Transcriptomic changes in eutopic endometrium and ectopic lesions during endometriosis progression in a mouse model.

OBJECTIVE: To identify the transcriptomic changes of ectopic lesions and eutopic endometrial tissues during the progression of endometriosis, we performed transcriptomic analysis in the eutopic endometrium and ectopic lesions. DESIGN: Laboratory study. SETTING: Academic medical center. ANIMALS: Four fertile and 4 subfertile Pgrcre/+Rosa26mTmG/+ mice with endometriosis, and 4 sham mice for each group of endometriosis mice as control. These mice underwent either surgery to induce endometriosis or sham surgery. Fertile sham and mice with endometriosis were used 1 month after surgery, whereas subfertile ones were used 3 months after surgery. INTERVENTIONS: Early and chronic effects of endometriosis on transcriptomics of ectopic lesions and eutopic endometrium. MAIN OUTCOME MEASURES: RNA-sequencing analysis and identification of differentially expressed genes and pathways in the ectopic lesions and eutopic uteri from mice with endometriosis and sham mice at day 3.5 of pregnancy. RESULTS: Our mouse model recapitulates the transcriptomic changes of ectopic lesions in humans. RNA-sequencing analysis was performed in ectopic lesions and eutopic uteri from mice with or without endometriosis during the progression of the disease. Estrogen activity, inflammation, angiogenesis, and fibrosis pathways were consistently elevated in all the ectopic lesions compared with eutopic endometrium. Cholesterol/glucose synthesis and stem cell pluripotency pathways were more enhanced in ectopic lesions from subfertile mice compared with their eutopic endometrium. Dysregulation of infiltration of macrophage, dendritic, T and B cells was validated with the use of immunohistochemistry in ectopic lesions. Multiple ligand-receptor pairs between the ectopic and eutopic endometrium were altered compared with the sham endometrium. Suppressed WNT and EGF pathways were only found in the eutopic endometrium from subfertile not fertile mice compared with sham. CONCLUSIONS: Our mouse endometriosis model recapitulates the transcriptomics of ectopic lesions in humans. Our transcriptomic analysis during endometriosis progression in our mouse model will help us understand the pathophysiology of endometriosis.

ARG1 Is a Potential Prognostic Marker in Metastatic Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy. While the majority of patients present with early-stage and low-grade EC and have an excellent prognosis, a subset has metastatic disease at presentation or develops distant recurrence after initial treatment of the primary. However, the lack of prognostic biomarkers for metastatic EC is a critical barrier. Arginase 1 (ARG1) regulates the last step of the urea cycle, and an increase in ARG1 has been correlated as a poor prognostic factor in a variety of cancers. In the present study, ARG1 expression was evaluated as a potential prognostic marker for metastatic EC in endometrial hyperplasia and cancer of mice with Pten mutation as well as Pten and Mig-6 double mutations. While Pten mutation in the uterus is not sufficient for distant metastasis, mice with concurrent ablation of Mig-6 and Pten develop distant metastasis. Our immunostaining and RT-qPCR analysis revealed that the expression of ARG1 in early stage of EC as well as endometrial hyperplasia from mice deficient in Mig-6 and Pten mutations significantly increased compared to Pten mutation in the uterus. The results suggest that a high level of ARG1 is associated with poor prognosis in association with EC of mouse.

ARG1 is a potential prognostic marker in metastatic and recurrent endometrial cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy. While the majority of patients present with early-stage and low-grade EC and have an excellent prognosis, a subset has metastatic disease at presentation, or develops distant recurrence after initial treatment of the primary. However, the lack of prognostic biomarkers for metastatic EC is a critical barrier. Arginase 1 (ARG1) regulates the last step of the urea cycle, and an increase in ARG1 has been correlated as a poor prognostic factor in a variety of cancers. In the present study, ARG1 expression was evaluated as a potential prognostic marker for metastatic EC in endometrial hyperplasia and cancer of mice with Pten mutation as well as Pten and Mig-6 double mutations. While Pten mutation in the uterus is not sufficient for distant metastasis, mice with concurrent ablation of Mig-6 and Pten develop distant metastasis. Our immunostaining and RT-qPCR analysis revealed that the expression of ARG1 in early stage of EC as well as endometrial hyperplasia from mice deficient in Mig-6 and Pten mutations significantly increased compared to Pten mutation in the uterus. The results suggest that a high level of ARG1 is associated with poor prognosis in association with EC of mouse.

Epigenetic Dysregulation in Endometriosis: Implications for Pathophysiology and Therapeutics.

Endometriosis is a prevalent gynecological condition associated with pelvic pain and infertility. Despite more than a century of research, the etiology of endometriosis still eludes scientific consensus. This lack of clarity has resulted in suboptimal prevention, diagnosis, and treatment options. Evidence of genetic contributors to endometriosis is interesting but limited; however, significant progress has been made in recent years in identifying an epigenetic role in the pathogenesis of endometriosis through clinical studies, in vitro cell culture experiments, and in vivo animal models. The predominant findings include endometriosis-related differential expression of DNA methyltransferases and demethylases, histone deacetylases, methyltransferases, and demethylases, and regulators of chromatin architecture. There is also an emerging role for miRNAs in controlling epigenetic regulators in the endometrium and endometriosis. Changes in these epigenetic regulators result in differential chromatin organization and DNA methylation, with consequences for gene expression independent of a genetic sequence. Epigenetically altered expression of genes related to steroid hormone production and signaling, immune regulation, and endometrial cell identity and function have all been identified and appear to play into the pathophysiological mechanisms of endometriosis and resulting infertility. This review summarizes and critically discusses early seminal findings, the ever-growing recent evidence of epigenetic contributions to the pathophysiology of endometriosis, and implications for proposed epigenetically targeted therapeutics.

Prenatal Octamethylcyclotetrasiloxane Exposure Impaired Proliferation of Neuronal Progenitor, Leading to Motor, Cognition, Social and Behavioral Functions.

Cyclic siloxane octamethylcyclotetrasiloxane (D4) has raised concerns as an endocrine-disrupting chemical (EDC). D4 is widely used in detergent products, cosmetics, and personal care products. Recently, robust toxicological data for D4 has been reported, but the adverse effects of D4 on brain development are unknown. Here, pregnant mice on gestational day 9.5 were treated daily with D4 to postnatal day 28, and the offspring mice were studied. The prenatal D4-treated mice exhibited cognitive dysfunction, limited memory, and motor learning defect. Moreover, prenatal D4 exposure reduced the proliferation of neuronal progenitors in the offspring mouse brain. Next, the mechanisms through which D4 regulated the cell cycle were investigated. Aberrant gene expression, such as cyclin-dependent kinases CDK6 and cyclin-dependent kinase inhibitor p27, were found in the prenatal D4-treated mice. Furthermore, the estrogen receptors ERa and ERb were increased in the brain of prenatal D4-treated mice. Overall, these findings suggest that D4 exerts estrogen activity that affects the cell cycle progression of neuronal progenitor cells during neurodevelopment, which may be associated with cognitive deficits in offspring.

Melatonin mitigates the adverse effect of hypoxia during myocardial differentiation in mouse embryonic stem cells.

BACKGROUND: Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. OBJECTIVES: This study aims to investigate the correlation between melatonin and hypoxia induction in cardiomyocytes differentiation. METHODS: Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated. RESULTS: Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects. CONCLUSIONS: This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway.

Loss of Nckx3 Exacerbates Experimental DSS-Induced Colitis in Mice through p53/NF-κB Pathway.

Inflammatory bowel diseases (IBDs) comprises a range of chronic inflammatory conditions of the intestinal tract. The incidence and prevalence of IBDs are increasing worldwide, but the precise etiology of these diseases is not completely understood. Calcium signaling plays a regulatory role in cellular proliferation. Nckx3, a potassium-dependent Na+/Ca2+ exchanger, is not only expressed in the brain but also in the aortic, uterine, and intestinal tissues, which contain abundant smooth muscle cells. This study investigated the role of Nckx3 in intestinal inflammation. Microarray analyses revealed the upregulation of the innate immune response-associated genes in the duodenum of Nckx3 knockout (KO) mice. The Nckx3 KO mice also showed an increase in IBD- and tumorigenesis-related genes. Using dextran sodium sulfate (DSS)-induced experimental colitis mice models, the Nckx3 KO mice showed severe colitis. Furthermore, the pathways involving p53 and NF-κB signaling were significantly upregulated by the absence of Nckx3. Overall, Nckx3 plays a critical role in the innate immune and immune response and may be central to the pathogenesis of IBD.

4-tert-Octylphenol Exposure Disrupts Brain Development and Subsequent Motor, Cognition, Social, and Behavioral Functions.

The endocrine-disrupting chemical 4-tert-octylphenol (OP) is a widespread estrogenic chemical used in consumer products such as epoxy resins and polycarbonate plastic. However, the effects of OP on brain development are unknown. The present study examined the effects of OP on neuron and neurobehavioral development in mice. By using primary cortical neuron cultures, we found that OP-treated showed a decreased length of axons and dendrites and an increased number of primary and secondary dendrites. OP reduced bromodeoxyuridine (BrdU), mitotic marker Ki67, and phospho-histone H3 (p-Histone-H3), resulting in a reduction of neuronal progenitor proliferation in offspring mouse brain. Moreover, OP induced apoptosis in neuronal progenitor cells in offspring mouse brain. Furthermore, offspring mice from OP-treated dams showed abnormal cognitive, social, and anxiety-like behaviors. Taken together, these results suggest that perinatal exposure to OP disrupts brain development and behavior in mice.

Perinatal Exposure to Triclosan Results in Abnormal Brain Development and Behavior in Mice.

Triclosan (TCS) is one of the most common endocrine-disrupting chemicals (EDCs) present in household and personal wash products. Recently, concerns have been raised about the association between abnormal behavior in children and exposure to EDC during gestation. We hypothesized that exposure to TCS during gestation could affect brain development. Cortical neurons of mice were exposed in vitro to TCS. In addition, we examined in vivo whether maternal TCS administration can affect neurobehavioral development in the offspring generation. We determined that TCS can impair dendrite and axon growth by reducing average length and numbers of axons and dendrites. Additionally, TCS inhibited the proliferation of and promoted apoptosis in neuronal progenitor cells. Detailed behavioral analyses showed impaired acquisition of spatial learning and reference memory in offspring derived from dams exposed to TCS. The TCS-treated groups also showed cognition dysfunction and impairments in sociability and social novelty preference. Furthermore, TCS-treated groups exhibited increased anxiety-like behavior, but there was no significant change in depression-like behaviors. In addition, TCS-treated groups exhibited deficits in nesting behavior. Taken together, our results indicate that perinatal exposure to TCS induces neurodevelopment disorder, resulting in abnormal social behaviors, cognitive impairment, and deficits in spatial learning and memory in offspring.

Effects of Bisphenol A and 4-tert-Octylphenol on Embryo Implantation Failure in Mouse.

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all human miscarriage cases. Calcium ion has been shown to be involved in many cellular signal transduction pathways as well as in the regulation of cell adhesion, which is necessary for the embryo implantation process. Exposure to endocrine-disrupting chemicals (EDs) during early gestation results in disruption of intrauterine implantation and uterine reception, leading to implantation failure. In this study, ovarian estrogen (E2), bisphenol A (BPA), or 4-tert-octylphenol (OP), with or without ICI 182,780 (ICI) were injected subcutaneously from gestation day 1 to gestation day 3 post-coitus. The expression levels of the calcium transport genes were assessed in maternal uteri and implantation sites. The number of implantation sites was significantly low in the OP group, and implantation sites were absent in the E2, ICI and EDs + ICI groups. There were different calcium transient transport channel expression levels in uterus and implantation site samples. The levels of TRPV5 and TRPV6 gene expression were significantly increased by EDs with/without ICI treatment in utero. Meanwhile, TRPV5 and TRPV6 gene expression were significantly lower in implantation sites samples. NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Compared to vehicle treatment in the uterus, both the MUC1 mRNA and protein levels were markedly high in all but the BPA group. Taken together, these results suggest that both BPA and OP can impair embryo implantation through alteration of calcium transport gene expressions and by affecting uterine receptivity.

Depletion of follicles accelerated by combined exposure to phthalates and 4-vinylcyclohexene diepoxide, leading to premature ovarian failure in rats.

Humans are at daily risk by simultaneous exposures to a broad spectrum of man-made chemicals in the commercial products. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to premature ovarian failure (POF) in mammals. We investigate the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalate, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP) and di-n-butyl phthalate (DBP) on POF. Combination exposure with VCD and phthalate significantly reduced the numbers of primary follicles. The expressions of Amh and Sohlh2 were significantly decreased in the combination groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of follicle-stimulating hormone were significantly increased in combination groups. Taken together, exposure to phthalates promotes the depletion of follicular follicles and consequently increases the risk of premature menopause, and combined exposure of phthalates and VCD to early menopausal women is likely to aggravate the POF syndrome.

Parabens Accelerate Ovarian Dysfunction in a 4-Vinylcyclohexene Diepoxide-Induced Ovarian Failure Model.

Parabens are widely used preservatives in basic necessities such as cosmetic and pharmaceutical products. In previous studies, xenoestrogenic actions of parabens were reported in an immature rat model and a rat pituitary cell line (GH3 cells). The relationship between parabens and ovarian failure has not been described. In the present study, the influence of parabens on ovarian folliculogenesis and steroidogenesis was investigated. A disruptor of ovarian small pre-antral follicles, 4-vinylcyclohexene diepoxide (VCD, 40 mg/kg), was used to induce premature ovarian failure (POF). Methylparaben (MP, 100 mg/kg), propylparaben (PP, 100 mg/kg), and butylparaben (BP, 100 mg/kg) dissolved in corn oil were treated in female 8-week-old Sprague-Dawley rat for 5 weeks. Estrus cycle status was checked daily by vaginal smear test. Ovarian follicle development and steroid synthesis were investigated through real-time PCR and histological analyses. Diestrus phases in the VCD, PP, and BP groups were longer than that in the vehicle group. VCD significantly decreased mRNA level of folliculogenesis-related genes (Foxl2, Kitl and Amh). All parabens significantly increased the Amh mRNA level but unchanged Foxl2 and Kitlg acting in primordial follicles. VCD and MP slightly increased Star and Cyp11a1 levels, which are related to an initial step in steroidogenesis. VCD and parabens induced an increase in FSH levels in serum and significantly decreased the total number of follicles. Increased FSH implies impairment in ovarian function due to VCD or parabens. These results suggest that VCD may suppress both formation and development of follicles. In particular, combined administration of VCD and parabens accelerated inhibition of the follicle-developmental process through elevated AMH level in small antral follicles.