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  • Author: P. C. O'Leary x
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T. C. Lavranos, P. C. O'Leary and R. J. Rodgers

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 numbers 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.

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A. E. Drummond, G. P. Risbridger, P. C. O'Leary and D. M. de Kretser

Summary. A single dose of EDS was given to mature male rats and interstitial fluid (IF) was collected to determine the potency of mitogenic and steroidogenic activities therein. The potency of the factor stimulating testosterone secretion in vitro by Percoll-purified Leydig cells was significantly elevated 2 weeks after EDS, whilst the potency of mitogenic activities (stimulation of DNA synthesis by BALB/c 3T3 cells) was not elevated until 4 weeks after EDS treatment.

This study suggests that two separate factors, one with mitogenic and the other steroidogenic activity, may be involved in the response of Leydig cells after EDS administration. The mitogenic factor may play a role in Leydig cell regeneration whereas the testosterone-stimulating factor may be involved in testicular testosterone homeostasis.

Keywords: mitogenesis; interstitial fluid; steroidogenesis; Leydig cell