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Differential neutrophil gene expression in early bovine pregnancy.

ABSTRACT: In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT)-stimulated gene expression in peripheral blood leukocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation.PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q) PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (MX) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analyzed in each fraction through day 28 of gestation using qPCR.Microarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and with density gradient separation.The expression profiles of ISG15, MX1, MX2, and OAS1 could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of these genes in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination.

SUBMITTER: Kizaki K

PROVIDER: S-EPMC3570308 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Differential neutrophil gene expression in early bovine pregnancy.

Kizaki Keiichiro K Shichijo-Kizaki Ayumi A Furusawa Tadashi T Takahashi Toru T Hosoe Misa M Hashizume Kazuyoshi K

Reproductive biology and endocrinology : RB&E 20130205

<h4>Background</h4>In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT)-stimulated gene expression in peripheral blood leukocytes ...[more]

PMID: 23384108

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Project description:Background: In food production animals, especially cattle, the diagnosis of gestation is an important issue because the timing of gestation has direct effects on the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection, or the accuracy, simplicity, or cost of the method, etc. A new method for detecting gestation, which involves assessing interferon-tau-related gene expression in peripheral blood leucocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. Methods: PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q)-PCR. PBL fractions were collected with flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solution. The expression levels of four interferon-tau-related genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (Mx) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analysed in each fraction until day 28 of gestation using qPCR. Results: Miroarray analysis detected 72 and 28 genes significantly higher in whole PBL on days 14 and 21 of gestation compare to those on day 0, respectively. They involved interferon-tau responded genes. The expression levels of these genes increased with the progression of gestation. In the flow cytometry experiment, on day 14 all of the genes displayed significantly higher expression levels in the granulocyte fraction than the other fractions, and their expression levels gradually decreased until day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and density gradient separation. Conclusions: The expression profile of the four abovementioned genes could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of ISG15, Mx1, Mx2 and OAS1 in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination. Gene expression profiles were analyzed in the peripheral blood leucocytes at days 0 (n=5), 14 (n=3) and 21 (n=5) after artificial insemination in cow.

Project description:A novel, non-terminal surgical procedure to remove a single placentome from the pregnant ewe for gene expression and histological analyses was recently developed in our laboratory. This technique allows for evaluation of nutritional insults on placental development at more than one stage of gestation using a single animal. Early attempts to develop a similar technique in cattle were met with complications due to inaccessibility of the gravid uterine horn because of its location and mass. One alternative is to collect a placentome from the contralateral uterine horn; however, the question remains as to whether gene expression varies among placentomes based on location relative to the fetus. Pregnant heifers were maintained on forage during early gestation and later moved into pens with a Calan gate system (American Calan, Northwood, NH). On gestational day (GD) 158, five heifers were assigned to receive a hay-based diet formulated to meet 100% of NRC requirements, and five heifers were fed 70% of NRC requirements until necropsy on GD244. At necropsy, a single representative placentome was selected for analysis from the antimesometrial side: (1) of the gravid uterine horn central to the amnion, (2) over the allantois immediately adjacent to the amnion, (3) in the tip of the gravid uterine horn, and (4) in the tip of the contralateral uterine horn. Mean placentome weight was greater (P < 0.05) for locations central to the amnion and allantois compared to locations within the tips of the ipsilateral and contralateral horns, respectively. Gene expression for angiogenic factors (FGF2, ODC1, VEGFA, and FLT1), nutrient transporters (SLC7A1 and SLC2A1), and factors associated with hormone action (ESR1, IGF1, IGFBP3, CSH1, and PAG1) were unaffected (P > 0.05) by dietary treatment or location of the placentome. Results indicate that location of the placentome in relation to the fetus does not impact gene expression, enhancing the efficacy of nonterminal methodologies for sampling gene expression in placentomes.

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Differential neutrophil gene expression in early bovine pregnancy.

Publications

Differential neutrophil gene expression in early bovine pregnancy.

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