Heart and neural crest derivatives expressed transcript 2 (HAND2) is a critical mediator of progesterone action in endometrial stromal cells. Silencing of Hand2 expression in mouse uterus leads to an unopposed FGFR-mediated action that causes female mice infertility. To investigate the involvement of HAND2-FGFR signaling in pathogenesis of adenomyosis, immunohistochemistry, in situ hybridization, and quantitative real-time PCR were employed to assess gene expression in the normal endometrium, the paired eutopic endometrium and ectopic lesions obtained from women with adenomyosis. DNA methylation in the regions of HAND2 promoter and the first exon was also monitored in these samples. Our results revealed that HAND2 expression were dramatically reduced, but FGF9 expression and FGFR-ERK1/2-mediated MAPK signaling pathway were enhanced in the eutopic endometrium and ectopic lesions of patients with adenomyosis compared to the normal controls. Interestingly, expression of HAND2-AS1, a long noncoding RNA that resides adjacent to HAND2 in genome, was also reduced in adenomyosis. DNA methylation analysis revealed that the bidirectional promoter between HAND2 and HAND2-AS1, and the first exon of HAND2 gene was heavily methylated in the eutopic endometrium and the ectopic lesions of adenomyosis. To investigate the regulation of gene expression by HAND2-AS1, HAND2-AS1 expression was silenced in human endometrial stromal cells. In contrast to the downregulation of HAND2 in response to HAND2-AS1 silencing, FGF9 expression was augmented significantly. Endometrial stromal cells lacking HAND2-AS1 exhibited enhanced proliferation and migration potentials. Collectively, our studies revealed a new molecular mechanism by which HAND2-AS1 is involved in the pathogenesis of adenomyosis via modulating HAND2-FGFR-mediated signaling.

碱性螺旋-环-螺旋(basie helix-loop helix, bHLH)蛋白是与各类细胞核组织的发育和功能相关的转录因子.1989年第1条bHLH被解析发现[1],至今已共有1000余条bHLH在不同物种中被鉴定[2].

HAND2 is a critical mediator of progesterone receptor signaling in endometrium. Silencing of HAND2 expression is associated with female infertility and endometrial cancers. We recently observed that lncRNA HAND2-AS1 and HAND2 are expressed coordinately in human endometrial stromal cells. To investigate involvement of HAND2-AS1 and HAND2 in pathogenesis of endometriosis, we employed immunohistochemistry, in situ hybridization, and quantitative real-time PCR to assess their expression in normal endometrium and the ectopic lesions obtained from patients with ovarian endometriosis. HAND2 promoter methylation was also monitored in these samples. Our results revealed that HAND2 and HAND2-AS1 expression levels were reduced but promoter methylation was enhanced significantly in ectopic endometrium when compared with the normal controls. Fluorescence in situ hybridization showed that HAND-AS1 is predominantly localized in the nuclei of endometrial stromal cells in contrast to the cytoplasmic distribution in epithelial cell compartment. To further investigate regulation of HAND2 expression by HAND2-AS1, HAND2-AS1 was silenced or overexpressed in human endometrial stromal cells. Our studies showed that expression levels of HAND2 and its direct target IL15 were attenuated markedly in HAND2-AS1 silenced cells but enhanced significantly in the overexpressed human endometrial stromal cells. Silencing of HAND2-AS1 also impaired endometrial stromal cell decidualization as indicated by downregulation of decidual biomarkers IGFBP1 and PRL. In addition, HAND2 promoter methylation was also enhanced upon HAND2-AS1 silencing. RNA immunoprecipitation studies further revealed that HAND2-AS1 is capable of binding to DNA methyltransferase DNMT1, indicating that HAND2-AS1 governs HAND2 expression epigenetically involving DNA methylation.

Background Chronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied. Methods Endometriosis was induced in female mice (45-day-old; n = 6–11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls ( n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning “Weka trainable segmentation” plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated. Results We observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw. Conclusion We believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis. Graphical Abstract

Failure in embryo implantation is a common issue in assisted reproduction technology, and establishment of endometrial receptivity plays a critical role in the process of embryo implantation. Although it was extensively studied in the endometrial physiology, the molecular mechanism underlying regulation of endometrial receptivity remains elusive. Ample clinical evidence suggests that endometriosis is closely associated with female infertility in reproductive age, it is still unclear how endometriosis adversely affects endometrial receptivity that leads to failure in embryo implantation. This literature review is mainly focusing on recent progress on adverse effect of the endometriosis on endometrial receptivity during early pregnancy.

Basigin (BSG) is a transmembrane glycoprotein involved in cell proliferation, angiogenesis, and tissue remodeling. BSG has been shown to be essential for male and female reproduction although little is known about its role in normal uterine function. To study the potential function of BSG in the female reproductive tract, we generated mice with conditional knockout of Bsg in uterine cells using progesterone receptor-Cre and hypothesized that BSG is required for normal pregnancy in mice. Fertility study data showed that the conditional knockout mice had significantly reduced fertility compared to controls. Ovarian function of the conditional knockout mice appeared normal with no difference in the number of superovulated oocytes collected or in serum progesterone levels between the conditional knockout and the control mice. Uterine tissues collected at various times of gestation showed increased abnormalities in implantation, decidualization, placentation, and parturition in the conditional knockout mice. Uterine cross sections on Day 5 of pregnancy showed implantation failure and abnormal uterine epithelial differentiation in a large proportion of the conditional knockout mice. There was a compromised decidual response to artificial decidualization stimuli and decreased mRNA and protein levels for decidualization genes in the uteri of the conditional knockout mice. We also observed altered protein expression of monocarboxylate transporter 1 (MCT1), as well as impaired angiogenesis in the conditional knockout uteri compared to the controls. These results support that BSG is required for successful pregnancy through its functions in implantation and decidualization.

转录因子 SRY-box containing gene 17 ( SOX17 )隶属于SOX 基因家族 F 亚族,定位于人8 号染色体 q11. 23,已知SOX17在胚胎发育过程中内胚层形成、干细胞分化、神经突胶质细胞发展及血管生成等方面起着十分重要的作用.大量证据表明SOX17作为一个新的抑癌基因与许多细胞的癌变过程密切有关,SOX17基因启动子序列的甲基化导致了SOX17基因表达含量的下降和WNT/β-catenin信号通路的持续激活,并进而诱导细胞周期因子和致癌基因的表达.最新研究发现SOX17在子宫内膜的表达与其生理功能有关, SOX17 表达水平的减少不仅与女性的不孕有关,与许多常见的妇科相关疾病如宫颈上皮内瘤变、宫颈癌、子宫内膜癌、子宫内膜异位症、子宫腺肌病存在着密切的关系.阐明SOX17在子宫内膜中的表达调控机理和作用有可能为诊断、治疗内膜相关疾病提供新的诊断标志物与治疗靶点.

Endometrial stromal cells differentiate to form decidual cells in a process known as decidualization, which is critical for embryo implantation and successful establishment of pregnancy. We previously reported that bone morphogenetic protein 2 (BMP2) mediates uterine stromal cell differentiation in mice and in humans. To identify the downstream target(s) of BMP2 signaling during decidualization, we performed gene-expression profiling of mouse uterine stromal cells, treated or not treated with recombinant BMP2. Our studies revealed that expression of Msx2, a member of the mammalian Msx homeobox gene family, was markedly upregulated in response to exogenous BMP2. Interestingly, conditional ablation of Msx2 in the uterus failed to prevent a decidual phenotype, presumably because of functional compensation of Msx2 by Msx1, a closely related member of the Msx family. Indeed, in Msx2-null uteri, the level of Msx1 expression in the stromal cells was markedly elevated. When conditional, tissue-specific ablation of both Msx1 and Msx2 was accomplished in the mouse uterus, a dramatically impaired decidual response was observed. In the absence of both Msx1 and Msx2, uterine stromal cells were able to proliferate, but they failed to undergo terminal differentiation. In parallel experiments, addition of BMP2 to human endometrial stromal cell cultures led to a robust enhancement of MSX1 and MSX2 expression and stimulated the differentiation process. Attenuation of MSX1 and MSX2 expression by small interfering RNAs greatly reduced human stromal differentiation in vitro, indicating a conservation of their roles as key mediators of BMP2-induced decidualization in mice and women.

Ample evidence suggests that environmental and occupational exposure to bisphenol A (BPA) and phthalate, two chemicals widely used in the plastics industry, disturbs homeostasis of innate immunity and causes inflammatory diseases. However, the underlying molecular mechanisms of these toxicants in the regulation of macrophage inflammatory functions remain poorly understood. In this study, we addressed the effect of chronic exposure to BPA or phthalate at levels relevant to human exposure, either in vitro or in vivo, on the inflammatory reprograming of peritoneal macrophages. Our studies revealed that BPA and phthalates adversely affected expression levels of the proinflammatory cytokines and mediators in response to lipopolysaccharide stimulation. Exposure to these toxicants also affected gene expression of scavenger receptors and phagocytic capacity of peritoneal macrophages. Our studies revealed that the epigenetic inhibitors differentially modulated target gene expression in these cells. Further analysis revealed that certain histone modification enzymes were aberrantly expressed in response to BPA or phthalate exposure, leading to alteration in the levels of H3K36 acetylation and dimethylation, two chromatin modifications that are critical for transcriptional efficacy and accuracy. Our results further revealed that silencing of H3K36-specific methyltransferase Smyd2 expression or inhibition of SMYD2 enzymatic activity attenuated H3K36 dimethylation and enhanced interleukin-6 and tumor necrosis factor-α expression but dampened the phagocytic capacity of peritoneal macrophages. In summary, our results indicate that peritoneal macrophages are vulnerable to BPA or phthalate at levels relevant to human exposure. These environmental toxicants affect phenotypic programming of macrophages via epigenetic mechanisms involving SMYD2-mediated H3K36 modification.

The peptide hormone prolactin (PRL) and certain members of the epidermal growth factor (EGF) family play central roles in mammary gland development and physiology, and their dysregulation has been implicated in mammary tumorigenesis. Our recent studies have revealed that the CUB and zona pellucida-like domain-containing protein 1 (CUZD1) is a critical factor for PRL-mediated activation of the transcription factor STAT5 in mouse mammary epithelium. Of note, CUZD1 controls production of a specific subset of the EGF family growth factors and consequent activation of their receptors. Here, we found that consistent with this finding, CUZD1 overexpression in non-transformed mammary epithelial HC11 cells increases their proliferation and induces tumorigenic characteristics in these cells. When introduced orthotopically in mouse mammary glands, these cells formed adenocarcinomas, exhibiting elevated levels of STAT5 phosphorylation and activation of the EGF signaling pathway. Selective blockade of STAT5 phosphorylation by pimozide, a small-molecule inhibitor, markedly reduced the production of the EGF family growth factors and inhibited PRL-induced tumor cell proliferation in vitro. Pimozide administration to mice also suppressed CUZD1-driven mammary tumorigenesis in vivo. Analysis of human MCF7 breast cancer cells indicated that CUZD1 controls the production of the same subset of EGF family members in these cells as in the mouse. Moreover, pimozide treatment reduced the proliferation of these cancer cells. Collectively, these findings indicate that overexpression of CUZD1, a regulator of growth factor pathways controlled by PRL and STAT5, promotes mammary tumorigenesis. Blockade of the STAT5 signaling pathway downstream of CUZD1 may offer a therapeutic strategy for managing these breast tumors.

Environmental and occupational exposure to endocrine disrupting chemicals (EDCs) is a major threat to female reproductive health. Bisphenol A (BPA), an environmental toxicant that is commonly found in polycarbonate plastics and epoxy resins, has received much attention due to its estrogenic activity and high risk of chronic exposure in human. Whereas BPA has been linked to infertility and recurrent miscarriage in women, the impact of its exposure on uterine function during early pregnancy remains unclear. In a recent publication in Endocrinology, we demonstrated that prolonged exposure to an environmental relevant dose of BPA disrupts progesterone receptor-regulated uterine functions, thus affecting uterine receptivity for embryo implantation and decidua morphogenesis, two critical events for establishment and maintenance of early pregnancy. In particular we reported a marked impairment of progesterone receptor (PGR) expression and its downstream effector HAND2 in the uterine stromal cells in response to chronic BPA exposure. In an earlier study we have shown that HAND2 controls embryo implantation by repressing fibroblast growth factor (FGF) expression and the MAP kinase signaling pathway, thus inhibiting epithelial proliferation. Interestingly we observed that downregulation of PGR and HAND2 expression in uterine stroma upon BPA exposure was associated with an enhanced activation of FGFR and MAPK signaling, aberrant proliferation, and lack of uterine receptivity in the epithelium. In addition, the proliferation and differentiation of endometrial stromal cells to decidual cells, an event critical for the maintenance of early pregnancy, was severely compromised in response to BPA. This research highlight will provide an overview of our findings and discuss the potential mechanisms by which chronic BPA impairs PGR-HAND2 pathway and adversely affects implantation and the establishment of pregnancy.

Environmental and occupational exposure to bisphenol A (BPA), a chemical widely used in polycarbonate plastics and epoxy resins, has received much attention in female reproductive health due to its widespread toxic effects. Although BPA has been linked to infertility and recurrent miscarriage in women, the impact of its exposure on uterine function during early pregnancy remains unclear. In this study, we addressed the effect of prolonged exposure to an environmental relevant dose of BPA on embryo implantation and establishment of pregnancy. Our studies revealed that treatment of mice with BPA led to improper endometrial epithelial and stromal functions thus affecting embryo implantation and establishment of pregnancy. Upon further analyses, we found that the expression of progesterone receptor (PGR) and its downstream target gene, HAND2 (heart and neural crest derivatives expressed 2), was markedly suppressed in BPA-exposed uterine tissues. Previous studies have shown that HAND2 controls embryo implantation by repressing fibroblast growth factor and the MAPK signaling pathways and inhibiting epithelial proliferation. Interestingly, we observed that down-regulation of PGR and HAND2 expression in uterine stroma upon BPA exposure was associated with enhanced activation of fibroblast growth factor and MAPK signaling in the epithelium, thus contributing to aberrant proliferation and lack of uterine receptivity. Further, the differentiation of endometrial stromal cells to decidual cells, an event critical for the establishment and maintenance of pregnancy, was severely compromised in response to BPA. In summary, our studies revealed that chronic exposure to BPA impairs PGR-HAND2 pathway and adversely affects implantation and the establishment of pregnancy.

Endometriosis, defined as growth of the endometrial cells outside the uterus, is an inflammatory disorder that is associated with chronic pelvic pain and infertility in women of childbearing age. Although the estrogen-dependence of endometriosis is well known, the role of progesterone in development of this disease remains poorly understood. In this study, we developed a disease model in which endometriosis was induced in the peritoneal cavities of immunocompetent female mice, and maintained with exogenous estrogen. The endometriosis-like lesions that were identified at a variety of ectopic locations exhibited abundant blood supply and extensive adhesions. Histological examination revealed that these lesions had a well-organized endometrial architecture and fibrotic response, resembling those recovered from clinical patients. In addition, an extensive proliferation, inflammatory response, and loss of estrogen receptor alpha (ERα) and progesterone receptor (PR) expression were also observed in these lesions. Interestingly, administration of progesterone before, but not after, lesion induction suppressed lesion expansion and maintained ERα and PR expressions. These progesterone-pretreated lesions exhibited attenuation in KI67, CD31, and pro-inflammatory cytokine expression as well as macrophage infiltration, indicating that progesterone ameliorates endometriosis progression by inhibiting cell proliferation, inflammation and neovascularization. Our studies further showed that suppression of global DNA methylation by application of DNA methyltransferase inhibitor to female mice bearing ectopic lesions restrained lesion expansion and restored ERα and PR expression in eutopic endometrium and ectopic lesions. These results indicate that epigenetic regulation of target gene expression via DNA methylation contributes, at least in part, to progesterone resistance in endometriosis.

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.