Structure-based pharmacophore modeling for precision inhibition of mutant ESR2 in breast cancer: A systematic computational approach.

BACKGROUND: Breast cancer, a leading cause of female mortality, is closely linked to mutations in estrogen receptor beta (ESR2), particularly in the ligand-binding domain, which contributed to altered signaling pathways and uncontrolled cell growth. OBJECTIVES/AIMS: This study investigates the molecular and structural aspects of ESR2 mutant proteins to identify shared pharmacophoric regions of ESR2 mutant proteins and potential therapeutic targets aligned within the pharmacophore model. METHODS: This study was initiated by establishing a common pharmacophore model among three mutant ESR2 proteins (PDB ID: 2FSZ, 7XVZ, and 7XWR). The generated shared feature pharmacophore (SFP) includes four primary binding interactions: Hydrogen bond donors (HBD), hydrogen bond acceptors (HBA), hydrophobic interactions (HPho), and Aromatic interactions (Ar), along with halogen bond donors (XBD) and totalling 11 features (HBD: 2, HBA: 3, HPho: 3, Ar: 2, XBD: 1). By employing an in-house Python script, these 11 features distributed into 336 combinations, which were used as query to isolate a drug library of 41,248 compounds and subjected to virtual screening through the generated SFP. RESULTS: The virtual screening demonstrated 33 hits showing potential pharmacophoric fit scores and low RMSD value. The top four compounds: ZINC94272748, ZINC79046938, ZINC05925939, and ZINC59928516 showed a fit score of more than 86% and satisfied the Lipinski rule of five. These four compounds and a control underwent molecular (XP Glide mode) docking analysis against wild-type ESR2 protein (PDB ID: 1QKM), resulting in binding affinity of -8.26, -5.73, -10.80, and -8.42 kcal/mol, respectively, along with the control -7.2 kcal/mol. Furthermore, the stability of the selected candidates was determined through molecular dynamics (MD) simulations of 200 ns and MM-GBSA analysis. CONCLUSION: Based on MD simulations and MM-GBSA analysis, our study identified ZINC05925939 as a promising ESR2 inhibitor among the top four hits. However, it is essential to conduct further wet lab evaluation to assess its efficacy.

Transcriptomic profiling of the oocyte-cumulus-granulosa cell complex from estrogen receptor ß knockout mice.

OBJECTIVE: To study the role of estrogen receptor ß in follicle development and maturation and the response to gonadotropin stimulation aiming at superovulation. DESIGN: Experimental study and transcriptomic analysis. SETTING: Karolinka Institutet, medical university. ANIMAL(S): Healthy wild-type (WT) and estrogen receptor ß knockout (Esr2-KO) female mice undergoing superovulation at 4 weeks, 7 weeks, and 6 months of age. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Oocyte yield after superovulation, transcriptomic profiling of cumulus-granulosa cell complexes and oocytes, and immunohistochemical analyses. RESULT(S): Superovulation of estrogen receptor ß (ERß) knockout mice resulted in reduced oocyte yield at 6 months of age compared with WT mice, but younger mice had similar yields. RNA-seq analysis of cumulus cells from superovulated WT and Esr2-KO mice identified genes and pathways associated with among others adhesion, proliferation, Wnt-signaling, and placed ERß in bipotential granulosa cell cluster. Loss of ERß increased expression of the other estrogen receptors Esr1 and Gper1. CONCLUSION(S): Our results show that ERß has an important role in regulating ovulation in response to exogenous gonadotropins in 6-month-old mice, but not in younger mice. Our transcriptomic and immunohistochemical observations suggest a dysregulation of the granulosa cell communication and lack of tight coordination between granulosa cell replication and antrum expansion. A significant upregulation of other estrogen receptors may support a compensatory mechanism sustaining fertility during younger age in Esr2-KO mice.

Genes Co-Expressed with ESR2 Influence Clinical Outcomes in Cancer Patients: TCGA Data Analysis.

ERß has been assigned a tumor suppressor role in many cancer types. However, as conflicting findings emerge, ERß's tissue-specific expression and functional role have remained elusive. There remains a notable gap in compact and comprehensive analyses of ESR2 mRNA expression levels across diverse tumor types coupled with an exploration of its potential gene network. In this study, we aim to address these gaps by presenting a comprehensive analysis of ESR2 transcriptomic data. We distinguished cancer types with significant changes in ESR2 expression levels compared to corresponding healthy tissue and concluded that ESR2 influences patient survival. Gene Set Enrichment Analysis (GSEA) distinguished molecular pathways affected by ESR2, including oxidative phosphorylation and epithelial-mesenchymal transition. Finally, we investigated genes displaying similar expression patterns as ESR2 in tumor tissues, identifying potential co-expressed genes that may exert a synergistic effect on clinical outcomes, with significant results, including the expression of ACIN1, SYNE2, TNFRSF13C, and MDM4. Collectively, our results highlight the significant influence of ESR2 mRNA expression on the transcriptomic landscape and the overall metabolism of cancerous cells across various tumor types.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Estrogen receptor beta expression and role in cancers.

Estrogen drives the growth of some cancers, such as breast cancer, via estrogen receptor alpha (ERα). Estrogen also activates ERß, but whether ERß is expressed and has a role in different cancers is debated. The use of nonspecific antibodies has contributed to the confusion, and this review delves into ERß's controversial role in cancer and focuses on tumor expression that can be supported by non-antibody-dependent assays. We discuss its expression at the transcript level and focus on its potential role in lymphoma, granulosa cell tumors, testicular, and adrenal cancers, emphasizing recent findings and the complexities that necessitate further research.

ESR2-HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture.

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding. Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid (IAA), which is polar-transported to excised regions, where cell fate transition leads to root founder cell specification to induce de novo root regeneration. The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies. Here, we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid (2,4-D)-rich callus-inducing medium (CIM). Notably, a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux; rather, it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture, delaying callus initiation. The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor, ENHANCER OF SHOOT REGENERATION 2 (ESR2), along with its interacting partner HISTONE DEACETYLASE 6 (HDA6). The ESR2-HDA6 complex binds directly to, and removes the H3ac mark from, the YUCCA1 (YUC1), YUC7, and YUC9 loci, consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM. These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

Regulation of Follicular Development in Chickens: WIF1 Modulates Granulosa Cell Proliferation and Progesterone Synthesis via Wnt/ß-Catenin Signaling Pathway.

Proliferation, apoptosis, and steroid hormone secretion by granulosa cells (GCs) and theca cells (TCs) are essential for maintaining the fate of chicken follicles. Our previous study showed that the Wnt inhibitor factor 1 (WIF1) plays a role in follicle selection. However, the significance of WIF1 in GC- and TC-associated follicular development was not explicitly investigated. This study found that WIF1 expression was strongly downregulated during follicle selection (p < 0.05) and was significantly higher in GCs than in TCs (p < 0.05). WIF1 inhibits proliferation and promotes apoptosis in GCs. Additionally, it promotes progesterone secretion in prehierarchal GCs (pre-GCs, 1.16 ± 0.05 ng/mg vs. 1.58 ng/mg ± 0.12, p < 0.05) and hierarchal GCs (hie-GCs, 395.00 ng/mg ± 34.73 vs. 527.77 ng/mg ± 27.19, p < 0.05) with the participation of the follicle-stimulating hormone (FSH). WIF1 affected canonical Wnt pathways and phosphorylated ß-catenin expression in GCs. Furthermore, 604 upregulated differentially expressed genes (DEGs) and 360 downregulated DEGs in WIF1-overexpressed GCs were found through RNA-seq analysis (criteria: |log2⁡(FoldChange)| > 1 and p_adj < 0.05). Cytokine-cytokine receptor interaction and the steroid hormone biosynthesis pathway were identified. In addition, the transcript of estrogen receptor 2 (ESR2) increased significantly (log2⁡(FoldChange) = 1.27, p_adj < 0.05). Furthermore, we found that WIF1 regulated progesterone synthesis by upregulating ESR2 expression in GCs. Additionally, WIF1 suppressed proliferation and promoted apoptosis in TCs. Taken together, these results reveal that WIF1 stimulates follicle development by promoting GC differentiation and progesterone synthesis, which provides an insight into the molecular mechanism of follicle selection and egg-laying performance in poultry.

Sex and interspecies differences in ESR2-expressing cell distributions in mouse and rat brains.

BACKGROUND: ESR2, a nuclear estrogen receptor also known as estrogen receptor ß, is expressed in the brain and contributes to the actions of estrogen in various physiological phenomena. However, its expression profiles in the brain have long been debated because of difficulties in detecting ESR2-expressing cells. In the present study, we aimed to determine the distribution of ESR2 in rodent brains, as well as its sex and interspecies differences, using immunohistochemical detection with a well-validated anti-ESR2 antibody (PPZ0506). METHODS: To determine the expression profiles of ESR2 protein in rodent brains, whole brain sections from mice and rats of both sexes were subjected to immunostaining for ESR2. In addition, to evaluate the effects of circulating estrogen on ESR2 expression profiles, ovariectomized female mice and rats were treated with low or high doses of estrogen, and the resulting numbers of ESR2-immunopositive cells were analyzed. Welch's t-test was used for comparisons between two groups for sex differences, and one-way analysis of variance followed by the Tukey-Kramer test were used for comparisons among multiple groups with different estrogen treatments. RESULTS: ESR2-immunopositive cells were observed in several subregions of mouse and rat brains, including the preoptic area, extended amygdala, hypothalamus, mesencephalon, and cerebral cortex. Their distribution profiles exhibited sex and interspecies differences. In addition, low-dose estrogen treatment in ovariectomized female mice and rats tended to increase the numbers of ESR2-immunopositive cells, whereas high-dose estrogen treatment tended to decrease these numbers. CONCLUSIONS: Immunohistochemistry using the well-validated PPZ0506 antibody revealed a more localized expression of ESR2 protein in rodent brains than has previously been reported. Furthermore, there were marked sex and interspecies differences in its distribution. Our histological analyses also revealed estrogen-dependent changes in ESR2 expression levels in female brains. These findings will be helpful for understanding the ESR2-mediated actions of estrogen in the brain.

Although the brain is a major target organ of estrogens, the distribution of estrogen receptors in the brain is not fully understood. ESR2, also known as estrogen receptor ß, is an estrogen receptor subtype; its localization in the brain has long been controversial because it has traditionally been difficult to detect. In the present study, we analyzed the expression sites of ESR2 in mouse and rat brains using immunohistochemistry with a well-validated antibody, PPZ0506. The immunohistochemical analysis revealed a more localized expression of ESR2 protein in brain subregions than has previously been reported. Additionally, there were clear sex and interspecies differences in the distribution of this protein. We also observed changes in ESR2 expression in the female brain in response to circulating estrogen levels. Our results, which show the precise expression profiles of ESR2 protein in rodent brains, will be helpful for understanding the ESR2-mediated actions of estrogen.

Ovarian ERß cistrome and transcriptome reveal chromatin interaction with LRH-1.

BACKGROUND: Estrogen receptor beta (ERß, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized. RESULTS: We here performed ERß ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERß knockout ovaries. By integrating the ERß cistrome and transcriptome, we identified its direct target genes and enriched biological functions in the ovary. This demonstrated its strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERß loss. Moreover, we identified a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1, Nr5a2) and showed that ERß and LRH-1 extensively bound to the same chromatin locations in granulosa cells. Using ChIP-reChIP, we corroborated simultaneous ERß and LRH-1 co-binding at the ERß-repressed gene Greb1 but not at the ERß-upregulated genes Cyp11a1 and Fkbp5. Transactivation assay experimentation further showed that ERß and LRH-1 can inhibit their respective transcriptional activity at classical response elements. CONCLUSIONS: By characterizing the genome-wide endogenous ERß chromatin binding, gene regulations, and extensive crosstalk between ERß and LRH-1, along with experimental corroborations, our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.

Estrogen Receptor-ß Gene Cytosine-Adenine (ESR2-CA) Repeat Polymorphism in Postmenopausal Colon Cancer.

The pathobiological role of estrogen is controversial in colorectal cancer. Cytosine-adenine (CA) repeat in the estrogen receptor (ER)-ß gene (ESR2-CA) is a microsatellite, as well as representative of ESR2 polymorphism. Though its function is unknown, we previously showed that a shorter allele (germline) increased the risk of colon cancer in older women, whereas it decreased it in younger postmenopausal women. ESR2-CA and ER-ß expressions were examined in cancerous (Ca) and non-cancerous (NonCa) tissue pairs from 114 postmenopausal women, and comparisons were made considering tissue types, age/locus, and the mismatch repair protein (MMR) status. ESR2-CA repeats <22/≥22 were designated as 'S'/'L', respectively, resulting in genotypes SS/nSS (=SL&LL). In NonCa, the rate of the SS genotype and ER-ß expression level were significantly higher in right-sided cases of women ≥70 (≥70Rt) than in those in the others. A decreased ER-ß expression in Ca compared with NonCa was observed in proficient-MMR, but not in deficient-MMR. In NonCa, but not in Ca, ER-ß expression was significantly higher in SS than in nSS. ≥70Rt cases were characterized by NonCa with a high rate of SS genotype or high ER-ß expression. The germline ESR2-CA genotype and resulting ER-ß expression were considered to affect the clinical characteristics (age/locus/MMR status) of colon cancer, supporting our previous findings.

Calponin 2 harnesses metabolic reprogramming to determine kidney fibrosis.

OBJECTIVE: In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy. However, how cellular mechanics and metabolism are connected remains unclear. METHODS: A multi-disciplinary approach was employed: the fibrotic kidney disease models were induced by renal ischemia-reperfusion injury and unilateral ureteral obstruction in Calponin 2 (CNN2) knockdown mice. Proteomics, bioinformatics, and in vivo and in vitro molecular experimental pathology studies were performed. RESULT: Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, CNN2 was predominantly expressed in fibroblasts and pericytes. In CKD patients, CNN2 levels was markedly induced in blood. In mice, CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in the diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway resulted in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2), binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions. CONCLUSIONS: Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Estrogen Signaling Influences Nephron Segmentation of the Zebrafish Embryonic Kidney.

Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17ß-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, esr2b, revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, irx3b and its target irx1a. These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis.

Bisphenol A and Di(2-Ethylhexyl) Phthalate promote pulmonary carcinoma in female rats via estrogen receptor beta: In vivo and in silico analysis.

The prevalence of lung cancer in women currently merits our attentions. However, cigarette exposure alone does not tell the whole story that lung cancer is more prevalent among non-smoking women. Since female lung cancer is closely linked to estrogen levels, many of endocrine disrupting chemicals (EDCs), as the substances similar to estrogen, affect hormone levels and become a potential risk of female lung cancer. Additionally, the combined toxicity of EDCs in daily environment has only been discussed on a limited scale. Consequently, this study explored the cancer-promoting effect of two representative substances of EDCs namely Bisphenol A (BPA) and Di(2-Ethylhexyl) Phthalate (DEHP) after their exposure alone or in combination, using a rat pulmonary tumor model published previously, combining bioinformatics analysis based on The Comparative Toxicogenomics Database (CTD) and The Cancer Genome Atlas (TCGA) databases. It demonstrated that BPA and DEHP enhanced the promotion of pulmonary tumor in female rats, either alone or in combination. Mechanistically, BPA and DEHP mainly directly bound and activated ESR2 protein, phosphorylated CREB protein, activated HDAC6 transcriptionally, induced the production of the proto-oncogene c-MYC, and accelerated the formation of pulmonary tumor in female rats. Remarkably, BPA, rather than DEHP, exhibited a much more critical effect in female lung cancer. Additionally, the transcription factor ESR2 was most affected in carcinogenesis, causing genetic disruption. Furthermore, the TCGA database revealed that ESR2 could enhance the promotion and progression of non-small cell lung cancer in females via activating the WNT/ß-catenin pathway. Finally, our findings demonstrated that BPA and DEHP could enhance the promotion of pulmonary carcinoma via ESR2 in female rats and provide a potential and valuable insight into the causes and prevention of lung cancer in non-smoking women due to EDCs exposure.

Sexually dimorphic effects of estrogen receptor 2 deletion in the dorsal raphe nucleus on emotional behaviors.

Sex differences in emotional behaviors and affective disorders have been widely noted, of which sexually dimorphic secretion of gonadal steroid hormones such as estrogen is suspected to play a role. However, the underlying neural mechanisms remain poorly understood. We noted that the expression of estrogen receptor 2 (Esr2, or ERß), a key mediator of estrogen signaling in the brain, was enriched in the dorsal raphe nucleus (DRN), a region involved in emotion regulation. To investigate whether DRN Esr2 expression confers sex-specific susceptibility or vulnerability in emotional behaviors, we generated a conditional allele of Esr2 that allowed for site-specific deletion of Esr2 in the DRN via local injection of Cre-expressing viruses. DRN-specific Esr2 deletion mildly increased anxiety behaviors in females, as shown by decreased time spent in the center zone of an open field in knockout females. By contrast, DRN Esr2 deletion had no effects on anxiety levels in males, as demonstrated by knockout males spending comparable time in the center zone of an open field and open arms of an elevated-plus maze. Furthermore, in the tail suspension test, DRN Esr2 deletion reduced immobility, a depression-like behavior, in a male-biased manner. Together, these results reveal sex-specific functions of DRN Esr2 in regulating emotional behaviors and suggest targeted manipulation of DRN Esr2 signaling as a potential therapeutic strategy to treat sex-biased affective disorders.

Estrogen receptor 1 appears essential for fetal viability in a murine model of premature birth.

PROBLEM: Protective effects for adult neurological disorders have been attributed to sex hormones. Using a murine model of prematurity, we evaluated the role of estrogen signaling in the process of perinatal brain injury following exposure to intrauterine inflammation. METHOD OF STUDY: Intrauterine lipopolysaccharide (LPS) was used to invoke preterm labor and fetal neuroinflammation. Fetal brains were analyzed for changes in Esr1, Esr2 and Cyp19. Dams heterozygous for the Esr1 knockout allele were also given intrauterine LPS to compare delivery and offspring viability to wild type controls. RESULTS: The upregulation in inflammatory cytokines was accompanied by an increase in Esr1 and Esr2 transcripts, though protein levels declined. Cyp19 did not differ by mRNA or protein abundance. Offspring from Esr1 mutants were larger, had a longer gestation and significantly greater mortality. CONCLUSIONS: Estrogen signaling is altered in the fetal brains of preterm offspring exposed to neuroinflammatory injury. The reduction of Esr1 and Esr2 proteins with LPS suggests that these proteins are degraded. It is possible that transcriptional upregulation of Esr1 and Esr2 occurs to compensate for the loss of these proteins. Alternatively, the translation of Esr1 and Esr2 mRNAs may be disrupted with LPS while a feedback mechanism upregulates transcription. Intact Esr1 signaling is also associated with early preterm delivery following exposure to intrauterine LPS. A loss of one Esr1 allele delays this process, but appears to do so at the cost of fetal viability. These results suggest estrogen signaling plays opposing roles between maternal and fetal responses to preterm birth.

Unhatched bovine blastocysts express all transcripts of the estrogen biosynthetic pathway, but steroid hormone synthesis could not yet be demonstrated.

Early embryos of rodent species and rabbits but also farm animals such as pigs, horses and cattle produce estrogens, which are considered important regulators of the implantation process. In cattle, the exact stage at which embryonic estrogen synthesis commences is yet unknown. However, this information is regarded as important to consider a possible role of embryonic estrogens in preimplantation development. Therefore, in this study, we first used quantitative reverse transcription PCR to examine the mRNA expression of the enzymes required for the conversion of cholesterol into free and sulfonated estrogens (CYP11A1, CYP17A1, HSD3B, CYP19A1, and SULT1E1), the cholesterol carrier protein STAR, and the estrogen receptors ESR1 and ESR2 in in vitro produced morulae and unhatched blastocysts (d 6-9). Only in the blastocysts, were the mRNAs of the entire estrogen biosynthesis chain and of both estrogen receptors clearly present, whereas mRNA specific to ESRs was already detectable in the morulae. We also examined the expression of the corresponding enzymes in blastocysts at the protein level. None of the enzymes were detectable by capillary-based western analysis. Immunofluorescence methods were established for the detection of CYP17A1, CYP19A1, and SULT1E1. CYP17A1 was observed in the inner cell mass and trophectoderm, whereas CYP19A1 and SULT1E1 were present only in trophectoderm. An attempt to detect estrogen sulfotransferase activity was unsuccessful. Despite clear evidence that some elements of the estrogen biosynthetic pathway are also present at the protein level, it remains to be clarified whether the enzyme cascade underlying estrogen production is already functional in unhatched blastocysts.

Expression of estrogen receptors, PELP1, and SRC in human spermatozoa and their associations with semen quality.

Sperm cells are target cells for both estrogens and xenoestrogens. Due to the specific structure of spermatozoa, these hormonal compounds may act on sperm in a non-genomic mechanism only. However, the ESR-mediated signaling pathways are still poorly understood. In this study, we obtained 119 samples from male participants of Caucasian descent who donated semen for standard analysis. We analyzed gene expression of estrogen receptors (ESR1 and ESR2) and their coregulators-proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and cellular kinase c-Src (SRC). RNA level was established using reverse-transcribed RNA as a template, followed by a polymerase chain reaction. Proteins' presence was confirmed by western blot and immunocytochemistry techniques. "Normal" values of semen parameters were defined as follows: > 32% sperm with progressive motility, > 4% sperm cells with normal morphology, > 15 × 106 sperm per mL, > 58% live spermatozoa and leukocyte amount < 106 cells per mL, according to WHO 2010 reference. Semen parameters that deviated from these "normal" values were labeled as "abnormal". Gene expression ratios revealed significant, moderate, and negative correlations for ESR1/ESR2 and weak, negative ESR2/PELP1 correlations in the subgroup of patients with abnormal values of semen parameters. In addition, SRC/PELP1 was moderately and positively correlated in the subgroup with parameters within the reference values established by WHO 2010. Our study showed that both PELP1 scaffolding protein and SRC kinase might influence semen quality via ESRs. It seems that not the expression of a single gene may affect the sperm quality, but more gene-to-gene mutual ratio. Characterization of estrogen-signaling pathway-related genes' modulated expression in sperm cells could aid in better understanding sperm biology and quality.

Isoliquiritigenin Inhibits the Growth of Colorectal Cancer Cells through the ESR2/PI3K/AKT Signalling Pathway.

Colorectal cancer (CRC) is one of the most common malignancies. Isoliquiritigenin (ISL), a flavonoid phytoestrogen, has shown anti-tumour activities against various cancers. However, its anti-CRC mechanism has not been clarified. In this study, the potential molecular mechanism of ISL against CRC was investigated through network pharmacological prediction and experimental validation. The results of the network prediction indicate that ESR2, PIK3CG and GSK3ß might be the key targets of ISL against CRC, which was verified by molecular docking, and that its anti-tumour mechanisms might be related to the oestrogen and PI3K/AKT signalling pathway. The experimental results show that ISL reduced the viability of SW480 and HCT116 cells, induced apoptosis, blocked the cell cycle in the G2 phase in vitro, and suppressed xenograft tumour growth in vivo. In addition, ISL significantly down-regulated the protein expression of PIK3CG, AKT, p-AKT, p-GSK3ß, CDK1, NF-κB and Bcl-2; up-regulated ESR2 and Bax; decreased the ratio of p-AKT/AKT and p-GSK3ß/GSK3ß; and increased the Bax/Bcl-2 ratio. This study indicates that ISL can inhibit the growth of CRC cells and induce apoptosis, which may be related to the up-regulation of ESR2 and inhibition of the PI3K/Akt signalling pathway.

Immunohistochemical Detection of Estrogen Receptor-Beta (ERß) with PPZ0506 Antibody in Murine Tissue: From Pitfalls to Optimization.

The estrogen receptor beta (ERß) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERß, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERß (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERß. Herein, we established an immunohistochemical detection protocol for ERß protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERß. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERß. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERß, is also suitable for reliable detection of murine ERß. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERß in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody.

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.