E2-treated Animals Research Articles

Abstract PO040: Chemoprevention and regression of estrogen-induced atypical endometrial hyperplasia by oral SHetA2 in a rat model

Abstract Introduction: The purpose was to evaluate the chemoprevention efficacy of SHetA2 (NSC 726189) in a rat model of atypical endometrial hyperplasia (AEH). Methods: Female Sprague Dawley rats were ovariectomized, rested for 2 weeks, and subcutaneously implanted with placebo or slow-release estrogen (E2; 1.5 mg/90-day) pellets on the lateral side of the neck. The rats were randomly divided for two studies: 1) AEH prevention, in which SHetA2 treatment was started one day after E2 pellet implantation; 2) AEH regression, in which SHetA2 treatment was started after AEH development (12 weeks after E2 pellet implantation) and continued for 4 weeks. SHetA2 was orally given as a suspension at three different doses of 30, 60 or 90 mg/kg/day. The suspension was prepared by first micronizing SHetA2 powder to < 63 µm and then ultrahomogenizing it in aqueous 30% Kolliphor HS15 until the size of drug particles in suspension was < 20 µm. The presence of AEH in H&E stained uterine sections was evaluated by two pathologists as the study endpoint by the presence of abnormal cells (nuclear enlargement, nuclear membrane irregularities, prominent nucleoli, nuclear hyperchromasia, nuclear pleomorphism, mitotic activity and/or loss of axial polarity) within tissue areas that exhibit at least one of the following features: increased gland to stroma ratio of >1:1, glandular complexity including irregular contours, and cribriform/papillary configurations. Results: E2-treated animals had reduced body weight gain compared to placebo-implanted animals (Repeated Measures ANOVA, p<0.0001). There were no effects of SHetA2 on rat body weights. In the prevention study, uterine to body weight ratios were significantly higher in E2-treated animals compared to placebo (Repeated Measures ANOVA, p<0.0001) and significantly reduced by 30 and 60 mg/kg/day SHetA2 doses (Repeated Measures ANOVA, p=0.0193 and 0.0108, respectively). Also, SHetA2 caused a dose-responsive decrease in AEH incidence in E2-implanted animals from 86.7% (13/15) in the untreated control to 53.3% (8/15), 26.7% (4/15) and 26.7% (4/15) in the 30, 60 and 90 mg/kg/day SHetA2 treatment groups, respectively (Chai Square Analysis p<0.0022). In the regression study, there were no significant differences in uterine to body weight ratios across all groups. Also, SHetA2 caused dose-responsive regression of AEH to normal histology in E2-implanted animals observed by lack of AEH in 18.2% (2/11) of the untreated control group and 83.3% (10/12), 81.8% (9/11) and 75% (9/12) in the 30, 60 and 90 mg/kg/day SHetA2 treatment groups, respectively (Chai Square Analysis p<0.0025). Conclusions: AEH can be induced by implantation of slow-release E2 pellets in ovariectomized rats. SHetA2 significantly prevented AEH development and caused AEH regression to normal endometrium histology in a dose-dependent manner. Further study is warranted to explore the mechanistic chemoprevention action of SHetA2 and translate these findings toward clinical trials. Funding: NCI PREVENT Program Contract HHSN26100008. Citation Format: Vishal Chandra, Rajani Rai, Stan S. Lightfoot, Manolya Kukut Hatipoglu, Lucila Garcia-Contreras, Doris M. Benbrook. Chemoprevention and regression of estrogen-induced atypical endometrial hyperplasia by oral SHetA2 in a rat model [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO040.

Abstract 27: Development of a rat model of atypical endometrial hyperplasia and a vaginal suppository formulation of SHetA2 for chemoprevention studies

Abstract Introduction: The purposes were to develop a rat model of atypical endometrial hyperplasia and a vaginal suppository of the drug SHetA2 (NSC 726189) for use in a chemoprevention study. Methods: Female Sprague Dawley rats (150-250 g) were ovariectomized to prevent production of endogenous estrogen and progesterone. After a 2 wks rest, placebo or slow release estrogen (E2) pellets were implanted subcutaneously on the lateral side of the neck in the ovariectomized rats randomly assigned to 3 groups (N=25): 1) placebo, 2) 0.5 mg E2/90-day, 3) 1.5 mg E2/90-day. Pellets were replaced after ~10 wks. Rats were weighed wkly. Three rats in each group were sacrificed at 2, 4, 6, 8, 10, 12, 14, and 16 wks after pellet implantation. Organs were collected and weighed. The dimensions of the vaginal canals of six rats were measured to design an aluminum mold to prepare vaginal suppositories containing SHetA2. The mold was custom-made to have a conical shape with a 5.5 mm diameter base (average vaginal canal diameter) and 5 mm height (half of the total length of the vaginal canal - 10.10 mm). A pilot batch of 20 SHetA2 suppositories was prepared using a cocoa butter base, stored at 4°C in an air-tight and light-tight container and evaluated for SHetA2 stability at 1, 2 and 3 months. Results: Rats with placebo implants exhibited continuous body weight gain. In comparison, both E2-treated groups had reduced weight gain (Repeated Measures ANOVA, p<0.0001). At each time point, average uterine horn weights from the E2-treated groups were significantly higher than the placebo group (Repeated Measures ANOVA, p<0.0001). Placebo group rats had normal endometrium with occasional glandular crowding. At 8-10 wks, focal atypia was observed in 1 of 6 rats in the 0.5 mg E2 group and in 4 of 6 rats in the 1.5 mg E2 group. At the 12-wk time point, atypical hyperplasia was observed in 7 of 9 rats in the 0.5 mg E2 group and in 8 out of 9 rats in the 1.5 g E2 group. Repeated measures ANOVA of organ/body weight ratios indicated that the E2-treated animals had significantly lower spleen weights (p=0.0019) and higher liver (p=0.0001), kidney (p<0.0001) and pancreas (p=0.0004) weights. The reduced organ weights may be due to the significant reduction in body weight in the E2-treated rats. The SHetA2 suppositories were stable at -20°C for 3 months. Storage of individual suppositories in wells of 48-well plates prevented the twining together that occurred when stored in bulk, and allowed removal from packaging without damaging the suppositories. Conclusions: A 1.5 mg slow release E2 pellet in ovariectomized rats caused development of atypical endometrial hyperplasia within a sufficient timeframe and incidence to allow a chemoprevention study. The mold was suitable to prepare rat-sized suppositories and the resulting SHetA2 suppositories can be safely stored for use within 3 months. Funding: NCI PREVENT Program Contract HHSN26100008 Citation Format: Vishal Chandra, Rajani Rai, Sanam Hussain, Lucila Garcia, Manolya Hatipoglu, Doris Benbrook. Development of a rat model of atypical endometrial hyperplasia and a vaginal suppository formulation of SHetA2 for chemoprevention studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 27.

Neonatal exposure to chlordecone alters female social behaviors and central estrogen alpha receptor expression in socially monogamous mandarin voles.

Chlordecone (CD) is one of the common persistent organic pollutants in nature and has a profound impact on the environment and on public health. Accumulating evidence has demonstrated that neonatal exposure of CD influences adult physiology and behavior due to its estrogenic properties. Using socially monogamous mandarin voles as an experimental animal model, the present study aimed to evaluate the impact of neonatal exposure to CD on female social behaviors and central estrogen receptor alpha (ERα) expression in adulthood. After receiving a single subcutaneous injection with sesame seed oil (female control group), 17 beta-estradiol (E2 group), or CD group on postnatal Day 1, the social behaviors of adult animals and ERα expression in specific brain regions were assessed. The data indicated that CD or E2-treated female animals displayed increased affiliative behaviors and decreased aggressive behaviors with regard to the unfamiliar females in the social interaction test. In addition, CD or E2-treated female voles exhibited significant preferences to females over males in the sexual preference test. Moreover, CD-treated female animals exhibited higher levels of ERα expression in the bed nucleus of the stria terminalis, the central amygdala, the medial amygdala and the medial preoptic area compared with those of the control voles. The results suggested that neonatal exposure to CD may masculinize female social behaviors, possibly via CD-induced changes in the ERα expression of relevant brain regions.

Abstract A44: Pharmacologic targeting of estrogen receptor in melanoma to enhance antitumor immunity

Abstract Metastatic melanoma is one of the most aggressive, morbid cancers with a median survival of 6-9 months. The development of MAPK-pathway inhibitors and immune checkpoint blockade (ICB) agents (α-PD1 and α-CTLA4) has significantly improved outcome in patients with melanoma. However, de novo and acquired resistance to these therapies remains a major impediment to achieving durable clinical responses in most patients. Epidemiologic studies have implicated sex steroids in melanoma etiology; however, the mechanisms by which these hormones impact the development and progression of melanoma are unclear. The aim of this study is to evaluate the extent to which estrogens, such as 17β-estradiol (E2), working through its cognate nuclear receptor(s), impact the pathobiology of melanoma. Using estrogen receptor alpha negative (ERα-) syngeneic (GEMM6, B16F10 and YuMM5.2 cells) and a transgenic B-RafCA/WT; Ptenf/f; mTyr-Cre/ERT2 (iBP) mouse model of melanoma, we have determined that manipulating circulating E2 levels in these mice models has a dramatic effect on tumor growth. This E2-mediated increase in tumor growth was not observed in NOD-scid/IL2null mice, suggesting that this hormone exerts its actions by modulating intratumoral immune cell repertoire and function. Indeed, multiparametric flow cytometry analysis of tumor-infiltrating immune cells indicates that E2 increases M2 polarization of macrophages in GEMM6 and iBP mouse models and decreases proinflammatory M1 macrophages in the B16F10 and YuMM 5.2 tumor models. Characterization of (a) tumor associated-macrophages (TAMs) from E2-treated mice and (b) bone marrow-derived macrophages (BMDM) from non-tumor bearing WT C57BL/6J mice differentiated in the presence of tumor conditioned media and E2, revealed increased expression of immune-suppressive cytokines (Tnf, Retnla, Il10 and Ptgs2). The E2 treated cells also demonstrated an enhanced ability to suppress CD8+ T-cell proliferation, an activity that could be reversed by addition of the ER antagonist ICI182780. Either pharmacologic depletion of TAMs by clodronate liposomes in GEMM6 tumor model or myeloid cell selective ablation of ERα expression negated the effects of E2 on tumor growth (B16F10, YuMM5.2). Further, CD8+T cell depletion (YuMM 5.2) increased tumor growth in ovariectomized animals to a level observed in E2-treated animals, suggesting that TAMs in E2-treated mice suppress CD8+ T-cell function and enhance tumor growth. Finally, treatment with ICI182780 inhibited the effects of E2 on tumor growth and immune cell repertoire (GEMM6, B16F10, and YuMM 5.2), confirming the role of ER in melanoma biology. Taken together, these results suggest that E2, working through ERα, promotes an immune-suppressive microenvironment during melanoma progression. These findings have informed the design of a clinical study that will assess the benefit of using antiestrogens, together with ICBs or standard of care, to treat melanoma. Citation Format: Binita Chakraborty, Jovita Bymerwa, Robert Baldi, Wen Liu, Ching-Yi Chang, Donald McDonnell. Pharmacologic targeting of estrogen receptor in melanoma to enhance antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A44.

Estrogen imprinting compromises male sexual behavior and affects the number of androgen-receptor-expressing hypothalamic neurons†.

Neonatal exposure to high-dose 17β-estradiol (E2) affects the morphology and physiology of sex and accessory sex organs in the long term. In this study, we examined the effects of E2 imprinting on male sexual behavior, fertility, and the number of androgen receptor (AR)-expressing cells in the hypothalamus. E2-treated males showed copulatory behavior represented by mounts and/or intromissions, demonstrating the preservation of aspects of male behavior. They had slightly increased latency for first intromission and a reduced number of ejaculations, associated with a 50% reduction in the fertility index. AR expression in the hypothalamus was assessed by RT-PCR, western blotting, and immunohistochemistry. Treated rats had a significantly lower ventral prostate (VP) weight, demonstrating the efficacy of the treatment. The AR mRNA and protein content in the hypothalamus of E2-treated animals was reduced to the levels of females. AR-expressing cell counts in the ventromedial, anterior medial preoptic, paraventricular nuclei, and preoptic areas were different from control males, and similar to those of females. In conclusion, E2 imprinting resulted not only in ill-developed sexual organs, but also affected sexual behavior, resulting in a female-type hypothalamus, at least with respect to the abundance of AR mRNA and protein and the number of AR-expressing cells in important regions/tracts.

Combined effects of rat Schwann cells and 17β-estradiol in a spinal cord injury model.

Spinal cord injury (SCI) is a devastating traumatic event which burdens the affected individuals and the health system. Schwann cell (SC) transplantation is a promising repair strategy after SCI. However, a large number of SCs do not survive following transplantation. Previous studies demonstrated that 17β-estradiol (E2) protects differentcell types and reduces tissue damage in SCI experimental animal model. In the current study, we evaluated the protective potential of E2 on SCs in vitro and investigated whether the combination of hormonal and SC therapeutic strategy has a better effect on the outcome after SCI. Primary SC cultures were incubated with E2 for 72h. In a subsequent experiment, thoracic contusion SCI was induced in male rats followed by sustained administration of E2 or vehicle. Eight days after SCI, DiI-labeled SCs were transplanted into the injury epicenter in vehicle and E2-treated animals. The combinatory regimen decreased neurological and behavioral deficits and protected neurons and oligodendrocytes in comparison to vehicle rats. Moreover, E2 and SC significantly decreased the number of Iba-1+ (microglia) and GFAP+ cells (astrocyte) in the SCI group. In addition, we found a significant reduction of mitochondrial fission-markers (Fis1) and an increase of fusion-markers (Mfn1 and Mfn2) in the injured spinal cord after E2 and SC treatment. These data demonstrated that E2 protects SCs against hypoxia-induced SCI and improves the survival of transplanted SCs.

Effect of 17β-estradiol on estrogen receptor negative breast cancer cells in an osteolytic mouse model

17β-Estradiol (E2) promotes metastasis of triple negative breast cancer cells to bone. Recent studies show many triple negative breast cancer cell lines lacking the 66 kDa estrogen receptor (ER) alpha (ERα66) or its splice variant ERα46, express another splice variant, ERα36 associated with membrane-mediated rapid actions of the hormone. qPCR and western blot confirmed that MCF7 cells possessed ERα splice variants ERα66, ERα46 and ERα36, while ER-negative breast cancer cells MDA-MB-231 possessed only ERα36. MDA-MB-231 breast cancer cells were implanted into medullary canals of ovariectomized female athymic nude mice femurs (N = 8 mice/treatment). To examine the effect of E2 on osteolysis, mice were treated with 0.72 mg E2 or placebo via 60 day release osmotic pumps implanted subcutaneously. Legs were examined by Faxitron through the course of the study, and by microCT and histology after 8 weeks. Greater occurrence of osteolysis and pathologic fracture was observed in E2-treated animals compared to placebo, and microCT demonstrated less bone volume remaining in MDA-MB-231 treated legs compared to contralateral control legs, as well as E2-treated animals compared to placebo. E2-treated animals had significantly greater tumor volume compared to placebo. Large nests of anaplastic tumor cells with eroded cortical margin were observed in E2-treated animals compared to placebo. MDA MB 231 breast cancer cells positive for ERα36 but negative for ERα46/66 had enhanced osteolysis, pathologic fractures, and tumor volume in an in vivo osteolytic mouse model when treated with 17β-estradiol compared to placebo, demonstrating a role for ERα36 in bone tumor progression.

Estrogen Attenuates Local Inflammasome Expression and Activation after Spinal Cord Injury.

17-estradiol (E2) is a neuroprotective hormone with a high anti-inflammatory potential in different neurological disorders. The inflammatory response initiated by spinal cord injury (SCI) involves the processing of interleukin-1beta (IL-1b) and IL-18 mediated by caspase-1 which is under the control of an intracellular multiprotein complex called inflammasome. We recently described in a SCI model that between 24 and 72h post-injury, most of inflammasome components including IL-18, IL-1b, NLRP3, ASC, and caspase-1 are upregulated. In this study, we investigated the influence of E2 treatment after spinal cord contusion on inflammasome regulation. After contusion of T9 spinal segment, 12-week-old male Wistar rats were treated subcutaneously with E2 immediately after injury and every 12h for the next 3days. Behavioral scores were significantly improved in E2-treated animals compared to vehicle-treated groups. Functional improvement in E2-treated animals was paralleled by the attenuated expression of certain inflammasome components such as ASC, NLRP1b, and NLRP3 together with IL1b, IL-18, and caspase-1. On the histopathological level, microgliosis and oligodendrocyte injury was ameliorated. These findings support and extend the knowledge of the E2-mediated neuroprotective function during SCI. The control of the inflammasome machinery by E2 might be a missing piece of the puzzle to understand the anti-inflammatory potency of E2.

Estrogen receptor-dependent attenuation of hypoxia-induced changes in the lung genome of pulmonary hypertension rats.

17β-estradiol (E2) exerts complex and context-dependent effects in pulmonary hypertension. In hypoxia-induced pulmonary hypertension (HPH), E2 attenuates lung vascular remodeling through estrogen receptor (ER)-dependent effects; however, ER target genes in the hypoxic lung remain unknown. In order to identify the genome regulated by the E2-ER axis in the hypoxic lung, we performed a microarray analysis in lungs from HPH rats treated with E2 (75 mcg/kg/day) ± ER-antagonist ICI182,780 (3 mg/kg/day). Untreated HPH rats and normoxic rats served as controls. Using a false discovery rate of 10%, we identified a significantly differentially regulated genome in E2-treated versus untreated hypoxia rats. Genes most upregulated by E2 encoded matrix metalloproteinase 8, S100 calcium binding protein A8, and IgA Fc receptor; genes most downregulated by E2 encoded olfactory receptor 63, secreted frizzled-related protein 2, and thrombospondin 2. Several genes affected by E2 changed in the opposite direction after ICI182,780 co-treatment, indicating an ER-regulated genome in HPH lungs. The bone morphogenetic protein antagonist Grem1 (gremlin 1) was upregulated by hypoxia, but found to be among the most downregulated genes after E2 treatment. Gremlin 1 protein was reduced in E2-treated versus untreated hypoxic animals, and ER-blockade abolished the inhibitory effect of E2 on Grem1 mRNA and protein. In conclusion, E2 ER-dependently regulates several genes involved in proliferative and inflammatory processes during hypoxia. Gremlin 1 is a novel target of the E2-ER axis in HPH. Understanding the mechanisms of E2 gene regulation in HPH may allow for selectively harnessing beneficial transcriptional activities of E2 for therapeutic purposes.

High-dose estrogen treatment at reperfusion reduces lesion volume and accelerates recovery of sensorimotor function after experimental ischemic stroke

Estrogens have previously been shown to protect the brain against acute ischemic insults, by potentially augmenting cerebrovascular function after ischemic stroke. The current study hypothesized that treatment with sustained release of high-dose 17β-estradiol (E2) at the time of reperfusion from middle cerebral artery occlusion (MCAO) in rats would attenuate reperfusion injury, augment post-stroke angiogenesis and cerebral blood flow, and attenuate lesion volume. Female Wistar rats underwent ovariectomy, followed two weeks later by transient, two-hour right MCAO (tMCAO) and treatment with E2 (n=13) or placebo (P; n=12) pellets starting at reperfusion. E2 treatment resulted in significantly smaller total lesion volume, smaller lesions within striatal and cortical brain regions, and less atrophy of the ipsilateral hemisphere after six weeks of recovery. E2-treated animals exhibited accelerated recovery of contralateral forelimb sensorimotor function in the cylinder test. Magnetic resonance imaging (MRI) showed that E2 treatment reduced the formation of lesion cysts, decreased lesion volume, and increased lesional cerebral blood flow (CBF). Ktrans, a measure of vascular permeability, was increased in the lesions. This finding, which represents lesion neovascularization, was not altered by E2 treatment. Ischemic stroke-related angiogenesis and vessel formation was confirmed with immunolabeling of brain tissue and was not altered with E2 treatment. In summary, E2 treatment administered immediately following reperfusion significantly reduced lesion size, cyst formation, and brain atrophy while improving lesional CBF and accelerating recovery of functional deficits in a rat model of ischemic stroke.

Estrogens down-regulate the stem cell factor (SCF)/c-KIT system in prostate cells: Evidence of antiproliferative and proapoptotic effects

The development of prostate cancer (PCa) is intimately associated with the hormonal environment, and the sex steroids estrogens have been implicated in prostate malignancy. However, if some studies identified estrogens as causative agents of PCa, others indicated that these steroids have a protective role counteracting prostate overgrowth. The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF), have been associated with the control of cell proliferation/apoptosis and prostate carcinogenesis, and studies show that estrogens regulate their expression in different tissues, though, in the case of prostate this remains unknown. The present study aims to evaluate the role of 17β-estradiol (E2) in regulating the expression of SCF/c-KIT in human prostate cell lines and rat prostate, and to investigate the consequent effects on prostate cell proliferation and apoptosis. qPCR, Western Blot, and immuno(cito)histochemistry analysis showed that E2-treatment decreased the expression of SCF and c-KIT both in human prostate cells and rat prostate. Furthermore, the diminished expression of SCF/c-KIT was underpinned by the diminished prostate weight and reduced proliferation index. On the other hand, the results of TUNEL labelling, the increased activity of caspase-3, and the augmented expression of caspase-8 and Fas system in the prostate of E2-treated animals indicated augmented apoptosis in response to E2. The obtained results demonstrated that E2 down-regulated the expression of SCF/c-KIT system in prostate cells, which was associated with antiproliferative and proapoptotic effects. Moreover, these findings support the protective role of estrogens in PCa and open new perspectives on the application of estrogen-based therapies.

Increased susceptibility of estrogen-induced bladder outlet obstruction in a novel mouse model

Disorders of the prostate and lower urinary tract are common in elderly men. We investigated the role of metallothionein-1 (MT1) in prostate carcinogenesis by generating a prostate-specific, MT1-expressing mouse. Unexpectedly, genomic analyses revealed that a 12.1-kb genomic region harboring several conserved noncoding elements was unintentionally deleted, upstream of the transgene integration site in the mouse, which we named it 12.1ΔMT1. Male 12.1ΔMT1 mice chronically treated with testosterone (T) plus 17β-estradiol (E2) to induce prostate cancer exhibited no evidence of precancerous or cancerous lesions. Instead, most of them exhibited a bladder outlet obstruction (BOO) phenotype not observed in treated wild-type (WT) mice. Thus, we hypothesized that 12.1ΔMT1 is a novel model for studying the hormonal requirement for BOO induction. Adult male 12.1ΔMT1 and WT mice were treated with T, E2, bisphenol A (BPA), T+E2, or T+BPA for up to 6 months. Histologic and immunohistochemical analysis of the prostate, bladder, and urethra were performed. No significant prostate pathologies were observed in WT or 12.1ΔMT1 mice treated with any of the hormone regimens. As expected, prostatic regression occurred in all E2-treated animals (WT and 12.1ΔMT1). Of great interest, despite a small prostate, 100% of E2-treated 12.1ΔMT1 mice, but only 40% of E2-treated WT mice, developed severe BOO (P<0.01). In contrast, T+E2 treatment was less effective than E2 treatment in inducing severe BOO in 12.1ΔMT1 mice (68%, P<0.05) and was completely ineffective in WT animals. Similarly, T, BPA, and T+BPA treatments did not induce BOO in either WT or 12.1ΔMT1 mice. The BOO pathology includes a thinner detrusor wall, narrowing of bladder neck and urethral lumen, and basal cell hyperplasia in the bladder body and urethra. These findings indicate that 12.1ΔMT1 mice exhibit enhanced susceptibility to E2-induced BOO that is independent of prostate enlargement but that is attenuated by the conjoint treatment with T.

17β-estradiol modulates gene expression in the female mouse cerebral cortex.

17β-estradiol (E2) plays critical roles in a number of target tissues including the mammary gland, reproductive tract, bone, and brain. Although it is clear that E2 reduces inflammation and ischemia-induced damage in the cerebral cortex, the molecular mechanisms mediating the effects of E2 in this brain region are lacking. Thus, we examined the cortical transcriptome using a mouse model system. Female adult mice were ovariectomized and implanted with silastic tubing containing oil or E2. After 7 days, the cerebral cortices were dissected and RNA was isolated and analyzed using RNA-sequencing. Analysis of the transcriptomes of control and E2-treated animals revealed that E2 treatment significantly altered the transcript levels of 88 genes. These genes were associated with long term synaptic potentiation, myelination, phosphoprotein phosphatase activity, mitogen activated protein kinase, and phosphatidylinositol 3-kinase signaling. E2 also altered the expression of genes linked to lipid synthesis and metabolism, vasoconstriction and vasodilation, cell-cell communication, and histone modification. These results demonstrate the far-reaching and diverse effects of E2 in the cerebral cortex and provide valuable insight to begin to understand cortical processes that may fluctuate in a dynamic hormonal environment.

Sex hormones selectively impact the endocervical mucosal microenvironment: implications for HIV transmission.

Several studies suggest that progesterone and estrogens may affect HIV transmission in different, possibly opposing ways. Nonetheless, a direct comparison of their effects on the mucosal immune system has never been done. We hypothesize that sex hormones might impact the availability of cells and immune factors important in early stages of mucosal transmission, and, in doing so influence the risk of HIV acquisition. To test this hypothesis, we employed 15 ovarectomized rhesus macaques: 5 were treated with Depot Medroxy Progesterone Acetate (DMPA), 6 with 17-β estradiol (E2) and 4 were left untreated. All animals were euthanized 5 weeks after the initiation of hormone treatment, a time post-DMPA injection associated with high susceptibility to SIV infection. We found that DMPA-treated macaques exhibited higher expression of integrin α4β7 (α4β7) on CD4+ T cells, the gut homing receptor and a marker of cells highly susceptible to HIV, in the endocervix than did the E2-treated animals. In contrast, the frequency of CCR5+ CD4+ T cells in DMPA-treated macaques was higher than in the E2-treated group in vaginal tissue, but lower in endocervix. α4β7 expression on dendritic cells (DCs) was higher in the DMPA-treated group in the endocervical tissue, but lower in vaginal tissue and on blood DCs compared with the E2-treated animals. Soluble MAdCAM-1, the α4β7 ligand, was present in the vaginal fluids of the control and E2-treated groups, but absent in the fluids from DMPA-treated animals. Both hormones modulated the expression and release of inflammatory factors and modified the distribution of sialomucins in the endocervix. In summary, we found that sex hormones profoundly impact mucosal immune factors that are directly implicated in HIV transmission. The effect is particularly significant in the endocervix. This may increase our understanding of the potential hormone-driven modulation of HIV susceptibility and potentially guide contraceptive policies in high-risk settings.

Chronic exposures to low levels of estradiol and their effects on the ovaries and reproductive hormones: Comparison with aging.

Aging in female rats is characterized by a state called "constant estrous" in which rats are unable to ovulate, have polycystic ovaries and moderately elevated estrogen levels. We hypothesized that chronic exposure of young animals to low levels of E2 can produce reproductive changes similar to that seen in aging animals. Adult female rats were sham-implanted (control) or implanted with slow-release E2 (20 ng/day) pellets for 30, 60, or 90 days. Old constant estrous (OCE) rats were used for comparison. Estrous cyclicity was monitored periodically. At the end of treatment, animals were sacrificed, trunk blood was collected for hormone measurements and ovaries for immunohistochemistry. Young animals became acyclic with increasing duration of E2 exposure while OCE rats were in a state of acyclicity. Ovaries became increasingly more cystic with E2 exposure, and were comparable to OCE rats; however, there was a marked reduction in interstitial tissue with exogenous E2 treatment. Exogenous E2 also decreased Mullerian inhibiting substance expression, increased infiltration of macrophages without much impact on apoptosis in the ovaries. Serum testosterone levels decreased in E2-treated young animals, while it increased significantly in OCE rats. There was a marked reduction in LH but not FSH levels with E2 exposure in both young and old animals. These results indicate that even very low doses of E2 are capable of inducing aging-like changes in young animals.

Estrogen Alters Remodeling of the Vaginal Wall after Surgical Injury in Guinea Pigs1

Loss of pelvic organ support (i.e., pelvic organ prolapse) is common in menopausal women. Surgical reconstruction of pelvic organ prolapse is plagued with high failure rates. The objective of this study was to determine the effects of estrogen on biomechanical properties, lysyl oxidase (LOX), collagen content, and histomorphology of the vagina with or without surgical injury. Nulliparous ovariectomized guinea pigs were treated systemically with either 50 μg/kg/day estradiol (E2,) or vehicle. After 2 wk, vaginal surgery was performed, and animals were treated with either beta-aminopropionitrile (BAPN, an irreversible LOX inhibitor), or vehicle to determine the role of LOX in recovery of the vaginal wall from injury with or without E2. Estradiol resulted in (i) significant growth, increased smooth muscle, and increased thickness of the vagina, (ii) increased distensibility without compromise of maximal force at failure, and (iii) increased total and cross-linked collagen. In the absence of E2, BAPN resulted in decreased collagen and vaginal wall strength in the area of the injury. In contrast, in E2-treated animals, increased distensibility, maximal forces, and total collagen were maintained despite BAPN. Interestingly, LOX mRNA was induced dramatically (9.5-fold) in the injured vagina with or without E2 at 4 days. By 21 days, however, LOX levels declined to near baseline in E2-deprived animals. LOX mRNA levels remained strikingly elevated (12-fold) at 21 days in the estrogenized vagina. The results suggest that prolonged E2 induced increases in LOX, and collagen cross-links may act to sustain a matrix environment that optimizes long-term surgical wound healing in the vagina.

17β-Estradiol Eye Drops Protect the Retinal Ganglion Cell Layer and Preserve Visual Function in an in Vivo Model of Glaucoma

Neuroprotection in glaucoma as a curative strategy complementary to current therapies to lower intraocular pressure (IOP) is highly desirable. This study was designed to investigate neuroprotection by 17β-estradiol (E2) to prevent retinal ganglion cell (RGC) death in a glaucoma model of surgically elevated IOP in rats. We found that daily treatment with E2-containing eye drops resulted in significant E2 concentration in the retina with concomitant profound neuroprotective therapeutic benefits, even in the presence of continually elevated IOP. The number of apoptotic cells in the RGC layer was significantly decreased in the E2-treated group, when compared to the vehicle-treated controls. Deterioration in visual acuity in these animals was also markedly prevented. Using mass spectrometry-based proteomics, beneficial changes in the expression of several proteins implicated in the maintenance of retinal health were also found in the retina of E2-treated animals. On the other hand, systemic side effects could not be avoided with the eye drops, as confirmed by the measured high circulating estrogen levels and through the assessment of the uterus representing a typical hormone-sensitive peripheral organ. Collectively, the demonstrated significant neuroprotective effect of topical E2 in the selected animal model of glaucoma provides a clear rationale for further studies aiming at targeting E2 into the eye while avoiding systemic E2 exposure to diminish undesirable off-target side effects.

Differential effects of estradiol on carotid artery inflammation when administered early versus late after surgical menopause

The aim of this study was to determine the effects of estrogen therapy (ET) on carotid artery inflammation when initiated early and late relative to surgical menopause. Female cynomolgus macaques consuming atherogenic diets were ovariectomized and randomized to control or oral estradiol (E2; human equivalent dose of 1 mg/d micronized E2) initiated at 1 month (early menopause, n = 24) or 54 months (late menopause, n = 40) after ovariectomy. The treatment period was 8 months. Carotid artery expression of the markers of monocyte/macrophages (CD68 and CD163), dendritic cells (CD83), natural killer cells (neural cell adhesion molecule-1), and interferon-γ was significantly lower in E2-treated animals in the early menopause group but not in the late menopause group (P < 0.05). In contrast, carotid artery transcripts for T-cell markers (CD3, CD4, CD8, and CD25), interleukin-10, type I collagen, monocyte chemoattractant protein-1, matrix metalloproteinase-9, and tumor necrosis factor-α were lower in E2-treated monkeys regardless of menopausal stage (P < 0.05). ET initiated soon after menopause inhibits macrophage accumulation in the carotid artery, an effect that is not observed when E2 is administered after several years of estrogen deficiency. No evidence for pro-inflammatory effects of late ET is observed. The results provide support for the timing hypothesis of postmenopausal ET with implications for the interpretation of outcomes in the Women's Health Initiative.

Oestrogens promote tumorigenesis in a mouse model for colitis-associated cancer

BackgroundHormone replacement therapy increases the risk of developing ulcerative colitis in postmenopausal women. Chronic intestinal inflammation predisposes to colon cancer development, but effects of female hormones on colitis-associated cancer development...

Chronic estradiol-17β exposure suppresses hypothalamic norepinephrine release and the steroid-induced luteinizing hormone surge: Role of nitration of tyrosine hydroxylase

Chronic exposure to estrogens is known to produce a variety of deleterious effects in women including breast and ovarian cancer and anovulation. In female rats, exposure to low levels of estradiol-17β (E2) decreases hypothalamic norepinephrine (NE) to suppress luteinizing hormone (LH) secretion and cause failure of ovulation. We hypothesized that E2 exposure most likely decreases NE release in the medial preoptic area (MPA) of the hypothalamus to produce this effect and that this may be due to E2-induced inflammatory changes in noradrenergic nuclei leading to nitration of an enzyme involved in NE synthesis. To test this, female Sprague Dawley rats were sham implanted or implanted with slow release E2 pellets (20ng/day) for 30, 60 or 90 days (E30, E60 and E90 respectively). At the end of the treatment period, the rats were implanted with a push–pull cannula in the MPA, ovariectomized and steroid primied to induce a LH surge and subjected to push-pull perfusion. Perfusates were analyzed for NE levels using HPLC-EC. Blood samples collected simultaneously were analyzed for LH levels. We measured interleukin-1β (IL-1β) and nitrate levels in brainstem noradrenergic nuclei that innervate the MPA. In control animals, there was a marked increase in NE levels in response to steroid priming at 1600h that was reduced in the E30 group, and completely abolished after 60 and 90 days of E2 exposure. LH profiles were similar to NE release profiles in control and E2-treated animals. We found that IL-1β levels increased in all three (A1, A2 and A6) noradrenergic nuclei with chronic E2 exposure, while nitrate levels increased only in the A6 region. There was an increase in the nitration of the NE synthesizing enzyme in the MPA in this group as well probably contributing to reduced NE synthesis. This could be a possible mechanism by which chronic E2 exposure decreases NE levels in the MPA to suppress the LH surge.