Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Seminal plasma and transforming growth factor-β regulate gene expression in human Ect1 ectocervical epithelial cells

ABSTRACT: In this study we examined the influence of seminal plasma on gene expression in human Ect1 ectocervical epithelial cells, and the extent to which recombinant TGFβ3 elicits comparable changes. Ect1 cells were incubated with recombinant human TGFβ3 (5 ng/ml), 10% pooled human seminal plasma (v/v), or medium alone for 10h. RNA was reverse transcribed into cDNA and hybridized to Affymetrix GeneChip® Human Genome U133 plus 2.0 microarrays (Affymetrix, Santa Clara, CA). Exposure of Ect1 cells to seminal plasma resulted in differential expression of a total of 3955 probe sets, identified using high stringency criteria with MAS 5.0 analysis. These corresponded to 1338 genes up-regulated and 1343 genes down-regulated by seminal plasma. TGFβ3 treatment of Ect1 cells resulted in differential expression of 884 probe sets, corresponding to 346 up-regulated genes and 229 down-regulated genes. The genes differentially regulated by seminal plasma included several genes associated with cytokine–cytokine receptor interaction, TGFβ signalling, JAK/STAT signalling or VEGF signalling pathways, as specified by the KEGG database. Of 47 genes in these families, 17 (36.1%) were similarly regulated by both seminal plasma and TGFβ3. These data, together with additional experiments showing all three TGFβ isoforms can regulate inflammatory cytokine expression in Ect1 cells, identify TGFβ isoforms as key agents in seminal plasma that signal induction of pro-inflammatory cytokine synthesis in cervical cells. RNA from each of four biological replicates, each comprising pooled material from separate sets of 4 replicate wells, was analysed for each treatment. Total RNA was reverse transcribed into cDNA and sent to the Australian Genome Research Facility (AGRF; Melbourne, Australia) for single-cycle labeling and hybridization to 12 Affymetrix GeneChip® Human Genome U133 plus 2.0 microarrays (Affymetrix, Santa Clara, CA).

ORGANISM(S): Homo sapiens

SUBMITTER: Sarah Robertson

PROVIDER: E-GEOD-35830 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

TGF-β mediates proinflammatory seminal fluid signaling in human cervical epithelial cells.

Sharkey David J DJ Macpherson Anne M AM Tremellen Kelton P KP Mottershead David G DG Gilchrist Robert B RB Robertson Sarah A SA

Journal of immunology (Baltimore, Md. : 1950) 20120615 2

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymet ...[more]

PMID: 22706080

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Project description: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 partner’s 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. Women aged 18-40 years, with regular menstrual cycles and a current sexual relationship with a proven fertile regular partner, volunteered for the study. All women had previously undergone tubal ligation and none of the women used steroidal contraceptives or an intrauterine device for a minimum of three months preceding the study. Subjects were screened to exclude bacterial or viral infection. A negative test result was a prerequisite for inclusion in the study. The study was approved by the local ethics committee of Karolinska University Hospital, Stockholm, Sweden.

Dataset Information

Seminal plasma and transforming growth factor-β regulate gene expression in human Ect1 ectocervical epithelial cells

Publications

TGF-β mediates proinflammatory seminal fluid signaling in human cervical epithelial cells.

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