Cloning and expression of two new prolactin-related proteins, prolactin-related protein-VIII and -IX, in bovine placenta

BACKGROUND: Prolactin-related proteins (PRPs) are specific proteins of the growth hormone/prolactin (GH/PRL) family in bovine placenta. This study reports the identification and sequencing of a full-length cDNA for two new members of bovine PRPs, bPRP-VIII and -IX, and their localization and quantitative expression in bovine placenta. METHODS: New bPRP-VIII and -IX were identified from bovine placentome. Localization and quantitative gene expression in the placenta were respectively investigated by in situ hybridization and real-time RT-PCR methods. Recombinant proteins of these genes were produced by a mammalian HEK293 cell expression system. RESULTS: Full-length bPRP-VIII and -IX cDNA were respectively cloned with 909 and 910 nucleotide open-reading-frames corresponding to proteins of 236 and 238 amino acids. The predicted bPRP-VIII amino acid sequence shared about 40 to 70% homology with other bPRPs, and bPRP-IX had about 50 to 80 % homology of others. The two new bPRPs were detected only in the placenta by RT-PCR. mRNA was primarily expressed in the cotyledon and intercotyledonary tissues throughout gestation. An in situ hybridization analysis revealed the presence of bPRP-VIII and -IX mRNA in the trophoblastic binucleate and/or trinucleate cells. bPRP-VIII mRNA was observed in the extra-embryonic membrane on Day 27 of gestation, however, no bPRP-IX mRNA was observed in the extra-embryonic membrane in the same stage of pregnancy by quantitative real-time RT-PCR analysis. Both new bPRP genes were possible to translate a mature protein in a mammalian cell expression system with approximately 28 kDa in bPRP-VIII and 38 kDa in bPRP-IX. CONCLUSION: We identified the new members of bovine prolactin-related protein, bPRP-VIII and -IX. Localization and quantitative expression were confirmed in bovine placenta by in situ hybridization or real-time PCR. Their different temporal and spatial expressions suggest a different role for these genes in bovine placenta during gestation

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries

<p>Abstract</p> <p>Background</p> <p>It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of <it>BMP15 </it>and <it>GDF9 </it>in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes.</p> <p>Methods</p> <p>Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and <it>in situ </it>hybridization. Immunohistochemical analysis was also performed.</p> <p>Results</p> <p>The expression of <it>BMP15 </it>and <it>GDF9 </it>in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. <it>GDF9 </it>expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, <it>BMP15 </it>expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry.</p> <p>Conclusions</p> <p>Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. <it>BMP15 </it>and <it>GDF9 </it>mRNA expression in oocytes and cumulus cells was different in calves and cows.</p

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

cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period

BACKGROUND: After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA) microarray. METHODS: Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR. RESULTS: In total, 1,773 individual genes were analyzed by complete k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs), prolactin-related proteins (PRPs), interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc) as well as novel candidate genes (AW464053, AW465434, AW462349, AW485575) related to already established trophoblast-specific genes such as PLs and PRPs. CONCLUSIONS: A large number of genes in extra-embryonic membrane increased up to implantation and these profiles provide information fundamental to an understanding of extra-embryonic membrane differentiation and development. Genes in significant expression suggest novel molecules in trophoblast differentiation

Reassociation of annelid giant hemoglobin from the polychaete Perinereis aibuhitensis

Annelid extracellular hemoglobin (Hb) is a supramolecule with molecular mass of ～3,500kDa. The giant Hb consists of 12 subassemblies (globin dodecamers, D) and 18 homodimeric linkers (L) of non-globin chain. The globin dodecamer and linker were isolated from the polychaete Perinereis aibuhittensis Hb separately. Subsequently, these two components were mixed in the presence of 1M urea at a neutral pH to reform a whole molecule of Hb. At first L was refined by reverse phase chromatography in organic solvent. On the other hand, Perinereis Hb was incubated in 4M urea solution at 4°C for 5 min, and applied to two amphoteric ion-exchange resin column to remove L stick to the resin, and to isolate only D. The eluate was condensed and subjected to gel filtration. As a result, an ingredient of molecule mass ～210 kDa, that is D, was provided in high yield. When D and L were mixed in the molar ratio of approximately 1:1 in 50mM phosphate buffer (pH 7.2) in the presence of 1 M urea at room temperature, most of the proteins met to natural Hb size again within about 20 hours. Furthermore, similar experiments were performed in 50 mM Tris-HCl buffer (pH 7.2) containing 1 M urea in the presence of 1 mM CaCl2 or 1mM EDTA. It was observed that the reassociation was affected substantially by the presence of Ca2+. In conclusion, the homodimeric linkers have the key role to form the gigantic Hb

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

Abstract 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.</p