Comparisons of the Ability of Follicular Fluid Glycosaminoglycans and Chemically Desulfated Heparin to Compete for Heparin-Binding Sites on Granulosa Cells1

Abstract

Glycosaminoglycans (GAGs) were purified from bovine follicular fluid, and their effectiveness to compete for heparin-binding sites in granulosa cells was evaluated. The GAGs dermatan sulfate (DS) and heparan sulfate (HS) were purified by anion-exchange high-performance liquid chromatography. Approximately 5 micrograms of protein from suspensions of bovine granulosa cells were incubated with 101 pmoles of [3H]heparin and 0.01-5.0 mg/ml of HS or DS for 2 h at 37 degrees C in 40 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.35. Heparan sulfate obtained from small and medium follicles displaced [3H]heparin in a dose-dependent manner from 0.1 to 5 mg/ml, but HS from large follicles did not displace [3H]heparin. The DS obtained from small, medium, and large follicles displaced [3H]heparin in a dose-dependent manner, and the potency of the DS to displace [3H]heparin increased as the size of the follicles from which the DS was purified increased. Those results were independent of the maturational state of the granulosa cells. In a separate experiment, heparin (17.1% sulfate) was N-desulfated (11.8%), and the desulfated heparin did not displace [3H]heparin. It was concluded that the effectiveness of follicular HS and DS to compete for heparin-binding sites on granulosa cells was dependent on the maturation of the follicle from which the fluid was obtained rather than on the source of granulosa cells. The binding interaction of the GAGs relies, to some extent, on the presence and positions of sulfate moieties.

Hypocoagulable State of Human Preovulatory Ovarian Follicular Fluid: Role of Sulfated Proteoglycan and Tissue Factor Pathway Inhibitor in the Fluid1

“…Specifically, they noted the absence of factor VIII and extremely low levels of factors IX and XI. The presence of sulfated proteoglycans in ovarian follicular fluid has been described in various animals, including cow [7][8][9], pig [10][11][12], sheep [13], horse [14], and rat [15][16][17]. The proteoglycans in the follicular fluid have been theorized to maintain the viscosity of the fluid in the extended follicles during follicular maturation [15].…”

Properties of heparin binding to purified plasma membranes from bovine granulosa cells

“…While one might expect a decrease in the net negative charge of the heparin or the membrane surface to result in increased binding, such calcium-induced inhibition has been observed in other heparin-protein interactions (Speight & Griffith, 1983). This is apparently due to interactions of the divalent cations with the negatively charged groups of the sulphated amino sugars, resulting in a conformational change which is unfavourable to binding (Boyd et al, 1980 (Beers, 1975 (Ax et al, 1984;Bushmeyer et al, 1985;Bellin et al, 1987b). Such a mechanism might remove the heparin-induced inhibition of progesterone secretion (Campbell & Valiquett, 1982;Bellin et al, 1987a;Ledwitz-Rigby et al, 1987).…”

“…Conversely, follicular GAGs inhibit gonadotrophin binding to cell surface receptors (Amsterdam et al, 1979;Nimrod & Lindner, 1980) possibly by steric specific, saturable, reversible, and dependent on the pH and ionic composition of the medium. Other factors which influence GAG binding to granulosa cells include the maturation state of the follicle from which the cells are obtained (Ax et al, 1984;Bushmeyer et al, 1985;Bellin et al, 1987b), and the presence of GAGs already attached to the cell surface (Ax et al, 1986). The degree of sulphation of the GAG has also been shown to affect binding to mouse tumour cells (Winterbourne & Mora, 1981) and granulosa cells (Bellin et al, 1987b).…”

“…Other factors which influence GAG binding to granulosa cells include the maturation state of the follicle from which the cells are obtained (Ax et al, 1984;Bushmeyer et al, 1985;Bellin et al, 1987b), and the presence of GAGs already attached to the cell surface (Ax et al, 1986). The degree of sulphation of the GAG has also been shown to affect binding to mouse tumour cells (Winterbourne & Mora, 1981) and granulosa cells (Bellin et al, 1987b).…”

Binding of bovine follicular fluid glycosaminoglycans to fibronectin, laminin and low-density lipoproteins

“…In contrast, production of heparan sulphate and dermatan sulphate and their associated proteoglycan cores is stimulated by FSH (Bellin et al, 1983;Ax et al, 1985). Grimek & Ax (1982) and Grimek et al (1984) Bushmeyer et al, 1985;Bellin et al, 1987a (Bellin et al, 1987b (Bellin et al, 1987b).…”