Equine Chorionic Gonadotrophin (eCG) is a heterodimeric glycoprotein produced by terminally differentiated, bi/multi-nucleate trophoblast cells in the placenta of horses and humans. It is responsible for the maintenance of early pregnancy via rescue of the corpus luteum and subsequently promotion of progesterone production. The beta subunit of eCG is expressed at levels ten-fold lower than that of the alpha subunit and confers the glycoproteins specificity to the placenta. Very little is known about the regulation of eCGβ. The aim of this study was to identify novel transcription factors that may regulate eCGβ expression in the chorionic girdle.
Conceptuses were obtained via non-surgical uterine lavage from mares between days 2734 of pregnancy, dissected into various tissue components and snap frozen. An Agilent 44K microarray was performed using RNA extracted from Chorionic Girdle and Chorion (control) samples from pregnancy days 27, 30, 31 and 34 (n=5 at each timepoint) and analysed using Genespring (Agilent) and Ingenuity Pathway Analysis (Qiagen) software. Predicted transcription factor binding sites were identified using Match.
Microarray analysis determined that 127 transcription factors were differentially regulated (fc>2) at day 31 compared to day 27 and were specific to the Chorionic Girdle (P<0.05). Nine of these transcription factors had predicted binding sites in a 2500bp length of the eCGβ promoter (core sequence match of 100%) and were also significantly correlated with eCGβ expression. These are CREB1 (FC=2.37, R2=0.59), ELF4 (FC=5.18, R2=0.62), CREB3L4 (FC=2.19, R2=0.71), ELF5 (FC=22.44, R2=0.82), NKX2-5 (FC=2.43, R2=0.86), SRF (FC=2.36, R2=0.78), SREBF1 (FC=2.29, R2=0.65), HNF4G (FC=2.26, R2=-0.54) and STAT5A (FC=2.51, R2=0.62). Current studies are investigating the capacity of these transcription factors to bind to the eCGβ promoter.
A greater understanding of the transcriptional regulation of eCG in the equine placenta will offer an insight into the maintenance of early pregnancy and where miss-regulation may occur in early pregnancy loss, which occurs in 15% of equine pregnancies.