Endocrine Abstracts

Equine chorionic gonadotrophin (eCG) is not only essential for the maintenance of early equine pregnancy but is also used therapeutically for synchronisation of farm and laboratory animals. Currently it is extracted from serum of farmed pregnant mares. We have recently reported that under normoxic conditions, BMP4 treatment of equine trophoblast resulted in a dose, and developmental stage-dependent increase in total number of differentiated binucleate cells that secrete eCG. Owing to the late implantation of the equine conceptus, we hypothesise that the binucleate trophoblast cells of the equine placenta are induced to differentiate in a low oxygen environment. The objective of this study was to observe the functional response of BMP4 treated trophoblast cells to reduced oxygen conditions. Normal conceptuses were obtained by non-surgical uterine lavage at days 30–31 of pregnancy. Pure populations of day 30 and 31 primary chorionic girdle trophoblast (corresponding to the initiation of terminal differentiation in vivo) were cultured in the presence/absence of 100 ng/ml human BMP4 in either 20% oxygen or in a modified gas composition of 5%CO₂, 5%O₂ and 90% N. Differentiation status was determined through quantification of nuclei number of each cell in five fields of view from each sample. We report, under normoxic conditions, at least a twofold increase in total number of differentiated binucleate cells in response to 100 ng/ml BMP4 in both day 30 and day 31 cells taken straight from the conceptus and cells passaged up to three times. Interestingly, we observed a fivefold increase in total number of binucleate cells cultured in the absence of BMP4 at 5% O₂ compared to 20% O₂ at day 30 of gestation. Furthermore, BMP4 had no additional effect on differentiation when cells were cultured at 5% O₂. In conclusion, these results show that hypoxic conditions alone can induce trophoblast differentiation in vitro.