OVX Female Mice Research Articles

Effects of E2 on the IDO1-mediated metabolic KYN pathway in OVX female mice.

The aim of this study was to investigate the role of 17β-estradiol (E2)-mediated oestrogen receptor (ER) in modulating the depressive-like behaviours of ovariectomy (OVX) mice and the associated mechanisms. E2 was administrated in OVX mice. The behaviour and physiological changes of OVX mice including immobility time in tail suspension test (TST) and forced swimming test (FST), levels of serum E2, inflammatory mediators, oxidative stress factors, indoleamine2,3-dioxygenase 1 (IDO1) and the neurotransmitters mediated by IDO1 activation were then recorded. Cell injury models established by lipopolysaccharide (LPS) or H2O2 stimulation in HT22 and BV2 cells were employed to further explore the mechanisms of E2's function. E2 treatment improved OVX-induced increase of immobility time in FST and TST. Meanwhile, E2 ameliorated the changes of inflammatory factors (NF-κB, TNF-α and IL-6), IDO1, IDO1-mediated TRP/KYN pathway and oxidative stress factors (iNOS, MDA, GSH and SOD) in the hippocampus of OVX mice. Interestingly, ERβ inhibitor abolished E2's inhibitory effects on the inflammation and IDO1-mediated TRP/KYN pathway; ERβ inhibitor also abolished E2's anti-oxidative stress effect. In cell experiments, ERβ small interfering RNA (siRNA) pretreatment reversed E2's anti-inflammatory effect on LPS-treated HT22 and BV2 cells and E2's inhibitory effect on IDO1 expression in LPS-treated BV2 cells. ERβ siRNA pretreatment also reversed E2's anti-oxidation effect on H2O2-treated HT22 cells. E2 exert the antidepressant function in OVX mice via ERβ-modulated suppression of NF-κB-mediated inflammatory pathway, oxidative stress factors and IDO1-mediated TRP/KYN pathway in the hippocampus.

Astrocytic glutamate transport is essential for the memory-enhancing effects of 17β-estradiol in ovariectomized mice

Infusion of 17β-estradiol (E2) into the dorsal hippocampus (DH) of ovariectomized (OVX) mice enhances memory consolidation, an effect that depends on rapid phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Astrocytic glutamate transporter 1 (GLT-1) modulates neurotransmission via glutamate uptake from the synaptic cleft. However, little is known about the contribution of DH astrocytes, and astrocytic glutamate transport, to the memory-enhancing effects of E2. This study was designed to test whether DH astrocytes contribute to estrogenic modulation of memory consolidation by determining the extent to which DH GLT-1 is necessary for E2 to enhance memory in object recognition and object placement tasks and trigger rapid phosphorylation events in DH astrocytes. OVX female mice were bilaterally cannulated into the DH or the DH and dorsal third ventricle (ICV). Post-training DH infusion of the GLT-1 inhibitor dihydrokainic acid (DHK) dose-dependently impaired memory consolidation in both tasks. Moreover, the memory-enhancing effects of ICV-infused E2 in each task were blocked by DH DHK infusion. E2 increased p42 ERK and Akt phosphorylation in DH astrocytes, and these effects were blocked by DHK. Results suggest the necessity of DH GLT-1 activity for object and spatial memory consolidation, and for E2 to enhance consolidation of these memories and to rapidly activate cell signaling in DH astrocytes. Findings indicate that astrocytic function in the DH of OVX females is necessary for memory formation and is regulated by E2, and suggest an essential role for DH astrocytic GLT-1 activity in the memory-enhancing effects of E2.

Accumulation of β-aminoisobutyric acid mediates hyperalgesia in ovariectomized mice through Mas-related G protein-coupled receptor D signaling

Hyperalgesia is typified by reduced pain thresholds and heightened responses to painful stimuli, with a notable prevalence in menopausal women, but the underlying mechanisms are far from understood. β-Aminoisobutyric acid (BAIBA), a product of valine and thymine catabolism, has been reported to be a novel ligand of the Mas-related G protein coupled receptor D (MrgprD), which mediates pain and hyperalgesia. Here, we established a hyperalgesia model in 8-week-old female mice through ovariectomy (OVX). A significant increase in BAIBA plasma level was observed and was associated with decline of mechanical withdrawal threshold, thermal and cold withdrawal latency in mice after 6 weeks of OVX surgery. Increased expression of MrgprD in dorsal root ganglion (DRG) was shown in OVX mice compared to Sham mice. Interestingly, chronic loading with BAIBA not only exacerbated hyperalgesia in OVX mice, but also induced hyperalgesia in gonadally intact female mice. BAIBA supplementation also upregulated the MrgprD expression in DRG of both OVX and intact female mice, and enhanced the excitability of DRG neurons in vitro. Knockout of MrgprD markedly suppressed the effects of BAIBA on hyperalgesia and excitability of DRG neurons. Collectively, our data suggest the involvement of BAIBA in the development of hyperalgesia via MrgprD-dependent pathway, and illuminate the mechanisms underlying hyperalgesia in menopausal women.

Sex-dependent cAMP signaling domains in vascular smooth muscle

The ubiquitous second messenger 3’,5’ monophosphate (cAMP) is essential for regulating vascular smooth muscle (VSM) function. Despite the overwhelming number of studies examining cAMP signaling compartmentalization in multiple cell types, no study has examined how biological sex regulates discrete cAMP pools and the functional implications in VSM. In this study, cAMP biosensors targeted to the cytosol (cyt), plasm membrane (PM), and sarcoplasmic reticulum (SR) were used to examine subcellular cAMP domains using live-cell FRET imaging. We uncovered a sex-specific distribution of cAMP signaling domains where cAMP pools are constrained in the SR of male but not in female mice. To test whether sexually dimorphic cAMP pools are mediated by sex hormones, we used WT male and sham or ovariectomized (OVX) WT female mice. cAMP subcellular distribution was like that of males in VSM from OVX female mice. Interestingly, aortic rings from males and OVX females showed less relaxation responses to isoproterenol compared to sham females. Our study highlights sex-dependent segregation cAMP pools in VSM that could contribute to the sex-specific regulation of vascular reactivity. American Heart Association award 852984 Research Supplement to Promote Diversity in Science 23DIVSUP1069345. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of GPER1 in macrophages ameliorates UUO-induced renal fibrosis

Numerous studies have proven the critical role of macrophages in the renal fibrosis process. Notably, G Protein-coupled Estrogen Receptor 1 (GPER1), a novel estrogen receptor, has been shown to play a ubiquitous role in regulating macrophage activities and proinflammatory pathways. However, the precise role of GPER1 in macrophage-mediated renal fibrosis is unknown. In this study, we aimed to investigate the function of macrophage GPER1 in the UUO-induced renal fibrosis model. Compared to vehicle-treated ovariectomized (OVX) female and male unilateral ureteral obstruction (UUO) models, we observed that G-1 (GPER1 agonist)-treated OVX female and male UUO mice had fewer renal fibrotic lesions and less M1 and M2 macrophage infiltration in the kidney tissues. Conversely, Gper1 deletion in male UUO mice accelerated renal fibrosis and increased inflammation. In vitro studies also revealed that GPER1 activation reduced M0 macrophage polarization towards M1 or M2 phenotypes. The RNA-sequencing analysis and immunoblotting indicated that GPER1 activation was primarily involved in downregulating immune pathways activation and inactivating MAPK pathways. Tubular epithelial cells co-cultured with G-1-pretreated M1 macrophages exhibited fewer injuries and immune activation. In addition, fibroblasts co-cultured with G-1-pretreated M2 macrophages showed downregulated extracellular matrix expression. Overall, this is the first study to demonstrate the effect of GPER1 on macrophage-mediated renal fibrosis via inhibition of M1 and M2 macrophage activation. These findings indicate that GPER1 may be a promising therapeutic target for treating renal fibrosis.

Diastolic dysfunction is associated with cardiac small vessel disease in ovariectomized females but not in males

Abstract Introduction Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature has still been poorly considered and investigated in animal models of HFpEF. Objective To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction. Method We assessed cardiac function and characterized the coronary microvasculature in two mouse models of HFpEF: mice fed with a high fat diet (HFD) + L-NAME regimen and Leptin receptor deficient (Leprdb/db) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dimorphisms. Results Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Leprdb/db mice, both Male and non OVX female mice develop diastolic dysfunction. Notably female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Leprdb/db mice. Conclusion: Diastolic dysfunction is not always associated with cardiac small vessel disease since Leprdb/db males and C57BL/6J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.

Abstract P3107: Sexual Dimorphism In Diastolic Dysfunction And Cardiac Small Vessel Disease In Heart Failure With Preserved Ejection Fraction Mouse Models

Introduction: Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature has still been poorly investigated in animal models of HFpEF and sex differences are not always considered. Objective: To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction in two HFpEF mouse models. Method: We assessed cardiac function and characterized the coronary microvasculature in mice fed with a high fat diet (HFD) + L-NAME regimen and in Leptin receptor deficient ( Lepr db/db ) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dimorphisms. Results: Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Lepr db/db mice, both Male and non OVX female mice develop diastolic dysfunction. Notably female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Lepr db/db mice. Conclusion: Diastolic dysfunction is not always associated with cardiac small vessel disease since Lepr db/db males and C57BL/6J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.

Diastolic dysfunction is associated with cardiac small vessel disease in ovariectomized females but not in males

Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature have still been poorly considered and investigated in animal models of HFpEF. To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction. We assessed cardiac function and characterized the coronary microvasculature in two mouse models of HFpEF: mice fed with a high fat diet (HFD) + L-NAME regimen and Leptin receptor deficient (Leprdb/db) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dysmorphism. Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Leprdb/db mice, both male and non OVX female mice develop diastolic dysfunction. Notably, female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Leprdb/db mice. Diastolic dysfunction is not always associated with cardiac small vessel disease since Leprdb/db males and C57BL/6 J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.

Abstract 11486: Hormone-Independent Sex Dimorphisms in Cardiac Reparative Functions of Bone Marrow Stem Cells: Underlying Epigenetic Insights

Introduction: This work was designed to identify the hormone-independent mechanisms that govern the superiority of female endothelial progenitor cells (F-EPC) for cardiac repair post myocardial infarction (MI). A direct comparison of therapeutic efficacy of gender-specific stem cells or estrogen-independent mechanisms of gender-specific dimorphism in the reparative properties of EPCs has not been established. Hypothesis: We hypothesized that epigenetic mechanisms contribute to the sex-specific functional dimorphisms of Sca-1+/CD31+ EPCs in regulating cardiac reparative functions post-MI. Methods: Male (M), female (F), and ovariectomized (OVX) mice derived Sca-1+/CD31+ EPCs were subjected through a series of cytokine quantifications and epigenetic CUT&Tag screening, followed by assessment of their therapeutic function in vitro and in vivo . Results: F-EPC and OVX EPC secrete high levels of proangiogenic factors and low amounts of pro-inflammatory cytokines compared to M-EPC. Further evaluation of the inflammasome showed that the M-EPC secreted the most elevated amounts of CCL3, and other inflammatory chemokines compared to F-EPC and OVX EPC. The secretome of F-EPC and OVX EPC was found to be substantially proangiogenic in vitro than the male EPCs. Post-MI injection of F-EPC and OVX EPC resulted in remarkable preservation of cardiac structure and functions. In contrast, post-MI injection of M-EPC resulted in an inflamed myocardium. Epigenetic sequencing of the sex differential EPCs for the H3K9me3 mark showed high methylation in M-EPC compared to F-EPC and OVX EPC. The inhibition of histone methyltransferase, G9a using BIX-01294 upregulated the secretion of inflammatory factors in all EPCs. The conditioned medium from all EPCs with high levels of CCL3 inhibited angiogenesis in vitro . Neutralizing CCL3 in the same medium restored in vitro angiogenesis. Conclusion: EPCs from both fertile and OVX female mice retain better cardiac reparative activity than their male counterparts irrespective of sex hormone influence. Mechanistically, we show that this gender-based functional dimorphism of EPCs is mediated, in part, by differential epigenetic regulation of chemokine CCL3.

Augmenting Skeletal Muscle Estrogen Does not Prevent or Rescue Obesity-linked Metabolic Impairments in Female Mice.

We developed a novel mouse model with increased skeletal muscle estrogen content via inducible, skeletal-muscle-specific aromatase overexpression (SkM-Arom↑). We proposed to examine the effect that increased skeletal muscle estrogen both in gonadally intact and ovariectomized (OVX) female mice has on preventing or rescuing high-fat diet (HFD)-induced obesity. In the prevention experiment, gonadally intact and OVX SkM-Arom↑ mice and littermate controls were fed a low-fat diet (LFD) or HFD for 13 weeks. SkM-Arom↑ was induced at the initiation of dietary treatment. In the intervention experiment, gonadally intact and OVX SkM-Arom↑ mice and littermate controls were fed an HFD for 14 weeks before induction of SkM-Arom↑ for 6 weeks. Glucose tolerance, insulin action, adipose tissue inflammation, and body composition were assessed. Liquid chromatography-mass spectrometry was used to determine circulating and skeletal muscle steroid content. SkM-Arom↑ significantly increased skeletal muscle 17β-estradiol (E2) and estrone (E1) in both experiments. Interestingly, this resulted in leakage of estrogens into circulation, producing a physiologically relevant E2 concentration. Consequently, bone mineral density (BMD) was enhanced and adipose tissue inflammation was reduced in the prevention experiment only. However, no benefits were seen with respect to changes in adiposity or metabolic outcomes. We show that increasing skeletal muscle estrogen content does not provide a metabolic benefit in gonadally intact and OVX female mice in the setting of obesity. However, a chronic physiological concentration of circulating E2 can improve BMD and reduce adipose tissue inflammation independently of a metabolic benefit or changes in adiposity.

Hypothalamic NPY-Y1R Interacts with Gonadal Hormones in Protecting Female Mice against Obesity and Neuroinflammation.

We previously demonstrated that Npy1rrfb mice, which carry the conditional inactivation of the Npy1r gene in forebrain principal neurons, display a sexually dimorphic phenotype, with male mice showing metabolic, hormonal and behavioral effects and females being only marginally affected. Moreover, exposure of Npy1rrfb male mice to a high-fat diet (HFD) increased body weight growth, adipose tissue, blood glucose levels and caloric intake compared to Npy1r2lox male controls. We used conditional knockout Npy1rrfb and Npy1r2lox control mice to examine whether forebrain disruption of the Npy1r gene affects susceptibility to obesity and associated disorders of cycling and ovariectomized (ovx) female mice in a standard diet (SD) regimen or exposed to an HFD for 3 months. The conditional deletion of the Npy1r gene increased body weight and subcutaneous white adipose tissue weight in both SD- and HFD-fed ovx females but not in cycling females. Moreover, compared with ovx control females on the same diet regimen, Npy1rrfb females displayed increased microglia number and activation, increased expression of Neuropeptide Y (NPY)-immunoreactivity (IR) and decreased expression of proopiomelanocortin-IR in the hypothalamic arcuate nucleus (ARC). These results suggest that in the ARC NPY-Y1R reduces the susceptibility to obesity of female mice with low levels of gonadal hormones and that this effect may be mediated via NPY-Y1R ability to protect the brain against neuroinflammation.

Sexual Dimorphism in Control of Heart Failure in Insulin Resistance

The incidence of cardiovascular diseases (CVD) is highly associated with type 2 diabetes (T2D). Women at an early age show reduced incidence of CVD and improved insulin sensitivity compared to men, but these disparities disappear during postmenopausal. Thus, ovary and ovarian hormone, such as estrogen, are expected to protect from these diseases. This study aimed to investigate the role of ovaries and ovarian hormone estrogen in cardiac function and energy metabolism upon loss‐of‐function of cardiac IRS1 and IRS2 in mice and investigate the underlying mechanisms. We found that all male heart‐specific IRS1 and IRS2 double knock‐out (H‐DKO) mice died of heart failure (HF) at 6‐8 weeks with severely impaired energy metabolism. However, all female H‐DKO mice survived more than 1 year and showed improvement in energy metabolism. Removal of ovaries in H‐DKO female mice led to cardiac dysfunction, disruption of energy metabolism, and ultimately death. However, E2 supplement significantly improved cardiac function, enhanced energy metabolism, and prolonged the lifespan in both male and OVX female H‐DKO mice. Finally, we provided evidence that the protective effect of E2 is partially mediated through activation of Akt‐Foxo1 signaling in H‐DKO heart. These results show that estrogen protects mice from heart insulin resistance induced cardiomyopathy. This study indicates that estrogen signaling could be a potential target to prevent insulin resistance induced heart dysfunction in humans. Estrogen signaling is mediated through estrogen receptors, such as estrogen receptor (ER) α, ERβ, and membrane estrogen receptor (GPER). In the future, we will further decipher the role of estrogen receptors in estrogen mediated protection against cardiac dysfunction during insulin resistance.

Protein kinase G phosphorylation of Chip protects ovariectomized females from myocardial infarction

Proteotoxicity from insufficient clearance of misfolded/damaged proteins exacerbates cardiac functional decline following a myocardial infarction (MI). Estrogen is known to play a crucial role in the survival rate of cardiomyocytes following a MI and is associated with increased protein degradation, however the mechanisms remain to be fully elucidated. Further estrogen has been shown to enhance myocardial protein kinase G (PKG) activity following an insult. In this study, we link this cardioprotective signaling pathway to the carboxyl terminus of Hsc70‐interacting protein (Chip), specifically via phosphorylation at serine 20 (pS20). Chip is a unique effector of proteostasisfunctioning as both a co‐chaperone and ubiquitin ligase. We test the role of Chip S20 with phospho‐mimetic (SE) and phospho‐null (SA) mice compared to WT mice in ovariectomized (OVX) and intact female mice following a MI. We did not observe anydifferences in cardiac function or proteostasis in sham mice regardless of genotype and OVX/intact surgery. Chip SE mice were protected against a MI in both OVX and intact female mice, as exhibited by maintained cardiac function and morphology. Chip WT, even moreso in Chip SA OVX mice, presented with decreased cardiac function, left ventricular chamber dilation and wall thinning, and myocardial fibrosis. Ubiquitinated proteins and protein aggregates were increased in the hearts of Chip SA OVX micewhile Chip SE OVX mice had little accumulation of either, both compared to Chip WT OVX mice. Notably the phospho‐status of Chip S20 did not alter the degradative capacities of the proteasome or lysosome, suggesting the underlying mechanism for enhanced proteostasis is due to increase functionality of the Chip complex. Collectively these dataindicate phosphorylation of Chip S20 is a critical regulator of cardiac proteostasis and is able to afford protection to OVX mice following a MI. These findings have clinical potential as post‐menopausal women are most at risk for deleterious outcomes following a heart attack.

Establishment of the Mouse Model of Social Avoidance Induced by Female-Directed Female Aggression

Background Most preclinical research on the effects of stress has been done on male subjects, even though women are more prone than men to experience stress-related problems. Chronic social defeat stress (CSDS) is a rodent model of psychosocial stress. However, this model has been challenged in female mouse studies since neither male nor female resident mice attack intruder females. A female-to-female CSDS model is needed to investigate the physiological and behavioral aspects. Methods The intruders were either male or female C57BL/6J mice, whereas the residents were male or ovariectomized (OVX) female CD-1 mice. The CD-1 aggressor mice had direct physical contact with the C57BL/6J mice for 10 min before initiating sensory contact with them for 24 h. Jump escape and freezing were evaluated during the social defeat of days 1 and 12. Experimental C57BL/6J mice underwent a social interaction test after suffering social defeat for 12 days. Results We found that the number of attack bites and attack latency had a significant negative correlation during the selection of aggressors. In the single-housed OVX mice, 34% of mice met the criterion of the selection of aggressors. However, single-housed OVX mice did not show sustained aggressive behavior (eg, attack bites) through the 12-day CSDS. As a result, we did not find susceptible mice during the social interaction test. In contrast, during the selection of aggressors, 42% of OVX mice housed with partners satisfied the criterion and displayed consistently aggressive behavior. CSDS produced susceptible (50%) and resilient (50%) phenotypes during the social interaction test. Notably, male and OVX female CD-1 mice housed with partners had similar amounts of attack bites and attack rates over the 12-day CSDS. Finally, we found that chronically socially defeated male and female mice displayed different coping behaviors (eg, active vs passive) with social defeat. Conclusions Our study demonstrates that OVX CD-1 mice housed with mates exhibited territorial aggression toward female intruders, producing susceptibility and resilience to social avoidance. Additionally, socially defeated male and female mice displayed different behavioral susceptibility to social defeat.

Sex differences in cocaine-associated memory: The interplay between CB1, mGluR5, and estradiol

We know surprisingly little about the sex differences in the neurobiology of cocaine addiction, except females are more susceptible to the rewarding effects of cocaine than their male counterparts. Only a handful of recent studies have examined the neurobiology of cocaine-induced conditioned place preference (CPP) memory among female rodents. We contribute to this emerging line of research by documenting sex differences in cocaine-associated memory and illustrating the underlying signaling pathways in five experiments. Rimonabant (Rim), a cannabinoid CB1 antagonist and inverse agonist, exerted a facilitating effect for low-dose cocaine and an impairing effect for high-dose cocaine CPP memory in male mice, as in our previous study, but not in female mice. Nor did we observe the effect exist among CB1 knockout male mice, which indicated that the CB1 receptors played a mediating role. We also found that the metabotropic glutamate receptor 5 (mGluR5) was located in the same signaling pathway as CB1 in male mice. To clarify the mechanisms behind the sex differences, we used ovariectomized (OVX) female mice with estradiol benzoate (EB) replacement. In the OVX female mice, we showed that Rim-alone and EB-alone, but not Rim-and-EB-combined, facilitated the low-dose cocaine CPP memory. Moreover, 4-hydroxytamoxifen (4-OHT), an estrogen receptor (ER) antagonist, blocked Rim's and EB's facilitating effect. Finally, 2-methyl-6-(phenylethynyl)pyridine (MPEP), an mGluR5 antagonist, partially blocked EB’s facilitating effect. In sum, we identified sex-specific effects of Rim on cocaine-induced CPP memory and the respective signaling pathways: mGluR5-CB1 for male mice and ER-mGluR5-CB1 for female mice. These findings may have merits for the development of sex-specific treatment for cocaine addiction.

Estrogen Enhances the Microvascular Reactivity Through RhoA-ROCK Pathway in Female Mice During Hemorrhagic Shock.

Vascular hypo-reactivity plays a critical role inducing organ injury during hemorrhagic shock. 17β-estradiol (E2) can induce vasodilation to increase blood flow in various vascular beds. This study observed whether E2 can restore vascular hypo-reactivity induced by hemorrhagic shock, and whether E2 effects are associated with RhoA-Rho kinase (ROCK)-myosin light chain kinase phosphatase (MLCP) pathway. The hemorrhagic shock model (40 ± 2 mm Hg for 1 h, resuscitation for 4 h) was established in ovary intact sham operation (OVI), ovariectomized (OVX), and OVX plus E2 supplement female mice. Intestinal microvascular loop was used to assess blood flow in vivo, mRNA expression and vascular reactivity in vitro. Hemorrhagic shock significantly reduced norepinephrine microvascular reactivity. Decreased microvascular reactivity was exacerbated by OVX and reversed by E2 supplement. U-46619 (RhoA agonist) increased microvascular reactivity, and C3 transferase (an ADP ribosyl transferase that selectively induces RhoA ribosylation) or Y-27632 (ROCK inhibitor) inhibited sham mice microvascular reactivity. Similarly, U-46619 increased microvascular reactivity in OVI and OVX mice following hemorrhagic shock, which was abolished by Y-27632 or concomitant incubation of okadaic acid (OA) (MLCP inhibitor) and Y-27632. In OVX plus E2 supplement mice with hemorrhagic shock, Y-27632 inhibited microvascular reactivity, which was abolished by concomitant U-46619 application. Lastly, hemorrhagic shock remarkably decreased intestinal loop blood flow, RhoA and ROCK mRNA expressions in vascular tissues in OVX females, but not in OVI females, which were reversed by E2 supplement. These results indicate that estrogen improves microvascular reactivity during hemorrhagic shock, and RhoA-ROCK signaling pathway may mediate E2 effects.

AhR/miR-23a-3p/PKCα axis contributes to memory deficits in ovariectomized and normal aging female mice

The mechanism of estrogen deficiency-induced cognitive impairment is still not fully elucidated. In this study, we assessed the effect of microRNA (miRNA) on the memory of long-term estrogen-deficient mice after ovariectomy (OVX) and normal aging. We observed that 5-month OVX and 22-month-old normal aging female mice showed significantly impaired spatial and object recognition memory, declined hippocampal long-term potentiation (LTP), and decreased hippocampal protein kinase C α (PKCα) protein. Quantitative real-time PCR analysis showed upregulated miRNA-23a-3p (miR-23a-3p) in the hippocampus of 5-month OVX and 22-month-old female mice. In vitro, overexpression of miR-23a-3p downregulated PKCα by binding the 3¢ UTRs of Prkca mRNAs, which was prevented by its antisense oligonucleotide AMO-23a. In vivo, adeno-associated virus-mediated overexpression of miR-23a-3p (AAV-pre-miR-23a-3p) suppressed hippocampal PKCα and impaired the memory of mice. Chromatin immunoprecipitation analysis showed that aryl hydrocarbon receptor (AhR) binds the promoter region of miR-23a-3p. The AhR-dependent downregulation of PKCα could be prevented by AMO-23a as well. Furthermore, knockdown of miR-23a-3p using AAV-AMO-23a rescued the cognitive and electrophysiological impairments of OVX and normal aging female mice. We conclude that long-term estrogen deficiency impairs cognition and hippocampal LTP by activating the AhR/miR-23a-3p/PKCα axis. The knockdown of miR-23a-3p may be a potentially valuable therapeutic strategy for estrogen deficiency-induced memory deficits.

Intact Female Mice Acquire Trace Eyeblink Conditioning Faster than Male and Ovariectomized Female Mice.

Female subjects have been widely excluded from past neuroscience work because of a number of biases, including the notion that cycling sex hormones increase variability. However, it is necessary to conduct behavioral research in mice that includes both sexes as mice are typically used for developing and evaluating future therapeutics. Understanding sex differences in learning is fundamental for the development of targeted therapies for numerous neurologic and neurodegenerative disorders, including Alzheimer’s disease, which is more prevalent in females than males. This study set out to confirm the role of sex and necessity of circulating ovarian hormones in the acquisition of the temporal associative memory task trace eyeblink conditioning (tEBC) in C57BL/6J mice. We present evidence that sex and ovarian hormones are important factors in learning. Specifically, intact female mice learn significantly faster than both male and ovariectomized (ovx) female mice. Data from pseudoconditioned control mice indicate that sex differences are because of differences in learned associations, not sensitization or spontaneous blink rate. This study strengthens the idea that ovarian hormones such as estrogen and progesterone significantly influence learning and memory and that further research is needed to determine the underlying mechanisms behind their effects. Overall, our findings emphasize the necessity of including both sexes in future behavioral studies.

TPH2 in the Dorsal Raphe Nuclei Regulates Energy Balance in a Sex-Dependent Manner

AbstractCentral 5-hydroxytryptamine (5-HT), which is primarily synthesized by tryptophan hydroxylase 2 (TPH2) in the dorsal Raphe nuclei (DRN), plays a pivotal role in the regulation of food intake and body weight. However, the physiological functions of TPH2 on energy balance have not been consistently demonstrated. Here we systematically investigated the effects of TPH2 on energy homeostasis in adult male and female mice. We found that the DRN harbors a similar amount of TPH2+ cells in control male and female mice. Adult-onset TPH2 deletion in the DRN promotes hyperphagia and body weight gain only in male mice, but not in female mice. Ablation of TPH2 reduces hypothalamic pro-opiomelanocortin (POMC) neuronal activity robustly in males, but only to a modest degree in females. Deprivation of estrogen by ovariectomy (OVX) causes comparable food intake and weight gain in female control and DRN-specific TPH2 knockout mice. Nevertheless, disruption of TPH2 blunts the anorexigenic effects of exogenous estradiol (E2) and abolishes E2-induced activation of POMC neurons in OVX female mice, indicating that TPH2 is indispensable for E2 to activate POMC neurons and to suppress appetite. Together, our study revealed that TPH2 in the DRN contributes to energy balance regulation in a sexually dimorphic manner.

Assessment of safety and efficacy of pine bark extract in normal and ovariectomized mice.

We evaluated the influence of pine bark extract (PBE) on organs, the cytochrome-P450 (CYP) activities in liver and estrogenic effects in normal and ovariectomized (OVX) female mice. The PBE did not affect organ weights and liver-function indexes (activities of alkaline phosphatase, aspartate amino transferase, and alanine amino transferase) at doses; 0.04%, 0.4%, and 2.0% PBE in the diet, in normal and OVX female mice. In the OVX mice, CYP1A1 activity was significantly higher in the 0.4% and 2.0% PBE groups than in the OVX control group, and in the 0.4% and 2.0% PBE groups were significantly higher than in the 0.04% PBE group. CYP1A2 and 3A4 activities were significantly higher in the 2.0% PBE group than in all other groups. The PBE did not affect uterine weight and femoral bone mineral density at all PBE doses. These results showed that the dose of PBE at the recommended human intake, had no toxic and estrogenic effects in normal female and OVX mice, however, it may need attention to use the excess intake of PBE with some drugs in postmenopausal women.