Cloning and expression of SOLD1 in ovine and caprine placenta, and their expected roles during the development of placentomes

<p>Abstract</p> <p>Background</p> <p>The Ly-6 (Ly-6/uPAR) superfamily members share the Ly-6 domain defined by distinct disulfide bonding patterns between 8 or 10 cysteine residues. They comprise membrane- and secretory-type proteins. We recently reported the gene and protein characterization of the bovine secreted protein of Ly-6 domain 1 (SOLD1). Bovine SOLD1 is expressed in trophoblast mononucleate cells (TMCs) and is localized in the cotyledonary mesenchyme. Here, we compared the expression and functionality of SOLD1 among the ruminants. We examined mRNA expression by chorionic fibroblasts as a measure of one of the SOLD1 functions.</p> <p>Results</p> <p>Ovine and caprine SOLD1 mRNAs have 303 bp open reading frames and encode for deduced SOLD1 proteins with 100 amino acids, including a 22-aa-long signal peptide at the N-terminal. Both of the SOLD1 amino acid sequences have high similarities with the bovine sequence. Both SOLD1 mRNAs were also expressed in TMCs of cotyledons and intercotyledonary membranes. The mature SOLD1 proteins were localized in the mesenchymal villi of cotyledons after secretion. Bovine, ovine and caprine SOLD1 affected gene expression in mesenchymal fibroblasts <it>in vitro</it>; nucleoredoxin expression was upregulated and BCL2-like 13 was downregulated. Thus, we suggest that SOLD1 acts as a modulator of cell proliferation and apoptosis.</p> <p>Conclusion</p> <p>Expressing cells and protein localization of SOLD1 coincided among the three ruminants. SOLD1 participated in regulating nucleoredoxin and BCL2-like 13 expression in chorionic fibroblasts. SOLD1 is produced specifically in the cotyledons and intercotyledonary membranes in ruminants and appears to be involved in the construction of the ruminant placenta.</p

Transforming growth factor beta family expression at the bovine feto-maternal interface

<p>Abstract</p> <p>Background</p> <p>Endometrial remodelling is necessary for implantation in all mammalian species. The TGF beta super-family plays a crucial role in this event in humans and mice. However, the role of TGF beta super-family members during implantation is still unclear in ruminants. In the present study, the spacio-temporal expression of TGF beta super-family members including activin was explored in bovine trophoblasts and endometrial tissue during the peri-implantation period in order to elucidate whether it is essential for promoting cell proliferation at the implantation site.</p> <p>Methods</p> <p>Gene expression in the fetal membrane and endometrium of the gravid and non-gravid horn around Day 35 of gestation were analyzed with a custom-made oligo-microarray in cattle. The expression of activin and its related genes was also analyzed with quantitative RT-PCR. Activin-like activity in trophoblastic tissue and BT-1 cells was examined using a fibroblast cell proliferation test and Western blotting.</p> <p>Results</p> <p>The expression of various TGF beta super-family related genes including activin was detected in trophoblasts and the endometrium in cattle. The most intensive activin expression was found in the gravid horn endometrium, and rather intense expression was detected in the non-gravid trophoblastic tissue. Extracts from the fetal membrane including trophoblasts and purified activin both stimulated fibroblast proliferation effectively, and activin was immunologically detected in BT-1 cells, which have trophoblastic features.</p> <p>Conclusions</p> <p>Specific expression of the activin gene (gene name: inhibin beta A) was found in the gravid horn endometrium during peri-implantation. An activin-like molecule, which was derived from the endometrium and trophoblasts, stimulated the proliferation of fibroblast cells. These results suggested that as in other species, the activity of TGF beta super-family members including activin-like molecules plays a pivotal role in endometrial remodelling, which is an essential process in implantation and placentogenesis during the peri-implantation period in cattle.</p

Characterization and Expression Analysis of SOLD1, a Novel Member of the Retrotransposon-Derived Ly-6 Superfamily, in Bovine Placental Villi

BACKGROUND:Ly-6 superfamily members have a conserved Ly-6 domain that is defined by a distinct disulfide bonding pattern between eight or ten cysteine residues. These members are divided into membrane-type and secretory-type proteins. In the present study, we report the identification of a novel Ly-6 domain protein, secreted protein of Ly-6 domain 1 (SOLD1), from bovine placenta. PRINCIPAL FINDINGS:SOLD1 mRNA was expressed in trophoblast mononucleate cells and the protein was secreted into and localized in the extracellular matrix of the mesenchyme in cotyledonary villi. SOLD1 bound mainly with type I collagen telopeptide. We confirmed secretion of SOLD1 from the basolateral surface of a bovine trophoblast cell line (BT-1). It may be related to the organization of the extra-cellular matrix in the mesenchyme of fetal villi. Since trophoblast mononucleate cells are epithelial cells, their polar organization is expected to have a crucial role in the SOLD1 secretion system. We established that SOLD1 is an intronless bovine gene containing the Alu retrotransposon, which was integrated via cytoplasmic reverse transcription. CONCLUSION:We identified a novel retrotransposon-like Ly-6 domain protein in bovine placenta. SOLD1 is a crucial secreted protein that is involved in the organization of the mesenchyme of the cotyledonary villi. Furthermore, the gene encoding SOLD1 has an interesting genomic structure

Global gene expression analysis and regulation of the principal genes expressed in bovine placenta in relation to the transcription factor AP-2 family

BACKGROUND: Cell-cell communication is an important factor in feto-maternal units during placentogenesis. The placenta produces pivotal hormones and cytokines for communication between cotyledonary villi and the maternal caruncle. Gene expression in bovine placenta throughout pregnancy was comprehensively screened by a cDNA microarray, and we searched for a common transcription factor in a gene cluster that showed increasing expression throughout gestation in cotyledonary villi and caruncle. METHODS: Placentomal tissues (villi and caruncle) were collected from Day 25 to Day 250 of gestation for microarray analysis. Global gene expression profiles were analyzed using the k-means clustering method. A consensus sequence cis-element that may control up-regulated genes in a characteristic cluster was examined in silico. The quantitative expression and localization of a specific transcription factor were investigated in each tissue using quantitative real-time RT-PCR and in situ hybridization. RESULTS: The microarray expression profiles were classified into ten clusters. The genes with most markedly increased expression became concentrated in cluster 2 as gestation proceeded. Cluster 2 included placental lactogen (CSH1), pregnancy-associated glycoprotein-1 (PAG1), and sulfotransferase family 1E estrogen-preferring member 1 (SULT1E1), which were mainly detected in giant trophoblast binucleate cells (BNC). Consensus sequence analysis identified transcription factor AP-2 binding sites in some genes in this cluster. Quantitative real-time RT-PCR analysis confirmed that high level expression of transcription factor AP-2 alpha (TFAP2A) was common to cluster 2 genes during gestation. In contrast, the expression level of another AP-2 family gene, transcription factor AP-2 beta (TFAP2B), was extremely low over the same period. Another gene of the family, transcription factor AP-2 gamma (TFAP2C), was expressed at medium level compared with TFAP2A and TFAP2B. In situ hybridization showed that TFAP2A, TFAP2B and TFAP2C mRNAs were localized in trophoblast cells but were expressed by different cells. TFAP2A was expressed in cotyledonary epithelial cells including BNC, TFAP2B was specifically expressed in BNC, and TFAP2C in mononucleate cells. CONCLUSION: We detected gestational-stage-specific gene expression profiles in bovine placentomes using a combination of microarray and in silico analysis. In silico analysis indicated that the AP-2 family may be a consensus regulator for the gene cluster that characteristically appears in bovine placenta as gestation progresses. In particular, TFAP2A and TFAP2B may be involved in regulating binucleate cell-specific genes such as CSH1, some PAG or SULT1E1. These results suggest that the AP-2 family is a specific transcription factor for clusters of crucial placental genes. This is the first evidence that TFAP2A may regulate the differentiation and specific functions of BNC in bovine placenta

Gene expression profiles of novel caprine placental prolactin-related proteins similar to bovine placental prolactin-related proteins

BACKGROUND: This study reports the identification of a full-length cDNA sequence for two novel caprine prolactin-related proteins (cPRP1 and cPRP6), and their localization and quantitative expression in the placenta. Caprine PRPs are compared with known bovine PRPs. We examined their evolution and role in the ruminant placenta. RESULTS: Full-length cPRP1 and cPRP6 cDNA were cloned with a 717- and 720- nucleotide open-reading frame corresponding to proteins of 238 and 239 amino acids. The cPRP1 predicted amino acid sequence shares a 72% homology with bovine PRP1 (bPRP1). The cPRP6 predicted amino acid sequence shares a 74% homology with bovine PRP6 (bPRP6). The two cPRPs as well as bPRPs were detected only in the placentome by RT-PCR. Analysis by in situ hybridization revealed the presence of both cPRPs mRNA in the trophoblast binucleate cells. These mRNA were quantified by real-time RT-PCR analysis of the placentome at 30, 50, 90 and 140 days of pregnancy. Both new cPRP genes were able to translate a mature protein in a mammalian cell-expression system. Western blotting established the molecular sizes of 33 kDa for cPRP1 with FLAG-tag and 45 kDa for cPRP6 with FLAG-tag. The sequence properties and localized expression of cPRP1 and cPRP6 were similar to those of bovine. However, their expression profiles differed from those in bovine placenta. Although this study demonstrated possible roles of PRPs in caprine placenta, PRPs may regulate binucleate-cell functions like those in bovine, but their crucial roles are still unclear. CONCLUSION: We have identified the novel PRPs in caprine placenta. Localization and quantitative expression of caprine PRPs were compared with bovine PRPs. The data indicate that PRP genes in caprine placenta have coordination functions for gestation, as they do in bovine. This is the first study of PRPs function in caprine placenta

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

<p>Abstract</p> <p>Background</p> <p>Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow.</p> <p>Methods</p> <p>In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis.</p> <p>Results</p> <p>EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium.</p> <p>Conclusion</p> <p>EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions.</p

Differential neutrophil gene expression in early bovine pregnancy

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 (<it>ISG15</it>), myxovirus-resistance (<it>MX</it>) <it>1</it> and <it>2</it>, and 2′-5′-oligoadenylate synthetase (<it>OAS1</it>), were then analyzed in each fraction through day 28 of gestation using qPCR.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>The expression profiles of <it>ISG15</it>, <it>MX1</it>, <it>MX2</it>, and <it>OAS1</it> 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.</p