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Endocrine Abstracts (2018) 56 GP220 | DOI: 10.1530/endoabs.56.GP220

ECE2018 Guided Posters Reproduction (10 abstracts)

Depletion of the primate-specific luteinizing-hormone receptor splice variant ‘exon-6A’ impairs LH-, but not hCG-mediated signaling in human primary granulosa cells

Livio Casarini 1, , Laura Riccetti 1 , Samantha Sperduti 1 , Clara Lazzaretti 1 , Alessia Masini 1 & Manuela Simoni 1,

1Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Center for Genomic Research, University of Modena and Reggio Emilia, Modena, Italy; 3Department of Medicine, Endocrinology, Metabolism and Geriatrics, Azienda Ospedaliero-Universitaria di Modena, Modena, Italy.

Introduction: A primate-specific luteinizing hormone (LH) receptor (LHCGR)-variant was suspected to discriminate between maternal choriogonadotropin (hCG)- and fetal LH-functioning. It is the so-called “LHCGRex6A”, truncated, intracellular receptor isoform produced by alternative cryptic exon stop-codons located between the 6th-7th out of 11 exons. Its function is unknown, although pseudohermaphroditism and 46-XY female-like phenotype were associated with LHCGRex6A mutations, in spite of intact LHCGR wild-type. LHCGR-6A was proposed as a key-mediator of hCG-signals in primates.

Aim: In order to address evolutionary issues related to LHCGRex6A, we evaluated the functions of this isoform in modulating LHCGR-driven LH- and hCG-signals in vitro.

Methods: Cell model is human primary granulosa lutein cells (hGLC), naturally expressing both the wild-type and LHCGRex6A, treated by siRNA for their selective depletion. siRNA efficacy was evaluated by qPCR, Western blotting and immunofluorescence, cell viability by MTT assay. Impact of LHCGRex6A on steroidogenic-signals was evaluated as LH- and hCG-dependent 2-h cAMP production by ELISA, 15-min CREB and ERK1/2 phosphorylation by Western blotting, 12-h STARD1- and CYP19A1-target gene expression by qPCR, 8-/24-h synthesis of progesterone by immunometric-assay. Forskolin-stimulated hGLC served as controls and data were normalized over cell number (5x104 cells-per-well).

Results: LHCGRex6A mRNA and protein analysis, and immunofluorescence under permeabilizing/non-permeabilizing conditions certified siRNA efficacy. Viability of siRNA- and mock-transfected hGLC confirmed experimental reliability. Equipotent, 500 pM LH and 100 pM hCG concentrations resulted in similar cAMP levels by mock-treated hGLC, while hCG induced 3-fold higher cAMP increase than LH in siRNA-treated hGLC (cAMP-LH=14.0±8.7 pmol/ml; cAMP-hCG=33.1±16.2; basal=0.5±0.3; means±S.E.M.; Mann-Whitney’s; P<0.05; n=6). Consistent with cAMP, hCG induced higher downstream pCREB activation than LH, while no different pERK1/2 activation was found, as well as STARD1 and CYP19A1 expression (ANOVA; P≥0.05; n=3), reflecting the qualitatively different LH/hCG-specific signal. While no different 8-h LH-/hCG-induced progesterone response was found, 24-h hormone production were about 2-fold higher upon hCG than LH exposure of siRNA-treated hGLC (progesterone-LH=35.7±5.5 ng/ml; progesterone-hCG=64.0±11.3; basal=24.2±3.2; means±S.E.M.; Mann-Whitney’s; P<0.05; n=5). Controls provided similar results.

Discussion: LHCGRex6A depletion impaired LH-, but not hCG-specific steroidogenic-signal in spite of functional LHCGR-expression, suggesting evolutionary relevance of the receptor-variant functioning during fetal stages in primates. LHCGRex6A roles would be investigated in the not-readily available primate male testis cells, providing wider picture of LH/hCG co-evolution.

Volume 56

20th European Congress of Endocrinology

Barcelona, Spain
19 May 2018 - 22 May 2018

European Society of Endocrinology 

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