Endocrine Abstracts

Placental chorionic gonadotrophin (CG) hormone secretion, critical for maintenance of early pregnancy, is dependent on differentiation of specialised CG-secreting binucleate trophoblast (horse) and syncytiotrophoblast (human). The most abundant genes expressed by binucleate cells are the α and β subunits of equine CG. We recently showed the transcription factor glial cells missing 1 (GCM1) is rapidly induced during differentiation of binucleate trophoblast and mRNA expression precedes and closely correlates with the expression of CGβ mRNA in vivo. Here, we confirmed by western blotting that GCM1 protein is predominantly expressed in the chorionic girdle of the equine conceptus and becomes up-regulated correlating with the induction of CGβ mRNA. Bioinformatic analysis of a 3000-bp fragment of equine CGβ promoter revealed five exact match consensus sites available for GCM1 binding; two located within 200 bp immediately upstream of the transcription start site. A 335 bp fragment, containing these two GCM1 sites, was cloned into pGL3 firefly luciferase vector (Promega) and sequenced. BeWo choriocarcinoma cells were transiently transfected with pGL3-335 or pGL3 basic (as a control) using, Lipofectamine 2000 (Invitrogen), in addition to pRL-SV40 Renilla luciferase vector (Promega) (transfection control). Promoter activity was measured using a Dual-Glo Luciferase Kit (Promega). BeWo cells transfected with pGL3-335 demonstrated a 37-fold increase in luciferase activity relative to control transfected cells at 24 h post transfection. Furthermore, co-transfection of a GCM1 expression vector (pEGFP-GCM1) in increasing amounts with pGL3-335 into BeWo cells resulted in a twofold increase in luciferase activity compared to control transfected cells (pGL3-335, pEGFPN1). In conclusion, these results support our hypothesis that GCM1 regulates equine CG expression, exerting its function via binding to the CGβ promoter. Ongoing studies are investigating the ability of GCM1 to bind to the CGβ promoter in vivo.