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

Gupta C, Chapekar T, Chhabra Y, Singh P, Sinha S, Luthra K - Indian J. Med. Res. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

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.

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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.
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Figure 7: 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.

Mentions: We studied the expression of total and phosphorylated ERK in the untreated hCG and LH treated cells by Western blot (Fig. 4 a, b) and flow cytometry (Fig. 4c–f. A similar increase was observed in phosphorylated ERK 1/2 (activation) in granulosa cells on hCG or LH stress in the cells in short term cultures by flow cytometry (Fig. 4 d). The expression of pERK1/2 was similar in hCG treated and LH treated cultures in short term cultures as shown by Western blot (Fig. 4 b). At two wk (long term culture), however, the activation of ERK 1/2 was maximum in LH treated cells as compared to those in hCG treated and untreated control cells on flow cytometric analysis, as shown in Fig. 4 g. Protein kinase C activity was found to be significantly (P<0.05) higher in LH treated cultures as compared to hCG treated short term cultures at 60 min of hormonal treatment. However, at 0 and 15 min, there was no difference in control or the treated groups (Fig. 3c). Both hCG and LH suppressed PKC expression in the long term cultures (Fig. 3d).


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

Gupta C, Chapekar T, Chhabra Y, Singh P, Sinha S, Luthra K - Indian J. Med. Res. (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3361869&req=5

Figure 7: 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.
Mentions: We studied the expression of total and phosphorylated ERK in the untreated hCG and LH treated cells by Western blot (Fig. 4 a, b) and flow cytometry (Fig. 4c–f. A similar increase was observed in phosphorylated ERK 1/2 (activation) in granulosa cells on hCG or LH stress in the cells in short term cultures by flow cytometry (Fig. 4 d). The expression of pERK1/2 was similar in hCG treated and LH treated cultures in short term cultures as shown by Western blot (Fig. 4 b). At two wk (long term culture), however, the activation of ERK 1/2 was maximum in LH treated cells as compared to those in hCG treated and untreated control cells on flow cytometric analysis, as shown in Fig. 4 g. Protein kinase C activity was found to be significantly (P<0.05) higher in LH treated cultures as compared to hCG treated short term cultures at 60 min of hormonal treatment. However, at 0 and 15 min, there was no difference in control or the treated groups (Fig. 3c). Both hCG and LH suppressed PKC expression in the long term cultures (Fig. 3d).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

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.

Show MeSH
Related in: MedlinePlus