes in sex hormones, inflammatory and oxidative status, BPA exposure could influence bone health in humans. This review aimed to summarize the current evidence on the relationship between BPA and bone health derived from cellular, animal and human studies. Exposure to BPA (0.5-12.5 µM) decreased the proliferation of osteoblast and osteoclast precursor cells and induce their apoptosis. Bisphenol AF (10 nM) enhanced transforming growth factor beta signalling but bisphenol S (10 nM) inhibited Wnt signalling involved in osteoblast differentiation in vitro. In animals, BPA and its derivatives demonstrated distinct effects in different models. In prenatal/postnatal exposure, BPA increased femoral bone mineral content in male rats (at 25 ug/kg/day) but decreased femoral mechanical strength in female mice (at 10 µg/kg/day). In oestrogen deficiency models, BPA improved bone mineral density and microstructures in aromatase knockout mice (at very high dose, 0.1% or 1.0% w/w diet) but decreased trabecular density in ovariectomized rats (at 37 or 370 ug/kg/day). In contrast, bisphenol A diglycidyl ether (30 mg/kg/day i.p.) improved bone health in normal male and female rodents and decreased trabecular separation in ovariectomized rodents. Two cross-sectional studies have been performed to examine the relationship between BPA level and bone mineral density in humans but they yielded negligible association. As a conclusion, BPA and its derivatives could influence bone health and a possible gender effect was observed in animal studies. However, its effects in humans await verification from more comprehensive longitudinal studies in the future.

Abnormal lipid/lipoprotein metabolism and high plasma testosterone levels in male but not female aromatase-knockout mice

Sex steroid hormones, such as estrogen and testosterone, are believed to play important roles in lipid metabolism. To elucidate the effects of estrogen depletion on lipid metabolism in male and female mice, we used aromatase-knockout (ArKO) mice, in which Cyp19 gene disruption prevented estrogen synthesis in vivo. These mice were divided into the following 4 groups: male and female ArKO mice and male and female wild-type (WT) mice. These mice were fed a normal-fat diet (13.6% fat) ad libitum. At 159 days after birth, the mice were tested for liver and plasma lipid content and hepatic hormone receptor- and lipid/lipoprotein metabolism-related gene expression. Interestingly, we found that hepatic steatosis was accompanied by markedly elevated plasma testosterone levels in male ArKO mice but not in female ArKO mice. Plasma lipoprotein profiles exhibited concurrent decreases in LDL- and small dense LDL-triglyceride (TG) levels in male ArKO mice. Moreover, male mice, but not female mice, exhibited marked elevations in androgen receptor (AR), sterol regulatory element-binding protein 1 (SREBP1), and CD36 expression. These results strongly suggest that Cyp19 gene disruption, which induces a sexually dimorphic response and high plasma testosterone levels in male mice, also induces hepatic steatosis.

Synaptopodin is regulated by aromatase activity.

Locally synthesized estradiol plays an important role in synaptic plasticity in the hippocampus. We have previously shown that in hippocampal neurons, activity of the enzyme aromatase, which converts testosterone into estradiol, is reduced via Ca2+ -dependent phosphorylation. Synaptopodin is a highly estrogen responsive protein, and it has been shown that it is an important regulator of synaptic plasticity, mediated by its close association with internal calcium stores. In this study, we show that the expression of synaptopodin is stronger in the hippocampus of female animals than in that of male animals. Phosphorylation of aromatase, using letrozole, however, down-regulates synaptopodin immunohistochemistry in the hippocampus of both male and females. Similarly, in aromatase knock-out mice synaptopodin expression in the hippocampus is reduced sex independently. Using primary-dissociated hippocampal neurons, we found that evoked release of Ca2+ from internal stores down-regulates aromatase activity, which is paralleled by reduced expression of synaptopodin. Opposite effects were achieved after inhibition of the release. Calcium-dependent regulation of synaptopodin expression was abolished when the control of aromatase activity by the Ca2+ transients was disrupted. Our data suggest that the regulation of aromatase activity by Ca2+ transients in neurons contributes to synaptic plasticity in the hippocampus of male and female animals as an on-site regulatory mechanism.

Estradiol-induced neurogenesis in the female accessory olfactory bulb is required for the learning of the male odor.

Odors processed by the main and accessory olfactory bulbs (MOB, AOB) are important for sexual behavior. Interestingly, both structures continue to receive new neurons during adulthood. A role for olfactory neurogenesis in sexual behavior in female mice has recently been shown and gonadal hormones such as estradiol can modulate adult neurogenesis. Therefore, we wanted to determine the role of estradiol in learning the odors of sexual partners and in the adult neurogenesis of female aromatase knockout mice (ArKO), unable to produce estradiol. Female wild-type (WT) and ArKO mice were exposed to male odors during 7days, and olfactory preferences, cell proliferation, cell survival and functional involvement of newborn neurons were analyzed, using BrdU injections, in combination with a marker of cell activation (Zif268) and neuronal fate (doublecortin, NeuN). Behavioral tasks indicated that both WT and ArKO females were able to discriminate between the odors of two different males, but ArKO mice failed to learn the familiar male odor. Proliferation of newborn cells was reduced in ArKO mice only in the dentate gyrus of the hippocampus. Olfactory exposure decreased cell survival in the AOB in WT females, suggesting a role for estradiol in a structure involved in sexual behavior. Finally, newborn neurons do not seem to be functionally involved in the AOB of ArKO mice compared with WT, when females were exposed to the odor of a familiar male, suggesting that estradiol-induced neurogenesis in the AOB is required for the learning of the male odor in female mice. Aromatase knockout mice (ArKO) presented deficits in olfactory preferences without affecting their olfactory discrimination abilities, and showed no functional involvement of newborn neurons in the accessory olfactory bulb (AOB) in response to the odor of a familiar male. These results suggest that estradiol-induced neurogenesis in the female AOB is required for the learning of the male odor.