Developmental programming: prenatal testosterone-induced epigenetic modulation and its effect on gene expression in sheep ovary†

Abstract Maternal perturbations or sub-optimal conditions during fetal development can predispose the offspring to diseases in adult life. Animal and human studies show that prenatal androgen excess may be an underlying cause of polycystic ovary syndrome (PCOS) later in life. In women, PCOS is a common fertility disorder with comorbid metabolic dysfunction. Here, using a sheep model of PCOS phenotype, we elucidate the epigenetic changes induced by prenatal (30–90 day) testosterone (T) treatment and its effect on gene expression in fetal day 90 (D90) and adult year 2 (Y2) ovaries. RNA-seq study shows 65 and 99 differentially regulated genes in prenatal T-treated fetal and adult ovaries, respectively. Interestingly, there were no differences in gene inducing histone marks H3K27ac, H3K9ac, and H3K4me3 or in gene silencing marks, H3K27me3 and H3K9me3 in the fetal D90 ovaries of control and excess T-exposed fetuses. In contrast, except for H3K4me3 and H3K27me3, all the other histone marks were upregulated in the prenatal T-treated adult Y2 ovary. Chromatin immunoprecipitation (ChIP) studies in adult Y2 ovaries established a direct relationship between the epigenetic modifications with the upregulated and downregulated genes obtained from RNA-seq. Results show increased gene inducing marks, H3K27ac and H3K9ac, on the promoter region of upregulated genes while gene silencing mark, H3K9me3, was also significantly increased on the downregulated genes. This study provides a mechanistic insight into prenatal T-induced developmental programming and its effect on ovarian gene expression that may contribute to reproductive dysfunction and development of PCOS in adult life.

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DNA methylation and mRNA expression of imprinted genes in blastocysts derived from an improved in vitro maturation method for oocytes from small antral follicles in polycystic ovary syndrome patients

Human Reproduction ◽

10.1093/humrep/dez121 ◽

2019 ◽

Vol 34 (9) ◽

pp. 1640-1649 ◽

Cited By ~ 8

Author(s):

M D Saenz-de-Juano ◽

E Ivanova ◽

S Romero ◽

F Lolicato ◽

F Sánchez ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Polycystic Ovary Syndrome ◽

Ovarian Stimulation ◽

Research Council ◽

Polycystic Ovary ◽

Rna Seq ◽

Methylation Analysis ◽

Ovary Syndrome ◽

Dna Methylation Analysis

Abstract STUDY QUESTION Does imprinted DNA methylation or imprinted gene expression differ between human blastocysts from conventional ovarian stimulation (COS) and an optimized two-step IVM method (CAPA-IVM) in age-matched polycystic ovary syndrome (PCOS) patients? SUMMARY ANSWER No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts. WHAT IS KNOWN ALREADY Animal models have revealed alterations in DNA methylation maintenance at imprinted germline differentially methylated regions (gDMRs) after use of ARTs. This effect increases as more ART interventions are applied to oocytes or embryos. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for patients. CAPA-IVM is an improved IVM system that includes a pre-maturation step (CAPA), followed by an IVM step, both in the presence of physiological compounds that promote oocyte developmental capacity. STUDY DESIGN, SIZE, DURATION For DNA methylation analysis 20 CAPA-IVM blastocysts were compared to 12 COS blastocysts. For RNA-Seq analysis a separate set of 15 CAPA-IVM blastocysts were compared to 5 COS blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS COS embryos originated from 12 patients with PCOS (according to Rotterdam criteria) who underwent conventional ovarian stimulation. For CAPA-IVM 23 women were treated for 3–5 days with highly purified hMG (HP-hMG) and no hCG trigger was given before oocyte retrieval. Oocytes were first cultured in pre-maturation medium (CAPA for 24 h containing C-type natriuretic peptide), followed by an IVM step (30 h) in medium containing FSH and Amphiregulin. After ICSI, Day 5 or 6 embryos in both groups were vitrified and used for post-bisulphite adaptor tagging (PBAT) DNA methylation analysis or RNA-seq gene expression analysis of individual embryos. Data from specific genes and gDMRs were extracted from the PABT and RNA-seq datasets. MAIN RESULTS AND THE ROLE OF CHANCE CAPA-IVM blastocysts showed similar rates of methylation and gene expression at gDMRs compared to COS embryos. In addition, expression of major epigenetic regulators was similar between the groups. LIMITATIONS, REASONS FOR CAUTION The embryos from the COS group were generated in a range of culture media. The CAPA-IVM embryos were all generated using the same sperm donor. The DNA methylation level of gDMRs in purely in vivo-derived human blastocysts is not known. WIDER IMPLICATIONS OF THE FINDINGS A follow-up of children born after CAPA-IVM is important as it is for other new ARTs, which are generally introduced into clinical practice without prior epigenetic safety studies on human blastocysts. CAPA-IVM opens new perspectives for patient-friendly ART in PCOS STUDY FUNDING/COMPETING INTEREST(S) IVM research at the Vrije Universiteit Brussel has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 project, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69Ref Nr 2016-119) and the Vrije Universiteit Brussel (IOF Project 4R-ART Nr 2042). Work in G.K.’s laboratory is supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. The authors have no conflicts of interest.

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Mesenchymal Stem Cell Therapy Ameliorates Metabolic Dysfunction and Restores Fertility in a PCOS Mouse Model Through Interleukin-10

10.21203/rs.3.rs-444566/v1 ◽

2021 ◽

Author(s):

Rishi Man Chugh ◽

Hang-soo Park ◽

Abdeljabar El Andaloussi ◽

Amro Elsharoud ◽

Sahar Esfandyari ◽

...

Keyword(s):

Gene Expression ◽

Polycystic Ovary ◽

Therapeutic Option ◽

Interleukin 10 ◽

Reproductive Age ◽

Metabolic Dysfunction ◽

Mesenchymal Stem Cell Therapy ◽

Anti Inflammatory

Abstract Background: Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder in reproductive-age women. Excessive inflammation and elevated androgen production from ovarian theca cells are key features of PCOS. Human bone marrow mesenchymal stem cells (BM-hMSC) and their secreted factors (secretome) exhibit robust anti-inflammatory capabilities in various biological systems. We evaluated the therapeutic efficacy of BM-hMSC and its secretome in both in vitro and in vivo PCOS models.Methods: For in vitro experiment, we treated conditioned media from BM-hMSC to androgen producing H293R cells, and analyzed androgen producing gene expression. For in vivo experiment, BM-hMSC were implanted into Letrozole (LTZ) induced mouse PCOS model. BM-hMSC effect in androgen producing cells or PCOS model mice was assessed by monitoring cell proliferation (immunohistochemistry), steroidogenic gene expression (quantitative real-time polymerase chain reaction [qRT-PCR] and Western blot, animal tissue assay (H&E staining), and fertility by pup delivery.Results: BM-hMSC significantly downregulate steroidogenic gene expression, curb inflammation, and restore fertility in treated PCOS animals. The anti-inflammatory cytokine interleukin-10 (IL-10) played a key role in mediating the effects of BM-hMSC in our PCOS models. We demonstrated that BM-hMSC treatment was improve in metabolic and reproductive markers in our PCOS model and able to restore fertility. Conclusion: Our study demonstrates for the first time the efficacy of intra-ovarian injection of BM-hMSC or its secretome to treat PCOS-related phenotypes, including both metabolic and reproductive dysfunction. This approach may represent a novel therapeutic option for women with PCOS. Our results suggest that BM-hMSC can reverse PCOS-induced inflammation through IL-10 secretion. BM-hMSC might be a novel and robust therapeutic approach for PCOS treatment.

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FBXO11 Activates Erythroid Gene Transcription By Degrading Heterochromatin-Associated Protein BAHD1

Blood ◽

10.1182/blood-2018-99-117362 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 529-529

Author(s):

Peng Xu ◽

Daniel C. Scott ◽

Xing Tang ◽

Yu Yao ◽

Yong-Dong Wang ◽

...

Keyword(s):

Gene Expression ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Sequencing Analysis ◽

Gene Promoters ◽

Rna Seq ◽

Histone Marks ◽

Erythroid Maturation ◽

Regulated Gene Expression

Abstract Mature red blood cells (RBC) contain approximately 95% cytosolic hemoglobin for the purpose of blood oxygen transport. This specialized state is achieved during erythropoiesis by regulated gene expression and protein degradation. During late-stage erythropoiesis, ubiquitin ligases eliminate unnecessary proteins and maintain quality control by degrading unstable proteins, including unpaired hemoglobin subunits. However, ubiquitin ligases are expressed at all stages of erythropoiesis and the functions of most are unknown. To study ubiquitin ligases involved in RBC formation, we performed a Cas9/single guide (sg) RNA screen for functional ubiquitin-proteasome components in HUDEP-2 cells, an immortalized human cell line that proliferates as immature erythroblasts and can be induced to undergo terminal maturation. We identified the E3 ubiquitin ligase FBXO11 as a top-ranked candidate. FBXO11 is a member of the F-box protein family that assembles into a SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. Depletion of FBXO11 by 2 different sgRNAs in HUDEP-2 cells inhibited erythroid maturation, as evidenced by reduced hemoglobinization, failure to induce the maturation marker Band3 and persistence of immature cell morphology. In primary human CD34+ cells, suppression of FBXO11 expression by Cas9 + two independent sgRNAs inhibited erythroid maturation, as evidenced by reduced Band3 expression (5.3% vs. 15.6% for non-targeting sgRNA, P < 0.001; n=4). RNA-seq analysis of FBXO11-depleted HUDEP-2 cells revealed 951 decreased transcripts (enriched for erythroid genes) and 339 increased transcripts (enriched for genes expressed in activated T-cells) compared to control cells expressing non-targeting gRNA (P < 0.05). Thus, FBXO11 is required for erythroid maturation and facilitates erythroid gene expression. We sought to establish how FBXO11 modulates erythropoiesis and erythroid gene expression by identifying the relevant ubiquitination substrate(s). Combined quantitative proteome analysis with RNA-seq of FBXO11-depleted HUDEP-2 cells identified several proteins that are upregulated with no change in their corresponding mRNA. We tested whether reduction of these candidate substrates could alleviate the erythroid maturation block conferred by FBXO11 depletion. In FBXO11 gene-disrupted HUDEP-2 cells, suppression of the heterochromatin-associated protein BAHD1 partially rescued hemoglobinization and Band3 expression (4.2% for Cas9 + non-targeting sgRNA vs. 21.7% for Cas9 + BAHD1 sgRNAs, P < 0.01; n=3) . Conversely, stable overexpression of V5-epitope-tagged BAHD1 in WT HUDEP-2 cells reduced Band3 expression from 25.0% to 11.4% (P < 0.001; n=3) and inhibited hemoglobinization. Transcriptome analysis demonstrated a significant inverse correlation between genes deregulated by BAHD1-V5 overexpression and FBXO11 deficiency in HUDEP-2 cells, particularly for erythroid genes that are downregulated (P < 0.0001). BAHD1, named after its bromo-adjacent homology domain that interacts with H3K27me3, is part of a transcriptional repressor complex. We showed that BAHD1 and FBXO11 co-immunoprecipitated in cells and that BAHD1 amino (N)-terminal segments of 188 or 240 amino acids were robustly modified with ubiquitin by SCFFBXO11 complex. Chromatin immunoprecipitation-sequencing analysis of BAHD1-V5-expressing WT HUDEP-2 cells showed strong enrichment for BAHD1 occupancy on erythroid gene promoters that were downregulated by FBXO11-deficiency (P < 0.0001). We next investigated whether a specific set of histone marks distinguish FBXO11-regulated genes in normal erythroblasts. We found that most FBXO11-regulated genes in both HUDEP-2 and primary CD34+ derived erythroblasts harbor histone marks H3K4me3 and H3K27me3, indicating a "bivalent" epigenetic state that supports low level transcription in stem or progenitor cells. Together, these data indicate that FBXO11 activates expression of erythroid genes by ubiquitinating and degrading bivalent promoter-bound BAHD1 repressor complexes with likely resolution to a monovalent transcriptionally active state. Overall, our findings identify FBXO11 as a ubiquitin ligase that utilizes a novel mechanism to activate erythroid genes during RBC formation. This newly identified pathway may contribute to known activities of FBXO11 as a tumor suppressor and developmental regulator in non-erythroid tissues. Disclosures No relevant conflicts of interest to declare.

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The Hepatokine Adropin Is Regulated by Estrogen

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1572 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A773-A773

Author(s):

Joshua Stokar ◽

Irina Gurt ◽

Oran Yakubovsky ◽

Einav Cohen-Kfir E ◽

Noa Hallak ◽

...

Keyword(s):

Gene Expression ◽

Weight Gain ◽

Metabolic Dysfunction ◽

Rna Seq ◽

Metabolic Derangement ◽

Endocrine Society ◽

Post Surgery ◽

Induced Changes ◽

Relative Change

Abstract Introduction: menopause is associated with weight gain, visceral adiposity and NAFLD. Rodent ovariectomy (OVX) is an accepted model for human menopause. Many OVX studies add high-fat diet or old-age to accentuate deranged phenotype. We have shown OVX alone induced weight gain and changes in liver transcriptome including downregulation of Enho, encoding for the hepatokine adropin (1). Here, we explore changes in VAT cytokine and adipokine genes, hepatic miRNA, and liver triglyceride content induced by OVX, in addition to estrogen’s role in regulation of adropin. Methods: 9-week-old C57BL/6J female mice underwent OVX or sham surgery. Groups of 10 mice were sacrificed at 6- and 12-weeks post-surgery and tissues harvested including mesenteric adipose tissue representing VAT. Liver TG was quantified using Cayman colorimetric assay. In-vitro studies performed in the murine hepatic cell-line, BNL1.ME. Adropin was measured using ELISA. Results: OVX induced adverse inflammatory cytokine & adipokine gene expression in VAT at 6-weeks post-surgery (Il18 1.1 p=0.01, Rares2 2.9, p=0.003, Retn 5.5, p=0.002) and 12-weeks post-surgery (Tnfa 2.3 p<0.001, Cxcl5 1.9 p=0.04). In the liver, OVX induced an increase in TG content at 12 weeks post-surgery (realtive increase vs sham 2.0 p=0.05). Hepatic Enho expression showed a strong inverse coorelation with total body weight gain (r= -0.7 p<0.001) and liver TG content (r=-0.4, p=0.04). In-vitro, estrogen induced an increase in Enho (relative mRNA change vs. growing medium 2.6, p=0.004); though protein level was unchanged, a trend for increased adropin was found in supernatant (relative change vs control 2.2 P=0.09). In-silico analysis of data from OVX mice treated with estrogen showed up-regulation of Enho (relative change vs vehicle, 6 p<0.001). At 6-weeks post-surgery OVX induced changes in hepatic miRNA profile with 48 miRNAs differentially expressed vs SHAM (24 up & 24 down). Integrating data from same sample RNA-SEQ and miRNA-SEQ created a network of differently expressed miRNA with oppositely differently expressed known specific mRNA targets. mIR-29, a known regulator of Enho in liver, was not found to be correlated with Enho expression in this context. Conclusions: OVX alone is sufficient to induce adverse changes in VAT gene expression and liver TG. Hepatic adropin gene expression is regulated by estrogen and its downregulation was strongly correlated to phenotypes relevant to menopause induced metabolic dysfunction, weight gain and increased liver fat. Thus, adropin should be further explored as a novel therapeutic and/or biomarker for menopause induced metabolic dysfunction. (1) Stokar, J., Gurt, I., Cohen-Kfir, E., Yakubovsky, O., Hanna, A., Assayag, E., & Dresner-Pollak, R. (2019). RNA-Seq Analysis of Ovariectomy-Induced Changes in Mouse Liver Reveals New Targets for Menopause-Associated Metabolic Derangement. Journal of the Endocrine Society, 3(Supplement_1), SUN-033.

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The Regulatory Role of Histone Modification on Gene Expression in the Early Stage of Myocardial Infarction

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2020.594325 ◽

2020 ◽

Vol 7 ◽

Author(s):

Jinyu Wang ◽

Bowen Lin ◽

Yanping Zhang ◽

Le Ni ◽

Lingjie Hu ◽

...

Keyword(s):

Gene Expression ◽

Myocardial Infarction ◽

Histone Modifications ◽

Gene Expression Regulation ◽

Transcriptional Regulatory Network ◽

Early Stage ◽

Regulation Of Gene Expression ◽

High Morbidity ◽

Rna Seq ◽

Histone Marks

Myocardial infarction (MI) is a fatal heart disease with high morbidity and mortality. Various studies have demonstrated that a series of relatively specific biological events occur within 24 h of MI. However, the roles of histone modifications in this pathological process are still poorly understood. To investigate the regulation of histone modifications on gene expression in early MI, we performed RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) on myocardial tissues 24 h after the onset of MI. The genome-wide profiles of five histone marks (H3K27ac, H3K9ac, H3K4me3, H3K9me3, and H3K27me3) were explored through ChIP-seq. RNA-seq identified 1,032 differentially expressed genes (DEGs) between the MI and sham groups. ChIP-seq analysis found that 195 upregulated DEGs were modified by change of at least one of the three active histone marks (H3K27ac, H3K9ac, and H3K4me3), and the biological processes and pathways analysis showed that these DEGs were significantly enriched in cardiomyocyte differentiation and development, inflammation, angiogenesis, and metabolism. In the transcriptional regulatory network, Ets1, Etv1, and Etv2 were predicted to be involved in gene expression regulation. In addition, by integrating super-enhancers (SEs) with RNA-seq data, 76 DEGs were associated with H3K27ac-enriched SEs in the MI group, and the functions of these SE-associated DEGs were mainly related to angiogenesis. Our results suggest that histone modifications may play important roles in the regulation of gene expression in the early stage of MI, and the early angiogenesis response may be initiated by SEs.

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Upregulated Ribosomal Pathway Impairs Follicle Development in a Polycystic Ovary Syndrome Mouse Model: Findings of Differential Gene Expression Analysis of Oocytes

10.21203/rs.3.rs-1155132/v1 ◽

2021 ◽

Author(s):

Natsuki Nakanishi ◽

Satoko Osuka ◽

Tomohiro Kono ◽

Hisato Kobayashi ◽

Shinya Ikeda ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Mouse Model ◽

Differential Gene Expression ◽

Polycystic Ovary ◽

Methylation Status ◽

Rna Seq ◽

Ovary Syndrome ◽

Pcos Group ◽

Differential Gene

Abstract Background: Polycystic ovary syndrome (PCOS), a common endocrinal disorder, is associated with impaired oocyte development, which leads to infertility. However, the pathogenesis of PCOS has not been completely elucidated. Limited studies have analyzed the pathological characteristics of oocytes in PCOS. This study aimed to analyze the differentially expressed genes (DEGs) and epigenetic changes in the oocytes of the PCOS mouse model to identify the etiological factors.Methods: C57BL/6J female mice were subcutaneously injected with vehicle or 5α-dihydrotestosterone (250 µg/day) on days 16–18 of pregnancy. Female offspring were used as the control or PCOS group. The oocytes were collected from mice aged 7–9 weeks. The DEGs between the control and PCOS groups were analyzed using RNA sequencing (RNA-Seq). Additionally, the DNA methylation status was analyzed using the post-bisulfite adaptor tagging method. The ovarian tissue sections were stained with hematoxylin and eosin to examine the morphological changes. The proteins, Rps21 and Rpl36, were measured using immunostaining.Results: Compared with the control group, the PCOS group exhibited impaired estrous cycle and polycystic ovary-like morphology. RNA-Seq analysis revealed that 90 DEGs were upregulated and 27 DEGs were downregulated in the PCOS mouse model. DNA methylation analysis revealed 30 hypomethylated and 10 hypermethylated regions in the PCOS group. However, the DNA methylation status was not correlated with differential gene expression. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that five DEGs (Rps21, Rpl36, Rpl36a, Rpl37a, and Rpl22l1) were enriched in ribosome-related pathways. The immunohistochemical analysis revealed that the expression levels of Rps21 and Rpl36 were significantly upregulated in the PCOS mouse model.Conclusions: These results suggest that differential gene expression in the oocytes of the PCOS mouse model is related to impaired folliculogenesis. These findings improved our understanding of the pathogenesis of PCOS.

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Establishment and Analysis of a Combined Diagnostic Model of Polycystic Ovary Syndrome with Random Forest and Artificial Neural Network

BioMed Research International ◽

10.1155/2020/2613091 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Ning-Ning Xie ◽

Fang-Fang Wang ◽

Jue Zhou ◽

Chang Liu ◽

Fan Qu

Keyword(s):

Neural Network ◽

Gene Expression ◽

Artificial Neural Network ◽

Random Forest ◽

Polycystic Ovary Syndrome ◽

Polycystic Ovary ◽

Diagnostic Model ◽

Rna Seq ◽

Ovary Syndrome ◽

Key Genes

Polycystic ovary syndrome (PCOS) is one of the most common metabolic and reproductive endocrinopathies. However, few studies have tried to develop a diagnostic model based on gene biomarkers. In this study, we applied a computational method by combining two machine learning algorithms, including random forest (RF) and artificial neural network (ANN), to identify gene biomarkers and construct diagnostic model. We collected gene expression data from Gene Expression Omnibus (GEO) database containing 76 PCOS samples and 57 normal samples; five datasets were utilized, including one dataset for screening differentially expressed genes (DEGs), two training datasets, and two validation datasets. Firstly, based on RF, 12 key genes in 264 DEGs were identified to be vital for classification of PCOS and normal samples. Moreover, the weights of these key genes were calculated using ANN with microarray and RNA-seq training dataset, respectively. Furthermore, the diagnostic models for two types of datasets were developed and named neuralPCOS. Finally, two validation datasets were used to test and compare the performance of neuralPCOS with other two set of marker genes by area under curve (AUC). Our model achieved an AUC of 0.7273 in microarray dataset, and 0.6488 in RNA-seq dataset. To conclude, we uncovered gene biomarkers and developed a novel diagnostic model of PCOS, which would be helpful for diagnosis.

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