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

ABSTRACT: 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.

SUBMITTER: Ushizawa K

PROVIDER: S-EPMC1325045 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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

Ushizawa Koichi K Takahashi Toru T Hosoe Misa M Kaneyama Kanako K Hashizume Kazuyoshi K

Reproductive biology and endocrinology : RB&E 20051207

<h4>Background</h4>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.<h4>Methods</h4>New bPRP-VIII and -IX were identified from bovine placentome. Localization and quantitative gene expression in the placenta were respectively ...[more]

PMID: 16332262

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Project description:BackgroundBovine trophoblast binucleate cells (BNC) express a plethora of molecules including bovine placental lactogen (bPL, gene name is bCSH1) and bovine prolactin-related protein-1 (bPRP1). BCSH1 and bPRP1 are members of the growth hormone (GH)/prolactin (PRL) gene family, which are expressed simultaneously in BNC and are central to placentation and the progression of pregnancy in cattle. However, there is a paucity of information on the transcriptional regulatory mechanisms of both the bCSH1 and bPRP1 genes. Recent studies, however, have demonstrated that the expression of a number of genes is controlled by the methylation status of their promoter region. In the present study, we examined the cell-type-specific epigenetic alterations of the 5'-flanking region of the bCSH1 and bPRP1 genes to gain an insight into their regulatory mechanisms.ResultsAnalysis of 5-aza-2'-deoxycytidine treatment demonstrated that bCSH1 expression is moderately induced in fibroblast cultures but enhanced in BT-1 cells. Sodium bisulfite based sequencing revealed that bCSH1 is hypomethylated in the cotyledonary tissue but not in the fetal skin, and this pattern was not altered with the progression of pregnancy. On the other hand, the methylation status of bPRP1 was similar between the cotyledon and fetal skin. The bPRP1 gene was exclusively hypermethylated in a bovine trophoblast cell-derived BT-1 cell-line. While the activity of bCSH1 was similar in both BT-1 and bovine fibroblast cells, that of bPRP1 was specific to BT-1. Treatment with a demethylating agent and luciferase assays provided in vitro evidence of the positive regulation of bCSH1 but not bPRP1.ConclusionThis is the first report to identify the differential regulatory mechanisms of the bCSH1 and bPRP1 genes and indicates that bCSH1 might potentially be the only transcript that is subject to DNA methyltransferase regulation. The data indicates the possibility of novel kinetics of induction of the synchronously expressed BNC-specific bCSH1 and bPRP1 transcripts, which may aid the understanding of the intricate regulation and specific role(s) of these important molecules in bovine placentogenesis and the progression of pregnancy.

Dataset Information

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

Publications

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

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