Endocrine Abstracts

Objective: Chorionic gonadotrophin (CG) is a hormone induced during terminal differentiation of trophoblast cells that significantly influences pregnancy outcome. The TGFβ superfamily SMAD2/3 pathway regulates placental function but the activity of the alternative pathway through SMAD1/5/8 in the placenta is unknown. This study investigated the role of BMP4 signalling through SMAD1/5 in terminal differentiation of primary chorionic gonadotrophin-secreting trophoblast cells.

Methods: A novel equine animal model of trophoblast differentiation was used to gain pure populations of primary CG-secreting trophoblast cells and placental tissue. Chorionic girdle was isolated from days 27 to 34 equine conceptuses. A 44 K gene probe equine expression array and RT-PCR was used to compare Type I and Type II serine/threonine kinase and accessory receptor expression. Cultured chorionic girdle trophoblast cells were supplemented with 1–100 ng/ml human BMP4. Differentiation was determined following dual-labelling of the cells with CellTrace BODIPY TR methyl ester and Hoeschst. eCG concentration was determined using ELISA. Total SMAD1/5, pSMAD1/5, total SMAD2 and pSMAD2 expression in the placenta was determined using western blotting.

Results: Chorionic girdle tissue and cultured CG-secreting trophoblast cells preferentially expressed receptors ALK3, BMPR-II, Dragon and Bambi that bind the ligand BMP4. Stimulation of chorionic girdle trophoblast cells with 1–100 ng/ml BMP4 resulted in a dose dependent increase in total number and proportion of terminally differentiated binucleate cells (P<0.001) and induced eCG secretion (P<0.01) in a developmental dependent manner. Phospho-SMAD1/5 expression, but not pSMAD2 was tightly regulated during CG-secreting trophoblast differentiation in vivo, with peak expression of pSMAD1/5 noted at gestation day 31 corresponding to maximal trophoblast differentiation.

Conclusion: Our findings support a role for TGFβ signalling in regulation of differentiation of primary trophoblast cells via BMP4 dependent binding to BMPR-II and ALK3 and activation of SMAD1/5. The observation of BMP4 signalling in primary trophoblast provides a previously unreported mechanism of TGFβ signalling in the placenta.

Declaration of funding: Wellcome Trust (grant number 091581).