Bone morphogenetic protein 4 accelerates the establishment of bovine trophoblastic cell lines

Abstract

Trophoblastic cells play a crucial role in implantation and placentogenesis. A large proportion of the failures of conception in cows occur in the peri-implantation period, which are known as early embryo losses. In exploring this critical phenomenon, trophoblastic cell lines can provide substantial information. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stock in the long term. In this study, 12 new cell lines were established using bone morphogenetic protein 4 (BMP4). BMP4 stimulated embryonic cells to enter the trophoblastic cell lineage but there were no significant differences between intact and BMP4-treated groups. Only one out of 49 embryos developed trophoblastic cells in the intact group. Finally, 12 cell lines were maintained for around 30 passages, and they retained trophoblastic characteristics and expressed bovine trophoblastic genes: placental lactogen, interferon-t, pregnancy-associated glycoprotein 1, and prolactin-related protein 1. Although the gene expression patterns were different among cell lines and depended on the cells, there was no significant relationship between the expression intensities of genes and the treatment dose of BMP4. All of them expressed bovine POU domain class 5 transcription factor 1 and caudal-type homeobox 2. The expression of these genes was confirmed by quantitative RT-PCR and immunohistochemical detection. These results suggest that BMP4 is involved in the raising of trophoblast cell lines from early embryonic cells and the newly developed cell lines can provide different types of bovine trophoblastic cells with different cell lineages. This may constitute a significant new tool for the examination of trophoblastic differentiation.

Bovine Trophoblastic Cell Differentiation and Binucleation Involves Enhanced Endogenous Retrovirus Element Expression.

“…BT cell lines (BT-1 and BT-A to BT-L) were established from in vitro matured and in vitro fertilized blastocysts and cultured, as described previously [31,33]. They were cultured and maintained according to a previously described method [32].…”

“…The RT cycle comprised 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 seconds in a thermal cycler, and the resultant cDNA were stored at −20°C. PCR primers that we had designed in previous studies [19,20,31] were used ( Table 1). The PCR reaction was performed at 95°C for 30 seconds for denaturing, 60°C for 30 seconds for annealing and 72°C for 1 minute for extension and was repeated for 30 cycles.…”

“…Several trophoblastic cell lines were confirmed to express bCSH1, bPRP-1, bPAG1, and interferon tau (IFNT). The primer pairs used in the SYBR Green assay were designed in previous reports [19,20,31] (Table 2). The standard curves for each gene were generated by the serial dilution of plasmids containing cDNA for one of these genes in order to quantify their mRNA concentrations.…”

“…We reported that binuc-leation was induced in a bovine trophoblastic cell line (BT-1) cultured on thick collagen gel (collagen I) [30]. Recently, we established twelve bovine trophoblastic cell lines (BT-A to BT-L) from in vitro fertilized embryos using bone morphogenetic protein-4 (BMP4) [31]. BT cells are used as a model trophoblastic cell lineage because certain cell culture conditions are known to enhance their differentiation from MNC to BNC [31,32].…”

“…Recently, we established twelve bovine trophoblastic cell lines (BT-A to BT-L) from in vitro fertilized embryos using bone morphogenetic protein-4 (BMP4) [31]. BT cells are used as a model trophoblastic cell lineage because certain cell culture conditions are known to enhance their differentiation from MNC to BNC [31,32]. The purpose of this study is to examine the expression of ERV elements in bovine trophoblastic cell lines under different in vitro cell culture conditions.…”