Bovine caruncular epithelial cell line (BCEC-1) isolated from the placenta forms a functional epithelial barrier in a polarised cell culture model.

In the bovine synepitheliochorial placenta key sites of fetal-maternal interaction are placentomes consisting of maternal caruncles interdigitating with fetal cotyledons. The aim of this study was to establish an epithelial cell line from caruncles of pregnant cows and to develop a model to study restricted trophoblast invasion, pathogenesis of pregnancy associated diseases and pathways of infection and transport. Primary epithelial cells were isolated, successfully subcultured for 32 passages and cryopreserved at various stages. The cultures were termed bovine caruncular epithelial cell line-1 (BCEC-1). Cytokeratin, zonula occludens-1 protein and vimentin but neither alpha-smooth muscle actin nor desmin were detected by immunofluorescence performed every 5 (+/-1) passages. These results were confirmed by Western blotting. BCEC-1 were then cultured either without matrix or on fibronectin or collagen coated Transwell polyester membrane inserts, respectively, enabling separate access to the basal or apical epithelial compartments. Transmission and scanning electron microscopy of BCEC-1 revealed ultrastructural features also observed in vivo, such as apical microvilli and junctional complexes. Transepithelial electrical resistance (TEER) was measured regularly and revealed an increase with advancing confluence in all cultures. Cultures on coated inserts reached confluence and corresponding TEER-levels at an earlier stage. In addition, the cells were tested negative for bovine virus diarrhoea (BVD) virus, but were permissive for the virus. In conclusion, the BCEC-1 cell line retained characteristics of maternal caruncular epithelial cells as observed in vivo and in primary cell cultures and thus will be a highly useful tool for future studies of pathways of invasion, fetal-maternal communication, transport and infection.

Expression of gap junctional connexins 26, 32 and 43 in bovine placentomes during pregnancy.

Gap junctional connexins (Cx) are induced in the endometrium during implantation in rodents, the human receptive window, and in the decidua Cx26 and Cx43 expression increases in response to trophoblast invasion. In contrast, this gap junctional response and decidualization is absent in non-invasive epitheliochorial placentae of pigs and horses. Bovine (syn)epitheliochorial placentation represents an intermediate type of trophoblast invasion, since it is characterized by the continuous migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells. Therefore the objective of the present study was to investigate the expression of Cx26, Cx32, and Cx43 in placental tissues during bovine pregnancy, to determine if Cx expression patterns correlate with the depth of trophoblast invasion. Cx26, Cx32, and Cx43 proteins were detected by immunohistochemistry and corresponding specific mRNAs were shown by RT-PCR and localized in tissue sections by in situ hybridization. Cx26 protein was detected at the feto-maternal contact interface and as cytoplasmic staining in TGC. Cx26 mRNA was located in maternal epithelium and in TGC. Cx32 protein expression was observed in the maternal epithelium exclusively on the tips of maternal septa, whereas Cx32 mRNA was detected in all maternal epithelial cells and single TGC. Cx43 protein and mRNA were coexpressed in TGC. Cx43 protein was present in maternal septal stroma and to a lesser extent in chorionic villous mesenchyme, while Cx43 mRNA was associated with the vasculature. In the course of gestation, expression of Cx26, Cx32, and Cx43 did not change. In conclusion, the intermediate invasive status of bovine trophoblast is supported by the fact that TGC coexpress Cx26, Cx32, and Cx43, which may be important for trophoblast migration (invasion), and fusion with maternal epithelial cells. Cx32 could be involved in the control of invasion.

Localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in bovine placentomes from implantation until term.

Interactions of vascular endothelial growth factor (VEGF) with its receptors VEGFR-1 and VEGFR-2 promoting angiogenesis have been described in placentation of human, mink and pig. The bovine placenta is multiplex, villous and synepitheliochorial due to migratory trophoblast giant cells (TGC). To determine the role of VEGF in bovine implantation and placentation, placentomes and interplacentomal areas from 33 cows from early implantation until near term were evaluated by immunohistochemistry. VEGF immunoreactivity was detected in fetal and maternal blood vessel tissues during implantation and throughout gestation, and in preimplantatory trophoblast cells and uterine epithelium. After implantation the immunoreaction was confined to TGC and uterine epithelium. An antibody against bovine VEGF revealed a strong reactivity in the stroma of maternal caruncular septa in early and mid-gestation, which distinctly decreased near term. In interplacentomal areas, VEGF was found in luminal and glandular epithelia as well as in trophoblast, with distinctly higher reactivity in giant cells. VEGFR-1 was observed in trophoblast and uterine epithelium around implantation. Later, in definite placentomes, VEGFR-1 was localized in TGC near the chorionic plate and in maternal endothelial cells in the center of the placentome. VEGFR-1 and VEGFR-2 were co-localized in uterine epithelium and trophoblast as well as in blood vessel tissue and uterine glands. The presence of VEGF, VEGFR-1 and VEGFR-2 at the feto-maternal interface and in vasculature indicates that in the bovine VEGF may have (1) classic functions in angiogenesis and vascular permeability, (2) growth factor properties, facilitating feto-maternal exchange via paracrine action, (3) chemotactic activity on capillary endothelium, and (4) an autocrine influence on TGC migratory activity.

Glucose metabolism is elevated and vascular resistance and maternofetal transfer is normal in perfused placental cotyledons from severely growth-restricted fetuses.

We hypothesized that placental resistance was elevated and transfer reduced in cotyledons from intrauterine growth-restricted (IUGR) fetuses. We perfused 10 cotyledons from term, normally grown fetuses, six from preterm, normally grown fetuses with normal umbilical arterial end-diastolic velocities (EDV), and six from preterm IUGR fetuses (<3rd centile) with absent or reversed umbilical arterial EDV. Perfused cotyledons were pressure-fixed, and villi were observed by scanning electron microscopy. The groups did not differ in fetoplacental resistance at baseline; neither did they differ in the change in resistance that followed the administration of nitroglycerin or angiotensin II. The increase in resistance during hypoxia was similar in the two preterm groups but greater in the term than in the preterm normally grown group (p < 0.05). Groups did not differ in net maternofetal transfer of oxygen or glucose, or in clearance of aminoisobutyric acid or antipyrine. However, glucose consumption was doubled in cotyledons of preterm IUGR versus preterm normally grown fetuses (p < 0.05). Terminal villi of perfused cotyledons from preterm IUGR fetuses displayed less terminal villous branching and budding than preterm controls, as anticipated from previous work. IUGR fetuses with absent or reversed umbilical arterial EDV in vivo may have high placental resistance due to a vasoconstrictive rather than anatomic abnormality and an elevated placental glucose consumption that may impair glucose transfer.