Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH.
Granulosa cells from fully differentiated bovine follicles were cultured in serum-free medium for 4 days. At the end of culture, the number of viable cells was low (10-15% of cells plated on day one) and only progesterone secretion responded to FSH. Insulin increased the number of viable cells at the end of culture (ED50 # 70 ng/ml) and stimulated progesterone secretion (ED50 # 50 ng/ml); the secretion of oestradiol-17 beta over basal value was evident only for concentrations of 1000 and 10,000 ng/ml. FSH acted synergistically with insulin to modify steroid secretion. In the presence of 50 ng/ml of insulin, dose-response studies indicated that secretion of progesterone was maximal at 10 ng/ml of FSH and plateaud thereafter, while oestradiol output peaked at 2 ng/ml of FSH, decreasing at higher concentrations. When cells were seeded in wells precoated with fibronectin, a comparison with cells cultured on plastic showed an increase (30-40%) in the number of viable cells at the end of culture and in oestradiol secretion but a decrease in progesterone output. These results indicate that granulosa cells from large bovine follicles, cultured in a serum-free medium containing insulin, maintain their steroidogenic potency for at least 4 days. Moreover, they show that oestradiol and progesterone synthesis are differentially sensitive to FSH concentrations and that fibronectin increases oestradiol secretion in response to FSH.
Saumande
J
J
INRA Station de Physiologie de la Reproduction, Monnaie, France.
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000008:administration & dosage / Q000494:pharmacology*; D005640:Follicle Stimulating Hormone / Q000008:administration & dosage / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D007328:Insulin / Q000008:administration & dosage / Q000494:pharmacology*; D011374:Progesterone / Q000378:metabolism
D005353:Fibronectins; D007328:Insulin; D011374:Progesterone; D004958:Estradiol; D005640:Follicle Stimulating Hormone