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It has now been about a century since a flurry of discoveries identified first the pituitary, then more specifically the anterior pituitary and soon thereafter the central nervous system as components regulating gonadal and downstream reproductive functions. This was an era of ablation/replacement designs using at first rudimentary and then increasingly pure preparations of gonadal and pituitary "activities" or transplanting actual glands, whole or homogenized, among subjects. There was, of course, controversy as is typical of lively and productive scientific debates to this day. The goals of this commentary are to briefly review the history of this work and how the terms referring to interactions among the components of the hypothalamo (as the central neural component was soon associated with)-pituitary-gonadal (HPG) axis evolved, and then to question if the current terms used have kept up with our understanding of the system. The focus in this review will be the actions of estradiol primarily upon the hypothalamus. Important actions of progesterone on the hypothalamus as well as both steroids on the pituitary response to hypothalamic factors are both acknowledged and largely ignored in this document, as are any sex differences as we focus on females., (© 2024 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

During pregnancy, physiological adaptations prepare the female body for the challenges of motherhood. Becoming a parent also requires behavioral adaptations. Such adaptations can occur as early as during pregnancy, but how pregnancy hormones remodel parenting circuits to instruct preparatory behavioral changes remains unknown. We found that action of estradiol and progesterone on galanin (Gal)-expressing neurons in the mouse medial preoptic area (MPOA) is critical for pregnancy-induced parental behavior. Whereas estradiol silences MPOA Gal neurons and paradoxically increases their excitability, progesterone permanently rewires this circuit node by promoting dendritic spine formation and recruitment of excitatory synaptic inputs. This MPOA Gal -specific neural remodeling sparsens population activity in vivo and results in persistently stronger, more selective responses to pup stimuli. Pregnancy hormones thus remodel parenting circuits in anticipation of future behavioral need.

Estradiol, a female sex hormone and the predominant form of estrogen, has diverse effects throughout the brain including in learning and memory. Estradiol modulates several types of learning that depend on the dorsomedial striatum (DMS), a subregion of the basal ganglia involved in goal-directed learning, cued action-selection, and motor skills. A cellular basis of learning is synaptic plasticity, and the presence of extranuclear estradiol receptors ERα, ERβ, and G-protein-coupled estrogen receptor (GPER) throughout the DMS suggests that estradiol may influence rapid cellular actions including those involved in plasticity. To test whether estradiol affects synaptic plasticity in the DMS, corticostriatal long-term potentiation (LTP) was induced using theta-burst stimulation (TBS) in ex vivo brain slices from intact male and female C57BL/6 mice. Extracellular field recordings showed that female mice in the diestrous stage of the estrous cycle exhibited LTP similar to male mice, while female mice in estrus did not exhibit LTP. Furthermore, antagonists of ERα or GPER rescued LTP in estrous females and agonists of ERα or GPER reduced LTP in diestrous females. In males, activating ERα but not GPER reduced LTP. These results uncover an inhibitory action of estradiol receptors on cellular learning in the DMS and suggest a cellular mechanism underlying the impairment in certain types of DMS-based learning observed in the presence of high estradiol. Because of the dorsal striatum's role in substance use disorders, these findings may provide a mechanism underlying an estradiol-mediated progression from goal-directed to habitual drug use., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Lewitus and Blackwell.)

To date, studies of development have mainly focused on the embryonic stage and a short time thereafter. There has been little research on the whole life of an individual from childhood to aging and death. For the first time, we used noninvasive urinary proteome technology to track changes in several important developmental time points in a group of rats, covering 10 time points from childhood, adolescence, young adulthood, middle adulthood, and near-death in old age. Similar to previous studies on puberty, proteins were detected and they are involved in sexual or reproductive maturation, mature spermatozoa in seminiferous tubules (first seen), gonadal hormones, decline of estradiol, brain growth, and central nervous system myelination, and our differential protein enrichment pathways also included reproductive system development, tube development, response to hormone, response to estradiol, brain development, and neuron development. Similar to previous studies in young adults, proteins were detected and they are involved in musculoskeletal maturity, peak bone mass, development of the immune system, and growth and physical development, and our differential proteins enrichment pathways also included skeletal system development, bone regeneration, system development, immune system processes, myeloid leukocyte differentiation, and developmental growth. Studies on aging-related changes in neurons and neurogenesis have been reported, and we also found relevant pathways in aged rats, such as regulation of neuronal synaptic plasticity and positive regulation of long-term neuronal synaptic plasticity. However, at all time points throughout life, there were many biological pathways revealed by differential urinary protein enrichment involving multiple organs, tissues, systems, etc. that have not been mentioned in existing studies. This study shows comprehensive and detailed changes in rat lifetime development through the urinary proteome, helping to fill the gap in development research. Moreover, it provides a new approach to monitoring changes in human health and diseases of aging using the urinary proteome., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The sex steroid hormone 17β-estradiol (estradiol) and its Estrogen Receptors (ERs) have been linked to modulation of anxiety-related and locomotor behaviors in female rodents. Research suggests that estradiol mitigates anxiety-related behaviors through activating Estrogen Receptor (ER)β and increases locomotor behaviors through ERα. The influence of ERs on these behaviors cannot always be detected. Here we discuss two experiments in which we tested the hypothesis that anxiety-related behaviors would decrease after ERβ activation and locomotor behaviors would increase after ERα activation, and also assessed the persistence of these behavioral effects by varying the timing of behavioral testing. Two cohorts of adult female ovariectomized rats were exposed to estradiol, the ERβ agonist DPN, the ERα agonist PPT, or oil for four consecutive days. Body mass was assessed throughout as a positive control. In both cohorts, open field behaviors were assessed on the first day of exposure. In one cohort (Experiment 1), open field, light/dark box, and elevated plus maze behaviors were assessed on the final day of injections. In the second cohort (Experiment 2), these behaviors were assessed 24 h after the final exposure. As expected, significant differences in body mass were detected in response to estradiol and PPT exposure, validating the estradiol and ER manipulation. No significant differences were observed in anxiety-related or locomotor behaviors across treatment groups, indicating that the efficacy of these agonists as therapeutic agents may be limited. We review these results in the context of previous literature, emphasizing relevant variables that may obscure ER-related actions on behavior., (Copyright © 2023 Elsevier Inc. All rights reserved.)

By targeting the endocannabinoid system, delta-9-tetrahydrocannabinol (THC) modulates female motivated behaviours, influenced by sex hormones. Both medial preoptic nucleus (MPN) and ventromedial nucleus of the hypothalamus (VMN) are involved in the modulation of female sexual responses. The first triggers proceptivity, whereas the ventrolateral division of the latter (VMNvl) triggers receptivity. These nuclei are modulated by glutamate, which inhibits female receptivity, and GABA, which has a dichotomous action in female sexual motivation. Here, we evaluated the action of THC on the modulation of social and sexual behaviours, on signalling pathways of MPN and VMNvl and how sex hormones influence these parameters. Young ovariectomized female rats, given sex hormones (oestradiol benzoate, EB, and progesterone, P) and THC were used for behavioural testing and for immunofluorescence analyses of vesicular glutamate transporter 2 (VGlut2) and GAD (glutamic acid decarboxylase)67 expression. Results showed that females given EB + P exhibited a higher preference for male partner, as well as higher proceptivity and a higher receptivity than control or females given only EB. Females treated with THC presented similar responses in control or EB + P female rats and even more facilitated behavioural responses in EB females than the ones that did not receive THC. Immunofluorescence results in the MPN exhibited a decreased expression of GAD67 and VGlut2 in EB + THC-treated female rats. Within VMNvl of EB-primed rats no changes in the expression of both proteins were observed after THC exposure. This study demonstrates how the possible outcomes of endocannabinoid system instability within hypothalamic neuron connectivity can modify female rat sociosexual behaviour., (© 2023 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Corticotropin-releasing hormone (CRH) neurons are the primary neural population controlling the hypothalamic-pituitary-adrenal (HPA) axis and the secretion of adrenal stress hormones. Previous work has demonstrated that stress hormone secretion can be regulated by circulating levels of estradiol. However, the effect of estradiol on CRH neuron excitability is less clear. Here, we show that chronic estradiol replacement following ovariectomy increases two types of potassium channel currents in CRH neurons: fast inactivating voltage-gated A-type K+ channel currents (IA) and non-inactivating M-type K+ channel currents (IM). Despite the increase in K+ currents following estradiol replacement, there was no overall change in CRH neuron spiking excitability assessed with either frequency-current curves or current ramps. Together, these data reveal a complex picture whereby ovariectomy and estradiol replacement differentially modulate distinct aspects of CRH neuron and HPA axis function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Introduction: Although the fluid inhibitory effects of estradiol are well characterized, a dipsogenic role of the hormone was recently identified. In ovariectomized (OVX) rats, unstimulated water intake, in the absence of food, was increased after estradiol treatment., Methods: The goals for these experiments were to further characterize the fluid enhancing effects of estradiol by determining the estrogen receptor subtype mediating the dipsogenic effect, examining saline intake, and testing for a dipsogenic effect of estradiol in male rats., Results: Pharmacological activation of estrogen receptor beta (ERβ) increased water intake, in the absence of food, and was associated with changes in postingestive feedback signals. Surprisingly, activation of ERα reduced water intake even in the absence of food. A follow-up study demonstrated that when food was available, co-activation of ERα and ERβ reduced water intake, but when food was not available water intake was increased. In addition, in OVX rats, estradiol increased saline intake through changes in postingestive and orosensory feedback signals. Finally, although estradiol decreased water intake in male rats with access to food, estradiol had no effect on water intake in the absence of food., Conclusions: These results demonstrate that the dipsogenic effect is mediated by ERβ, the fluid enhancing effects of estradiol generalize to saline, and is limited to females, which implies that a feminized brain is necessary for estradiol to increase water intake. These findings will aid in guiding future studies focused on elucidating the neuronal mechanisms that allow estradiol to both increase and decrease fluid intake., (© 2023 S. Karger AG, Basel.)

Introduction: The nucleus accumbens core (NAcc) is a sexually differentiated brain region that is modulated by steroid hormones such as 17β-estradiol (estradiol), with consequential impacts on relevant motivated behaviors and disorders such as addiction, anxiety, and depression. NAcc estradiol levels naturally fluctuate, including during the estrous cycle in adult female rats, which is analogous to the menstrual cycle in adult humans. Across the estrous cycle, excitatory synapse properties of medium spiny neurons rapidly change, as indicated by analysis of miniature excitatory postsynaptic currents (mEPSCs). mEPSC frequency decreases during estrous cycle phases associated with high estradiol levels. This decrease in mEPSC frequency is mimicked by acute topical exposure to estradiol. The identity of the estrogen receptor (ER) underlying this estradiol action is unknown. Adult rat NAcc expresses three ERs, all extranuclear: membrane ERα, membrane ERβ, and GPER1., Methods: In this brief report, we take a first step toward addressing this challenge by testing whether activation of ERs via acute topical agonist application is sufficient for inducing changes in mEPSC properties recorded via whole-cell patch clamp., Results: An agonist of ERα induced large decreases in mEPSC frequency, while agonists of ERβ and GPER1 did not robustly modulate mEPSC properties., Conclusions: These data provide evidence that activation of ERα is sufficient for inducing changes in mEPSC frequency and is a likely candidate underlying the estradiol-induced changes observed during the estrous cycle. Overall, these findings extend our understanding of the neuroendocrinology of the NAcc and implicate ERα as a primary target for future studies., (© 2023 S. Karger AG, Basel.)

The development of oestrogen positive feedback is a hallmark of female puberty. Both oestrogen and progesterone signalling are required for the functioning of this neuroendocrine feedback loop but the physiological changes that underlie the emergence of positive feedback remain unknown. Only after puberty does oestradiol (E2) facilitate progesterone synthesis in the rat female hypothalamus (neuroP), an event critical for positive feedback and the LH surge. We hypothesize that prior to puberty, these astrocytes have low levels of membrane oestrogen receptor alpha (ERα), which is needed for facilitation of neuroP synthesis. Thus, we hypothesized that prepubertal astrocytes are unable to respond to E2 with increased neuroP synthesis due a lack of membrane ERα. To test this, hypothalamic tissues and enriched primary hypothalamic astrocyte cultures were acquired from prepubertal (postnatal week 3) and post-pubertal (week 8) female mice. E2-facilitated neuroP was measured in the hypothalamus pre- and post-puberty, and hypothalamic astrocyte responses were measured after treatment with E2. Prior to puberty, E2-facilitated neuroP synthesis did not occur in the hypothalamus, and mERα expression was low in hypothalamic astrocytes, but E2-facilitated neuroP synthesis in the rostral hypothalamus and mERα expression increased post-puberty. The increase in mERα expression in hypothalamic astrocytes corresponded with a post-pubertal increase in caveolin-1 protein, PKA phosphorylation, and a more rapid [Ca 2+ ] i flux in response to E2. Together, results from the present study indicate that E2-facilitated neuroP synthesis occurs in the rostral hypothalamus, develops during puberty, and corresponds to a post-pubertal increase in mERα levels in hypothalamic astrocytes., (© 2021 British Society for Neuroendocrinology.)

For about two decades, recordings of identified gonadotropin-releasing hormone (GnRH) neurons have provided a wealth of information on their properties. We describe areas of consensus and debate the intrinsic electrophysiologic properties of these cells, their response to fast synaptic and neuromodulatory input, Ca 2+ imaging correlates of action potential firing, and signaling pathways regulating these aspects. How steroid feedback and development change these properties, functions of GnRH neuron subcompartments and local networks, as revealed by chemo- and optogenetic approaches, are also considered., (© 2021 British Society for Neuroendocrinology.)

Anxiety-related behaviours as well as the prevalence of anxiety disorders show a large sex difference in humans. Clinical studies in humans as well as behavioural studies in rodents suggest that estradiol may have anxiolytic properties. In line with this, anxiety symptoms fluctuate with estradiol levels along the menstrual cycle. However, the influence of estradiol on subjective, behavioural, as well as physiological correlates of anxiety has never been systematically addressed in humans. We ran a double-blind, randomized, placebo-controlled study (N = 126) to investigate the effects of estradiol on anxiety in men and women. In healthy volunteers, circulating estradiol levels were elevated through estradiol administration over two consecutive days to simulate the rise in estradiol levels around ovulation. Subjective, behavioral, as well as, physiological correlates of anxiety were assessed using a virtual reality elevated plus-maze (EPM). Estradiol treatment reduced the physiological stress response with blunted heart rate response and lower cortisol levels compared to placebo treatment in both sexes. In contrast, respiration frequency was only reduced in women after estradiol treatment. Behavioural measures of anxiety as well as subjective anxiety on the EPM were not affected by estradiol treatment. In general, women showed more avoidance and less approach behavior and reported higher subjective anxiety levels on the EPM than men. These results highlight the limited anxiolytic properties of circulating levels of estradiol in humans, which influence physiological markers of anxiety but not approach and avoidance behaviour or subjective anxiety levels., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of β-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Estradiol (E2) is a major hormone controlling women fertility, in particular folliculogenesis. This steroid, which is locally produced by granulosa cells (GC) within ovarian follicles, controls the development and selection of dominant preovulatory follicles. E2 effects rely on a complex set of nuclear and extra-nuclear signal transduction pathways principally triggered by its nuclear receptors, ERα and ERβ. These transcription factors are differentially expressed within follicles, with ERβ being the predominant ER in GC. Several ERβ splice isoforms have been identified and display specific structural features, which greatly complicates the nature of ERβ-mediated E2 signaling. This review aims at providing a concise overview of the main actions of E2 during follicular growth, maturation, and selection in human. It also describes the current understanding of the various roles of ERβ splice isoforms, especially their influence on cell fate. We finally discuss how E2 signaling deregulation could participate in two ovarian pathogeneses characterized by either a follicular arrest, as in polycystic ovary syndrome, or an excess of GC survival and proliferation, leading to granulosa cell tumors. This review emphasizes the need for further research to better understand the molecular basis of E2 signaling throughout folliculogenesis and to improve the efficiency of ovarian-related disease therapies.

Women are vulnerable to developing mental disorders that are associated with circulating estrogens. Estrogens, especially 17β-estradiol (E2), have a wide array of effects on the brain, affecting many behavioral endpoints associated with mental illness. By using a total estrogen receptor (ER) α knockout (KO), an ERα knock in/knock out (KIKO) that lacks a functional DNA-binding domain, and wild type (WT) controls treated with either oil or E2, we evaluated ERα signaling, dependent and independent of the estrogen response element (ERE), on avoidance behavior, social interactions and memory, and palatable ingestive behavior using the open field test, the elevated plus maze, the light dark box, the 3-chamber test, and palatable feeding. We found that ERα does not mediate control of anxiety-like behaviors but rather yielded differences in locomotor activity. In evaluating social preference and social recognition memory, we observed that E2 may modulate these measures in KIKO females but not KO females, suggesting that ERE-independent signaling is likely involved in sociability. Lastly, observations of palatable (high-fat) food intake suggested an increase in palatable eating behavior in oil-treated KIKO females. Oil-treated KO females had a longer latency to food intake, indicative of an anhedonic phenotype compared to oil-treated WT and KIKO females. We have observed that social-related behaviors are potentially influenced by ERE-independent ERα signaling and hedonic food intake requires signaling of ERα., (Copyright © 2021. Published by Elsevier Inc.)

The menopause transition, which constitutes the five or so years surrounding the final menstrual period, has been established as a time of increased risk for depressive symptoms. While mounting research suggests that exposure to more extreme and fluctuating levels of estradiol (E2) plays a role, it remains unclear which specific trigger is most strongly implicated in the development of depressive mood: acute E2 withdrawal or extreme increases in E2. The current review summarises the literature supporting the role of each, considering research pertaining to perimenopausal depression as well as other reproductive mood disorders in which ovarian hormone change is believed to play a key role, namely premenstrual dysphoric disorder and postpartum depression. Taking together the available research pertaining to the various reproductive mood disorders, we propose that women may exhibit one of four E2 sensitivity profiles, each of which may have important implications for the expected timing and severity of depressive mood during the menopause transition: the E2-increase sensitive profile, developing depressive mood in response to elevations in E2, the E2-decrease sensitive profile, for whom E2 withdrawal triggers negative mood, the E2-change sensitive profile, characterised by mood sensitivity to E2 change in either direction, and the E2 insensitive profile for whom changes in E2 have negligible psychological effects. The evidence supporting the existence of such profiles are summarised, potential biological mechanisms are briefly highlighted, and implications for future research are discussed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Emerging human studies demonstrate that theta oscillations in the dorsal anterior cingulate cortex are enhanced during fear recall (enhanced fear expression) and reduced during successful extinction recall (reduced fear expression). Although evidence suggests sex differences in fear recall and extinction recall, there are currently no human studies examining the oscillatory foundations of these memory processes separately in men and women., Methods: Because previous studies suggest that estradiol partially mediates these sex differences, we examined 20 men (low estradiol and low progesterone), 20 women using oral contraceptives (low estradiol and low progesterone), and 20 free-cycling women during midcycle (high estradiol and low progesterone). We used a fear-conditioning procedure, allowing us to separately assess fear recall and extinction recall 24 hours after fear and extinction learning. Skin conductance responses and electroencephalography were recorded during fear recall and extinction recall, and prefrontal oscillations were source localized., Results: We found elevated fear expression during fear recall and impaired extinction recall, as indicated by increased peripheral arousal (skin conductance responses) and fronto-central theta oscillations, source localized in the dorsal anterior cingulate cortex and dorsomedial prefrontal cortex. Importantly, peripheral arousal and dorsal anterior cingulate cortex theta oscillations were stronger in men and women on oral contraceptives than in women from the midcycle group., Conclusions: Our data show that neural oscillatory and peripheral correlates of heightened fear expression during fear recall and (impaired) extinction recall do not simply differ between sexes but depend on hormonal fluctuations within women., (Copyright © 2021 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Polycomb complexes have traditionally been prescribed roles as transcriptional repressors, though increasing evidence demonstrate they can also activate gene expression. However, the mechanisms underlying positive gene regulation mediated by Polycomb proteins are poorly understood. Here, we show that RING1B, a core component of Polycomb Repressive Complex 1, regulates enhancer-promoter interaction of the bona fide estrogen-activated GREB1 gene. Systematic characterization of RNA:DNA hybrid formation (R-loops), nascent transcription and RNA Pol II activity upon estrogen administration revealed a key role of RING1B in gene activation by regulating R-loop formation and RNA Pol II elongation. We also found that the estrogen receptor alpha (ERα) and RNA are both necessary for full RING1B recruitment to estrogen-activated genes. Notably, RING1B recruitment was mostly unaffected upon RNA Pol II depletion. Our findings delineate the functional interplay between RING1B, RNA and ERα to safeguard chromatin architecture perturbations required for estrogen-mediated gene regulation and highlight the crosstalk between steroid hormones and Polycomb proteins to regulate oncogenic programs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Estrogen hormones are implicated in a majority of breast cancers and estrogen receptor alpha (ER), the main nuclear factor mediating estrogen signaling, orchestrates a complex molecular circuitry that is not yet fully elucidated. Here, we investigated genome-wide DNA methylation, histone acetylation and transcription after estradiol (E2) deprivation and re-stimulation to better characterize the ability of ER to coordinate gene regulation. We found that E2 deprivation mostly resulted in DNA hypermethylation and histone deacetylation in enhancers. Transcriptome analysis revealed that E2 deprivation leads to a global down-regulation in gene expression, and more specifically of TET2 demethylase that may be involved in the DNA hypermethylation following short-term E2 deprivation. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER connection mainly with two partner TF families, AP-1 and FOX. These interactions take place in the proximity of E2 deprivation-mediated differentially methylated and histone acetylated enhancers. Finally, while most deprivation-dependent epigenetic changes were reversed following E2 re-stimulation, DNA hypermethylation and H3K27 deacetylation at certain enhancers were partially retained. Overall, these results show that inactivation of ER mediates rapid and mostly reversible epigenetic changes at enhancers, and bring new insight into early events, which may ultimately lead to endocrine resistance., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Previous research indicates that worry is associated with poorer working memory performance. Moreover, prior work demonstrates that estradiol relates to both worry and working memory performance. In the present study, we sought to further examine interrelations between worry, estradiol and working memory by testing whether estradiol moderates the association between worry and working memory in females. We hypothesized that worry would be associated with poorer working memory performance at higher levels of estradiol. We also conducted exploratory analyses to examine the role of progesterone as a moderator of the association between worry and working memory. Participants were 97 naturally-cycling females who attended four lab sessions across their menstrual cycles. Consistent with predictions, higher average levels of worry were associated with lower working memory accuracy on particularly difficult trials when average levels of estradiol were also high. The same association between higher worry and lower working memory accuracy emerged when average levels of progesterone were high. Findings highlight the importance of considering ovarian hormones in future studies and current theories of anxiety and cognition., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Previous midlife estradiol treatment, like continuous treatment, improves memory and results in lasting increases in hippocampal levels of estrogen receptor (ER) α and ER-dependent transcription in ovariectomized rodents. We hypothesized that previous and continuous midlife estradiol act to specifically increase levels of nuclear ERα, resulting in transcriptional regulation of proteins that mediate estrogen effects on memory. Ovariectomized middle-aged rats received estradiol or vehicle capsule implants. After 40 days, rats initially receiving vehicle received another vehicle capsule (ovariectomized controls). Rats initially receiving estradiol received either another estradiol (continuous estradiol) or a vehicle (previous estradiol) capsule. One month later, hippocampi were dissected and processed. Continuous and previous estradiol increased levels of nuclear, but not membrane or cytosolic ERα and had no effect on Esr1. Continuous and previous estradiol impacted gene expression and/or protein levels of mediators of estrogenic action on memory including ChAT, BDNF, and PSD-95. Findings demonstrate a long-lasting role for hippocampal ERα as a transcriptional regulator of memory following termination of previous estradiol treatment in a rat model of menopause., Competing Interests: Disclosure statement None., (Copyright © 2021. Published by Elsevier Inc.)

Objective: Sex steroid hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism. In transwomen, gender-affirming hormone therapy like estradiol (in combination with antiandrogenic compounds) could affect metabolism as well. Given that the underlying pathophysiological mechanisms are not fully understood, this study assessed circulating estradiol-driven microRNAs (miRs) in transwomen and their regulation of genes involved in metabolism in mice., Methods: Following plasma miR-sequencing (seq) in a transwomen discovery (n = 20) and validation cohort (n = 30), we identified miR-224 and miR-452. Subsequent systemic silencing of these miRs in male C57Bl/6 J mice (n = 10) was followed by RNA-seq-based gene expression analysis of brown and white adipose tissue in conjunction with mechanistic studies in cultured adipocytes., Results: Estradiol in transwomen lowered plasma miR-224 and -452 carried in extracellular vesicles (EVs) while their systemic silencing in mice and cultured adipocytes increased lipogenesis (white adipose) but reduced glucose uptake and mitochondrial respiration (brown adipose). In white and brown adipose tissue, differentially expressed (miR target) genes are associated with lipogenesis (white adipose) and mitochondrial respiration and glucose uptake (brown adipose)., Conclusion: This study identified an estradiol-drive post-transcriptional network that could potentially offer a mechanistic understanding of metabolism following gender-affirming estradiol therapy.

Lung adenocarcinoma is the most common type of lung cancer in women. Our previous studies demonstrated that 17β-estradiol (E2) promoted lung adenocarcinoma cell proliferation and tumor growth through estrogen receptor ERα. Transcriptomic analysis suggested that E2 potentiated TNFα-NFκB signaling in ERα-expressing lung adenocarcinoma cells. This study further demonstrated that E2 increased TNFα receptor expression and TNFα-triggered NFκB activity in ERα-expressing cells. E2-activated ERα had no physical association with NFκB p65/p50 heterodimer but facilitated TNFα-initiated IκBα degradation, NFκB nuclear translocation, and S468/S536 phosphorylation of p65 essential for NFκB activity. While knockdown of ERα prevented E2 from boosting NFκB activity, antiestrogen ICI 182,780 stimulated NFκB activity like E2. Inhibition of GSK3β hampered E2:ERα-promoted NFκB activity and abolished S468 phosphorylation of p65, suggesting that GSK3β played a role in the E2-TNFα signaling crosstalk. In ERα-expressing cells, E2 and TNFα synergistically regulated many genes that were not typically responsive to either E2 or TNFα. Functional analysis of microarray data inferred that E2/TNFα-induced transcriptomic changes improved cell survival and movement. Viability and colony formation assays validated that E2 and TNFα together increased cisplatin tolerance of ERα-expressing cells. Wound healing assays also confirmed that E2/TNFα cotreatment increased cell migration in an ERα-dependent manner. E2/TNFα-induced dysregulation of genes such as cell survival and movement-associated genes, proto-oncogenes, metallothioneins and histone core genes was correlated with poor overall survival in patients. In summary, E2 and TNFα engaged in an ERα-dependent positive crosstalk in lung adenocarcinoma cells, consequently increasing NFκB activation, cisplatin tolerance and cell migration and worsening prognosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Sex hormones have been implicated in pH regulation of numerous physiological systems. One consistent factor of these studies is the sodium-hydrogen exchanger 1 (NHE1). NHE1 has been associated with pH homeostasis at epithelial barriers. Hormone fluctuations have been implicated in protection and risk for breaches in blood brain barrier (BBB)/blood endothelial barrier (BEB) integrity. Few studies, however, have investigated BBB/BEB integrity in neurological disorders in the context of sex-hormone regulation of pH homeostasis., Methods//results: Physiologically relevant concentrations of 17-β-estradiol (E2, 294 pM), progesterone (P, 100 nM), and testosterone (T,3.12 nM) were independently applied to cultured immortalized bEnd.3 brain endothelial cells to study the BEB. Individual gonadal hormones showed preferential effects on extracellular pH (E2), 14 C-sucrose uptake (T), stimulated paracellular breaches (P) with dependence on functional NHE1 expression without impacting transendothelial resistance (TEER) or total protein expression. While total NHE1 expression was not changed as determined via whole cell lysate and subcellular fractionation experiment, biotinylation of NHE1 for surface membrane expression showed E2 reduced functional expression. Quantitative proteomic analysis revealed divergent effects of 17-β-estradiol and testosterone on changes in protein abundance in bEnd.3 endothelial cells as compared to untreated controls., Conclusions: These data suggest that circulating levels of sex hormones may independently control BEB integrity by 1) regulating pH homeostasis through NHE1 functional expression and 2) modifying the endothelial proteome., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oxygen causes white matter damage in preterm infants and male sex is a major risk factor for poor neurological outcome, which speculates the role of steroid hormones in sex-based differences. Preterm birth is accompanied by a drop in 17β-estradiol (E2) and progesterone along with increased levels of fetal zone steroids (FZS). We performed a sex-based analysis on the FZS concentration differences in urine samples collected from preterm and term infants. We show that, in preterm urine samples, the total concentration of FZS, and in particular the 16α-OH-DHEA concentration, is significantly higher in ill female infants as compared to males. Since we previously identified Nup133 as a novel target protein affected by hyperoxia, here we studied the effect of FZS, allopregnanolone (Allo) and E2 on differentiation and Nup133 signaling using mouse-derived primary oligodendrocyte progenitor cells (OPCs). We show that the steroids could reverse the effect of hyperoxia-mediated downregulation of Nup133 in cultured male OPCs. The addition of FZS and E2 protected cells from oxidative stress. However, E2, in presence of 16α-OH-DHEA, showed a negative effect on male cells. These results assert the importance of sex-based differences and their potential implications in preterm stress response.

Most broadcast spawner corals have a vitellogenic phase that lasts at least 6 months. It is established that estrogen regulates vitellogenin synthesis in vertebrates. Although some research have been conducted on the physiological role of sex steroids in corals, little is known about their involvement in oocyte development. This study aimed to detect steroid hormones - progesterone, testosterone, and estradiol-17β (E2) - in Acropora tenuis and study the relationships between vitellogenesis/vitellogenin synthesis and these steroids. This study also investigated the effect of E2 on vitellogenin synthesis in corals and identified steroidogenic enzymes in A. tenuis genome. Branches from tagged coral colonies were collected monthly from March to November. Histological observations showed that oocytes were vitellogenic from March to May (Stage IV and V), but not in June, and that gonads were occupied by immature oocytes in September (Stage I). Real-time qPCR revealed that vitellogenin (vg1 and vg2) transcript levels in coral branches were high in April and May, implying that corals actively underwent vitellogenesis during these months, and spawned before June. Liquid chromatography-mass spectrometry revealed that E2 could be detected in coral branches in March, April, and May, but not in June, whereas testosterone and progesterone did not fluctuate much in the same months. Immersing branches in E2-containing seawater failed to increase vitellogenin transcript levels. The results indicate that E2 is involved in oogenesis but does not positively regulate vitellogenin synthesis. Steroidogenic enzymes (except CYP19A) were identified in A. tenuis, suggesting that corals may endogenously synthesize progestogens and androgens from cholesterol., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Increasing age, male gender, and metabolic syndrome are associated with kidney stone formation. As sex hormones change with age, gender, and metabolic syndrome, we hypothesized that sex hormones may underlie the physiologic changes affecting stone formation., Methods: We analyzed the relationships between testosterone, estradiol, and history of kidney stones using data from 10,193 participants in the Continuous National Health and Nutrition Examination Survey (NHANES) database from 2013-2016. We performed logistic regression analysis to analyze the predictive value of low testosterone and low estradiol on the history of kidney stones in both males and females. Self-reported history of kidney stone diagnosis was the outcome., Results: After adjusting for risk factors known to be associated with nephrolithiasis such as age, race, BMI, and medical comorbidities including: gout, angina, coronary disease, stroke, asthma, hypertension, and diabetes, multiple regression analysis demonstrated that there is no independent association between sex hormones (testosterone and estradiol) and history of kidney stones in either males or females., Conclusions: There appears to be no association between sex hormones and history of kidney stones. Whether there is a more complex interaction of sex hormone levels and the shared association with factors such as metabolic syndrome requires additional investigation. Further studies matching menopausal status for women are necessary to further investigate the potential relationship between estrogen and kidney stones.

The corpus luteum (CL) is a temporary endocrine gland vital for pregnancy establishment and maintenance. Estradiol-17β (E2) is the major embryonic signal in pigs supporting the CL's function. The mechanisms of the luteoprotective action of E2 are still unclear. The present study aimed to determine the effect of E2 on luteal expression of factors involved in CL function. An in vivo model of intrauterine E2 infusions was applied. Gilts on day 12 of pregnancy and the estrous cycle were used as referential groups. Concentrations of E2 and progesterone were elevated in CLs of gilts receiving E2 infusions, compared to placebo-treated gilts. Estradiol-17β stimulated luteal expression of DNA-methyltransferase 1 (DNMT1), but decreased expression of DNMT3B gene and protein, as well as DNMT3A protein. Similar results for DNMT3A and 3B were observed in CLs on day 12 of pregnancy compared to day 12 of the estrous cycle. Intrauterine infusions of E2 altered luteal expression of the genes involved in CL function: PTGFR , PTGES , STAR , HSD17B1 , CYP19A1, and PGRMC1 . Our findings indicate a role for E2 in expression regulation of factors related to CL function and a novel potential for E2 to regulate DNA methylation as putative physiological mechanisms controlling luteal gene expression.

Lactobacilli and estrogens play essential roles in vaginal homeostasis. We investigated the potential direct effect of 17β-estradiol on a vaginal strain of Lactobacillus crispatus, the major bacterial species of the vaginal microbiota. 17β-estradiol (10 -6 to 10 -10  M) had no effect on L. crispatus growth, but markedly affected the membrane dynamics of this bacterium. This effect appeared consistent with a signal transduction process. The surface polarity and aggregation potential of the bacterium were unaffected by exposure to 17β-estradiol, but its mean size was significantly reduced. 17β-estradiol also promoted biosurfactant production by L. crispatus and adhesion to vaginal VK2/E6E7 cells, but had little effect on bacterial biofilm formation activity. Bioinformatic analysis of L. crispatus identified a membrane lipid raft-associated stomatin/prohibitin/flotillin/HflK domain containing protein as a potential 17β-estradiol binding site. Overall, our results reveal direct effects of 17β-estradiol on L. crispatus. These effects are of potential importance in the physiology of the vaginal environment, through the promotion of lactobacillus adhesion to the mucosa and protection against pathogens.

Neurones in the arcuate nucleus co-expressing kisspeptin, neurokinin B (NKB) and dynorphin (KNDy) play a critical role in the control of gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) secretion. In sheep, KNDy neurones mediate both steroid-negative- and -positive-feedback during pulsatile and preovulatory surge secretions of GnRH/LH, respectively. In addition, KNDy neurones receive glutamatergic inputs expressing vGlut2, a glutamate transporter that serves as a marker for those terminals, from both KNDy neurones and other populations of glutamatergic neurones. Previous work reported higher numbers of vGlut2-positive axonal inputs onto KNDy neurones during the LH surge than in luteal phase ewes. In the present study, we further examined the effects of the ovarian steroids progesterone (P) and oestradiol (E 2 ) on glutamatergic inputs to KNDy neurones. Ovariectomised (OVX) ewes received either no further treatment (OVX) or steroid treatments that mimicked the luteal phase (low E 2  + P), and early (low E 2 ) or late follicular (high E 2 ) phases of the oestrous cycle (n = 4 or 5 per group). Brain sections were processed for triple-label immunofluorescent detection of NKB/vGlut2/synaptophysin and analysed using confocal microscopy. We found higher numbers of vGlut2 inputs onto KNDy neurones in high E 2 compared to the other three treatment groups. These results suggest that synaptic plasticity of glutamatergic inputs onto KNDy neurones during the ovine follicular phase depend on increasing levels of E 2 required for the preovulatory GnRH/surge. These synaptic changes likely contribute to the positive-feedback action of oestrogen on GnRH/LH secretion and thus the generation of the preovulatory surge in the sheep., (© 2021 British Society for Neuroendocrinology.)

The roles of sex and sex-hormones on the metabolic consequences of intermittent hypoxia (IH, a reliable model of sleep apnea) are unknown. We used intact male or female mice and ovariectomized (OVX) females treated with vehicle (Veh) or estradiol (E 2 ) and exposed to normoxia (Nx) or IH (6% O 2 , 10 cycles/h, 12 h/day, 2 wk). Mice were then fasted for 6 h, and we measured fasting glucose and insulin levels and performed insulin or glucose tolerance tests (ITT or GTT). We also assessed liver concentrations of glycogen, triglycerides (TGs), and expression levels of genes involved in aerobic or anaerobic metabolism. In males, IH lowered fasting levels of glucose and insulin, slightly improved glucose tolerance, but altered glucose tolerance in females. In OVX-Veh females, IH reduced fasting glucose and insulin levels and strongly impaired glucose tolerance. E 2 supplementation reversed these effects and improved homeostasis model assessment of β-cell function (HOMA-β), a marker of pancreatic glucose-induced insulin released. IH decreased liver TG concentration in males and slightly increased glycogen in OVX-Veh females. Liver expression of glycolytic ( Ldha ) and mitochondrial (citrate synthase, Pdha1 ) genes was reduced by IH in males and in OVX-Veh females, but not in intact or OVX-E 2 females. We conclude that 1 ) IH reduced fasting levels of glycemia in males and in ovariectomized females. 2 ) IH improves glucose tolerance only in males. 3 ) In females IH decreased glucose tolerance, this effect was amplified by ovariectomy, and reversed by E 2 supplementation. 4 ) During IH exposures, E 2 supplementation appears to improve pancreatic β cells functions. NEW & NOTEWORTHY We assessed fasting glycemic control, and tolerance to insulin and glucose in male and female mice exposed to intermittent hypoxia. IH improves glucose tolerance in males but had opposite effects in females. This response was amplified following ovariectomy in females and prevented by estradiol supplementation. Metabolic consequences of IH differ between males and females and are regulated by estradiol in female mice.

Animal taxa show remarkable variability in sexual reproduction, where separate sexes, or gonochorism, is thought to have evolved from hermaphroditism for most cases. Hermaphroditism accounts for 5% in animals, and sequential hermaphroditism has been found in teleost. In this study, we characterized a novel form of the transient hermaphroditic stage in little yellow croaker ( Larimichthys polyactis ) during early gonadal development. The ovary and testis were indistinguishable from 7 to 40 days post-hatching (dph). Morphological and histological examinations revealed an intersex stage of male gonads between 43 and 80 dph, which consist of germ cells, somatic cells, efferent duct, and early primary oocytes (EPOs). These EPOs in testis degenerate completely by 90 dph through apoptosis yet can be rescued by exogenous 17- β -estradiol. Male germ cells enter the mitotic flourishing stage before meiosis is initiated at 180 dph, and they undergo normal spermatogenesis to produce functional sperms. This transient hermaphroditic stage is male-specific, and the ovary development appears to be normal in females. This developmental pattern is not found in the sister species Larimichthys crocea or any other closely related species. Further examinations of serum hormone levels indicate that the absence of 11-ketotestosterone and elevated levels of 17- β -estradiol delineate the male intersex gonad stage, providing mechanistic insights on this unique phenomenon. Our research is the first report on male-specific transient hermaphroditism and will advance the current understanding of fish reproductive biology. This unique gonadal development pattern can serve as a useful model for studying the evolutionary relationship between hermaphroditism and gonochorism, as well as teleost sex determination and differentiation strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xie, Li, Zhan, Liu, Tan, Wang and Lou.)

While human vaginal pH in childbearing-age women is conclusively acidic, the mouse vaginal pH is reported as being near neutral. However, this information appears to be somewhat anecdotal with respect to vulvovaginal candidiasis, as such claims in the literature frequently lack citations of studies that specifically address this physiological factor. Given the disparate pH between mice and humans, the role of exogenous hormones and colonization by the fungal pathogen Candida albicans in shaping vaginal pH was assessed. Use of a convenient modified vaginal lavage technique with the pH indicator dye phenol red demonstrated that indeed vaginal pH was near neutral (7.2 ± 0.24) and was not altered by delivery of progesterone or estrogen in C57BL/6 mice. These trends were conserved in DBA/2 and CD-1 mouse backgrounds, commonly used in the mouse model of vaginitis. It was also determined that vaginal colonization with C. albicans did not alter the globally neutral vaginal pH over the course of one week. Construction and validation of a C. albicans reporter strain expressing GFPy, driven by the pH-responsive PHR1 promoter, confirmed the murine vaginal pH to be at least ≥6.0. Collectively, our data convincingly demonstrate a stable and conserved near neutrality of the mouse vaginal pH during vulvovaginal candidiasis and should serve as a definitive source for future reference. Implications and rationale for disparate pH in this model system are also discussed., (Copyright © 2021 American Society for Microbiology.)

Both estrogen and hydrogen sulfide (H2S) inhibit the proliferation of vascular smooth muscle cells (SMCs) and development of atherosclerosis. In the absence of endogenous H2S as occurred in CSE-knockout (KO) mouse, however, estrogen stimulates the proliferation of vascular SMCs. The underlying mechanisms for this seemingly controversial vascular effect of estrogen are unclear. In the present study, we demonstrated that the stimulatory effect of estrogen on the proliferation of CSE-KO SMCs was suppressed by the inhibitor of insulin-like growth factor-1 receptor (IGF-1R) or knockdown of IGF-1R protein expression. Estrogen downregulated the expression of insulin-like growth factor-1 (IGF-1) and IGF-1R in aortic tissues or aortic SMCs isolated from WT and CSE-KO mice. Furthermore, endogenous H2S downregulated IGF-1R, but upregulated estrogen receptor (ER)-α, in aortic tissues or SMCs. ER-α and IGF-1R were co-located in SMCs and co-immunoprecipitated, which was decreased by H2S. Finally, both endogenous and exogenous H2S induced the S-sulfhydration of IGF-1R, but not ER-α, in WT-SMCs and CSE-KO SMCs, which underlies the decreased formation of IGF-1R/ER-α hybrid in the presence of H2S. Thus, the absence of H2S favors the interaction of estrogen with IGF-1R/ER-α hybrid to stimulate SMCs proliferation. The appreciation of a critical role of H2S in preventing estrogen-induced SMCs proliferation will help better understand the regulation of complex vascular effects of estrogen and sex-related cardiovascular diseases.

Genomic sequence and gene expression association studies in animals and humans have identified genes that may be integral in the pathogenesis of various diseases. CD14 (cluster of differentiation 14)-a cell surface protein involved in innate immune system activation-is one such gene associated with cardiovascular and hypertensive disease. We previously showed that this gene is upregulated in renal macrophages of Dahl salt-sensitive animals fed a high-salt diet; here we test the hypothesis that CD14 contributes to the elevated pressure and renal injury observed in salt-sensitive hypertension. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), we created a targeted mutation in the CD14 gene on the Dahl SS (SS/JrHSDMcwi) background and validated the absence of CD14 peptides via mass spectrometry. Radiotelemetry was used to monitor blood pressure in wild-type and CD14 -/ - animals challenged with high salt and identified infiltrating renal immune cells via flow cytometry. Germline knockout of CD14 exacerbated salt-sensitive hypertension and renal injury in female animals but not males. CD14 -/- females demonstrated increased infiltrating macrophages but no difference in infiltrating lymphocytes. Transplant of CD14 +/+ or CD14 -/- bone marrow was used to isolate the effects of CD14 knockout to hematopoietic cells and confirmed that the differential phenotype observed was due to knockout of CD14 in hematopoietic cells. Ovariectomy was used to remove the influence of female sex hormones, which completely abrogated the effect of CD14 knockout. These studies provide a novel treatment target and evidence of a new dichotomy in immune activation between sexes within the context of hypertensive disease where CD14 regulates immune cell activation and renal injury.

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17α-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17α-estradiol elicits these benefits remain unresolved. Herein, we show that 17α-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor α (ERα) to that of 17β-estradiol. In addition, we show that the ablation of ERα completely attenuates the beneficial metabolic effects of 17α-E2 in male mice. Our findings suggest that 17α-E2 may act through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17α-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17α-E2 are not limited to mice. Collectively, these studies suggest ERα may be a drug target for mitigating chronic diseases in male mammals., Competing Interests: SM, NH, MN, AR, RS, SA, MA, AU, MS, JH, DH, WF, MS No competing interests declared, (© 2020, Mann et al.)

Epithelial ovarian cancer (EOC) is the most lethal estrogen-sensitive gynecological cancer. Studies have reported that estrogen induces rapid cellular calcium mobilization in cells and can determine the fate of a cell. We found that estrogen increased the calcium release-activated calcium channel modulator 1 (Orai1) protein expression levels in SK-OV-3 cells. However, to date, there has been no research on the functional relationship and molecular mechanism of estrogen-regulating Orai1 during EOC development. In our study, Orai1 had a high expression level in high-grade serous ovarian tumor tissues and SK-OV-3 cells. Estrogen promoted cell proliferation and migration while inhibiting cell apoptosis in SK-OV-3 cells. Orai1 silencing suppressed estrogen-induced cell migration and proliferation. Overexpression of Orai1, however, enhanced the ability of 17β-estradiol (E2) to exert its function. Estrogen induced rapid calcium influx in SK-OV-3 cells. Knockdown of Orai1 in SK-OV-3 cells blocked E2-induced stored-operated Ca 2+ influx. The messenger RNA expression of caspase 3, matrix metallopeptidase 1, and cyclin-dependent kinase 6 were regulated via Orai1 under E2 treatment. Our results suggest that estrogen, by regulating Orai1, induced calcium influx to determine cell fate., (© 2020 Wiley Periodicals LLC.)

Ovarian hormones exert an influence on social information processing, in which, however, the exact roles of estradiol and progesterone remain unclear. This study examines the specific influences of these two ovarian hormones on social information processing across the menstrual cycle using the emotional face flanker task and attentional network test (ANT). Twenty-six naturally cycling, healthy women were tested thrice: during menses, in the follicular phase, and in the luteal phase. In the emotional face flanker task, a significant positive relation was found between progesterone levels and reaction times (RTs) for sad faces, suggesting that high progesterone levels may activate the social monitoring system and allocate more attention to the social stimulus, which benefits individuals' survival and adaptation. In the ANT, a significant increase was found in RTs and accuracy during the luteal phase, suggesting that luteal women increase this accuracy by exerting a relatively conservative strategy of allocating more attention to the targets. Taken together, these findings indicate that high levels of progesterone may facilitate social information processing by optimizing attention allocation. Moreover, overactivation of the social monitoring system may make women more susceptible to stressors and promote affective disturbances, which may provide underlying pathophysiology of the premenstrual dysphoric disorder., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Estrogen protects animals from obesity through estrogen receptor α (ERα), partially by inhibiting overeating in animals fed ad libitum. However, the effects of estrogen on feeding behavior in hungry animals remain unclear. In this study, we examined the roles of 17β-estradiol (E2) and ERα in the regulation of feeding in hungry female animals and explored the underlying mechanisms., Methods: Wild-type female mice with surgical depletion of endogenous estrogens were used to examine the effects of E2 supplementation on acute refeeding behavior after starvation. ERα-C451A mutant mice deficient in membrane-bound ERα activity and ERα-AF2 0 mutant mice lacking ERα transcriptional activity were used to further examine mechanisms underlying acute feeding triggered by either fasting or central glucopenia (induced by intracerebroventricular injections of 2-deoxy-D-glucose). We also used electrophysiology to explore the impact of these ERα mutations on the neural activities of ERα neurons in the hypothalamus., Results: In the wild-type female mice, ovariectomy reduced fasting-induced refeeding, which was restored by E2 supplementation. The ERα-C451A mutation, but not the ERα-AF2 0 mutation, attenuated acute feeding induced by either fasting or central glucopenia. The ERα-C451A mutation consistently impaired the neural responses of hypothalamic ERα neurons to hypoglycemia., Conclusion: In addition to previous evidence that estrogen reduces deviations in energy balance by inhibiting eating at a satiated state, our findings demonstrate the unexpected role of E2 that promotes eating in hungry mice, also contributing to the stability of energy homeostasis. This latter effect specifically requires membrane-bound ERα activity., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Ventromedial hypothalamic nucleus (VMN) gluco-regulatory transmission is subject to sex-specific control by estradiol. The VMN is characterized by high levels of aromatase expression., Methods: The aromatase inhibitor letrozole (LZ) was used with high-resolution microdissection/Western blot techniques to address the hypothesis that neuroestradiol exerts sex-dimorphic control of VMN neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase 65/67 (GAD) protein expression. Glycogen metabolism impacts VMN nNOS and GAD profiles; here, LZ treatment effects on VMN glycogen synthase (GS) and phosphorylase brain- (GPbb; glucoprivic-sensitive) and muscle (GPmm; norepinephrine-sensitive) variant proteins were examined., Results: VMN aromatase protein content was similar between sexes. Intracerebroventricular LZ infusion of testes-intact male and ovariectomized, estradiol-replaced female rats blocked insulin-induced hypoglycemic (IIH) up-regulation of this profile. LZ exerted sex-contingent effects on basal VMN nNOS and GAD expression, but blocked IIH-induced NO stimulation and GAD suppression in each sex. Sex-contingent LZ effects on basal and hypoglycemic patterns of GPbb and GPmm expression occurred at distinctive levels of the VMN. LZ correspondingly down- or up-regulated baseline pyruvate recycling pathway marker protein expression in males (glutaminase) and females (malic enzyme-1), and altered INS effects on those proteins., Conclusions: Results infer that neuroestradiol is required in each sex for optimal VMN metabolic transmitter signaling of hypoglycemic energy deficiency. Sex differences in VMN GP variant protein levels and sensitivity to aromatase may correlate with sex-dimorphic glycogen mobilization during this metabolic stress. Neuroestradiol may also exert sex-specific effects on glucogenic amino acid energy yield by actions on distinctive enzyme targets in each sex.

Gender and menopause influence the severity and development manner of nonalcoholic steatohepatitis (NASH). Male p62/Sqstm1 and nuclear factor E2-related factor-2 (p62 and Nrf2) double-knockout (DKO) mice exhibit severe steatohepatitis caused by hyperphagia-induced obesity, overload of lipopolysaccharide (LPS) into the liver, and potentiation of the inflammatory response in Kupffer cells. However, the pathogenetic phenotype of steatohepatitis in female DKO mice remains unknown. Phenotypic changes of steatohepatitis in DKO mice were compared in terms of gender differences. Compared with DKO male mice, DKO female mice exhibited later onset of steatohepatitis with obesity after 30 weeks of age, as well as milder severity of hepatic inflammation and fibrosis. Serum estradiol was higher in female than male mice, with levels increasing up to 30 weeks of age before decreasing until 50 weeks of age (corresponding to the post-menopausal period). Fecal and serum LPS were lower in female mice than male mice, and inflammatory signaling in the liver was attenuated in female compared with male mice. Correlating with LPS levels, the composition of intestinal microbiota in female mice was different from male mice. Gender differences were observed for the development of steatohepatitis in DKO mice. Low-grade inflammatory hit in the liver under in vivo conditions of high estradiol may be attributable to the milder pathological features of steatohepatitis in female mice.

The estrogen membrane receptor GPR30 (also known as G-protein coupled receptor 30) has recently been shown to be involved in the regulation of oocyte maturation and cumulus expansion. However, whether GPR30 expression is regulated by gonadotropin stimulation and how it participates in the regulation of the maturation process is still not clear. In this study, we explored the mechanism underlying the synergy between luteinizing hormone and 17β-estradiol (17β-E 2 ) to improve the epidermal growth factor (EGF) response in cumulus oocyte complexes (COCs) during oocyte maturation in mice. The expression and distribution of GPR30, EGFR, and EGF-like growth factors were examined by real-time quantitative PCR, western blot, and immunofluorescence staining. Lyso-Tracker Red labeling was performed to detect the lysosomal activity in follicle granular cells (FGCs). Cumulus expansion of COCs was evaluated after in vitro maturation for 16 h. We found that EGF-like growth factors transmit LH signals to increase GRP30 levels by inhibiting protein degradation in lysosomes. Meanwhile, 17β-E 2 stimulates the GPR30 signaling pathway to increase EGF receptor levels, enhancing the response ability of EGF signaling in COCs and thus promoting cumulus expansion. In conclusion, our study reveals the synergistic mechanism between LH and estrogen in the regulation of cumulus expansion during oocyte maturation process.

Although hormones such as glucocorticoids have been broadly accepted in recent decades as general neuromodulators of memory processes, sex steroid hormones such as the potent oestrogen 17β-oestradiol have been less well recognized by the scientific community in this capacity. The predominance of females in studies of oestradiol and memory and the general (but erroneous) perception that oestrogens are 'female' hormones have probably prevented oestradiol from being more widely considered as a key memory modulator in both sexes. Indeed, although considerable evidence supports a crucial role for oestradiol in regulating learning and memory in females, a growing body of literature indicates a similar role in males. This Review discusses the mechanisms of oestradiol signalling and provides an overview of the effects of oestradiol on spatial, object recognition, social and fear memories. Although the primary focus is on data collected in females, effects of oestradiol on memory in males will be discussed, as will sex differences in the molecular mechanisms that regulate oestrogenic modulation of memory, which may have important implications for the development of future cognitive therapeutics.

The Fenton reaction (Fe 2+ +H 2 O 2 →Fe 3+ + • OH+OH - ) results in strong oxidative damage to macromolecules when iron (Fe) or hydrogen peroxide (H 2 O 2 ) are in excess. This study aims at comparing Fe 2+ +H 2 O 2 -induced oxidative damage to membrane lipids (lipid peroxidation, LPO) and protective effects of 17β-estradiol (a potential antioxidant) in porcine ovary and thyroid homogenates. Iron, as one of the Fenton reaction substrates, was used in the highest achievable concentrations. Thyroid or ovary homogenates were incubated in the presence of: (1st) FeSO 4 +H 2 O 2 with/without 17β-estradiol (1 mM; 100, 10.0, 1.0 µM; 100, 10.0, 1.0 nM; 100, 10.0, 1.0 pM); five experiments were performed with different FeSO 4 concentrations (2400, 1200, 600, 300, 150 µM); (2nd) FeSO 4 (2400, 1200, 600, 300, 150 µM)+H 2 O 2 with/without 17β-estradiol; three experiments were performed with three highest 17β-estradiol concentrations; (3rd) FeSO 4 (2400, 1200, 1100, 1000, 900, 800, 700, 600, 300, 150, 75 µM)+H 2 O 2 (5 mM). LPO level [MDA+4-HDA/mg protein] was measured spectrophotometrically. The basal LPO level is lower in ovary than in thyroid homogenates. However, experimentally-induced LPO was higher in the former tissue, which was confirmed for the three highest Fe 2+ concentrations (2400, 1200, 1100 µM). Exogenous 17β-estradiol (1 mM, 100, and 10 µM) reduced experimentally-induced LPO independently of iron concentration and that protective effect did not differ between tissues. The ovary, compared to the thyroid, reveals higher sensitivity to prooxidative effects of iron, however, it showed similar responsivity to protective 17β-estradiol activity. The therapeutic effect of 17β-estradiol against iron overload consequences should be considered with relation to both tissues.

Women acquire cocaine habits faster and are more motivated to obtain drug than men. In general, female rodents acquire intravenous cocaine self-administration (SA) faster and show greater locomotor responses to cocaine than males. Sex differences are attributed to differences in circulating estradiol. We used the four core genotype (FCG) mouse to ask whether sex chromosome complement influences vulnerability to cocaine's reinforcing and/or locomotor-activating effects. The FCG cross produces ovary-bearing mice with XX or XY genotypes (XXF, XYF) and testes-bearing mice with XX or XY genotypes (XXM, XYM). A greater percentage of gonadal females acquired cocaine SA via infusions into jugular catheters as compared with XYM mice, but XXM mice were not significantly different than any other group. Discrimination of the active versus inactive nose poke holes and cocaine intake were in general greater in gonadal females than in gonadal males. Progressive ratio tests for motivation revealed an interaction between sex chromosomes and gonads: XYM mice were more motivated to self-administer cocaine taking more infusions than mice in any other group. Locomotor responses to cocaine exposure revealed effects of sex chromosomes. After acute exposure, activity was greater in XX than in XY mice and the reverse was true for behavioral sensitization. Mice with XY genotypes displayed more activity than XX mice when given cocaine after a 10-day drug-free period. Our data demonstrate that sex chromosome complement alone and/or interacting with gonadal status can modify cocaine's reinforcing and locomotor-activating effects. These data should inform current studies of sex differences in drug use., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The behavioral activation system (BAS) and the behavioral inhibition system (BIS) have been proposed to relate to stable traits that predict inter-individual differences in motivation. Prior reports point dopamine (DA) pathways, mainly including ventral tegmental area (VTA) and substantia nigra (SN), implicate in subserving reward-related functions associated with BAS and inhibitory functions related with BIS. However, as an important factor that affects DA releasing, it remains an open question whether the ovarian hormones may also be related to BIS/BAS. Here, to investigate effects of the estradiol (E2) and progesterone (PROG) on BIS/BAS and related DA pathways, we employed a BIS/BAS scale and the resting-state functional magnetic resonance imaging (fMRI) during the late follicular phase (FP) and the mid-luteal phase (LP). On the behavioral level, when women had high PROG levels, their E2 levels were found positively correlated with BIS scores, but those women whose PROG levels were low, their E2 levels were negative correlation with BIS scores. On the neural level, we demonstrated BAS was related with the VTA pathway, included brain reward regions of nucleus accumbens (NAc) and orbitofrontal cortex (OFC). Meanwhile, the BIS was correlated with the SN-dorsolateral prefrontal cortex (dlPFC) pathway. ROI-based resting-state functional connectivity (RSFC) analyses further revealed that, RSFC between the SN and dlPFC was modulated by ovarian hormones. With higher PROG levels, increased E2 levels among women were accompanied by stronger RSFC of the SN-dlPFC, but when PROG levels were low, E2 levels were negatively correlated with the SN-dlPFC RSFC. These findings revealed a combined enhancement effect of E2 and PROG on BIS, and the SN-dlPFC pathway was mainly involved in this process., Competing Interests: No authors have competing interests.

Naturally occurring cyclical changes in sex steroid hormones such as 17β-estradiol and progesterone can modulate neuron function and behavior in female mammals. One example is the estrous cycle in rats, which is composed of multiple phases. We previously reported evidence of differences between estrous cycle phases in excitatory synapse and intrinsic electrophysiological properties of rat nucleus accumbens core (AcbC) medium spiny neurons (MSNs). The AcbC is a nexus between the limbic and premotor systems and is integral for controlling motivated and reward-associated behaviors and disorders, which are sensitive to the estrous cycle and hormones. The present study expands our prior findings by testing whether circulating levels of estradiol and progesterone correlate with changes in MSN electrophysiology across estrous cycle phases. As part of this project, the excitatory synapse and intrinsic excitability properties of MSNs in late proestrus of adult female rats were assessed. Circulating levels of estradiol correlate with resting membrane potential, the time constant of the membrane, and rheobase. Circulating levels of progesterone correlate with miniature excitatory postsynaptic current (mEPSC) frequency and amplitude. Circulating levels of estradiol and progesterone together correlate with mEPSC amplitude, resting membrane potential, and input resistance. The late proestrus phase features a prominent and unique decrease in mEPSC frequency. These data indicate that circulating levels of estradiol and progesterone alone or in combination interact with specific MSN electrophysiological properties, indicating differential and synergistic roles of these hormones. Broadly, these findings illustrate the underlying endocrine actions regarding how the estrous cycle modulates MSN electrophysiology. NEW & NOTEWORTHY This research indicates that estradiol and progesterone act both differentially and synergistically to modulate neuron physiology in the nucleus accumbens core. These actions by specific hormones provide key data indicating the endocrine mechanisms underlying how the estrous cycle modulates neuron physiology in this region. Overall, these data reinforce that hormones are an important influence on neural physiology.

The menstrual cycle in humans and its analogous cycle in rodents, the estrous cycle, modulate brain function and behavior. Both cycles are characterized by the cyclical fluctuation of ovarian hormones including estrogens such as estradiol. Estradiol induces cycle- and sex-dependent differences in the phenotype and incidence of many behaviors, including those related to reward and motivation. The nucleus accumbens core (AcbC), a limbic and premotor system nexus region, directly regulates these behaviors. We previously showed that the estrous cycle modulates intrinsic excitability and excitatory synapse properties of medium spiny neurons (MSNs) in the AcbC. The identity of the underlying hormone mechanism is unknown, with estradiol being a prime candidate. The present study tests the hypothesis that estradiol induces estrous cycle-relevant differences in MSN electrophysiology. To accomplish this goal, a time- and dose-dependent estradiol replacement paradigm designed to simulate the rise of circulating estradiol levels across the estrous cycle was employed in ovariectomized adult female rats as well as a vehicle control group. Estradiol replacement decreased MSN excitability by modulating properties such as resting membrane potential, input resistance in both the linear and rectified ranges, and rheobase compared with vehicle-treated females. These differences in MSN excitability mimic those previously described regarding estrous cycle effects on MSN electrophysiology. Excitatory synapse properties were not modulated in response to this estradiol replacement paradigm. These data are the first to demonstrate that an estrous cycle-relevant estradiol exposure modulates MSN electrophysiology, providing evidence of the fundamental neuroendocrine mechanisms regulating the AcbC. NEW & NOTEWORTHY The present study shows, for the first time, that an estrous cycle-relevant estradiol exposure modulates nucleus accumbens neuron excitability. This evidence provides insight into the neuroendocrine mechanisms by which estradiol cyclically alters neuron properties during the estrous cycle. Overall, these data emphasize the significant influence of hormone action in the brain and especially individual neuron physiology.

Hypothalamic neurons show sex differences in neuritogenesis, female neurons have longer axons and higher levels of the neuritogenic factor neurogenin 3 (Ngn3) than male neurons in vitro. Moreover, the effect of 17-β-estradiol (E2) on axonal growth and Ngn3 expression is only found in male-derived neurons. To investigate whether sex chromosomes regulate these early sex differences in neuritogenesis by regulating the E2 effect on Ngn3, we evaluated the growth and differentiation of hypothalamic neurons derived from the "four core genotypes" mouse model, in which the factors of "gonadal sex" and "sex chromosome complement" are dissociated. We showed that sex differences in neurite outgrowth are determined by sex chromosome complement (XX > XY). Moreover, E2 increased the mRNA expression of Ngn3 and axonal length only in XY neurons. ERα/β expressions are regulated by sex chromosome complement; however, E2-effect on Ngn3 expression in XY neurons was only fully reproduced by PPT, a specific ligand of ERα, and prevented by MPP, a specific antagonist of ERα. Together our data indicate that sex chromosomes regulate early development of hypothalamic neurons by orchestrating not only sex differences in neuritogenesis, but also regulating the effect of E2 on Ngn3 expression through activation of ERα in hypothalamic neurons.

This review highlights fifty years of progress in research on estradiol's role in regulating behavior(s). It was initially thought that estradiol was only involved in regulating estrus/menstrual cycles and concomitant sexual behavior, but it is now clear that estradiol also influences the higher order neural function of cognition. We provide a brief overview of estradiol's regulation of memory and some mechanisms which underlie its effects. Given systemically or directly into the hippocampus, to ovariectomized female rodents, estradiol or specific agonists, enhance learning and/or memory in a variety of rodent cognitive tasks. Acute (within minutes) or chronic (days) treatments enhance cognitive functions. Under the same treatment conditions, dendritic spine density on pyramidal neurons in the CA1 area of the hippocampus and medial prefrontal cortex increase which suggests that these changes are an important component of estrogen's ability to impact memory processes. Noradrenergic, dopaminergic and serotoninergic activity are also altered in these areas following estrogen treatments. Memory enhancements and increased spine density by estrogens are not limited to females but are also present in castrate males. In the next fifty years, neuroscientists need to determine how currently described neural changes mediate improved memory, how interactions among areas important for memory promote memory and the potential significance of neurally derived estrogens in normal cognitive processing. Answering these questions may provide significant advances for treatment of dementias as well as age and neuro-degenerative disease related memory loss., (Copyright © 2020 Elsevier Inc. All rights reserved.)