Analysis of circulating microRNA during early gestation in Japanese black cattle.

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

Newly identified interferon tau-responsive Hes family BHLH transcription factor 4 and cytidine/uridine monophosphate kinase 2 genes in peripheral blood granulocytes during early pregnancy in cows.

In cattle, interferon-stimulated genes (ISGs) such as ISG15, MX1, MX2, and OAS1 are known as classic ISGs that are highly involved in the implantation process. Various molecules play a crucial role in the mechanisms underlying ISG effects. Although microarray analyses have highlighted the expression of various molecules during the implantation period, these molecules remain incompletely characterized. In the present study, various specifically expressed genes were selected and their characteristics were examined. The microarray data from peripheral blood leukocytes derived from artificially inseminated cows and granulocytes obtained from embryo-transferred cows, respectively, were used to identify new ISG candidates. Seven common genes, including ISG15 and OAS1, were confirmed, but only 4 of the 5 genes were amplified by reverse transcription quantitative polymerase chain reaction. In addition, 3 expressed sequence tags (ESTs) exhibited significantly greater expression in granulocytes from pregnant cows than that observed in bred nonpregnant cows, and the expression in granulocytes increased after interferon-tau stimulation. Sequence alignment revealed similar sequences within 2 ESTs on the Hairy and enhancer of split (Hes) family basic helix-loop-helix transcription factor 4 (HES4) gene. An additional EST was identified as cytidine/uridine monophosphate kinase 2 (CMPK2). In silico analysis facilitated the identification of transcription factor-binding sequences, including an interferon-stimulated response element and interferon regulatory factor-binding sites, within the promoter region of HES4 and CMPK2. These genes may function as new ISGs in the context of implantation and may participate in the coordination of the feto-maternal interface in cows.

Evaluation of interferon-stimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals.

Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status.