Differential gene expression in decidualized human endometrial stromal cells induced by different stimuli.

Decidualization can be induced by culturing human endometrial stromal cells (ESCs) with several decidualization stimuli, such as cAMP, medroxyprogesterone acetate (MPA) or Estradiol (E2). However, it has been unclear how decidualized cells induced by different stimuli are different. We compared transcriptomes and cellular functions of decidualized ESCs induced by different stimuli (MPA, E2 + MPA, cAMP, and cAMP + MPA). We also investigated which decidualization stimulus induces a closer in vivo decidualization. Differentially expressed genes (DEGs) and altered cellular functions by each decidualization stimuli were identified by RNA-sequence and gene-ontology analysis. DEGs was about two times higher for stimuli that use cAMP (cAMP and cAMP + MPA) than for stimuli that did not use cAMP (MPA and E2 + MPA). cAMP-using stimuli altered the cellular functions including angiogenesis, inflammation, immune system, and embryo implantation whereas MPA-using stimuli (MPA, E2 + MPA, and cAMP + MPA) altered the cellular functions associated with insulin signaling. A public single-cell RNA-sequence data of the human endometrium was utilized to analyze in vivo decidualization. The altered cellular functions by in vivo decidualization were close to those observed by cAMP + MPA-induced decidualization. In conclusion, decidualized cells induced by different stimuli have different transcriptome and cellular functions. cAMP + MPA may induce a decidualization most closely to in vivo decidualization.

Analysis of cell-cell interaction between mural granulosa cells and cumulus granulosa cells during ovulation using single-cell RNA sequencing data of mouse ovary.

Purpose: We investigated the interactions between mural granulosa cells (MGCs) and cumulus granulosa cells (CGCs) during ovulation after the LH surge. Methods: We performed clustering, pseudotime, and interactome analyses utilizing reported single-cell RNA sequencing data of mouse ovary at 6 h after eCG-hCG injection. Results: Clustering analysis classified granulosa cells into two distinct populations, MGCs and CGCs. Pseudotime analysis divided granulosa cells into before and after the LH surge, and further divided them into two branches, the ovulatory MGCs and the ovulatory CGCs. Interactome analysis was performed to identify the interactions between MGCs and CGCs. Twenty-six interactions were acting from CGCs toward MGCs, involving ovulation and steroidogenesis. Thirty-six interactions were acting from MGCs toward CGCs, involving hyaluronan synthesis. There were 25 bidirectional interactions, involving the EGFR pathway. In addition, we found three novel interactions: Ephrins-Ephs pathway and Wnt-Lrp6 pathway from CGCs to MGCs, associated with steroidogenesis and lipid transport, respectively, and TGF-ß-TGFBR1 pathway from MGCs to CGCs, associated with hyaluronan synthesis. Conclusions: MGCs and CGCs interact with each other in the preovulatory follicle after the LH surge, and their interactions have roles in corpus luteum formation, oocyte maturation, and follicle rupture.

Combined histological and DNA methylome profiling approaches may provide insights into the pathophysiology of ovarian endometriomas.

Purpose: To test the theory that invaginated ovarian surface epithelium and endometrial implants on the ovary form ovarian endometriomas. Methods: Adhesion sites of ovarian endometrioma on the peritoneum and consecutive ovarian endometrioma cyst wall, called non-adhesion sites, were histologically examined. DNA methylomes of the adhesion sites, non-adhesion sites, and blueberry spots were compared with those of ovary, endometrium, and peritoneum. Results: The non-adhesion sites showed an ovarian surface epithelium-like structure near the adhesion site, which continued to a columnar epithelium-like structure. Calretinin staining was strong in the ovarian surface epithelium-like structure but weak in the columnar epithelium-like structure. Estrogen receptors were absent in the ovarian surface epithelium-like structure, but present in the columnar epithelium-like structure. The adhesion sites had endometrial gland-like structures that expressed estrogen receptors. Analyses of DNA methylomes classified the non-adhesion sites and ovaries into the same group, suggesting that ovarian endometriomas originate from the ovarian surface epithelium. The adhesion sites, blueberry spots and peritoneum were classified in the same group, suggesting that the adhesion sites and blueberry spots originate from the peritoneum. Conclusions: The present results support the invagination theory. Ovarian endometriomas consist of invaginated ovarian surface epithelium with celomic metaplasia and endometrium implants on the peritoneum.

Genome-wide analysis of histone modifications that underlie the dynamic changes in gene expression during decidualization in human endometrial stromal cells.

Human endometrial stromal cells (hESCs) undergo a differentiation process with dramatic changes in cell functions during the menstrual cycle, which is called decidualization. This is an important event for implantation of the embryo and successful pregnancy. Defective decidualization can cause implantation failure, miscarriage, and unexplained infertility. A number of genes are upregulated or downregulated during decidualization. Recent studies have shown that epigenetic mechanisms are involved in the regulation of decidualization-related genes and that histone modifications occur throughout the genome during decidualization. The present review focuses on the involvement of genome-wide histone modifications in dramatic changes in gene expression during decidualization. The main histone modifications are the increases of H3K27ac and H3K4me3, which activate transcription. C/EBPß works as a pioneer factor throughout the genome by recruiting p300. This is the main cause of the genome-wide acetylation of H3K27 during decidualization. Histone modifications were observed in both the proximal promoter and distal enhancer regions. Genome editing experiments show that the distal regions have transcriptional activities, which suggests that decidualization induces the interactions between proximal promoter and distal enhancer regions. Taken together, these findings show that gene regulation during decidualization is closely associated with genome-wide changes of histone modifications. This review provides new insights regarding the cases of implantation failure in terms of decidualization insufficiency owing to epigenetic dysregulation, and may lead to novel treatment options for women with implantation failure.

Glucose and lipid metabolisms in human endometrial stromal cells during decidualization.

Decidualization is a process of differentiation of human endometrial stromal cells (hESCs) accompanied by dramatic changes in cellular functions. This process is critical for embryo implantation and the establishment of pregnancy. Impairment of decidualization of hESCs leads to implantation failure, miscarriage, and unexplained infertility. The present review focuses on the metabolic changes in hESCs during decidualization. One of the changes taking place is in the glucose metabolism. Glucose uptake increases during decidualization because glucose is essential for the decidualization of hESCs. In hESCs, GLUT1 is highly expressed and involved in the increase of glucose uptake during decidualization. The up-regulation of GLUT1 is mediated by an epigenetic mechanism, which is regulated by CCAAT enhancer-binding protein ß (C/EBPß) and Wilms tumor 1 (WT1). Another metabolic change is in the lipid metabolism. Lipid accumulation in hESCs increases during decidualization. This increase is mediated by very low-density lipoprotein receptor (VLDLR). The up-regulation of VLDLR is regulated by WT1. In contrast to glucose, lipid is not essential for decidualization of hESCs. Endometrial cells have been implicated as important sources of nutrition for the embryo. hESCs may increase glucose and lipid storage so that they can supply them to the embryo during the implantation process. Taken together, decidualization is the process accompanied by metabolic changes, which may be associated with successful implantation.

Effects of Melatonin on the Transcriptome of Human Granulosa Cells, Fertilization and Blastocyst Formation.

Melatonin is a promising reagent that can improve assisted reproductive technology (ART) outcomes in infertility patients. However, melatonin is not effective for all infertile patients, and it remains unclear for which patients melatonin would be effective. This study examined the effects of melatonin on ART outcomes and examined its mechanisms. Melatonin increased the fertilization rate in patients whose fertilization rates in the previous cycle were less than 50%, but not in patients whose fertilization rates were more than 50% in the previous cycle. Melatonin increased the blastocyst formation rate in patients whose embryo development rates in the previous cycle were less than 50%, but not in patients whose embryo development rates were more than 50% in the previous cycle. To clarify its mechanisms, transcriptome changes by melatonin treatment in granulosa cells (GCs) of the patients were examined by RNA-sequence. Melatonin treatment altered the transcriptomes of GCs of patients with poor ART outcomes so that they were similar to the transcriptomes of patients with good ART outcomes. The altered genes were associated with the inhibition of cell death and T-cell activity, and the activation of steroidogenesis and angiogenesis. Melatonin treatment was effective for patients with poor fertilization rates and poor embryo development rates in the previous ART cycle. Melatonin alters the GCs transcriptome and, thus, their functions, and this could improve the oocyte quality, leading to good ART outcomes.

Transcriptional coactivator PGC-1α contributes to decidualization by forming a histone-modifying complex with C/EBPß and p300.

We previously reported that CCAAT/enhancer-binding protein beta (C/EBPß) is the pioneer factor inducing transcription enhancer mark H3K27 acetylation (H3K27ac) in the promoter and enhancer regions of genes encoding insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) and that this contributes to decidualization of human endometrial stromal cells (ESCs). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α; PPARGC1A) is a transcriptional coactivator known to regulate H3K27ac. However, although PGC-1α is expressed in ESCs, the potential role of PGC-1α in mediating decidualization is unclear. Here, we investigated the involvement of PGC-1α in the regulation of decidualization. We incubated ESCs with cAMP to induce decidualization and knocked down PPARGC1A to inhibit cAMP-induced expression of IGFBP-1 and PRL. We found cAMP increased the recruitment of PGC-1α and p300 to C/EBPß-binding sites in the promoter and enhancer regions of IGFBP-1 and PRL, corresponding with increases in H3K27ac. Moreover, PGC-1α knockdown inhibited these increases, suggesting PGC-1α forms a histone-modifying complex with C/EBPß and p300 at these regions. To further investigate the regulation of PGC-1α, we focused on C/EBPß upstream of PGC-1α. We found cAMP increased C/EBPß recruitment to the novel enhancer regions of PPARGC1A. Deletion of these enhancers decreased PGC-1α expression, indicating that C/EBPß upregulates PGC-1α expression by binding to novel enhancer regions. In conclusion, PGC-1α is upregulated by C/EBPß recruitment to novel enhancers and contributes to decidualization by forming a histone-modifying complex with C/EBPß and p300, thereby inducing epigenomic changes in the promoters and enhancers of IGFBP-1 and PRL.

The essential glucose transporter GLUT1 is epigenetically upregulated by C/EBPß and WT1 during decidualization of the endometrium.

Human endometrial stromal cells (ESCs) differentiate into decidual cells by the action of progesterone, which is essential for implantation and maintenance of pregnancy. We previously reported that glucose uptake by human ESCs increases during decidualization and that glucose is indispensable for decidualization. Although glucose transporter 1 (GLUT1) is upregulated during decidualization, it remains unclear whether it is involved in glucose uptake. Here, we attempted to determine the role of GLUT1 during decidualization as well as the factors underlying its upregulation. ESCs were incubated with cAMP to induce decidualization. Knockdown of GLUT1 suppressed cAMP-increased glucose uptake and the expressions of specific markers of decidualization, IGF-binding protein-1 (IGFBP-1), and prolactin (PRL). To investigate the regulation of GLUT1 expression, we focused on CCAAT enhancer-binding protein ß (C/EBPß) and Wilms' tumor 1 (WT1) as the upstream transcription factors regulating GLUT1 expression. Knockdown of either C/EBPß or WT1 suppressed cAMP-increased GLUT1 expression and glucose uptake. cAMP treatment also increased the recruitment of C/EBPß and WT1 to the GLUT1 promoter region. Interestingly, cAMP increased the H3K27 acetylation (H3K27ac) and p300 recruitment in the GLUT1 promoter region. Knockdown of C/EBPß or WT1 inhibited these events, indicating that both C/EBPß and WT1 contribute to the increase of H3K27ac by recruiting p300 to the GLUT1 promoter region during decidualization. These findings indicate that GLUT1 is involved in glucose uptake in ESCs during decidualization, thus facilitating the establishment of pregnancy.

Relationship between follicular size and developmental capacity of oocytes under controlled ovarian hyperstimulation in assisted reproductive technologies.

PURPOSE: We investigate the relationships between oocyte developmental capacity and follicular size of its origin in Japanese women: those undergoing conventional IVF (cIVF) and ICSI, respectively. METHODS: A total of 3377 follicles were punctured separately and were classified into three groups (large, medium, and small) by their diameters. A total of 1482 retrieved oocytes were individually cultured and received cIVF or ICSI. The oocytes receiving ICSI were denuded and the number of mature (MII) oocytes was counted. RESULTS: The oocyte retrieval rates and the proportion of MII oocytes were significantly lower in small follicles than in large follicles. Under cIVF, the fertilization rate was significantly lower in oocytes from small follicles than large follicles. Under ICSI, the fertilization rate for MII oocytes was not significantly related to follicular size. Follicular size was not significantly related to the development potential to blastocyst and pregnancy rate for either the cIVF oocytes or the ICSI oocytes. CONCLUSIONS: Although the fertilization rate by cIVF is low in oocytes from small follicles due to the lower proportion of mature oocytes, their development potential is comparable to that of oocytes from larger follicles if they could be fertilized. Under ICSI using mature oocytes, their development potential is not related to follicular size.

Long-term melatonin treatment attenuates body weight gain with aging in female mice.

Women usually experience body weight gain with aging, which can put them at risk for many chronic diseases. Previous studies indicated that melatonin treatment attenuates body weight gain and abdominal fat deposition in several male animals. However, it is unclear whether melatonin affects female animals in the same way. This study investigated whether long-term melatonin treatment can attenuate body weight gain with aging and, if it does, what the mechanism is. Ten-week-old female ICR mice were given melatonin-containing water (100 µg/mL) or only water until 43 weeks. Melatonin treatment significantly attenuated body weight gain at 23 weeks (control; 57.2 ± 2.0 g vs melatonin; 44.4 ± 3.1 g), 33 weeks (control; 65.4 ± 2.6 g vs melatonin; 52.2 ± 4.2 g) and 43 weeks (control; 66.1 ± 3.2 g vs melatonin; 54.4 ± 2.5 g) without decreasing the amount of food intake. Micro-CT analyses showed that melatonin significantly decreased the deposition of visceral and s.c. fat. These results suggested that melatonin attenuates body weight gain by inhibiting abdominal fat deposition. Metabolome analysis of the liver revealed that melatonin treatment induced a drastic change in the metabolome with the downregulation of 149 metabolites, including the metabolites of glucose and amino acids. Citrate, which serves as a source of de novo lipogenesis, was one of the downregulated metabolites. These results show that long-term melatonin treatment induces drastic changes in metabolism and attenuates body weight gain and fat deposition with aging in female mice.

Integrated Analysis of Transcriptome and Histone Modifications in Granulosa Cells During Ovulation in Female Mice.

The ovulatory luteinizing hormone (LH) surge induces rapid changes of gene expression and cellular functions in granulosa cells (GCs) undergoing luteinization. However, it remains unclear how the changes in genome-wide gene expression are regulated. H3K4me3 histone modifications are involved in the rapid alteration of gene expression. In this study, we investigated genome-wide changes of transcriptome and H3K4me3 status in mouse GCs undergoing luteinization. GCs were obtained from mice treated with equine chorionic gonadotropin (hCG) before, 4 hours, and 12 hours after human chorionic gonadotropin injection. RNA-sequencing identified a number of upregulated and downregulated genes, which could be classified into 8 patterns according to the time-course changes of gene expression. Many genes were transiently upregulated or downregulated at 4 hours after hCG stimulation. Gene Ontology terms associated with these genes included steroidogenesis, ovulation, cumulus-oocyte complex (COC) expansion, angiogenesis, immune system, reactive oxygen species (ROS) metabolism, inflammatory response, metabolism, and autophagy. The cellular functions of DNA repair and cell growth were newly identified as being activated during ovulation. Chromatin immunoprecipitation-sequencing revealed a genome-wide and rapid change in H3K4me3 during ovulation. Integration of transcriptome and H3K4me3 data identified many H3K4me3-associated genes that are involved in steroidogenesis, ovulation, COC expansion, angiogenesis, inflammatory response, immune system, ROS metabolism, lipid and glucose metabolism, autophagy, and regulation of cell size. The present results suggest that genome-wide changes in H3K4me3 after the LH surge are associated with rapid changes in gene expression in GCs, which enables GCs to acquire a lot of cellular functions within a short time that are required for ovulation and luteinization.

Transcription factor C/EBPß induces genome-wide H3K27ac and upregulates gene expression during decidualization of human endometrial stromal cells.

We previously reported that H3K27 acetylation (H3K27ac) increases throughout the genome during decidualization of human endometrial stromal cells (ESCs). However, its mechanisms have not been clarified. We also reported that C/EBPß acts as a pioneer factor initiating chromatin remodeling by increasing H3K27ac of IGFBP-1 and PRL promoters. Therefore, C/EBPß may be involved in the genome-wide increase of H3K27ac during decidualization. In this study, we investigated whether C/EBPß causes genome-wide H3K27ac modifications and regulates gene expressions during decidualization. cAMP was used to induce decidualization. Three types of cells (control cells, cAMP-treated cells, and cAMP-treated + C/EBPß-knockdowned cells by siRNA) were generated. Of 4190 genes that were upregulated by cAMP, C/EBPß knockdown inhibited these upregulation in 2239 genes (53.4%), indicating that they are under the regulation of C/EBPß. cAMP increased H3K27ac in 1272 of the 2239 genes. C/EBPß knockdown abolished the increase of H3K27ac in almost all genes (1263 genes, 99.3%), suggesting that C/EBPß can upregulate gene expression by increasing H3K27ac. To investigate how C/EBPß regulates H3K27ac throughout the genome, we tested the hypothesis that C/EBPß binds to its binding regions and recruits cofactors with histone acetyltransferase activities. To do this, we collated our ChIP-sequence data with public ChIP-sequence database of transcription factors, and found that p300 is the most likely cofactor that binds to the H3K27ac-increased-regions with C/EBPß. ChIP-qPCR of several genes confirmed that C/EBPß binds to the target regions, recruits p300, and increases H3K27ac. Our genome-wide analysis revealed that C/EBPß induces H3K27ac throughout the genome and upregulates gene expressions during decidualization by recruiting p300 to the promoters.

An Integrated Genomic Approach Identifies HOXC8 as an Upstream Regulator in Ovarian Endometrioma.

PURPOSE: To identify the upstream regulators (URs) involved in the onset and pathogenesis of ovarian endometrioma. METHODS: Recently, a method called Significance-based Modules Integrating the Transcriptome and Epigenome (SMITE) that uses transcriptome data in combination with publicly available data for identifying URs of cellular processes has been developed. Here, we used SMITE with transcriptome data from ovarian endometrioma stromal cells (ovESCs) and eutopic endometrium stromal cells (euESCs) in combination with publicly available gene regulatory network data. To confirm the URs identified by SMITE, we developed a Boolean network simulation to see if correcting aberrant expressions of the identified genes could restore the entire gene expression profile of ovESCs to a profile similar to that of euESCs. We then established euESCs overexpressing the identified gene and characterized them by cell function assays and transcriptome analysis. RESULTS: SMITE identified 12 potential URs in ovarian endometrioma that were confirmed by the Boolean simulation. One of the URs, HOXC8, was confirmed to be overexpressed in ovESCs. HOXC8 overexpression significantly enhanced cell proliferation, migration, adhesion, and fibrotic activities, and altered expression statuses of the genes involved in transforming growth factor (TGF)-ß signaling. HOXC8 overexpression also increased the expression levels of phosphorylated SMAD2/SMAD3. The increased adhesion and fibrosis activities by HOXC8 were significantly inhibited by E-616452, a selective inhibitor of TGF-ß receptor type I kinases. MAIN CONCLUSIONS: Integrated genomic approaches identified HOXC8 as an UR in ovarian endometrioma. The pathological features of ovarian endometrioma including cell proliferation, adhesion, and fibrosis were induced by HOXC8 and its subsequent activation of TGF-ß signaling.

Importance of Melatonin in Assisted Reproductive Technology and Ovarian Aging.

Melatonin is probably produced in all cells but is only secreted by the pineal gland. The pineal secretion of melatonin is determined by the light-dark cycle, and it is only released at night. Melatonin regulates biological rhythms via its receptors located in the suprachiasmatic nuclei of the hypothalamus. Melatonin also has strong antioxidant activities to scavenge free radicals such as reactive oxygen species (ROS). The direct free radical scavenging actions are receptor independent. ROS play an important role in reproductive function including in the ovulatory process. However, excessive ROS can also have an adverse effect on oocytes because of oxidative stress, thereby causing infertility. It is becoming clear that melatonin is located in the ovarian follicular fluid and in the oocytes themselves, which protects these cells from oxidative damage as well as having other beneficial actions in oocyte maturation, fertilization, and embryo development. Trials on humans have investigated the improvement of outcomes of assisted reproductive technology (ART), such as in vitro fertilization and embryo transfer (IVF-ET), by way of administering melatonin to patients suffering from infertility. In addition, clinical research has examined melatonin as an anti-aging molecule via its antioxidative actions, and its relationship with the aging diseases, e.g., Alzheimer's and Parkinson's disease, is also underway. Melatonin may also reduce ovarian aging, which is a major issue in assisted reproductive technology. This review explains the relationship between melatonin and human reproductive function, as well as the clinical applications expected to improve the outcomes of assisted reproductive technology such as IVF, while also discussing possibilities for melatonin in preventing ovarian aging.

Wilms tumor 1 regulates lipid accumulation in human endometrial stromal cells during decidualization.

We previously reported that the transcription factor Wilms tumor 1 (WT1) regulates the expression of insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) during decidualization of human endometrial stromal cells (ESCs). However, other roles of WT1 in decidualization remain to be fully clarified. Here, we investigated how WT1 regulates the physiological functions of human ESCs during decidualization. We incubated ESCs isolated from proliferative-phase endometrium with cAMP to induce decidualization, knocked down WT1 with siRNA, and generated three types of treatments (nontreated cells, cAMP-treated cells, and cAMP-treated + WT1-knockdown cells). To identify WT1-regulated genes, we used gene microarrays and compared the transcriptome data obtained among these three treatments. We observed that WT1 up-regulates 121 genes during decidualization, including several genes involved in lipid transport. The WT1 knockdown inhibited lipid accumulation (LA) in the cAMP-induced ESCs. To examine the mechanisms by which WT1 regulates LA, we focused on very low-density lipoprotein receptor (VLDLR), which is involved in lipoprotein uptake. We found that cAMP up-regulates VLDLR and that the WT1 knockdown inhibits it. Results of ChIP assays revealed that cAMP increases the recruitment of WT1 to the promoter region of the VLDLR gene, indicating that WT1 regulates VLDLR expression. Moreover, VLDLR knockdown inhibited cAMP-induced LA, and VLDLR overexpression reverted the suppression of LA caused by the WT1 knockdown. Taken together, our results indicate that WT1 enhances lipid storage by up-regulating VLDLR expression in human ESCs during decidualization.

SATB2 and NGR1: potential upstream regulatory factors in uterine leiomyomas.

PURPOSE: We attempted to identify the genes involved in the pathogenesis of uterine leiomyomas, under a hypothesis that the aberrant expression of upstream regulatory genes caused by aberrant DNA methylation is involved in the onset and development of uterine leiomyomas. METHODS: To find such genes, we compared genome-wide mRNA expression and DNA methylation in uterine leiomyomas and adjacent normal myometrium. Analysis of the data by Ingenuity Pathway Analysis software identified SATB2 which is known to be an epigenetic regulator, and NRG1 as candidate upstream regulatory genes. To infer the functions of these genes, human uterine smooth muscle cell lines overexpressing SATB2 or NRG1 genes were established (SATB2 or NRG1 lines), and their transcriptomes and pathways were analyzed. RESULTS: SATB2 and NRG1 were confirmed to be hypermethylated and upregulated in most uterine leiomyoma specimens (nine to 11 of the 11 cases). Among the established cell lines, morphological changes from spindle-like forms to fibroblast-like forms with elongated protrusions were observed in only the SATB2 line. Pathway analysis revealed that WNT/ß-catenin and TGF-ß signaling pathways which are related to the pathogenesis of uterine leiomyomas were activated in both SATB2 and NRG1 lines. In addition, signaling of growth factors including VEGF, PDGF, and IGF1, and retinoic acid signaling were activated in the SATB2 and NRG1 lines, respectively. CONCLUSIONS: These results indicate that SATB2 and NRG1 overexpression induced many of the signaling pathways that are considered to be involved in the pathogenesis of uterine leiomyomas, suggesting that these genes have roles as upstream regulatory factors.

Glucose regulates the histone acetylation of gene promoters in decidualizing stromal cells.

Decidualization stimuli activate the insulin signaling pathway and increase the glucose uptake in human endometrial stromal cells (ESCs). The inductions of prolactin (PRL) and IGF-binding protein-1 (IGFBP1), specific markers of decidualization, were inhibited by incubating ESCs under low glucose concentrations. These results suggested that decidualization stimuli activate the insulin signaling pathway, which contributes to decidualization through the increase of glucose uptake. Here, we investigated the mechanisms by which glucose regulates decidualization. ESCs were incubated with cAMP to induce decidualization. We examined whether low glucose affects the expression levels of transcription factors that induce decidualization. Forkhead box O1 (FOXO1) expression was significantly suppressed under low glucose conditions. Knockdown of FOXO1 by siRNA inhibited the expression levels of PRL and IGFBP1 during decidualization. Taken together, our results showed that low glucose inhibits decidualization by decreasing FOXO1 expression. We also examined the levels of histone H3K27 acetylation (H3K27ac), which is related to active transcription, of the promoter regions of FOXO1, PRL and IGFBP1 by ChIP assay. The H3K27ac levels of these promoter regions were increased by decidualization under normal glucose conditions, but not under low glucose conditions. Thus, our results show that glucose is indispensable for decidualization by activating the histone modification status of the promoters of PRL, IGFBP1 and FOXO1.

Aberrant DNA methylation suppresses expression of estrogen receptor 1 (ESR1) in ovarian endometrioma.

BACKGROUND: In ovarian endometriomas (OE), the expression statuses of various steroid hormone receptors are altered compared with their expression statuses in eutopic endometrium (EE). For example, in OE, the expressions of estrogen receptor 1 (ESR1), which encodes ERα, and progesterone receptor (PGR) are downregulated, while the expression of ESR2, which encodes ERß, is upregulated. The causes of these changes are unclear. DNA methylation of a specific region of a gene can result in tissue-specific gene expression. Such regions are called tissue-dependent and differentially methylated regions (T-DMRs). We previously reported that the tissue-specific expression of ESR1 is regulated by DNA methylation of a T-DMR in normal tissues. In the present study, we examined whether aberrant DNA methylation of the T-DMR is associated with the altered expressions of ESR1, ESR2 and PGR in OE. RESULTS: Gene expression levels of ESR1, ESR2 and PGR were measured by quantitative RT-PCR. The expression levels of ESR1 and PGR were significantly lower and the expression level of ESR2 was significantly higher in OE than in EE. DNA methylation statuses were examined with an Infinium HumanMethylation450K BeadChip and sodium bisulfite sequencing. DNA methylation at the T-DMRs of ESR1 were significantly higher in OE than in EE, but no significant differences were observed in the DNA methylation statuses of ESR2 and PGR. CONCLUSIONS: Aberrant DNA methylation of the T-DMR was associated with the impaired expression of ESR1, but not the altered expressions of ESR2 and PGR, in OE.

C/EBPß regulates Vegf gene expression in granulosa cells undergoing luteinization during ovulation in female rats.

The ovulatory LH-surge increases Vegf gene expression in granulosa cells (GCs) undergoing luteinization during ovulation. To understand the factors involved in this increase, we examined the roles of two transcription factors and epigenetic mechanisms in rat GCs. GCs were obtained from rats treated with eCG before, 4 h, 8 h, 12 h and 24 h after hCG injection. Vegf mRNA levels gradually increased after hCG injection and reached a peak at 12 h. To investigate the mechanism by which Vegf is up-regulated after hCG injection, we focused on C/EBPß and HIF1α. Their protein expression levels were increased at 12 h. The binding activity of C/EBPß to the Vegf promoter region increased after hCG injection whereas that of HIF1α did not at this time point. The C/EBPß binding site had transcriptional activities whereas the HIF1α binding sites did not have transcriptional activities under cAMP stimulation. The levels of H3K9me3 and H3K27me3, which are transcriptional repression markers, decreased in the C/EBPß binding region after hCG injection. The chromatin structure of this region becomes looser after hCG injection. These results show that C/EBPß regulates Vegf gene expression with changes in histone modifications and chromatin structure of the promoter region in GCs undergoing luteinization during ovulation.

The distal upstream region of insulin-like growth factor-binding protein-1 enhances its expression in endometrial stromal cells during decidualization.

We have previously shown that decidualization of human endometrial stromal cells (ESCs) causes a genome-wide increase in the levels of acetylation of histone-H3 Lys-27 (H3K27ac). We also reported that the distal gene regions, more than 3 kb up- or downstream of gene transcription start sites have increased H3K27ac levels. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a specific decidualization marker and has increased H3K27ac levels in its distal upstream region (-4701 to -7501 bp). Here, using a luciferase reporter gene construct containing this IGFBP-1 upstream region, we tested the hypothesis that it is an IGFBP-1 enhancer. To induce decidualization, we incubated ESCs with cAMP and found that cAMP increased luciferase expression, indicating that decidualization increased the transcriptional activity from the IGFBP-1 upstream region. Furthermore, CRISPR/Cas9-mediated deletion of this region in HepG2 cells significantly reduced IGFBP-1 expression, confirming its role as an IGFBP-1 enhancer. A ChIP assay revealed that cAMP increased the recruitment of the transcriptional regulators CCAAT enhancer-binding protein ß (C/EBPß), forkhead box O1 (FOXO1), and p300 to the IGFBP-1 enhancer in ESCs. Of note, C/EBPß knockdown inhibited the stimulatory effects of cAMP on the levels of H3K27ac, chromatin opening, and p300 recruitment at the IGFBP-1 enhancer. These results indicate that the region -4701 to -7501 bp upstream of IGFBP-1 functions as an enhancer for IGFBP-1 expression in ESCs undergoing decidualization, that C/EBPß and FOXO1 bind to the enhancer region to up-regulate IGFBP-1 expression, and that C/EBPß induces H3K27ac by recruiting p300 to the IGFBP-1 enhancer.