Luteinizing hormone/chorionic gonadotrophin receptor overexpressed in granulosa cells from polycystic ovary syndrome ovaries is functionally active Venkateswarlu.

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Luteinizing hormone/chorionic gonadotrophin receptor overexpressed in granulosa cells from polycystic ovary syndrome ovaries is functionally active Venkateswarlu Kanamarlapudi, Uma D. Gordon, Andrés López Bernal Reproductive BioMedicine Online Volume 32, Issue 6, Pages 635-641 (June 2016) DOI: 10.1016/j.rbmo.2016.03.003 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 The expression of luteinizing hormone/chorionic gonadotrophin receptor (LHCGR) in granulosa cells (GC) from normal and polycystic ovarian syndrome (PCOS) ovaries. Immunoblot analysis of LHCGR expression in GC isolated from normal and PCOS ovaries (A). GC were cultured in vitro for 4 days, lysed and the lysates were immunoblotted using a rabbit polyclonal LHCGR antibody (α-tubulin was used as loading control). Corresponding quantitative data for A (B). The intensity of the bands in A was quantified and normalized to the expression of loading control (α-tubulin). Values are mean ± SEM from five individual experiments (***, P < 0.001). Reproductive BioMedicine Online 2016 32, 635-641DOI: (10.1016/j.rbmo.2016.03.003) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 The activity of luteinizing hormone/chorionic gonadotrophin receptor (LHCGR) in normal and polycystic ovarian syndrome (PCOS) granulosa cells (GC). Dose-dependent (A) and time-dependent (B) accumulation of cyclic adenosine monophosphate (cAMP) in normal (Δ) and PCOS (▲) GC. GC cultured in a 24-well plate were serum starved for 2 h and stimulated with 1–1000 IU/ml of human chorionic gonadotrophin for 30 min or 10 IU/ml for 0–60 min and cAMP levels were measured in the cultured media. Values are mean ± SEM from five individual experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Reproductive BioMedicine Online 2016 32, 635-641DOI: (10.1016/j.rbmo.2016.03.003) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 The expression and activity of ARF6 in granulosa cells (GC) from normal and polycystic ovarian syndrome (PCOS). Immunoblot analysis of ARF1 and ARF6 protein expression in GC isolated from normal and PCOS women (A). The intensity of the bands was quantified, normalized to the expression of loading control (α-tubulin) and shown below the respective blots. The basal and human chorionic gonadotrophin (HCG)-induced ARF6 activation (ARF6-GTP) in PCOS and normal GC (B(i)). GC isolated from PCOS and normal women were serum starved for 2 h and stimulated without or with HCG 10 IU/ml for 30 min at 37°C. The cell lysates were incubated with GST-GGA3 PBD resin and the protein bound to the resin was analysed by immunoblotting using an anti-ARF6 antibody. The intensity of the bands in B(i) was quantified and normalized to the expression of total ARF6 (B(ii)). Values are mean ± SEM from three individual experiments (*, P < 0.05 compared with basal value in the normal group; ***, P < 0.001 compared with basal value in the same group). Reproductive BioMedicine Online 2016 32, 635-641DOI: (10.1016/j.rbmo.2016.03.003) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 The effect of the activation inhibitors of ARF6 (Myr-ARF6 or SecinH3) and ARF1 (Myr-ARF1p or BFA) on human chorionic gonadotrophin (HCG)-induced cyclic adenosine monophosphate (cAMP) accumulation in normal and polycystic ovarian syndrome (PCOS) granulosa cells (GC). The inhibitory peptide Myr-ARF1p or Myr-ARF6p (A) and BFA or SecinH3 (B) effect on cAMP accumulation in HCG-stimulated GC from normal (upper panel) and PCOS (lower panel) women was assessed. The GC from normal and PCOS women cultured in a 24-well plate were incubated in triplicate with indicated peptide (penetratin or Myr-ARF1p or Myr-ARF6p) or chemical (DMSO [solvent] or BFA or SecinH3) in Dulbecco's modified Eagle's medium (DMEM) for 2 h at 37°C and stimulated with HCG 10 IU/ml for 30 min. The cAMP levels were then measured in the culture media. Values are mean ± SEM from three individual experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with no HCG [basal] value in the same group). Reproductive BioMedicine Online 2016 32, 635-641DOI: (10.1016/j.rbmo.2016.03.003) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions