Estradiol-mediated protection against high-fat diet induced anxiety and obesity is associated with changes in the gut microbiota in female mice.

Decreased estrogens during menopause are associated with increased risk of anxiety, depression, type 2 diabetes and obesity. Similarly, depleting estrogens in rodents by ovariectomy, combined with a high-fat diet (HFD), increases anxiety and adiposity. How estrogens and diet interact to affect anxiety and metabolism is poorly understood. Mounting evidence indicates that gut microbiota influence anxiety and metabolism. Here, we investigated the effects of estradiol (E) and HFD on anxiety, metabolism, and their correlation with changes in gut microbiota in female mice. Adult C57BL/6J mice were ovariectomized, implanted with E or vehicle-containing capsules and fed a standard diet or HFD. Anxiety-like behavior was assessed and neuronal activation was measured by c-fos immunoreactivity throughout the brain using iDISCO. HFD increased anxiety-like behavior, while E reduced this HFD-dependent anxiogenic effect. Interestingly, E decreased neuronal activation in brain regions involved in anxiety and metabolism. E treatment also altered gut microbes, a subset of which were associated with anxiety-like behavior. These findings provide insight into gut microbiota-based therapies for anxiety and metabolic disorders associated with declining estrogens in menopausal women.

Differential effects of Akkermansia-enriched fecal microbiota transplant on energy balance in female mice on high-fat diet.

Estrogens protect against weight gain and metabolic disruption in women and female rodents. Aberrations in the gut microbiota composition are linked to obesity and metabolic disorders. Furthermore, estrogen-mediated protection against diet-induced metabolic disruption is associated with modifications in gut microbiota. In this study, we tested if estradiol (E2)-mediated protection against obesity and metabolic disorders in female mice is dependent on gut microbiota. Specifically, we tested if fecal microbiota transplantation (FMT) from E2-treated lean female mice, supplemented with or without Akkermansia muciniphila, prevented high fat diet (HFD)-induced body weight gain, fat mass gain, and hyperglycemia in female recipients. FMT from, and cohousing with, E2-treated lean donors was not sufficient to transfer the metabolic benefits to the E2-deficient female recipients. Moreover, FMT from lean donors supplemented with A. muciniphila exacerbated HFD-induced hyperglycemia in E2-deficient recipients, suggesting its detrimental effect on the metabolic health of E2-deficient female rodents fed a HFD. Given that A. muciniphila attenuates HFD-induced metabolic insults in males, the present findings suggest a sex difference in the impact of this microbe on metabolic health.

Distinct Changes in Gut Microbiota Are Associated with Estradiol-Mediated Protection from Diet-Induced Obesity in Female Mice.

A decrease in ovarian estrogens in postmenopausal women increases the risk of weight gain, cardiovascular disease, type 2 diabetes, and chronic inflammation. While it is known that gut microbiota regulates energy homeostasis, it is unclear if gut microbiota is associated with estradiol regulation of metabolism. In this study, we tested if estradiol-mediated protection from high-fat diet (HFD)-induced obesity and metabolic changes are associated with longitudinal alterations in gut microbiota in female mice. Ovariectomized adult mice with vehicle or estradiol (E2) implants were fed chow for two weeks and HFD for four weeks. As reported previously, E2 increased energy expenditure, physical activity, insulin sensitivity, and whole-body glucose turnover. Interestingly, E2 decreased the tight junction protein occludin, suggesting E2 affects gut epithelial integrity. Moreover, E2 increased Akkermansia and decreased Erysipleotrichaceae and Streptococcaceae. Furthermore, Coprobacillus and Lactococcus were positively correlated, while Akkermansia was negatively correlated, with body weight and fat mass. These results suggest that changes in gut epithelial barrier and specific gut microbiota contribute to E2-mediated protection against diet-induced obesity and metabolic dysregulation. These findings provide support for the gut microbiota as a therapeutic target for treating estrogen-dependent metabolic disorders in women.

Dopamine-induced interactions of female mouse hypothalamic proteins with progestin receptor-A in the absence of hormone.

Neural progestin receptors (PR) function in reproduction, neural development, neuroprotection, learning, memory and the anxiety response. In the absence of progestins, PR can be activated by dopamine (DA) in the rodent hypothalamus to elicit female sexual behaviour. The present study investigated mechanisms of DA activation of PR by testing the hypothesis that proteins from DA-treated hypothalami interact with PR in the absence of progestins. Ovariectomised, oestradiol-primed mice were infused with a D1-receptor agonist, SKF38393 (SKF), into the third ventricle 30 minutes prior to death. Proteins from SKF-treated hypothalami were pulled-down with glutathione S-transferase-tagged mouse PR-A or PR-B and the interactomes were analysed by mass spectrometry. The largest functional group to interact with PR-A in a DA-dependent manner was synaptic proteins. To test the hypothesis that DA activation of PR regulates synaptic proteins, we developed oestradiol-induced PR-expressing hypothalamic-like neurones derived from human-induced pluripotent stem cells (hiPSCs). Similar to progesterone (P4), SKF treatment of hiPSCs increased synapsin1/2 expression. This SKF-dependent effect was blocked by the PR antagonist RU486, suggesting that PR are necessary for this DA-induced increase. The second largest DA-dependent PR-A protein interactome comprised metabolic regulators involved in glucose metabolism, lipid synthesis and mitochondrial energy production. Interestingly, hypothalamic proteins interacted with PR-A, but not PR-B, in an SKF-dependent manner, suggesting that DA promotes the interaction of multiple hypothalamic proteins with PR-A. These in vivo and in vitro results indicate novel mechanisms by which DA can differentially activate PR isoforms in the absence of P4 and provide a better understanding of ligand-independent PR activation in reproductive, metabolic and mental health disorders in women.

Daily Vaginal Microbiota Fluctuations Associated with Natural Hormonal Cycle, Contraceptives, Diet, and Exercise.

The microorganisms of the vaginal tract are critical for vaginal and reproductive health. However, the regulation of these microorganisms is not well understood. Therefore, we investigated whether different factors regulate the vaginal microbiota of healthy college-aged women (n = 26) with high temporal resolution by collecting daily self-administered vaginal swabs and using 16S rRNA sequencing for bacterial identification. As expected, vaginal microbiota clustered into five predefined community state types. Vaginal microbial diversity, stability, and Lactobacillus abundances were associated with the menstrual cycle and hormonal contraceptive use. Vaginal microbial diversity, as measured using the Shannon index, increased during menses (P < 0.001), while Lactobacillus abundances decreased (P = 0.01). The covariance of these microbial measures with previously established estradiol levels suggests that estrogens can regulate vaginal microbiota. Moreover, the use of hormonal contraceptives may alter the temporal dynamics of the vaginal microbiota and decrease Lactobacillus abundances, depending on hormonal content and release method. Interestingly, intrasample diversity was greater in participants on a vegetarian diet (P = 0.004) and among participants who exercised more (P = 0.04). These findings indicate that ovarian hormones, diet, and exercise can regulate vaginal microbial composition and stability and may impact vaginal and reproductive health.IMPORTANCE The vaginal microbiome is a critical component of women's sexual and reproductive health, with variations in microbial composition, particularly the loss of Lactobacillus species, being implicated in gynecologic and obstetric diseases. Given that the vaginal microbiome is so crucial, why do vaginal microbial profiles vary strikingly from person to person and even change over time within the same person? In the present study, which tracked the daily vaginal microbiomes of young healthy women through different lifestyles, we found that use of a locally released progestin contraceptive, a vegetarian diet, and intense exercise appear to lead to vaginal microbiome alterations and loss of Lactobacillus species. The impact of these vaginal microbiome changes on immediate and long-term health remain to be investigated.

Estradiol and high fat diet associate with changes in gut microbiota in female ob/ob mice.

Estrogens protect against diet-induced obesity in women and female rodents. For example, a lack of estrogens in postmenopausal women is associated with an increased risk of weight gain, cardiovascular diseases, low-grade inflammation, and cancer. Estrogens act with leptin to regulate energy homeostasis in females. Leptin-deficient mice (ob/ob) exhibit morbid obesity and insulin resistance. The gut microbiome is also critical in regulating metabolism. The present study investigates whether estrogens and leptin modulate gut microbiota in ovariectomized ob/ob (obese) or heterozygote (lean) mice fed high-fat diet (HFD) that received either 17ß-Estradiol (E2) or vehicle implants. E2 attenuated weight gain in both genotypes. Moreover, both obesity (ob/ob mice) and E2 were associated with reduced gut microbial diversity. ob/ob mice exhibited lower species richness than control mice, while E2-treated mice had reduced evenness compared with vehicle mice. Regarding taxa, E2 was associated with an increased abundance of the S24-7 family, while leptin was associated with increases in Coriobacteriaceae, Clostridium and Lactobacillus. Some taxa were affected by both E2 and leptin, suggesting these hormones alter gut microbiota of HFD-fed female mice. Understanding the role of E2 and leptin in regulating gut microbiota will provide important insights into hormone-dependent metabolic disorders in women.

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent.

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase-tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I-positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

Adult Neurogenesis in the Female Mouse Hypothalamus: Estradiol and High-Fat Diet Alter the Generation of Newborn Neurons Expressing Estrogen Receptor α.

Estrogens and leptins act in the hypothalamus to maintain reproduction and energy homeostasis. Neurogenesis in the adult mammalian hypothalamus has been implicated in the regulation of energy homeostasis. Recently, high-fat diet (HFD) and estradiol (E2) have been shown to alter cell proliferation and the number of newborn leptin-responsive neurons in the hypothalamus of adult female mice. The current study tested the hypothesis that new cells expressing estrogen receptor α (ERα) are generated in the arcuate nucleus (ARC) and the ventromedial nucleus of the hypothalamus (VMH) of the adult female mouse, hypothalamic regions that are critical in energy homeostasis. Adult mice were ovariectomized and implanted with capsules containing E2 or oil. Within each hormone group, mice were fed an HFD or standard chow for 6 weeks and treated with BrdU to label new cells. Newborn cells that respond to estrogens were identified in the ARC and VMH, of which a subpopulation was leptin sensitive, indicating that the subpopulation consists of neurons. Moreover, there was an interaction between diet and hormone with an effect on the number of these newborn ERα-expressing neurons that respond to leptin. Regardless of hormone treatment, HFD increased the number of ERα-expressing cells in the ARC and VMH. E2 decreased hypothalamic fibroblast growth factor 10 (Fgf10) gene expression in HFD mice, suggesting a role for Fgf10 in E2 effects on neurogenesis. These findings of newly created estrogen-responsive neurons in the adult brain provide a novel mechanism by which estrogens can act in the hypothalamus to regulate energy homeostasis in females.

Estradiol Preferentially Induces Progestin Receptor-A (PR-A) Over PR-B in Cells Expressing Nuclear Receptor Coactivators in the Female Mouse Hypothalamus

Estrogens act in brain to profoundly influence neurogenesis, sexual differentiation, neuroprotection, cognition, energy homeostasis, and female reproductive behavior and physiology through a variety of mechanisms, including the induction of progestin receptors (PRs). PRs are expressed as two isoforms, PR-A and PR-B, that have distinct functions in physiology and behavior. Because these PR isoforms cannot be distinguished using cellular resolution techniques, the present study used isoform-specific null mutant mice that lack PR-A or PR-B for the first time to investigate whether 17ß-estradiol benzoate (EB) regulates the differential expression of the PR isoforms in the ventromedial nucleus of the hypothalamus (VMN), arcuate nucleus, and medial preoptic area, brain regions that are rich in EB-induced PRs. Interestingly, EB induced more PR-A than PR-B in all three brain regions, suggesting that PR-A is the predominant isoform in these regions. Given that steroid receptor coactivator (SRC)-1 and SRC-2 are important in estrogen receptor (ER)-dependent transcription in brain, including PR induction, we tested whether the expression of these coactivators was correlated with PR isoform expression. The majority of EB-induced PR cells expressed both SRC-1 and SRC-2 in the three brain regions of all genotypes. Interestingly, the intensity of PR-A immunoreactivity correlated with SRC-2 expression in the VMN, providing a potential mechanism for selective ER-mediated transactivation of PR-A over PR-B in a brain region-specific manner. In summary, these novel findings indicate that estrogens differentially regulate PR-A and PR-B expression in the female hypothalamus, and provide a mechanism by which steroid action in brain can selectively modulate behavior and physiology.

Sexually dimorphic expression and estradiol mediated up-regulation of a sex-linked ribosomal gene, RPS6, in the zebra finch brain.

Sex-linked genes are considered to be a major contributor to neural sex differences in zebra finches. While several candidates have been identified, additional ones are continuously being discovered. Here we report on a novel Z-linked ribosomal gene (rpS6) that is enhanced in the male brain as compared to the female's throughout life. In both sexes, expression of rpS6 is highest at P3 and P8 (just before the onset of morphologically detectable sex differences), decreases around P15, and then remains decreased through adulthood. Analysis of rpS6 mRNA revealed widespread distribution throughout the brain. However, within song regions HVC and RA, mRNA containing cells were greater in males as compared to females. Hormones are also involved in the development of neural dimorphisms, so we additionally investigated whether rpS6 might interact with estradiol (E2 ). An up-regulation of rpS6 gene was observed in both sexes following treatment with E2 and the effect was approximately twice as large in males as compared with females. These data suggest that rpS6 may be involved in sexual differentiation of the zebra finch brain, and that the effect is facilitated by E2 .

Characterization of the G-protein-coupled membrane-bound estrogen receptor GPR30 in the zebra finch brain reveals a sex difference in gene and protein expression.

Estradiol-induced structural dimorphisms exist in the songbird brain. However, how they arise is not clear since there is a scarce distribution of ERα and lack of ERß in song control nuclei. This suggests that other receptors are involved. The G-protein coupled membrane-bound estrogen receptor, GPR30, is a candidate but has never been investigated in songbirds. In this study, we characterized its gene and protein in the zebra finch brain. Analysis of the putative GPR30 protein sequence revealed a strong similarity to avian and mammalian homologues. Quantitative PCR indicated that the gene was elevated in the telencephalon of both sexes from posthatching day (P) 15 to P45, with a male-biased sex difference at P21 and P30. In comparison, expression at younger posthatching ages and in adults was significantly less. At P21, GRP30 protein was widespread, nonuniform, and overlapped with song control nuclei. Of particular interest, the number of immunoreactive cells was greatest in HVC and RA, but less in LMAN and Area X. Labeling in HVC was also dimorphic; with more cells present in males than in females. In parallel with the gene, by adulthood, protein expression was reduced across most brain regions. Taken together these data suggest that GPR30 may contribute to differences in song system development by mediating dimorphic responses to estrogens. In addition, the extensive protein distribution indicates that it may also have a role in general brain development in both sexes.