Action Of Estradiol Research Articles

Independent effects of testosterone, estradiol, and sex chromosomes on gene expression in immune cells of trans- and cisgender individuals.

The origins of sex differences in human disease are elusive, in part because of difficulties in separating the effects of sex hormones and sex chromosomes. To separate these variables, we examined gene expression in four groups of trans- or cisgender individuals: XX individuals treated with exogenous testosterone (n=21), XY treated with exogenous estradiol (n=13), untreated XX (n=20), and untreated XY (n=15). We performed single-cell RNA-sequencing of 358,426 peripheral blood mononuclear cells. Across the autosomes, 8 genes responded with a significant change in expression to testosterone, 34 to estradiol, and 32 to sex chromosome complement with no overlap between the groups. No sex-chromosomal genes responded significantly to testosterone or estradiol, but X-linked genes responded to sex chromosome complement in a remarkably stable manner across cell types. Through leveraging a four-state study design, we successfully separated the independent actions of testosterone, estradiol, and sex chromosome complement on genome-wide gene expression in humans.

Estrous cycle-dependent modulation of sexual receptivity in female mice by estrogen receptor beta-expressing cells in the dorsal raphe nucleus

The sexual receptivity of female mice, shown as lordosis response, is mainly regulated by estradiol action on estrogen receptor alpha (ERα) and beta (ERβ), depending on the day of the estrous cycle. Previous studies revealed that ERα in the ventromedial nucleus of the hypothalamus (VMH) plays an essential role in the induction of lordosis on the day of estrus (Day 1). However, the mechanisms of the transition to non-receptive states on the day after estrus (Day 2) are not completely understood. In the present study, we investigated the possible role of ERβ, which is highly expressed in the dorsal raphe nucleus (DRN), in lordosis expression. We found that ERβ-Cre female mice, which were ovariectomized and primed with estradiol and progesterone to mimic the estrous cycle, showed high levels of lordosis on Day 2 when ERβ-expressing DRN (DRN-ERβ+) neuronal activity was chemogenetically suppressed. This finding suggests that excitation of DRN-ERβ+ neurons is necessary for the decline of lordosis on Day 2. Fiber photometry recordings during female-male behavioral interactions revealed that DRN-ERβ+ neuronal activation in response to male intromission was significantly more prolonged on Day 2 compared to Day 1. Chemogenetic over-stimulation of DRN-ERβ+ neurons induced c-Fos expression in brain areas known to be inhibitory for lordosis expression, even though they did not express anterogradely labeled fibers of DRN-ERβ+ cells. These findings collectively suggest that DRN-ERβ+ neuronal excitation serves as an inhibitory modulator and is responsible for the decline in receptivity during non-estrus phases.Significance StatementIn females, switching from a sexually receptive state to a non-receptive phase during the estrous cycle is essential for effective behavioral interactions with males and successful reproduction. Here, we delineate the possible brain regions that regulate the decline in receptive behavioral responses, such as lordosis, from those shown on the day of estrus to those on the day after estrus. We found that excitation of neurons expressing estrogen receptor (ER) β in the midbrain dorsal raphe nucleus is crucial for the suppression of lordosis during the day after estrus. This is contrasted with the facilitatory action of ERα, another type of ER, and provides new insights for understanding the neural basis of the adaptive expression of female reproductive behavior.

A sustainable and innovative method to determine parabens in body creams for exposure and risk assessment

Methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), and butylparaben (BuP) are among the most widely used preservatives in cosmetics, drugs, and foods. These compounds have been associated with toxic effects due to the overuse of products with parabens in their formulation. The toxicity of parabens may be correlated to endocrine disruption, owing to their ability to mimic the actions of estradiol. In this paper, a simple, sustainable, robust, and innovative dispersive liquid-liquid microextraction (DLLME) technique was developed and employed to extract these xenobiotics from body cream samples, aiming to calculate the margin of safety (MoS) to assess the risk of exposure. The validated method presented suitable linearity (r > 0.99), lower limits of detection (ranging from 0.01 to 0.04 % w/w), and satisfactory precision and accuracy (ranging from 4.33 to 10.47, and from −14.25 to 13.85, respectively). Seven of the ten analysed samples presented paraben contents within the acceptable concentration according to European legislation. The MoS value obtained for PrP (37.58) suggested its reduced safety, indicating that PrP may significantly contribute to systemic exposure resulting from the use of personal care products.

Sexual Dimorphism in Mitochondrial Metabolism and Insulin Sensitivity Is Controlled by HSP72-ERa Crosstalk

HSP72 is highly induced during exercise, and muscle expression is reduced in obese and diabetic men. Muscle-specific HSP72 overexpression protects male mice against diet-induced obesity and insulin resistance. The current study objective was to determine the mechanisms by which HSP72 controls metabolic function and to ascertain whether these mechanisms are impacted by biological sex. We interrogated the impact of biological sex and estradiol action in the context of HSP72 loss and gain of expression in mice. HSP72 is a binding partner of the mitochondrial quality control protein Parkin. HSP72-KO led to metabolic dysfunction via impaired Parkin action, by promoting the retention of enlarged, fission incompetent mitochondria with reduced respiratory capacity. In contrast, female KOs remained lean and insulin sensitive with small spherical muscle mitochondria. Muscle estrogen receptor alpha transcript and protein (Esr1/ERa) was induced in female HSP72-KO vs. wildtype (WT), and this enhanced Parkin-DRP1-induced fatty acid oxidation and underpinned improvements in exercise capacity. The phenotype of female HSP72-KO mice recapitulates that of muscle-specific ESR1 overexpression animals. Because muscle ERa expression was identical between male genotypes, KO vs. WT, we treated male KO mice with 17β-estradiol and the treatment restored mitochondrial form/function as well as insulin sensitivity. In summary, biological sex is critical for understanding the impact of HSP72 in governing metabolic health and insulin sensitivity. National Institutes of Health DK060484 and P30DK063491. 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.

Estradiol action in the female hypothalamo-pituitary-gonadal axis.

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.

Alterations in aquaporin 4 expression in the chicken ovary following tamoxifen treatment

Previously, we demonstrated the follicular size- and a layer of the follicular wall-dependend expression of aquaporin 4 (AQP4) in the chicken ovary. In this study, we aimed to examine the mRNA and protein expression of aquaporin 4 (AQP4) in the chicken ovary during a pause in laying induced by a tamoxifen (TMX, oestrogen receptor modulator) treatment. Ovarian white and yellowish follicles and the granulosa and theca layers of the largest yellow preovulatory follicles were harvested from control and TMX-treated (daily until the cessation of egg-laying) hens. It was found that the TMX treatment lowered the AQP4 transcript abundance in the white follicles, but increased it in the theca layer of the F3-F1 follicles. Moreover, the TMX caused a decrease in AQP4 protein abundance in the theca layer of the F2 follicle. We propose that AQP4 plays a role in the ovarian follicle development and that there is a relationship between oestradiol action and the AQP4 gene and protein expression in chicken ovarian follicles.

Abstract 4390: IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells

Abstract Insulin-like growth factor binding protein-6 (IGFBP-6) is induced by progesterone (P4) in T-47D breast cancer cells. IGFBP-6 regulates insulin-like growth factor 2 (IGF2) by sequestering it to inhibit receptor binding and extend its extracellular half-life. IGF2 binds to insulin-like growth factor 1 receptor or insulin receptor A to promote breast cancer cell proliferation and survival. Progesterone is an ovarian steroid hormone which has complex roles in breast cancer through the actions of progesterone receptor A and B (PR-A and B). Progesterone antagonizes the actions of estradiol (E2), another steroid hormone which promotes proliferation and survival in breast cancer. T-47D cells treated with both P4 and E2 exhibit significantly reduced proliferation compared to treatment with E2 alone. To better understand the role of IGFBP-6 in progesterone signaling, T-47D cells which over-express IGFBP-6 were treated with P4, E2, or both. Overexpressing cells exhibited stronger antagonism compared to controls when co-treated with P4 and E2. RNA-interference was performed to knockdown IGFBP-6 induction in the presence of P4 and E2. Knockdown of IGFBP-6 in the presence of P4 and E2 decreases the antagonistic effects of P4. Expression of both PR-A and PR-B decreases by about 50% when IGFBP-6 is knocked down. These results indicate that IGFBP-6 increases PR expression in response to P4 stimulation. Citation Format: Francisco Javier Lariz, Pacha Botero, Kevin Houston. IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4390.

Estradiol increases cortical and trabecular bone accrual and bone strength in an adolescent male-to-female mouse model of gender-affirming hormone therapy

The effects of gender-affirming hormone therapy on the skeletal integrity and fracture risk in transitioning adolescent trans girls are unknown. To address this knowledge gap, we developed a mouse model to simulate male-to-female transition in human adolescents in whom puberty is first arrested by using gonadotrophin-releasing hormone analogs with subsequent estradiol treatment. Puberty was suppressed by orchidectomy in male mice at 5 weeks of age. At 3 weeks post-surgery, male-to-female mice were treated with a high dose of estradiol (~0.85 mg) by intraperitoneal silastic implantation for 12 weeks. Controls included intact and orchidectomized males at 3 weeks post-surgery, vehicle-treated intact males, intact females and orchidectomized males at 12 weeks post-treatment. Compared to male controls, orchidectomized males exhibited decreased peak bone mass accrual and a decreased maximal force the bone could withstand prior to fracture. Estradiol treatment in orchidectomized male-to-female mice compared to mice in all control groups was associated with an increased cortical thickness in the mid-diaphysis, while the periosteal circumference increased to a level that was intermediate between intact male and female controls, resulting in increased maximal force and stiffness. In trabecular bone, estradiol treatment increased newly formed trabeculae arising from the growth plate as well as mineralizing surface/bone surface and bone formation rate, consistent with the anabolic action of estradiol on osteoblast proliferation. These data support the concept that skeletal integrity can be preserved and that long-term fractures may be prevented in trans girls treated with GnRHa and a sufficiently high dose of GAHT. Further study is needed to identify an optimal dose of estradiol that protects the bone without adverse side effects.

Prelimbic and infralimbic responsivity to amygdala input is modified by gonadal hormones in parallel to low anxiety-like behavior in ovariectomized rats

Gonadal hormones may influence sexual activity by reducing anxiety. The basolateral amygdala (BLA) and prelimbic (PL) and infralimbic (IL) cortical regions comprise a loop that is related to fear, anxiety, and social behavior. In female ovariectomized rats, actions of estradiol, progesterone, and sequential estradiol and progesterone administration were explored in the open field test (OFT) and plus maze test (PMT) to evaluate signs of anxiety-like behavior. The three hormonal treatments reduced indicators of anxiety in the PMT but did not influence behavior in the OFT. In the same behaviorally tested rats under urethane anesthesia, single-unit extracellular recordings were obtained from the PL and IL during electrical stimulation of the BLA. The analysis of 250 ms peristimulus histograms showed that BLA stimulation produced two kinds of response. A small group of neurons increased their firing rate after BLA stimulation. Most neurons exhibited a reduction of spiking. Neurons that increased their firing rate after BLA stimulation did not show any difference with the hormonal treatments. In neurons that were inhibited by BLA stimulation, estradiol reduced the neuronal firing rate in the PL and IL, and progesterone alone and the sequential administration of estradiol followed by progesterone administration 24 h later (priming) increased the firing rate during the 240 ms before BLA stimulation. Analyses of responsivity of the PL and IL during electrical stimulation of the BLA indicated that estradiol, progesterone, and estradiol followed by progesterone administration 24 h later (priming) reduced inhibitory actions of the BLA on the PL but not IL. In the BLA-IL connection, progesterone exacerbated the inhibitory response. These findings indicate that anxiolytic actions of estradiol, progesterone, and estradiol followed by progesterone administration 24 h later (priming) correspond to lower BLA-PL responsivity. Actions of progesterone on BLA-IL responsivity appear to contribute to sexual activity by interacting with other forebrain structures that are also related to sexual receptivity.

Sex steroid hormones, the estrous cycle, and rapid modulation of glutamatergic synapse properties in the striatal brain regions with a focus on 17β-estradiol and the nucleus accumbens

The striatal brain regions encompassing the nucleus accumbens core (NAcc), shell (NAcs) and caudate-putamen (CPu) regulate cognitive functions including motivated behaviors, habit, learning, and sensorimotor action, among others. Sex steroid hormone sensitivity and sex differences have been documented in all of these functions in both normative and pathological contexts, including anxiety, depression and addiction. The neurotransmitter glutamate has been implicated in regulating these behaviors as well as striatal physiology, and there are likewise documented sex differences in glutamate action upon the striatal output neurons, the medium spiny neurons (MSNs). Here we review the available data regarding the role of steroid sex hormones such as 17β-estradiol (estradiol), progesterone, and testosterone in rapidly modulating MSN glutamatergic synapse properties, presented in the context of the estrous cycle as appropriate. Estradiol action upon glutamatergic synapse properties in female NAcc MSNs is most comprehensively discussed. In the female NAcc, MSNs exhibit development period-specific sex differences and estrous cycle variations in glutamatergic synapse properties as shown by multiple analyses, including that of miniature excitatory postsynaptic currents (mEPSCs). Estrous cycle-differences in NAcc MSN mEPSCs can be mimicked by acute exposure to estradiol or an ERα agonist. The available evidence, or lack thereof, is also discussed concerning estrogen action upon MSN glutamatergic synapse in the other striatal regions as well as the underexplored roles of progesterone and testosterone. We conclude that there is strong evidence regarding estradiol action upon glutamatergic synapse function in female NAcs MSNs and call for more research regarding other hormones and striatal regions.

Estradiol-mediated small GTP-binding protein GDP dissociation stimulator induction contributes to sex differences in resilience to ferroptosis in takotsubo syndrome

BackgroundDeclining beneficial cardiovascular actions of estradiol (E2) have been associated with disproportionate susceptibility to takotsubo syndrome (TTS) in postmenopausal women. However, the underlying mechanisms between E2 and this marked disproportion remain unclear. SmgGDS (small GTP-binding protein GDP dissociation stimulator), as a key modulator of cardiovascular disease, plays protective roles in reducing oxidative stress and exerts pleiotropic effects of statins. Whether SmgGDS levels are influenced by E2 status and the effect of SmgGDS on sex differences in TTS are poorly understood. MethodsClinical data were reviewed from TTS inpatients. Echocardiography, immunofluorescence, and immunohistochemistry were performed together with expression analysis to uncover phenotypic and mechanism changes in sex differences in TTS-like wild-type (WT) and SmgGDS± mice. HL-1 cardiomyocytes were used to further examine and validate molecular mechanisms. ResultsIn 14 TTS inpatients, TTS had a higher incidence in postmenopausal women as compared to premenopausal women and men. In murine TTS, female WT mice exhibited higher cardiac SmgGDS levels than male WT mice. Ovariectomy reduced SmgGDS expression in female WT mice similar to that observed in male mice, whereas E2 replacement in these ovariectomized (OVX) female mice reversed this effect. The physiological importance of this sex-specific E2-mediated SmgGDS response is underscored by the disparity in cardiac adaptation to isoproterenol (ISO) stimulation between both sexes of WT mice. E2-mediated SmgGDS induction conferred female protection against TTS-like acute cardiac injury involving ferritinophagy-mediated ferroptosis. No such cardioprotection was observed in male WT mice and OVX female. A causal role for SmgGDS in this sex-specific cardioprotective adaptation was indicated, inasmuch as SmgGDS deficiency abolished E2-modulated cardioprotection against ferritinophagy and aggravates TTS progression in both sexes. Consistently, knockdown of SmgGDS in HL-1 cardiomyocytes exacerbated ferroptosis in a ferritinophagy-dependent manner and abrogated the protective role of E2 against ferritinophagy. Mechanistically, our findings revealed that SmgGDS regulated E2-dependent cardioprotective effects via AMPK/mTOR signaling pathway. SmgGDS deficiency abolished E2-conferred protection against ferritinophagy through activating AMPK/mTOR pathway, while treatment with recombinant SmgGDS in HL-1 cells significantly mitigated this pathway-associated ferritinophagy activity. ConclusionsThese results demonstrate that SmgGDS is a central mediator of E2-conferred female cardioprotection against ferritinophagy-mediated ferroptosis in TTS.

The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Quantification of Urinary Exosome Sex Hormone Binding Globulin Levels during Estrus and Diestrus Stages of Buffaloes

Background: The efficiency of reproduction in buffaloes can be increased by being able to spot buffaloes in estrus, as silent heat is a severe problem in this species. Proteins of urinary exosomes are identified to be better physiological biomarkers when compared to cell free proteins as these extracellular vesicles (EVs) are tiny in size, have additional protection from degradation by endogenous protease activity and are involved in intercellular communication. Hence, the present study was aimed to quantify the urinary exosome sex hormone binding globulin (SHBG) levels during estrus and diestrus stages of buffaloes to determine its suitability as a marker for identifying buffaloes in estrus. Methods: The grouping of animals as mid-diestrus (Group-I/G-I/Control group), regular estrus (Group-II/G-II) and silent estrus (Group-III/G-III) buffaloes was done using a combinatorial approach. The levels of SHBG in urinary exosomes of three groups of animals was quantified using the bovine SHBG ELISA kit. Result: The present study’s findings showed significantly higher urinary exosome SHBG concentration (p less than 0.05) in G-I in comparison to G-II and G-III. The present study revealing for decreased concentration of urinary exosome SHBG in G-II and G-III animals when compared to G-I animals probably construes the physiological role of SHBG in the female reproductive tract at the estrus stage by mediating the action of estradiol 17 â (Eα) at the target site. However, for urinary exosome SHBG to be considered as a biomarker, there is need for additional research on a bigger population.

Hormone-mediated neural remodeling orchestrates parenting onset during pregnancy.

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 MPOAGal neurons and paradoxically increases their excitability, progesterone permanently rewires this circuit node by promoting dendritic spine formation and recruitment of excitatory synaptic inputs. This MPOAGal-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.

FRI419 Progesterone Regulation Of The GnRH Pulse Generator

Abstract Disclosure: E.G. Wall: None. A group of kisspeptin neurons located in the arcuate nucleus (ARN) of the hypothalamus represent the GnRH pulse generator and their activity is thought to be controlled primarily by the feedback actions of estradiol and progesterone (P4). In a healthy menstruating woman, following the mid-cycle ovulation, P4 levels dramatically increase and exert a negative feedback action onto the hypothalamus to slow pulsatile LH secretion. However, the exact mechanism by which P4 negatively feeds back to the hypothalamus remains unknown. The ARN kisspeptin neurons display synchronized episodes (SEs) of activity approximately every hour for most of the cycle but these dramatically reduce in number following the LH surge. As these ARN kisspeptin neurons express the progesterone receptor (PR) it is hypothesized that they may be the site at which P4 negative feedback occurs. Given the lack of detailed information in the mouse, we first assessed the profile of circulating P4 levels in mice at 8-hr intervals throughout the estrous cycle. Terminal blood samples were taken from individual mice along the 4 stages of the estrous cycle at 3-time points 8 hrs apart and the gonadal hormones were analyzed using LC-MS. The concentration of P4 was found to peak around the time of the LH surge at 6 pm on proestrus (21.94 ± 3.78 ng/ml) and was at its lowest at diestrus 10 am (0.42 ± 0.11 ng/ml). We next assessed the impact of circulating P4 on the activity of the ARN kisspeptin pulse generator using GCaMP fibre photometry in freely behaving mice. A prior study found that IP administration of 8 mg/kg P4 resulted in circulating P4 concentrations reaching 34 ng/ml (Wong et al., J Pharm Pharmacol 64, 2012). Hence, in this study, diestrus mice equipped for monitoring the activity of the kisspeptin pulse generator were injected with either 8 or 4 mg/kg P4 at 10 am and SEs monitored for 24 hrs. Both doses resulted in a significantly decreased frequency of SEs compared to the vehicle (p<0.05), which persisted for 6 hrs. To next examine whether P4 may be acting directly on the ARN kisspeptin neurons to slow pulse generation, P4 (25 ng/ml) was infused directly into the ARN while the activity of the pulse generator was monitored. Again, this resulted in a decreased frequency of SEs compared to the vehicle with the effects lasting approximately 5 hrs (p<0.05).These results indicate that P4 exerts powerful inhibitory control over the activity of the kisspeptin pulse generator and that these effects may be direct. Elucidating the mechanisms of P4 negative feedback may provide a platform for the development of new therapeutic strategies in the infertility clinic. Presentation: Friday, June 16, 2023

SAT434 Estradiol Controls GnRH-II Gene Expression In The Hypothalamus And Pituitary Gland Of Female Rhesus Macaques (Macaca Mulatta)

Abstract Disclosure: A. Lomniczi: None. M. Appleman: None. H.F. Urbanski: None. It has been known for many years that rhesus macaques express two forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) in the medial basal hypothalamus (MBH), and that both forms are capable of stimulating the release of luteinizing hormone (LH) in vivo. However, while much has been published about the role of GnRH-I in reproduction, very little is known about the function of GnRH-II. To shed light on this issue, we studied the expression pattern of these two genes in different parts of the monkey hypothalamus, median eminence and pituitary gland. Quantitative PCR (qPCR) analysis revealed that while GnRH-I is preferentially expressed in the Arcuate Nucleus (ARC), Median Eminence (ME), Supraoptic Nucleus (SON) and Suprachiasmatic Nucleus (SCN); GnRH-II was highly expressed in the SON, ARC-ME and Paraventricular Nucleus (PVN), confirming our previously published in situ hybridization studies. Strikingly, GnRH-II mRNA was also detected in the pituitary gland, a site where GnRH-I was not found. We have previously demonstrated that maximum hypothalamic GnRH-II expression occurs around the time of the preovulatory LH surge, when estrogen (E2) levels are maximal, but no significant changes in GnRH-I mRNA levels occur. Here using an ovariectomized (OVX) macaque model subjected to E2 replacement paradigm, designed to mimic the plasma E2 levels observed during the preovulatory estradiol peak, we performed RNAseq and qPCR from the PVN and pituitary gland to identify genes differentially regulated by E2. While GnRH-I mRNA was not affected by E2 treatment in any of the tissues studied, pituitary GnRH-II expression highly correlated with estrogen levels (r>0.8; p<0.01), with over a hundred-fold increase in mRNA expression. Taken together, these data suggest that pituitary GnRH-II (but not GnRH-I) gene expression is induced by a positive feedback action of estradiol. This novel finding raises the possibility that GnRH-II plays a major role in triggering the preovulatory LH surge in primates, not only at the ARC-ME but also at the pituitary level. Presentation Date: Saturday, June 17, 2023

Distinct roles of estrone and estradiol in endothelial colony-forming cells.

Our current understanding of the relationship between estrogen and human endothelial colony-forming cell (hECFC) function is based almost exclusively on studies investigating estradiol action at nuclear estrogen receptors. In the current study the hypothesis was tested that the less potent estrogen receptor agonist, estrone, affects hECFC proliferation, migration, secretion, and tube formation in a way that is unique from that of estradiol. The relationship between the estrogens, estradiol and estrone, is clinically important, particularly in postmenopausal women where estradiol levels wane and estrone becomes the predominant estrogen. Cultured hECFCs from peripheral blood mononuclear cell fractions were treated with concentrations of estradiol and estrone ranging from 1 nM to 1 μM separately and in combination. Following treatment, proliferation, migration, ability to attract other hECFCs (autocrine secretion), and ability to enhance endothelial cell tube formation (tubulogenesis) were tested. Functional assays revealed unique, concentration-dependent physiological effects of estrone and estradiol. Estradiol exposure resulted in increased hECFC proliferation, migration, secretion of chemoattractant, and enhancement of tube formation as expected. As with estradiol, hECFC secretion of chemoattractant increased significantly with each increase in estrone exposure. Estrone treatment produced a biphasic, concentration-dependent relationship with proliferation and tube formation and relatively no effect on hECFC migration at any concentration. The quantitative relationship between the effects of estrone and estradiol and each hECFC function was analyzed. The extent to which estrone was similar in effect to that of estradiol was dependent on both the concentrations of estradiol and estrone and the hECFC function measured. Interestingly, when the two estrogens were present, differing ratios resulted in unique functional responses. hECFCs that were treated with combinations of estrone and estradiol with high estrone to estradiol ratios showed decreased proliferative capacity. Conversely, hECFCs that were treated with combinations that were relatively high in estradiol, showed increased proliferative capacity. Cells that were treated with estrone and estradiol in equal concentrations showed an attenuated proliferative response that was decreased compared to the proliferation that either estrone or estradiol produced when they were present alone. This co-inhibitory relationship, which has not been previously reported, challenges the prevailing understanding of estrone as solely a weak agonist at estrogen receptors. This study provides evidence that estrone signaling is distinct from that of estradiol and that further investigation of estrone's mechanism of action and the biological effect may provide important insight into understanding the dysfunction and decreased number of hECFCs, and the resulting cardiovascular disease risk observed clinically in menopausal women and women undergoing hormone replacement therapy.

Estrogen-induced signalling and the renal contribution to salt and water homeostasis

The kidney is considered to be one of the most estrogen-responsive, not reproductive organs in the body. Different estrogen receptors (ERs) show sex-specific differences in expression along the nephron and the expression of different ERs also changes with the estrous cycle of the female. The kidney becomes more estrogen-sensitive when estradiol levels are at their highest, just prior to ovulation. This review discusses the different mechanisms by which estradiol can modify the salt and water conservation processes of the kidney through transporter regulation to support the fluid and electrolyte homeostasis changes required in mammalian reproduction. The kidney plays a critical role in regulating blood pressure by controlling fluid homeostasis, and so protects the female cardiovascular system from dramatic changes in whole body fluid volume that occur at critical points in the human menstrual cycle and in pregnancy. This is augmented by the direct actions of estradiol on the cardiovascular system, for example through the direct stimulation of endothelial nitric oxide (NO) synthase, which releases NO to promote vasodilation. This and other mechanisms are less evident in the male and give women a degree of cardiovascular protection up until menopause, when the risks of cardiovascular disease and chronic kidney disease begin to match the risks experienced by males.

Endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle

The estrogen receptor (ER) designated ERα has actions in many cell and tissue types that impact glucose homeostasis. It is unknown if these include mechanisms in endothelial cells, which have the potential to influence relative obesity, and processes in adipose tissue and skeletal muscle that impact glucose control. Here we show that independent of impact on events in adipose tissue, endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle. Endothelial ERα-deficient male mice are glucose intolerant and insulin resistant, and in females the antidiabetogenic actions of estradiol (E2) are absent. The glucose dysregulation is due to impaired skeletal muscle glucose disposal that results from attenuated muscle insulin delivery. Endothelial ERα activation stimulates insulin transcytosis by skeletal muscle microvascular endothelial cells. Mechanistically this involves nuclear ERα-dependent upregulation of vesicular trafficking regulator sorting nexin 5 (SNX5) expression, and PI3 kinase activation that drives plasma membrane recruitment of SNX5. Thus, coupled nuclear and non-nuclear actions of ERα promote endothelial insulin transport to skeletal muscle to foster normal glucose homeostasis.

Psoralen prevents the inactivation of estradiol and treats osteoporosis via covalently targeting HSD17B2.

Psoralea corylifolia L. seeds (P. corylifolia), popularly known as Buguzhi in traditional Chinese medicine, are often used to treat osteoporosis in China. Psoralen (Pso) is the key anti-osteoporosis constituent in P. corylifolia, however, its targets and mechanism of action are still unclear. The purpose of this study was to explore the interaction between Pso and 17-β hydroxysteroid dehydrogenase type 2 (HSD17B2), an estrogen synthesis-related protein that inhibits the inactivation of estradiol (E2) to treat osteoporosis. Tissue distribution of Pso was analyzed by in-gel imaging after oral administration of an alkynyl-modified Pso probe (aPso) in mice. The target of Pso in the liver was identified and analyzed using chemical proteomics. Co-localization and cellular thermal shift assays (CETSA) were used to verify the key action targets. To detect the key pharmacophore of Pso, the interaction of Pso and its structural analogs with HSD17B2 was investigated by CETSA, HSD17B2 activity assay, and in-gel imaging determination. Target competitive test, virtual docking, mutated HSD17B2 activity, and CETSA assay were used to identify the binding site of Pso with HSD17B2. A mouse model of osteoporosis was established by ovariectomies, and the efficacy of Pso in vivo was confirmed by micro-CT, H&E staining, HSD17B2 activity, and bone-related biochemical assays. Pso regulated estrogen metabolism by targeting HSD17B2 in the liver, with the α, β-unsaturated ester in Pso being the key pharmacophore. Pso significantly suppressed HSD17B2 activity by irreversibly binding to Lys236 of HSD17B2 and preventing NAD+ from entering the binding pocket. In vivo studies in ovariectomized mice revealed that Pso could inhibit HSD17B2 activity, prevent the inactivation of E2, increase levels of endogenous estrogen, improve bone metabolism-related indices, and play a role in anti-osteoporosis. Pso covalently binds to Lys236 of HSD17B2 in hepatocytes to prevent the inactivation of E2, thereby aiding in the treatment of osteoporosis.