title: Differential response to sustained stimulation by hCG & LH on goat ovarian granulosa cells.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Chanchal
authors.initials: C
authors.lastname: Gupta
authors.forename: Trimbak
authors.initials: T
authors.lastname: Chapekar
authors.forename: Yash
authors.initials: Y
authors.lastname: Chhabra
authors.forename: Prashant
authors.initials: P
authors.lastname: Singh
authors.forename: Subrata
authors.initials: S
authors.lastname: Sinha
authors.forename: Kalpana
authors.initials: K
authors.lastname: Luthra
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D006041:Goats; D006107:Granulosa Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D007986:Luteinizing Hormone / Q000494:pharmacology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D061251:Primary Cell Culture; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D006063:Chorionic Gonadotropin; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D048052:Mitogen-Activated Protein Kinase 3