Endocrine Abstracts

Chorionic Gonadotrophin (CG) is secreted by both horse and human placenta and is critical to maintenance of early pregnancy. Production of CG is dependent on the differentiation of specialised CG-secreting cells, binucleate (horse) and syncytiotrophoblast (human). We recently showed the transcription factor Glial Cells Missing 1 (GCM1) is rapidly induced in vivo during differentiation of equine binucleate trophoblast. Studies in human syncytiotrophoblast have shown cAMP regulates trophoblast differentiation via the induction of GCM1. The aim of this study was to determine the function of GCM1 during in vitro differentiation of binucleate trophoblast. Pure populations of chorionic girdle trophoblast cells were isolated from day 31–35 equine conceptuses. Cells were grown in the presence or absence of 25–100 µM Forskolin (0.02% DMSO) for up to 72 h. Differentiation was determined following labelling of the cells with CellTrace™ BODIPY^® TR methyl ester and the nuclear stain Hoechst. The number of uni-, bi- and multinucleated cells was quantified in 5 randomly selected fields under an inverted fluorescent microscope. GCM1 and eCGβ mRNA expression was determined by qRT-PCR. In contrast to untreated and vehicle treated cells, Forskolin treatment resulted in a 10-fold increase in multinucleated cells. Forskolin induced differentiation occurred either directly around the edge of a uninucleate cluster (25 µM) or in discrete zones (≥50 µM). In addition, Forskolin significantly decreased total cell number at 50 µM (P<0.05), 75 and 100 µM (P<0.001). We observed no change in eCGβ mRNA expression and a total absence of eCG secretion determined by immunoassay. Preliminary experiments confirmed an induction of GCM1 mRNA at higher concentrations of Forskolin treatment (100 µM P<0.05). In conclusion, Forskolin treatment of equine chorionic girdle trophoblast cells increased levels of GCM1 mRNA expression, either inhibited cell proliferation or promote cell apoptosis and promoted an altered differentiation pattern that is eCG independent.

Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding: Declaration of Funding: Wellcome Trust WT091581.