Endocrinology, Vol 119, 481-489, Copyright © 1986 by Endocrine Society

ARTICLES

Stimulation of endothelial cell proliferation by rat granulosa cell- conditioned medium

RD Koos

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum- free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8- fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.

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