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 factor binding protein (IGFBP)-2 and particularly IGFBP-5 has been shown to increase dramatically in apoptotic granulosa cells from atretic follicles. The aim of this work was to study the relationship between apoptosis induced by serum starvation in vitro and expression of IGFBP-2 and -5 by ovine granulosa cells. For this purpose, granulosa cells from 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 viability was assessed by studying DNA fragmentation, and IGFBPs expression was studied by quantitative autoradiography, Western-ligand blotting, immunoblotting, and quantitative in situ hybridization. In vitro, IGFBP-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 IGFBP-5 associated with cell membranes and with the walls of culture wells, but no change in culture medium. The increase in the amount of cell- and wall-associated IGFBP-5 after serum starvation was essentially due to the consecutive decrease in cell density rather than to an increase in cell apoptosis. Indeed, irrespective of the presence or absence of serum, the amount of IGFBP-5 associated to cell membranes was inversely correlated to cell density. In contrast, the amount of IGFBP-5 present in culture medium was positively correlated to cell density. Furthermore, expression of IGFBP-5 mRNA was shown to increase with both cell density and cell death. Indeed, the expression of IGFBP-5 mRNA dramatically increased with cell density, irrespective of the presence or absence of serum, but at a similar cell density, expression was higher in serum-free than in serum conditions. Overall, these results indicate that, in vitro, the localization of IGFBP-5 on ovine granulosa cell membranes and in culture medium, respectively, was mainly dependent on cell density, whereas expression of IGFBP-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.
Monget
P
P
Station INRA de Physiologie de la Reproduction des Mammifères Domestiques, Nouzilly, France. monget@tours.inra.fr
Pisselet
C
C
Monniaux
D
D
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001798:Blood Proteins / Q000494:pharmacology; D015153:Blotting, Western; D002452:Cell Count; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D053938:DNA Fragmentation; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012756:Sheep
D001798:Blood Proteins; D003470:Culture Media; D018975:Insulin-Like Growth Factor Binding Protein 5; D012333:RNA, Messenger