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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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Effect of LH and hCG on progesterone secretion in granulosa cells. Values are mean ± SEM (n=3).
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Figure 1: Effect of LH and hCG on progesterone secretion in granulosa cells. Values are mean ± SEM (n=3).

Mentions: A distinct morphology of the granulosa cells subjected to LH stress was observed, which differed from that of the hCG and untreated cell controls, respectively. Both hCG and LH treated cells in short term (5 day) cultures exhibited similar functional characteristics, assessed by their progesterone secretion (Fig. 1a). A distinct differential response of the ovarian granulosa cells was observed in both short (passage 0, P0, 5th day) and long term cultures (passage 1, P1, two wk) on subjecting these to sustained LH and hCG stimulation, respectively. Cell counts from day 0 to 4 revealed no significant difference in cell number after treatment with LH and hCG (Fig. 1b). Granulosa cells in primary cultures on sustained stimulation with LH began to proliferate and continued to grow at the same rate with subsequent subculture (P1-P3 passages) and were monitored up to 8 wk. On the other hand, granulosa cells in primary culture when subjected to hCG stress, on subculture, became slow growing after P1 as the number of days taken to become confluent increased. The untreated cells also became slow growing after subculture (P1 & P2) and began to degenerate after the P2 stage (data not shown). Since the cells subjected to hCG stress and untreated cells decreased in number in the long term cultures, further studies to assess the short term and long term effect of sustained hormonal stimulation on the downstream signaling molecules were conducted in 2 wk old (long term, P1) cultures and on day 5 of the primary culture (short term, P1) of granulosa cells.


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)

Effect of LH and hCG on progesterone secretion in granulosa cells. Values are mean ± SEM (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effect of LH and hCG on progesterone secretion in granulosa cells. Values are mean ± SEM (n=3).
Mentions: A distinct morphology of the granulosa cells subjected to LH stress was observed, which differed from that of the hCG and untreated cell controls, respectively. Both hCG and LH treated cells in short term (5 day) cultures exhibited similar functional characteristics, assessed by their progesterone secretion (Fig. 1a). A distinct differential response of the ovarian granulosa cells was observed in both short (passage 0, P0, 5th day) and long term cultures (passage 1, P1, two wk) on subjecting these to sustained LH and hCG stimulation, respectively. Cell counts from day 0 to 4 revealed no significant difference in cell number after treatment with LH and hCG (Fig. 1b). Granulosa cells in primary cultures on sustained stimulation with LH began to proliferate and continued to grow at the same rate with subsequent subculture (P1-P3 passages) and were monitored up to 8 wk. On the other hand, granulosa cells in primary culture when subjected to hCG stress, on subculture, became slow growing after P1 as the number of days taken to become confluent increased. The untreated cells also became slow growing after subculture (P1 & P2) and began to degenerate after the P2 stage (data not shown). Since the cells subjected to hCG stress and untreated cells decreased in number in the long term cultures, further studies to assess the short term and long term effect of sustained hormonal stimulation on the downstream signaling molecules were conducted in 2 wk old (long term, P1) cultures and on day 5 of the primary culture (short term, P1) of granulosa cells.

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