Introduction: Reproduction exhibit a regular cyclicity and is regulated by complex interactions between circadian signals, gonadotropins and sex steroid hormones. Peripheral circadian rhythm is generated upon cyclic transcription of clock genes expressed in several cells, e.g. granulosa cells in the ovary. Here we investigated whether cyclic intracellular events occurring in response to gonadotropins are linked to expression of clock genes.
Methods: Primary human granulosa cells (hGLC), naturally expressing luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR), were maintained under continuous exposure to LH/hCG over 24 h, and the expression of clock genes (PER1, PER2, ARNTL, CLOCK), gonadotropin receptors (LHCGR and LHCGR-exon 6A variants, FSHR) and genes regulating steroidogenesis (STARD1, CYP19A1, etc.) was analysed by real-time PCR. Cyclicity of cAMP production, G proteins and β-arrestins and ERK1/2 phosphorylation were evaluated by ELISA, Western blotting and immunofluorescence.
Results: Treatments by gonadotropins de-synchronize CLOCK and ARNTL gene expression, as well as LHCGR mRNA isoforms, STARD1 and CYP19A1 gene expression (two-way Anova; P<0.05; n=3). Immunostainings revealed a 45 h-rhythmic, up- and down-regulation of Gαs protein and β-arrestin1/2, as well as G protein coupling/uncoupling to LHCGR upon gonadotropin treatment. 17 h-treatment is linked to rounded shape of the cells and intracytoplasmic vacuoles, LHCGR internalization and translocation of Gαs protein to cytoplasm. These gonadotropins-induced changes are accompanied by 35 h-oscillatory intracellular cAMP production over 24 h, in spite of LHCGR internalization.
Discussion: We found a link between circadian rhythm, LHCGR up-/down-regulation and interaction with Gαs protein and β-arrestin1/2, resulting in the modulation of cAMP-dependent signalling.
Conclusions: These data suggest that the kinetics of LHCGR activation and downstream events are influenced by circadian rhythm, which modulates the cell response to gonadotropins and secondary waves of cell signalling activation.
20 - 23 May 2017
European Society of Endocrinology