Effects of insulin-like growth factors and binding protein 1 on bovine granulosa cell division in anchorage-independent culture.
Granulosa cells can exhibit the properties of stem cells and tumour cells. Contact with neighbouring cells does not inhibit their replication in vivo and they can divide in vitro while embedded in agar and thus without anchorage on a substratum. By culturing granulosa cells without anchorage, those cells that do not require anchorage, and thus exhibit at least one property of stem cells, divide. The effects of insulin-like growth factors (IGFs) and insulin-like growth factor binding protein 1 (IGFBP-1) on the replication of such cells was investigated by isolating granulosa cells from follicles (3-5 mm diameter) from cyclic cows and culturing them in soft agar-methylcellulose solution. Cell division was measured as [3H]thymidine incorporation into DNA, total DNA, or as the amount of the nuclear La antigen. This antigen is involved in RNA synthesis and is expressed ubiquitously; here it was used to estimate the number of cells indirectly. Both IGF-I (100 ng ml-1) and IGF-II (100 ng ml-1) and their respective analogues, as well as insulin, all at the same concentration, significantly increased DNA synthesis as determined by [3H]thymidine incorporation (n = 5). An increase in the number of cells in the presence of IGF-I was also confirmed by DNA measurement (P < 0.05, n = 5) and by western immunoblotting analyses of La antigen (n = 3). IGFBP-1 significantly inhibited cell division stimulated by IGF-I (P < 0.001) and IGF-II (P < 0.001), but not that stimulated by the analogue, LR3IGF-I, which has low affinity for IGFBPs. Other factors also known to affect IGF synthesis or effectiveness (FSH, oestradiol, growth hormone) did not appear to influence division of granulosa cells when cultured under anchorage-independent conditions, while dibutyryl cAMP significantly inhibited cell division (P < 0.01, n = 5). In conclusion, IGFs have a role to play in stimulating division of stem-cell-like granulosa cells during follicular development and IGFBP-1 can specifically inhibit the mitogenic effects of IGFs.
Lavranos
T C
TC
Department of Medicine, Flinders University of South Australia, Bedford Park, Australia.
O'Leary
P C
PC
Rodgers
R J
RJ
D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002455:Cell Division / Q000187:drug effects; D005260:Female; D006107:Granulosa Cells / Q000166:cytology* / Q000187:drug effects; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000494:pharmacology*; D013002:Somatomedins / Q000494:pharmacology*; D013268:Stimulation, Chemical
D018970:Insulin-Like Growth Factor Binding Protein 1; D013002:Somatomedins