Comment[ArrayExpressAccession] E-GEOD-56392 MAGE-TAB Version 1.1 Public Release Date 2014-04-01 Investigation Title Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau Comment[Submitted Name] Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau Experiment Description The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endo- metria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status. mRNA profiles of pregnant vs not pregnant cross-bred beef heifers at days 13 and 16 (n=5 per group) Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name McGettigan Forde Duffy McGettigan Browne Mehta Kelly Mansouri-Attia Sandra Loftus Crowe Fair Roche Lonergan Evans Person First Name Paul Niamh Gillian Paul John Jai A N O Brendan Mark Trudee James Patrick Alexander Person Mid Initials B A A P K J A F C Person Email paul.mcgettigan@ucd.ie Person Affiliation University College Dublin Person Address School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Veterinary Sciences Centre, UCD, Belfield, Co Dublin, Ireland Person Roles submitter Protocol Name P-GSE56392-4 P-GSE56392-1 P-GSE56392-3 P-GSE56392-2 Protocol Description Image analysis and base calling was carried out using Illumina IPAR1.01 software Alignment to bt4 genome was carried out with Illumina ELAND software Read counts for each transcript was generated by Illumina CASAVA software Genome_build: bt4 Supplementary_files_format_and_content: raw counts for each transcript for each sample Thirty-eight heifers were inseminated, and 16 were left as noninsemi- nated cyclic controls. Heifers were then randomly assigned to be slaughtered on day 13 or 16 of early pregnancy/estrous cycle corre- sponding to the initiation of conceptus elongation (day 13) and maternal recognition of pregnancy (day 16) in pregnant heifers, respectively. All reproductive tracts were processed <30 min post- slaughter, whereby the uterine horn ipsilateral to the CL was flushed with phosphate-buffered saline containing 5% fetal calf serum. In inseminated heifers, the presence of the conceptus was determined by examination under a stereomicroscope. Tissue was collected only from heifers from which a conceptus at the appropriate stage of development was recovered. After being flushed, the uterine horn ipsilateral to the CL was opened longitudinally, and 300 mg strips of endometrium (predominantly inter- caruncular tissue) were immersed in 1:5 wt/vol of RNAlater. This tissue was transported back to the laboratory on ice, stored at 4°C for 24 h, removed from RNAlater, and stored at <80°C prior to RNA extrac- tion. RNA libraries were prepared for sequencing using standard Illumina protocols for single read libraries. In brief, the estrus cycles of cross-bred beef heifers (predominantly Charolais and Limousin cross) were synchronized using a controlled internal drug release (CIDR) device (1.94 g pro- gesterone; InterAg, Hamilton, New Zealand) placed into the vagina for 8 days with intramuscular administration of a prostaglandin F2α analog (equivalent to 0.5 mg of PG, Estrumate; Shering-Plough Animal Health, Hertfordshire, UK) 3 days prior to CIDR removal. Only heifers detected in standing estrous were used (n = 54). Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name PREGNANCY_STATUS DAY Experimental Factor Type pregnancy_status day Publication Title Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau. Publication Author List Forde N, Duffy GB, McGettigan PA, Browne JA, Mehta JP, Kelly AK, Mansouri-Attia N, Sandra O, Loftus BJ, Crowe MA, Fair T, Roche JF, Lonergan P, Evans AC PubMed ID 22759920 Publication DOI 10.1152/physiolgenomics.00067.2012 Comment[SecondaryAccession] GSE56392 Comment[GEOReleaseDate] 2014-04-01 Comment[ArrayExpressSubmissionDate] 2014-03-31 Comment[GEOLastUpdateDate] 2014-04-01 Comment[AEExperimentType] RNA-seq of coding RNA Comment[SecondaryAccession] SRP040747 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1210436-SRR1210456 SDRF File E-GEOD-56392.sdrf.txt