title: An improved method for primary culture of ovarian androgen-producing cells in serum-free medium: effect of lipoproteins, insulin, and insulinlike growth factor-I.
abstract: Although luteinizing hormone (LH) alone stimulates ovarian interstitial cells cultured in serum-free medium to synthesize large amounts of androgens, there seem to be additional factors in vivo that modulate the time course and magnitude of the cellular responses to LH. In an attempt to develop a more nearly physiologic cell culture model, lipoproteins, insulin, and insulinlike growth factor-I (IGF-I) were added to the serum-free medium. The effects of these modifications on androgen biosynthesis by dispersed cells from ovaries of hypophysectomized immature rats cultured in 96-well tissue culture plates were examined. A saturating dose of LH stimulated a 25-fold increase in androsterone synthesis at 2 d, which decreased at 4 and 6 d. Addition of human high density (hHDL) or human low density lipoprotein (hLDL) caused a 2.5-fold increase in LH-stimulated androsterone synthesis. Cells were approximately twice as sensitive to hHDL (ED50 = 5.5 +/- 0.5 micrograms cholesterol/ml) compared to hLDL (ED50 = 9.1 +/- 1.1 micrograms cholesterol/ml). Surprisingly, rat HDL caused only a 40% increase in LH-stimulated androsterone synthesis. When insulin alone was added to cells cultured with a saturating dose of LH, there was a 2.8-fold increase in androsterone synthesis. Addition of hHDL and insulin together caused a synergistic increase in LH-stimulated androsterone synthesis. In contrast to hHDL, which did not change the time course of LH-stimulated androsterone production, insulin prolonged maximal LH-stimulated androsterone synthesis at 4 and 6 d. Inasmuch as the ED50 for insulin action (1.3 +/- 0.1 micrograms/ml) was supraphysiologic, the effects of IGF-I on LH-stimulated androgen synthesis were examined. IGF-I mimicked all of the effects of insulin, but at a physiologic concentration (ED50 = 2.5 +/- 0.3 ng/ml). Ovarian cells cultured in serum-free medium supplemented with hHDL and insulin or IGF-I exhibit responses that closely approximate the physiologic responses observed in vivo. These results suggest that lipoproteins and IGF-I are important physiologic stimulators of ovarian theca-interstitial cell androgen biosynthesis which, when added to the serum-free medium, make the cellular responses in this in vitro model more nearly approximate the responses in vivo.
mesh_terms: D000728:Androgens / Q000096:biosynthesis*; D000738:Androsterone / Q000096:biosynthesis; D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D005260:Female; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008075:Lipoproteins, HDL / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D007986:Luteinizing Hormone / Q000494:pharmacology; D008954:Models, Biological; D010053:Ovary / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013002:Somatomedins / Q000494:pharmacology*; D013799:Theca Cells / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
chemical_list: D000728:Androgens; D003470:Culture Media; D007328:Insulin; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007986:Luteinizing Hormone; D000738:Androsterone