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ISSN : 2508-755X(Print)
ISSN : 2288-0178(Online)
Journal of Embryo Transfer Vol.33 No.4 pp.195-203
DOI : https://doi.org/10.12750/JET.2018.33.4.195

Analysis of Bovine Interferon-tau Gene subtypes Expression in the Trophoblast and Non-trophoblast cells

Min-Su Kim1, Hyun-Joo Lim1, Ji Hwan Lee1, Soo Bong Park1, Jeong-Il Won2, Hyun Jong Kim1
1Dairy Science Division, National Institute of Animal Science, RDA
2Hanwoo Research Institue, National Institute of Animal Science, RDA
Correspondence: Hyun Jong Kim Tel: +82-41-580-3386, Fax: +82-41-580-3491 E-mail address: hyunjongnt@korea.kr
28/11/2018 17/12/2018 19/12/2018

Abstract


Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.



초록


    Rural Development Administration
    PJ01268901

    INTRODUCTION

    Interferon-tau (IFNT) is known as a major conceptus protein that initiates the process of maternal recognition of pregnancy in ruminant ungulates (Godkin et al., 1982;Imakawa et al., 1987;Roberts et al., 1992). The expression of IFNT is subject to temporal and spatial limits since its production is restricted to TE during peri-implantation period (Farin et al., 1989;Guillomot et al., 1990;Demmers et al., 2001). IFNT, a major cytokine involved in the process of maternal recognition of pregnancy, is secreted into the uterine lumen by the mononuclear trophectoderm of the conceptus (Roberts et al., 1992;Imakawa et al., 2004). This factor is secreted from the embryo to mother around day 12 and the timeing of the signal production is very critical. Martal and coworkers have identified that days 14-16, not day 25 conceptuses. During the peri-implantation period of pregnancy in sheep, endometrial activity and their epithelial secretions are largely regulated by progesterone from the corpus luteum(CL) and cytokines/hormones from the conceptus such as interferon tau (IFNT) (Spencer et al., 2004, 2007).

    IFNs are divided into two groups, sud-resistant type I and acid-sensitive type II IFNs (Peskta et al., 1987). Type I IFNs is induced by virus infection and includes two families, IFNA and IFNB, which are produced by leukocytes and fibroblasts, respectively. IFNG is the only member in type II IFN and produced by T cells or NK cells under mitogen treatment. This conceptus IFN consists of four cysteine residues, which are conserved across Type I IFN, and therefore this IFN belongs to a family of type I IFN. However, the observations that this IFN is not secreted by blood cells and more importantly, it can be serologically distinguished from other Type I IFNs, lead to a new classification, ovine IFNT (Roberts et al., 1992;Yamaguchi et al 2001). IFNA consists of two subtypes as IFNAI and IFNAII (=IFNW). oIFNT cDNA shares 55-65% similarity to IFNAI and is the closest to IFNW with approximately 85% identity (Imakawa et al., 1987). IFNT gene was originated from a single duplication event from an IFNW gene about 36 million years ago in the mammalian lineage, leading to the present day pecoran ruminants, a suborder comprised of cattle, deer, giraffes and their relatives (Robertset al., 1997;Roberts et al., 1998). It has been continued to duplicate since then, and it has been estimated that there may be as many as ten IFNT in bovine, with all of them clustered within or in close proximity to the genetic locus that contains the other Type I IFN genes (Ryan et al., 1993). Presently, multiple ovine and bovine IFNT are transcribed during early pregnancy and encode proteins that can possess different biological activities (Ealy et al., 1998;Winkelman et al., 1999). But it has remained unclear how many are expressed, the extent of their variation, and whether different genes exhibit similar patterns of expression and code for proteins with similar biological activities. In ruminant ungulates, large amount of an anti-luteolytic substance secreted bythe bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein-1 (bIFNT1), a new type I (bIFNTc1, bIFNTc2, and bIFNTc3) genes (Kim et al., 2016). However, molecular mechanisms of these bIFNT genes have not been characterized because of their expressionlevel. The objectives of the present study were, therefore, to determine the differences in IFNT gene expression, to identify differences in IFNT genes expression level, and trophoblast of cattle during the peri-implantation period and non-trophoblast cells.

    MATERIALS AND METHODS

    Animals and experimentation and sampling

    Estrous synchronization, super-ovulation and embryo transfer processes were performed as previously described (Ideta et al., 2007). Seven-day embryos (Day 0=day of estrus) were collected from superovulated cattle. Twelve embryos derived from the superovulation were transferred nonsurgically into the uterine horn of three Holstein heifers (n=4 each), ipslateral to the CL on the day 7 of the estrous cycle. Elongated conceptuses were collected nonsurgically by uterine flushing on the days 17, 20, or 22.

    DNA isolation and plasmids construction

    Genomic DNA was isolated from pooled bovine trophoblast tissues using the Genomic DNA Purification Kit (Promega, Madison, WI), according to the protocol provided by the manufacturer. The quality and integrity of genomic DNA was determined by agarose gel (1%) electrophoresis and visualization under UV light after ethidium bromide staining. Bovine genomic DNA isolated from conceptus tissues was used as the template for amplifying the IFNT genes. In short, the upstream regions (positions -1000 to JET-33-195_image2.gif51) of bIFNT1, bIFNTc1, bIFNTc2, and bIFNTc3 (GenBank accession numbers bIFNT1; M60903: bIFNTc1; AF238613: bIFNTc2; AF238612:bIFNTc3; AF238611, respectively) were PCR amplified through PCR with specific primers in Table 1, and then inserted into the KpnI/NheI sites of pGL3 basic vector (Promega, Madison, WI). CDX2, ETS2, JUN and CREBBP expression plasmids were described previously (Imakawa et al., 2006). All expression vectors, pSG5 (Agilent)-based constructs, were driven by the SV40 promoter/enhancer. These plasmid constructs were each confirmed to have expected nucleotide sequences by dideoxy sequencing.

    RNA extraction and RT-PCR

    Total RNA (80-100 mg) was extracted from an individual trophoblast tissues on days 17, 20, and 22 with Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF) and endometrium (Endo) non-trophoblast cells. Using the Isogen Reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. The RNA extracted from each sample then underwent reverse transcription (RT) into cDNA using oligo (dT) 12-18 primers and SuperScript II (Gibco BRL Life Technologies, Rockville, MD) according to the protocol suggested by the manufacturer, and these RT products were used as templates for PCR analysis. The PCR mixture consisted of 1JET-33-195_image1.gifl of RT product, 1JET-33-195_image1.gifl of 10X PCR buffer, 0.4JET-33-195_image1.gifl each of forward and reverse primers (10 pM), 0.2JET-33-195_image1.gifl of dNTP mixture (10 mM), 0.3JET-33-195_image1.gifl of MgCl2 (50 mM), 6.6JET-33-195_image1.gifl of ddH2O, and 0.1JET-33-195_image1.gifl of Taq DNA polymerase (5U/JET-33-195_image1.gifl; Invitrogen, Carlsbad, CA). The PCR was performed under the following conditions: 94°C for 5 min, followed by 30 cycles of 94°C for 1min, 57°C for 1min, and 72°C for 1min. The PCR product was analyzed by electrophoresis on 1% agarose gel stained with ethidium bromide. The cDNA fragment was extracted from agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan) and then cloned into pGEM T-easy vector (Promega, Madison, WI), and nucleotide sequences were determined by DNA sequencing (ABI-PRISM, Foster City, CA). β-actin mRNA was used as an internal control.

    Sequencing and sequence analysis

    The positive clones as well as the direct PCR product were sequenced using an ABI PRISM automatic sequencer (ABI-PRISM, Foster City, CA) using standard cycle conditions by Sanger’s dideoxy chain termination method with standard sequencing primers (viz., M13F and M13R) and the primers employed for amplification, respectively. The sequences were subjected to BLAST analysis (www.ncbi.nlm.nih.gov/BLAST). The nucleotides as well as deduced amino acid sequences of bIFNT were aligned with that of ruminant ungulate species available in the GenBank database using the Clustal method of MegAlign Program of Lasergene Software (DNASTAR). A phylogram was also constructed to analyze the evolutionary significance.

    Cell culture

    Ear-derived fibroblast (EF) cells were obtained from Animal Genetic Resources Research Center, Korean. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 5% FBS (JRH Biosciences) and antibiotics (Invitrogen) at 37°C in air with 5% CO2.

    Transient transfection

    EF cells were cultured in the medium described in the cell culture section, and plated on a 24-well plate for subsequent transfection (Imakawa et al., 2006). At 60 80% confluence, transient transfection was performed using HilyMax reagents (Dojin Chemicals, Kumamoto, Japan) according to the manufacturer’s protocol. In brief, 2 mg total plasmid DNAs including bIFNTreporter (1.5 mg), expression plasmids (total of 0.5 mg) and 4ml HilyMax were prepared in 30ml DMEM with no supplements (plasmid mixture). Amounts of total plasmids for each transfection were adjusted with the inclusion of pSG5 only (empty vector). After 15min, plated cells were overlaid with the plasmid mixture and incubated at 37°C for 48hr under 5% CO2 in air. Forty-eight hours after transfection, the cells were lysed by the addition of 100ml Passive Lysis Buffer (Promega). Luciferase assay was performed using Dual-Luciferase Reporter Assay System as described previously (Imakawa et al., 2006).

    Statistical analysis

    The results of luciferase assays were expressed as means ±SEM. Differences in fold activation (luciferase activity) were examined by ANOVA, followed by multiple comparison tests of Fisher’s LSD.

    RESULTS

    Sequences analysis of IFNT subtype genes

    To determine whether IFNT from different phylogenetic groupings had conserved or very different promoter sequences, particularly within the main enhancer regions, an extensive series of IFNT clones were analyzed after PCR amplification from genomic DNA. The primers were designed to provide approximately- 1000bp of DNA upstream of the transcription start site, the +51bp untranslated region and the entire open reading frame (Table 1). This upstream region is well conserved across ruminant ungulates (Leaman et al., 1992) and is both necessary and sufficient to provide full expression from IFNT promoters in Jar choriocarcinoma cells (Cross et al., 1991;Leaman et al., 1994). Four of the ten were bi-directionally sequenced. The upstream regions of these four genes are compared of the IFNT gene (GenBank Acc. No. bIFNT1; M60903: bIFNTc1; AF238613: bIFNTc2;AF238612: bIFNTc3; AF238611) in Figure 1. All sequences were very similar (GenBank Database). The ETS2 promoter region at (between -70 to -63) (Ezashi et al., 1998, 2001) was completely conserved in all four genes analyzed. A modified TATA box (TATTTAA) starting at (between -33 to-26) bases promotor from the transcription start site (the position +1 adenine), 69 bases up from the ATG start codon; the consensus start site for transcription of IFNT was present in all clones.

    Luciferase activity of upstream regions by bIFNT subtype

    The wild-type 5’-upstream sequences of bIFNT1, c1, c2, and c3 genes were examined for the degrees of transactivation using a transient transfection method into EF cell and luciferase activities were measured (Figure 2). In these data sets, similarly to its expression in vivo, the reporter plasmid of the upstream region of the bIFNT1, c2, and c3 genes exhibited high luciferase assay whereas the c1 gene had very low activity, less than of that expressed by bIFNT1, c2, and c3 (Figure 2).

    Transactivation of bIFNT subtype genes in EF cells

    To examine the responsiveness of bIFNT1, c1, c2, and c3-Luc constructs to transcription factor JUN, ETS2, CREBBP and/or CDX2 the bIFNT1, c1, c2, and c3-Luc reporter plasmid with each or combination of transcription factor, was cotransfected into EF cells, and luciferase activities were measured (Figure 3). When EF cells were cotransfected with the bIFNT1, c1, c2, and c3-Luc constructs and JUN, ETS2, CREBBP and/or CDX2 expression plasmid, Luc activity was enhanced approximately 10 and 18 fold more than twice of those cotransfected with bIFNT1, c1, c2, and c3-Luc construct and PSG5 empty plasmid (Con) (Figure 3). Furthermore, The degree of transcriptional activation by a combination of the JUN, ETS2, CREBBP and/or CDX2 expression vectors was similar to that of CDX2 along plasmid. However, expression patterns of these Luc activity differented. The degree of transcriptional activation of the bIFNT1 gene was similar to that c2 and c3 genes by a combination of JUN, ETS2, and/or CDX2 expression plasmid. Whereas bIFNTc1- Luc showed lowest antivity had than bIFNT1, c2, and c3-Luc reports. Although, lowest antivity had of the bIFNTcl Luc report, cotransfected with the bIFNTc1-Luc construct and JUN or/and ETS2 expression plasmid, Luc activity was enhanced approximately 2 and 4-fold more than the bIFNT1, c2, and c3-Luc. Furthermore, along CDX2 expression factor had high effect on activity of bIFNT1, c2, and c3-Luc reporter than the c1 gene in EF cells.

    Expression levels of bIFNT subtype mRNA in trophoblast and non-trophoblast cells.

    The regions of these four form genes are compared of the IFNT gene in Figure 1. For assay levels expression of bIFNT1, c1, c2, and c3 mRNA in bovine day 17, 20, and 22 bovine trophoblasts and non-trophoblast cells, oligonucleotides identical sequence primer used for bIFNT1, c1, c2, and c3 mRNAs levels analysis (Table 2). Relative amounts of bIFNT1, c1, c2, and c3 mRNAs on day 17, 20, and 22 bovine trophoblasts and nontrophoblast cells. RNA extracted from frozen bovine trophoblasts (n=3 each), was analyzed for the presence of these transcripts using RT-PCR. Agarose gel electrophoresis of the amplicon expected to reveal an amplification of a fragment of approximately 364 bp. The size of the amplicon was further confirmed by the nucleotide sequencing. A total of thirty prospective recombinant colonies that contained the 364 bp insert were sequenced. Sequence analyzed of bIFNT1 and related type c1, c2, and c3 genes by Escherichia coli cells (n=3 each). In order to characterize the difference on the levels of expression of bIFNT1, c1, c2, and c3, the IFNT mRNA in the nucleotides region of bIFNT1, c1, c2, and c3 the IFNT were analyzed.

    The levels of expression of bIFNT1, c2, and c3 were shown higher on day 17, 20, and 22 bovine trophoblasts. However, the other bIFNTc1 gene transcripts were expressions of transcripts became weaker on day 17, 20, and 22 bovine trophoblasts (Figure 4A). In addition, Sequence analyzed of bIFNT1, c1, c2, and c3 in MDBK, EF, and Endo of non-trophoblast cells. The bIFNT1, c1, c2, and c3 mRNA were present in the tree type non-trophoblast cells. The levels of expression of bIFNT1 and c2 were shown higher on the tree type non-trophoblast cells, weakly detectable on bIFNTc1 and c3 (Figure 4B).

    DISCUSSION

    The present study, four forms of bovine IFNT transcripts in trophoblast and non- trophoblast cells were found through the use of sequence. It was previously predicted that several IFNTcDNAs of cattle could exist. There have been 12 IFNT cDNAs already identified (Ealy et al., 2001). Moreover, evaluation by (Walker et al., 2009) found six more IFNT cDNA variants from bovine conceptuses and trophophectoderm cells, indicating that at least 18 different IFNT cDNAs exist. At present, there are 4 novel sequences that we have been working to identify different bIFNT sequences andexpression levels. To date, none of the four full-length bIFNT genes isolated and three genes have the similar nucleotide sequence. Like its progenitor IFNW, the 585bp ORF of the IFNTgene encodes for a 195 amino acid pre-protein of which the first 23 residues is the signal peptide that is cleaved off to yield a mature protein of 172 amino acids (Roberts et al., 1997). Therefore, even though the expressed bIFNT represent a rapidly evolving group of genes, it is likely that the majority of them are represented in all bos taurus breeds. Their uniformity contrasts with the diversity of the ovine IFNT, which differ as much as 10% in nucleotide sequence (Ealy et al., 1998). It is well known that IFN type I genes are found in various mammals, but IFNTgenes are found only in ruminants (Robert et al., 1992). These IFN type I genes are known to be located tandemly in the chromosome 8, suggesting that duplication events lead the emergence of several IFNTgenes in the bovine genome (Walker et al., 2009, Sakurai et al., 2013).

    In this study, the relative abundance of transcription factor transcripts, which have been demonstrated to regulate four forms IFNT gene transcription, were evaluated. Luciferase activity assay resulted from the upstream region of wild type bIFNT1, c1, c2, and c3 genes transfected to EF cell reflected the expression levels of these genes in the utero. These results indicated that the expressions of bIFNT1, c1, c2, and c3 genes are regulated at transcriptional level and transient transfection analyses using EF cell and also demonstrated that high and low expression levels from bIFNT1, c1, c2, and c3 genes, respectively. CDX2functions as a transcription factor that is required for IFNT transcription (Imakawa et al., 2006;Sakurai et al., 2009). Although the molecular mechanisms by which CDX2 gene transcription subsides are unknown, similar regulatory mechanism might exist between bIFNT1, c1, c2, and c3 genes with CDX2 factor in the bovine. Importantly, CDX2 exhibited effects on bIFNT1, c1, c2, and c3 genes transcription only when CDX2 was transfected intoEF cells. As CDX2 expression increases, CDX2 binds, allowing ETS2 binding to the JUN, CREBBP complex formation and the activation of bIFNT1, c1, c2, and c3 genes transcription.

    In addition, the sequence analysis also revealed that only fourkinds of bovine IFNT transcripts were found in day 17, 20 and 22 trophoblastsof the peri-implantation period, and in MDBK, EF, and Endo ofnon-trophoblast cells. Coexpression of the four forms bIFNT mRNA was evident (trophoblast and non-trophoblast), bIFNT1, c2, and c3 were highly expressed on day 17, 20, and 22 bovine trophoblasts. However, the transcrips of other bIFNTc1 gene was weakly detectable on bovine trophoblasts. Also, expression of bIFNT1, c1, c2, and c3 in MDBK, EF, and Endo of non-trophoblast cells. The bIFNT1, c1, c2, and c3 werepresent in the tree type non-trophoblast cells. The levels of expression of bIFNT1 and c2 were shown higher on the tree type non-trophoblast cells, weakly detectable on bIFNTc1 and c3. Collectively, perhaps more interestingly, a trophoblast and non-trophoblast cells temporal pattern and levelof gene expression was observed in this study.

    In conclusion, Co-expression of the four forms bIFNT mRNA was evident in trophoblast peri-implantation period and nontrophoblast cells. The subtype IFNT genes had similar activity, regulated by the transcription factor complex and/or maternal factors, however, not the same, resulting in different degree of their expression.

    ACKNOWLEDGEMENTS

    This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of data gathering and evaluation system for reproduction and milk component traits on dairy cattle in Korea, Project No. PJ01268901)" Rural Development Administration, Republic of Korea and supported by 2018 Postdoctoral Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korean.

    Figure

    JET-33-195_F1.gif

    Differences in nucleotide structures between 4 subunits. Comparison of nucleotide sequences of the bIFNT1 promoter/enhancer region and related type bIFNTc1 and bIFNTc2 and bIFNTc3 genes. Multiple alignment of all known members of the bovine IFNT subtype. Alignment of nucleotides represents the open-reading frame only. This sequence was originally deposited to GenBank an accession number bIFNT1;M60903: bIFNTc1;AF238613: bIFNTc2;AF238612: bIFNTc3;AF238611. TATA box are underlined. Transcription start site (), and initiation codon (=) are indicated.

    JET-33-195_F2.gif

    Change in the degree of the bIFNT1-reporter and related type bIFNTc1 and bIFNTc2 and bIFNTc3 repoters -transcription in bovine non-trophoblast EF cells. Luciferase-reporter plasmid (-1000-Luc) containing the upstream region (-1000 to +51) of the wild type bIFNT1 and bIFNTc1 and bIFNTc2 and bIFNTc3 genes were transfected into ear-derived fibroblast (EF) cells, and luciferase activity was determined. Results were expressed as relative luciferase activity to that of the bIFNT1-reporter and related type bIFNTc1 and bIFNTc2 and bIFNTc3-reporter constructs without any expression plasmid. Luci activities are expressed relative to that of the control (pGL3-Control vecter) and the data are show as means±SEM (n=4 each) Data from bIFNT-reporter constructs are presented as means±SEM from four independent experiments.

    JET-33-195_F3.gif

    Change in the degree of the bIFNT1 promoter/enhancer region and related type bIFNTc1 and bIFNTc2 and bIFNTc3 gene repoter transactivation by CDX2, ETS2, JUN and/or coactivator CREBBP. Luciferase-reporter plasmid (-1000-Luc) containing the upstream region (-1000 to +51) of the wild type bIFNT1 and bIFNTc1 and bIFNTc2 and bIFNTc3 genes was cotransfected into EF cells with CDX2, ETS2, JUN, CREBBP and ETS2 plus JUN and ETS2 plus JUN plus CREBBP and CDX2 plus ETS2 plus JUN and CDX2 plus ETS2 plus JUN plus CREBBP. Left; Presence (+) or absence (-) of expression plasmids. Luci activities are expressed relative to that of the control (-1000-Luc) and the data are show as means±SEM (n=4 each).

    JET-33-195_F4.gif

    Levels of expression of bIFNT subtypes, mRNA. Expression of of bIFNT1, bIFNTc1, bIFNTc2, and bIFNTc3 mRNA in bovine trophoblast and non-trophoblast cells. Relative amounts of bIFNT1, bIFNTc1, bIFNTc2, and bIFNTc3 mRNAs in on the days 17, 20, and 22 bovine trophoblast and, Madin-Darby bovine kidney (MDBK), Ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. RNA extracted from frozen bovine trophoblast and non-trophoblast cells (n=3 each), were analyzed for the presence of these transcripts using RT-PCR. Changes in bIFNT1 and related type bIFNTc1, bIFNTc2, and bIFNTc3 genes mRNAs levels were determined in mRNAs isolated from on trophoblast and non-trophoblast cells. Sequence analyses of bIFNT1 and related type bIFNTc1, bIFNTc2, and bIFNTc3 genes by Escherichia coli cells (n=3 each).

    Table

    Oligonucleotides primer sequence used for probe

    Oligonucleotides primer sequence used for bIFNTs-ORF assay

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