Abstract
Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between −119 bp and −335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.
Highlights
Equine chorionic gonadotrophin is a heterodimeric glycoprotein critical for early equine pregnancy
A significant increase in glial cells missing 1 (GCM1) expression was observed at day (11-fold, p = 0.002) and day (24-fold, p < 0.0001) compared to day 27 chorionic girdle (ChG), preceding the increase in eCGB expression by 24 h
Results above indicate that GCM1 binds to GCM1 binding site 1 located within 119 bp of the translational start site but activity required the upstream segment of the CGB promoter between −119 and −335
Summary
Equine chorionic gonadotrophin (eCG) is a heterodimeric glycoprotein critical for early equine pregnancy. ECG extracted from pregnant mare sera is utilized therapeutically in cows and laboratory species to support follicle development and reproductive activity [6]. Corresponding to the development of the ChG between days 30 and 36 of pregnancy, eCG can be detected in the sera of pregnant mares from around day 40. While structural modifications that regulate eCG protein expression and activity are well defined [9], surprisingly little is known about the factors that initially induce and maintain the expression of eCG transcripts in equine trophoblast. The β-subunit of eCG/ LH is shown to demonstrate expression levels 100 times lower than that of the α-subunit [13], suggesting LHB is the limiting factor in the glycoproteins expression and subsequent activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
