EXPRESSION OF INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-5 BY OVINE GRANULOSA-CELLS IS REGULATED BY CELL-DENSITY AND PROGRAMMED CELL-DEATH IN-VITRO

In vivo, in the sheep ovary, the expression of insulin-like growth fac tor binding protein (IGFBP)-2 and particularly IGFBP-5 has been shown to increase dramatically in apoptotic granulosa cells from atretic fol licles. The aim of this work was to study the relationship between apo ptosis induced by serum starvation in vitro and expression of IGFBP-2 and -5 by ovine granulosa cells. For this purpose, granulosa cells fro m follicles 1-3 mm in diameter were cultured in the presence of serum for 2 days, then cultured in the presence or absence of serum for 24, 48, or 72 hr. At the end of the culture, cells were counted, cell viab ility was assessed by studying DNA fragmentation, and IGFBPs expressio n was studied by quantitative autoradiography, Western-ligand blotting , immunoblotting, and quantitative in situ hybridization. In vitro, IG FBP-2 and particularly IGFBP-5 were the main IGFBPs secreted by ovine granulosa cells. Serum starvation provoked (i) apoptosis of granulosa cells within 48 hr, (ii) a marked decrease in cell density, and (iii) a marked increase in the amount of ICFBP-5 associated with cell membra nes and with the walls of culture wells, but no change in culture medi um. The increase in the amount of cell- and wall-associated IGFBP-5 af ter serum starvation was essentially due to the consecutive decrease i n cell density rather than to an increase in cell apoptosis. Indeed, i rrespective of the presence or absence of serum, the amount of ICFBP-5 associated to cell membranes was inversely correlated to cell density . In contrast, the amount of IGFBP-5 present in culture medium was pos itively correlated to cell density. Furthermore, expression of IGFBP-5 mRNA was shown to increase with both cell density and cell death. Ind eed, the expression of ICFBP-5 mRNA dramatically increased with cell d ensity, irrespective of the presence or absence of serum, but at a sim ilar cell density, expression was higher in serum-free than in serum c onditions. Overall, these results indicate that, in vitro, the localiz ation of IGFBP-5 on ovine granulosa cell membranes and in culture medi um, respectively, was mainly dependent on cell density, whereas expres sion of ICFBP-5 mRNA was related to both cell density and cell death. These data suggest that IGFBP-5 is involved in both growth arrest and apoptosis of granulosa cells in the sheep. I. Cell. Physiol. 177:13-25 , 1998. (C) 1998 Wiley-Liss, Inc.