Differential response to sustained stimulation by hCG & LH on goat ovarian granulosa cells

Abstract

Background & objectives: Chapekar established a model of ovarian tumourigenesis in mice by splenic transplantation of ovaries, resulting in sustained luteinizing hormone (LH) levels because of absence of feedback inhibition. There is increasing evidence of the differential response to LH or hCG under various experimental conditions. The effect of sustained hormonal stimulation in long term cultures is sparsely investigated. The study is aimed to determine the role of hCG and LH stress on caprine ovarian granulosa cells and their downstream signaling in short and long term cultures.

Methods: To study the response of hCG and LH stress and downstream signaling, short term cultures were set up by exposing goat ovarian granulosa cells in primary cultures to hCG and LH stress (levels beyond their physiological doses) for 5 days (P0). Cells were sub-cultured at sixth day and subjected to prolonged LH/ hCG stress for two weeks in passage 1(P1) (long term cultures). Downstream cell signaling molecules were assessed. Intracellular cAMP was estimated by ELISA. For PKA and PKC, activity assays were performed. pERK protein expressions in short term cultures were assessed by Western blot and flowcytometry; in long term cultures, pERK expression was analyzed by flowcytometry.

Results: Differential effects on cell proliferation were observed in long term cultures, where the untreated and hCG exposed cells showed markedly reduced cell proliferation after second week of exposure while LH treated cells continued to proliferate. Different levels of cAMP, PKA, PKC and phosphorylated ERK1/2 were observed on short term and long term LH stimulation. On sustained hormonal stimulation, cAMP levels were significantly (P<0.05) higher in hCG treated cultures as compared to controls and LH treated cultures. LH led to maximal elevation of ERK in long term cultures.

Interpretation & conclusions: As pERK1/2 promotes cellular proliferation, activation of ERK1/2 in LH treated cultures may be responsible for sustained growth. Prolonged LH treatment promoted growth and proliferation in caprine ovarian granulosa cells whereas prolonged exposure to hCG led to elevated levels of cAMP and decreased the rate of proliferation. Defining the signals and second messengers that act as survival or apoptotic mediators may help in elucidation of the mechanisms controlling proliferation or programmed cell death in granulosa cells.

Fig. 1a

Effect of LH and hCG on progesterone secretion in granulosa cells. Values are mean ± SEM (n=3).

Fig. 1b

Effect of LH and hCG on cell proliferation and growth rate of granulosa cells in short term culture. Data expressed as mean ± SEM.

Fig. 2a

Effect of LH/hCG on intracellular cAMP in granulosa cells in short term culture after hormonal stimulation. Data expressed as mean ± SEM. **P<0.001 compared to controls and LH cultures. b. Effect of LH/hCG on levels of intracellular cAMP in granulosa cells in long term culture subjected to prolonged hormonal stimulation (2 wk). Data expressed as mean ± SEM (n=3). *P<0.05 compared to LH.

Fig. 3a

LH and hCG increased PKA activity in a time dependent manner in short term cultures of granulosa cells (day 5). Data expressed as mean ± SEM (n=3). Increase in PKA activity is seen after stimulation with LH as well as hCG, with maximum elevation seen in LH treated cultures. **P<0.001. b. LH and hCG increased PKA activity in a time dependent manner in long term cultures of granulosa cells (2 wk). Data expressed as mean ± SEM (n=3). *P<0.05 compared to LH treated cultures in a time dependent manner. c. LH and hCG stress increased PKC activity in a time dependent manner in short term cultures of granulosa cells (day 5). Data is expressed as mean±SEM. Increase in PKC activity in a time dependent manner is seen after stimulation with LH or hCG (*P<0.05). d. PKC activity is more in untreated cells as compared to hCG treated and LH treated cultures after long term stimulation (2 wk). Data expressed as mean ± SEM. Increase in PKC activity is maximum in untreated cells (P=ns).

Figs. 4a and 4b

Western blot of total ERK1/2 (4a) and pERK1/2 (4b) in untreated, hCG treated and LH treated cultures at different time points. hCG and LH treated cultures show similar expression whereas there is no expression of pERK1/2 in untreated cultures at 0 and 40 min (L-ladder, C-positive control). Anti-β actin antibody was used to ensure equal loading.

Figs. 4c & 4d

Flowcytometric analysis of ERK after short term stimulation with LH or hCG for 30 min. hCG and LH treated cultures show similar increase in total ERK (4c) and phosphorylated ERK (4d) expression. Values are mean ± SEM of 3 observations.

Figs. 4e, 4f and 4g

Flowcytometric analysis of total (4e) and phosphorylated ERK (4 f) after sustained stimulation with LH or hCG. Expression of phosphorylated ERK in LH treated culture is significantly higher as compared to hCG treated cultures (4 g). Values given as mean ± SEM of 3 observations. *P<0.05 compared to untreated control cells.