P43 - Rekovelle® vs Gonal-F®: in vitro characterization of intracellular signals induced by the two FSH preparations
G Mercanile, C Lazzaretti, S Sperduti, C Perri, C Fusco, E Mascolo, M Simoni, L CasariniAbstract
Background/Introduction
Rekovelle® and Gonal-F® are two commercial preparations of recombinant FSH used in assisted reproduction. These preparations differ in originator cells (PER.C6® cell line of human fetal retinal origin for Rekovlle®; modified Chinese Hamster Ovary cell line for Gonal-F®) and in glycosylation profiles impacting their mode of action.
Purpose
The aim of this study is to investigate different intracellular events mediated by Gonal-F® and Rekovelle® in vitro, likely originated by different glycan profiles and possibly impacting the ovarian response during assisted reproduction.
Methods
FSHR-expressing HEK293 cells were treated with increasing concentrations of GonalF® and Rekovelle® (pM-µM) to evaluate receptor-receptor interaction and trafficking, intracellular cyclic adenosine monophosphate (cAMP) and Ca2+ increase by bioimaging techniques, as well as phospho-protein by Western blotting, and genomic effects by CRE-luciferase reporter gene activity.
Results
Both the preparations induced similar, dose-dependent increase of receptor trafficking through intracellular organelles. These data were obtained by evaluation of the interaction between FSHR and endosomal markers RAB-GTPases (Rab) 5 and 7. Rekovelle® induced more pronounced steroidogenic signals than Gonal-F®, detected as FSHR-FSHR interactions at the cell membrane, cAMP accumulation, cAMP- responsive elements binding protein (CREB) phosphorylation and CRE promoter activity. Interestingly, while cell treatment by Gonal-F® mediated complete dose- response of cAMP, consisting in the continuous increase of the second messenger together with the increase of FSH concentration, Rekovelle® had biphasic action where cAMP increased from 0 to 100 nM although decreased at 1 µM Rekovelle® dose. Moreover, Rekovelle® was 10-times more potent than Gonal-F® in activating intracellular Ca2+ activation. The two preparations induced similar pattern of extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation.
Conclusion(s)
In conclusion, Rekovelle® has higher potency than Gonal-F® in activating cAMP- and Ca2+-dependent intracellular events, potentially resulting in preparation-specific steroidogenic pattern.