ECE2017 Eposter Presentations: Reproductive Endocrinology Female Reproduction (62 abstracts)
Heterogeneous hCG and hMG commercial preparations result in biased intracellular signaling but induce similar progesterone response in vitro
Laura Riccetti 1 , Danièle Klett 2 , Mohammed Akli Ayoub 2, , Thomas Boulo 2 , Julie Mariot 2 , Elisa Pignatti 1, , Simonetta Tagliavini 5 , Manuela Varani 5 , Tommaso Tranti 5 , Alessia Nicoli 6 , Giovanni Battista La Sala 6 , Yves Combarnous 2 , Eric Reiter 2 , Manuela Simoni 1, & Livio Casarini 1,
1Unit of Endocrinology, Department Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Biologie et Bioinformatique des Systèmes de Signalisation (BIOS) group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements; CNRS, UMR7247, F-37380 Nouzilly, Nouzilly, France; 3Biology Department, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates; 4Center for Genomic Research, University of Modena and Reggio Emilia, Modena, Italy; 5Azienda Ospedaliero-Universitaria di Modena, Modena, Italy; 6Unit of Obstetrics and Gynecology, IRCCS-Arcispedale Santa Maria Nuova, Reggio Emilia, Italy.
Human chorionic gonadotropin (hCG) is the pregnancy hormone marketed as a drug for assisted reproduction technologies to support follicle-stimulating hormone action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition. In this study, we investigated the molecular features and the intracellular signaling mediated by five urinary and recombinant hCGs. The drug comparisons were performed by quantifying the hormones by immunoassay, calibrated against the hCG standard (5th IS; NIBSC 07/364). Immunoreactivity pattern, isoelectric point and oligosaccharide content of hCGs were evaluated by reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production, as well as β-arrestin two recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and LH/hCG receptor (LHCGR)-transfected HEK293 cells stimulated by increasing hormone doses. Heterogeneous profiles were found among preparations, revealing hormone-specific molecular weight patterns (20–75 kDa range), isoelectric points (4.0–9.0 pI range) and lectin binding (Two-way ANOVA and Bonferroni post-test; P<0.05; n=5). These drug-specific compositions are linked to different potencies on cAMP production (EC50 1.0–400 ng/ml range) and partial agonism on β-arrestin 2 recruitment (EC50 0.03–2.0 μg/ml) in hGLC and transfected HEK293 cells (Mann-Whitney’s U test; P<0.05; n=3 or 5). However, the treatment of hGLC with hCGs resulted in similar progesterone production after 24 h of exposure (Mann-Whitney’s U test; P≥0.05; n=3). Therefore, although hCG preparations consist of different glycosylated isoforms and mediate drug-specific early signaling, they result in similar downstream steroidogenesis in vitro. Commercial gonadotropins calibration relies on their in vivo activity (Pharmacopea), which can be reached with different mixtures of isoforms with various half-lives and receptor binding activities. Here, we demonstrate that commercial hCG preparations, likely with different assortments of isoforms, exhibit similar steroidogenic activity in vitro, by triggering biased intracellular signaling pathways.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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