Cultivation of rat granulosa cells in a serum-free chemically defined medium--a useful model to study lipoprotein metabolism.
We have developed a chemically defined, serum-free medium for the culture of rat granulosa cells. This medium contains Dulbecco's modified Eagle's medium/Ham's nutrient F12 (DME:F12) (1:1) plus insulin (2 micrograms/ml), hydrocortisone (100 ng/ml), transferrin (5 micrograms/ml) and fibronectin (2 micrograms/cm2). Granulosa cells grown in this medium have an absolute requirement for added cholesterol-rich lipoproteins for steroidogenesis. When cells are cultured in basal medium, progestin production is low; when cells are cultured in the presence of follicle-stimulating hormone (FSH) or dibutyryl cAMP [Bu)2 cAMP), progestin secretion is increased 10-100-fold. Both heterologous and homologous lipoproteins synergistically increased the effects of (Bu)2 cAMP or FSH: e.g., addition to the medium of human (h)-HDL3 produced a significant increase in both basal (approx. 15-fold) and (Bu)2 cAMP-stimulated (approx. 1000-2000-fold) progestin production. LDL were less effective than HDL at equivalent concentrations of lipoprotein cholesterol. FSH invoked changes similar to that of (Bu)2 cAMP, although the magnitude of the FSH-induced change was less dramatic than that seen with (Bu)2 cAMP. The effect of h-HDL3 and h-LDL on both basal and hormone-stimulated progestin production was concentration- and time-dependent. The maximum effect of h-HDL3 was achieved at a protein concentration of 500 micrograms/ml, with an ED50 of approx. 90 micrograms/ml. In contrast, h-LDL was most effective at a concentration of 30-40 micrograms protein/ml. Likewise, rat (r-)HDL and r-LDL supported steroidogenesis in a concentration-dependent manner. Maximal responses to all additions were observed after 72 h of treatment. Granulosa cells secreted 20 alpha-hydroxypregn-4-ene-3-one as the predominant steroid in response to (Bu)2 cAMP. However, with the addition of h-HDL3, the major secreted product was progesterone. In conclusion, rat granulosa cells maintained in the described serum-free medium are exquisitely sensitive to supplied cholesterol-rich lipoproteins. When cultured in the presence of both lipoproteins and stimulatory agents, they produce from 1000-2000-times the progestins made by comparable cells maintained in medium alone. This responsiveness of the cells to both lipoprotein and hormone stimulation makes them uniquely suitable for studies involving the uptake and metabolism of lipoproteins during steroidogenesis.
Azhar
S
S
Department of Medicine, Stanford University School of Medicine, Palo Alto, CA.
Tsai
L
L
Maffe
W
W
Reaven
E
E
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D005260:Female; D005353:Fibronectins / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008074:Lipoproteins / Q000097:blood / Q000378:metabolism*; D008954:Models, Biological; D011372:Progestins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014168:Transferrin / Q000494:pharmacology
D003470:Culture Media; D005353:Fibronectins; D007328:Insulin; D008074:Lipoproteins; D011372:Progestins; D014168:Transferrin; D003994:Bucladesine; D005640:Follicle Stimulating Hormone; D006854:Hydrocortisone