An improved method for primary culture of ovarian androgen-producing cells in serum-free medium: Effect of lipoproteins,insulin, and insulinlike growth factor-I |
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Authors: | Denis A Magoffin Gregory F Erickson |
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Institution: | (1) Department of Reproductive Medicine, School of Medicine, University of California, San Diego, 92093 La Jolla, California |
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Abstract: | Summary 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 μg cholesterol/ml) compared to hLDL (ED50=9.1±1.1 μg 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 μg/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.
This research was supported by research center grant HD 12303 from the National Institute of Child Health and Human Development,
Bethesda, MD, and USCD Academic Senate grant RM-169M |
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Keywords: | ovary theca cell interstitial cell androgen |
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