Activation of the LH receptor up regulates the type 2 adiponectin receptor in human granulosa cells

Abstract

Purpose: Adiponectin is a predominantly adipocyte-derived hormone which influences insulin sensitivity and energy homeostasis through at least two receptors, AdipoR1 and AdipoR2. In animal models, adiponectin may regulate ovarian steroidogenesis, folliculogenesis, and ovulation. The receptors AdipoR1 and AdipoR2 are present in the human ovary, but their regulation is unknown. In these studies, we determined the effects of LH receptor activation on the expression and function of the two adiponectin receptors in human granulosa cells.

Methods: Granulosa cells were obtained at the time of oocyte retrieval in women undergoing in vitro fertilization (IVF). Cells were isolated and cultured for 48 h in DMEM/F12 medium with 5 % FBS and 50 ug/ml gentamicin. Medium was changed to low serum for 12 h and cells were treated with hCG (100 ng/ml), forskolin (30 μMol/L), or FSH (1 IU/ml) for 24 h for mRNA experiments. mRNA was isolated and RT PCR was performed using Taqman assays and quantification with the delta delta CT method. For immunocytochemistry, cells were grown on chamber slides and treated with hCG for 1 to 24 h and fixed with acetone. ICC was performed with polyclonal rabbit primary antibodies followed by alexa fluor goat anti-rabbit antibody and imaging with a fluorescence microscope and Zeiss software analysis. 3β-hydroxysteroid dehydrogenase (3βHSD) enzyme activity was determined by measuring the progesterone produced when cells were provided with an excess of 22-hydroxy-cholesterol as substrate following an incubation with hCG (1 IU/ml) and/or adiponectin (10 ng/ml). Progesterone content in the media was determined by ELISA.

Results: Messenger RNA for the two Adiponectin receptors is differentially regulated by activation of LHR with hCG treatment. AdipoR2 was increased nearly 4-fold (p < 0.05), whereas AdipoR1 expression was not changed by hCG treatment. Treatment with either FSH or forskolin (an activator of cAMP) had similar effects. Basal AdipoR2 protein was fairly low in granulosa cells in culture however treatment of cells with hCG resulted in a discernible increase in immunodetectable cytoplasmic protein as early as 6 h after treatment and was maintained for at least 24 h. The number of cells positive for AdipoR2 at 6 h increased from a basal of 20 % to almost 60 % (p < 0.05). Adiponectin treatment of hCG-primed cells resulted in increased 3βHSD activity by approximately 60 % over hCG alone and more than 3-fold over basal levels.

Conclusions: AdipoR2 is regulated by the LH receptor function via a cAMP dependant mechanism. Increased expression of adipoR2 prior to and following ovulation may contribute to enhanced 3βHSD activity and increased progesterone secretion by the corpus luteum of the ovary. Dysregulation of adiponectin that may occur with PCOS may impair normal progesterone production.

Fig. 1

AdipoR1 (gray bars) and AdipoR2 (blue bars) mRNA expression in human granulosa cells cultured for 24 h with or without hCG (10 IU), Forskolin (10 uM) or FSH (10 IU). Experiment was repeated at least 7 times with different cell isolations. Bars represent average with SEM. * represents difference from control (p < 0.05)

Fig. 2

Fluorescence (a, c, e) and phase contrast (b, d, f) micrographs of human granulosa cells cultured with hCG (10 IU) at 0 h (a, b), 6 h (c, d), and 24 h (e, f). Cells were incubated with an antibody for AdipoR2 and imaged as described in materials and methods. The positive signal is seen as red. In Fig. 2g, at least 100 cells from 4 different cell isolations were imaged and counted for positive staining for AdipoR2 with defined thresholds as described in materials and methods. Bars represent averages with SEM. * represents significantly different from control (p < 0.05)

Fig. 3

3-β-hydroxysteroid dehydrogenase activity of cells incubated for 2 h with or without hCG (10 IU) and adiponectin (25 ng/ml). a is different from b which is different from c (p < 0.05)

Fig. 4

A schematic of the events within the granulosa cell depicting how the interactions of LHR and adiponectin might occur. LH or hCG binds and activates the LH receptor which increases cAMP and activates the PKA pathway of signal transduction. This increases AdipoR2 mRNA expression and also results in the translocation of R2 to the cell membrane. Adiponectin binding of AdipoR2 activates an as-yet undetermined signaling pathway and increases the production of Progesterone from the 22OH-cholesterol substrate