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Effect of intrauterine infusion of IFNT, PAG, or PAG and IFNT on luteal gene expression [in vivo]

ABSTRACT: The effect of interferon tau (IFNT) on the uterus is critical for maternal recognition of pregnancy in ruminants, while its direct role in luteal function is less well understood. To address this, we performed two experiments. In experiment 1, cattle received intrauterine infusions of one of four treatments: bovine serum albumin (BSA; vehicle) from day 14 to 16 of the estrous cycle, vehicle with IFNT from day 14 to 16 (3-day IFNT), vehicle with IFNT from day 14 to 19 (6-day IFNT), and vehicle with IFNT from day 14 to 16 followed by vehicle with IFNT and pregnancy associated glycoprotein (PAG) from day 17 to 19 (IFNT+PAG). Corpora lutea (CL) were collected on day 17 or 20 and RNAseq was performed. In experiment 2, cultured luteal steroidogenic cells from cyclic (day 10-12) cattle were treated with IFNT and RNAseq was performed. In both experiments, IFNT resulted in luteal changes (in vivo: 130 transcripts; in vitro: 2981 transcripts), while additional treatment with PAG resulted in only 13 changed transcripts. Only 31% of the genes that changed in the CL during early pregnancy (Hughes et al., 2020) were regulated by IFNT. Gene ontology analysis revealed that these genes were regulators of antiviral and immune responses. In contrast, 50% of the genes that changed during early pregnancy were not regulated by IFNT in vivo or in vitro and were associated with cellular proliferation and extracellular matrix organization. It is suggested that the temporal changes in the CL during early pregnancy are only partially regulated directly by IFNT, drawing into question identities of other luteal regulators or the effect of age of CL.

ORGANISM(S): Bos taurus

PROVIDER: GSE279381 | GEO | 2025/03/24

REPOSITORIES: GEO

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Project description:To explore chorionic gonadotropin (CG)-regulated gene expression in the primate corpus luteum (CL), adult female rhesus macaques were treated with a model of simulated early pregnancy (SEP). Total RNA was isolated from individual CL and hybridized to AffymetrixM-bM-^DM-" GeneChip Rhesus Macaque Genome Arrays The level of 1192 transcripts changed expression > 2-fold (one-way ANOVA, FDR correction; P<0.05) during SEP when compared to Day 10 untreated controls, and the majority of changes occurred between Days 10 and 12 of SEP. To compare transcript levels between SEP rescued and regressing CL, previously banked rhesus GeneChip array data from the mid- to late and very late luteal phase were analyzed with time-matched intervals in SEP. Comparing RMA-normalized transcripts from the natural cycle with those from luteal rescue revealed 7677 transcripts changing in expression pattern >2 fold (one-way ANOVA, FDR correction; P<0.05) between the two groups. Clustering of samples revealed that the SEP samples possessed the most related transcript expression profiles. Regressed CL (days 18-19, around menses) were the most unlike all other CL. The most affected KEGG pathway was Steroid Biosynthesis, and most significantly absent pathways following SEP treatment includes groups of genes whose products promote cell-death. By further comparing the genome-wide changes in luteal gene expression during rescue in SEP, with those in CL during luteolysis in the natural menstrual cycle, it is possible to identify key regulatory pathways promoting fertility. Simulated early pregnancy (SEP) treatment was begun on day 9 as Duffy and Stouffer (1997) by treatment of females with recombinant human chorionic gonadotropin (hCG; NovarelM-bM-^DM-", Ferring Pharmaceuticals Inc. Parsippany, NJ, USA) in increasing dosages (15, 30,45,90,180,360,720,1440, and 2880 IU) twice daily by intramuscular injection. CL were collected by laparotomy on days 10, 12, 15, and 18, representing 1, 3, 6 and 9 days of hCG treatment (n=4 CL/day). Additionally, luteal day 10 untreated CL were collected to serve as baseline controls for SEP CL. All CL were dissected away from luteal tissue, sectioned, and snap-frozen in liquid nitrogen and stored at -80M-BM-0C until RNA and protein isolation by TRIzolM-BM-. extraction (Invitrogen, Carlsbad, CA, USA) according to manufacturerM-bM-^@M-^Ys protocols.

Project 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.

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Effect of intrauterine infusion of IFNT, PAG, or PAG and IFNT on luteal gene expression [in vivo]

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