In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. Here we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partners seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the peri-ovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. One pair of first biopsy and second biopsy RNA samples from each treatment group were reverse transcribed into cDNA and hybridized to Affymetrix Human Gene 1.0 ST arrays. A total of 713 probe sets were identified as differentially expressed (fold change >2) between first and second biopsies after unprotected coitus, with 436 genes upregulated and 277 genes downregulated. Ingenuity Pathway Analysis revealed that gene pathways including inflammatory response, immune response, immune cell trafficking, cellular movement and antigen presentation were significantly affected by seminal fluid exposure. Amongst these were genes encoding several chemokines which target granulocytes, monocyte/macrophages, dendritic cells and lymphocytes, proinflammatory cytokines and regulators of cytokine synthesis, prostaglandin pathway gene including PTGS2; COX-2) and several matrix metalloproteinases (MMPs). Of these genes, no change or a substantially smaller change was seen between first and second biopsies obtained after coitus with condom use, or abstinence. An increase in CSF2, IL6, IL8 and IL1A expression was confirmed by qRT-PCR in larger sets of duplicate biopsies (n=6-7 per group). We conclude that seminal fluid introduced at intercourse elicits expression of pro-inflammatory cytokines and chemokines which underpins the accompanying recruitment of macrophages, dendritic cells and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens, and potentially susceptibility to cervical metaplasia.
Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus.
Treatment
View SamplesCell-type specific RNA-seq is a powerful approach for unravelling molecular processes of endometrial receptivity, and to detect novel sensitive biomarkers of receptivity. Overall design: 16 paired endometrial tissue samples from pre-receptive (defined as LH2) and receptive phase endometria (defined as LH8) from Estonia (defined as E) and Spain (defined as S) were collected. CD9-positive epithelial cells (defined as epithelium) and CD13-positive stromal cells (defined as stroma) were isolated with fluorescent activated cell sorting (FACS) and full transcriptome analysis was performed by RNA-seq.
Meta-signature of human endometrial receptivity: a meta-analysis and validation study of transcriptomic biomarkers.
Specimen part, Subject
View SamplesCRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for delivery in adult animal models. Here we created an S. aureus Cas9-based transcriptional repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for targeting an endogenous gene in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) resulted in significant reductions of serum PCSK9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, PCSK9 repression was maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 genetic perturbation toolbox for basic research and gene therapy applications. Overall design: C57Bl/6 wild-type mice were treated with AAVs expressing dSaCas9-KRAB and/or a Pcsk9-targeting gRNA by tail-vein injection. Six weeks after treatment, we harvested the livers of treated mice and performed mRNA-sequencing.
RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.
Specimen part, Cell line, Subject
View SamplesSexual differentiation in zebrafish is complex. Although zebrafish sex determination is primarily genetic, hormonal and environmental factors can influence sexual development. 17 alpha-methyltestosterone (MT), a synthetic androgen, induces female-to-male sex reversal in zebrafish. MT treatment is routinely used in aquaculture for production of all-male populations. However, the molecular mechanisms underlying 17 alpha-methyltestosterone induced gonad masculinisation in fish are poorly understood.In this study, we analysed gonad transcriptomes of zebrafish treated with 17 alpha-methyltestosterone during gonadal development (from 20 dpf to 40 dpf and 60 dpf) and compared them with testis and ovary transcriptomes of untreated zebrafish. These data improve our understanding of the role of androgens in teleost sex differentiation.
Histological and transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development.
No sample metadata fields
View SamplesBy contrast with mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of spared cardiomyocytes. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. While it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. In this study, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin-1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration. Overall design: Deep sequencing of isolated single epicardial cells
Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration.
Age, Specimen part, Cell line, Subject
View SamplesAcquisition of a new strain of non-typeable Haemophilus influenzae (NTHi) is often associated with exacerbation of chronic obstructive pulmonary disease (COPD). We have previously reported that COPD patients who are homozygous null for SIGLEC14 gene is less susceptible to COPD exacerbation than those who have wild-type allele with functional SIGLEC14 gene.
Association of serum interleukin-27 with the exacerbation of chronic obstructive pulmonary disease.
Cell line
View SamplesTo investigate the time-dependent and coordinated sequence of inflammation-related events, and the dynamic features of macrophage polarisation/activation, we build and validated an in vitro model based on primary human monocytes
Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro.
Specimen part
View SamplesGenome-wide studies have identified abundant small, non-coding RNAs including snRNAs, snoRNAs, cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs) that are transcribed by RNA polymerase II (pol II) and terminated by a Nrd1-dependent pathway. Here, we show that the prolyl isomerase, Ess1, is required for Nrd1-dependent termination of ncRNAs. Ess1 binds the carboxy terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of ~10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, SUTs and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase, and we provide evidence for a competition between Nrd1 and Pcf11 for CTD-binding that is regulated by Ess1-dependent isomerization. This is the first example of a prolyl isomerase required for interpreting the CTD code.
The Ess1 prolyl isomerase is required for transcription termination of small noncoding RNAs via the Nrd1 pathway.
No sample metadata fields
View SamplesWe compared transcriptional profiles of regenerating zebrafish caudal fins following fin amputation with profiles from uninjured zebrafish caudal fins Overall design: Examination of whole fin transcriptional profiles from regenerating fins (2 pools of 10 fins) and uninjured fins (2 pools of 10 fins)
Modulation of tissue repair by regeneration enhancer elements.
No sample metadata fields
View SamplesWe compared transcriptional and chromatin profiles of regenerating zebrafish hearts following genetic ablation with profiles from uninjured zebrafish hearts. Overall design: Examination of whole heart transcriptional profiles from ablated hearts (2 pools of 10 hearts) and uninjured hearts (2 pools of 10 hearts). Examination of differential H3K27Ac marks following genetic ablation of cardiomyocytes (regenerating hearts) and uninjured hearts.
Modulation of tissue repair by regeneration enhancer elements.
No sample metadata fields
View Samples