Development of a culture system for bovine granulosa cells: effects of growth hormone, estradiol, and gonadotropins on cell proliferation, steroidogenesis, and protein synthesis
1991
Langhout, D.J. | Spicer, L.J. | Geisert, R.D.
The objectives of the present studies were 1) to develop a culture system that has the positive effect of serum on granulosa cell attachment and allows subsequent expression of hormonal effects in serum-free medium and 2) to determine the effect of insulin, epidermal growth factor (EGF), estradiol (E2), and growth hormone (GH) on growth, steroidogenesis, and(or) protein synthesis of bovine granulosa cells. Cells from small (1 to 5 mm) and large (> 8 mm) follicles were collected from cattle and cultured for either 4 or 6 d. When cells from small follicles were cultured, insulin (5 micrograms/ml) increased (P < .05) cell numbers (cells X 10(5)/well) severalfold compared with controls. Alone, EGF (10 ng/ml), FSH (200 ng/ml), LH (200 ng/ml), E2 (2 micrograms/ml), or GH (0 to 1,000 ng/ml) had no effect on cell numbers. However, when included with insulin, 30, 100, and 300 ng/ml of GH increased (P < .05) granulosa cell numbers on d 4 of culture. Insulin alone increased (P < .05) progesterone production (ng.10(5) cells-1.24 h-1) by severalfold on d 4, but EGF, FSH, LH, or GH alone had no effect and E2 inhibited progesterone production. In the presence of insulin, FSH and GH (100 ng/ml) increased (P < .05) progesterone production on d 4 of culture, whereas EGF (10 ng/ml) elicited a decrease (P < .05) in production. In cells from both sizes of follicles, GH (300 ng/ml) increased synthesis of cellular proteins (> 10 kDa). In cells from only large follicles, LH (200 ng/ml) decreased synthesis and secretion of proteins (greater than or equal to 3.5 kDa). These results support the hypothesis that GH may have direct effects on bovine ovarian function.
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