title: Modulation of human granulosa cell steroid production in vitro by tumor necrosis factor alpha: implications of white blood cells in culture.
abstract: OBJECTIVE
  To test the hypothesis that the macrophage cytokine, tumor necrosis factor-alpha (TNF-alpha), directly inhibits progesterone, estrone (E1), and estradiol (E2) synthesis by human granulosa cells in vitro in the presence and absence of white blood cells.
  
  
  METHODS
  Granulosa cells from follicle aspirates of patients undergoing in vitro fertilization (IVF) were separated from red blood cells on 50% Percoll columns. Such preparations contained numerous white blood cells (lymphocytes, 40-52%, and macrophages, 6-14%) as determined with immunocytochemistry. In some studies, anti-CD45 magnetic beads followed by an additional adherence step and media change were used to remove white blood cells from granulosa cell cultures. Granulosa cells with and without associated white blood cells were cultured in basal and hCG-supplemented media. Androstenedione (40 ng/mL) and/or recombinant TNF-alpha (0.5-50 ng/mL) were added to triplicate wells. Media were harvested for radioimmunoassay of progesterone, E1, and E2 after 24 and 48 hours of incubation.
  
  
  RESULTS
  The effects of TNF-alpha on progesterone production in white blood cell-associated cultures were inconsistent when 0.5 ng/mL TNF-alpha was added under basal conditions. At higher TNF-alpha doses (5-50 ng/mL) and under hCG-stimulated conditions, there was a consistent decrease in progesterone production, but the effect was not clearly dose-dependent. It was possible to remove white blood cells effectively from granulosa cell cultures. In granulosa cell cultures without associated white blood cells, 0.5 ng/mL of TNF-alpha at 48 hours produced an increase in progesterone, whereas 50 ng/mL of TNF-alpha decreased progesterone (P < .001). Estrone and E2 were both decreased by TNF-alpha regardless of whether white blood cells were present in culture, without clear evidence of dose-dependency. Granulosa cell viability and proliferation were unaffected by TNF-alpha as demonstrated by direct cell counts, trypan blue exclusion, and tetrazolium salt viability assays.
  
  
  CONCLUSIONS
  In the normal ovary, TNF-alpha may influence the development of the dominant follicle by inhibiting aromatase activity. It may also mediate oocyte maturation disorders and ovarian endocrine dysfunction in some pathologic states. White blood cells can be effectively removed from granulosa cell cultures. Application of this removal technique will facilitate future granulosa cell studies by allowing more precise determination of direct granulosa cell function.
authors.affiliation: Fearing Research Laboratory, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.
authors.forename: C L
authors.initials: CL
authors.lastname: Best
authors.forename: J
authors.initials: J
authors.lastname: Pudney
authors.forename: D J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: J A
authors.initials: JA
authors.lastname: Hill
mesh_terms: D000735:Androstenedione; D001141:Aromatase; D002452:Cell Count; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000032:analysis / Q000378:metabolism* / Q000502:physiology*; D004970:Estrone / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D017493:Leukocyte Common Antigens; D007962:Leukocytes / Q000502:physiology*; D008264:Macrophages / Q000502:physiology; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development; D011374:Progesterone / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011863:Radioimmunoassay; D013778:Tetrazolium Salts; D014343:Trypan Blue; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D006063:Chorionic Gonadotropin; D003470:Culture Media; D013778:Tetrazolium Salts; D014409:Tumor Necrosis Factor-alpha; D004970:Estrone; D000735:Androstenedione; D011374:Progesterone; D004958:Estradiol; D001141:Aromatase; D017493:Leukocyte Common Antigens; D014343:Trypan Blue