FOLLICLE-STIMULATING-HORMONE AND HUMAN CHORIONIC-GONADOTROPIN INDUCEDCHANGES IN GRANULOSA-CELL GLYCOSYL-PHOSPHATIDYLINOSITOL CONCENTRATION

In the present investigation, a hCG sensitive glycosyl-phosphatidylino sitol (GPI) was isolated from cultured rat granulosa cells obtained fr om the ovaries of diethylstilbestrol (DES) implanted immature rats. Th e inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galact ose, glucosamine, and myoinositol as demonstrated by metabolic labelli ng of granulosa cells for different time periods (5-96 h) with [H-3]ga lactose, [H-3]glucosamine, or [H-3]myoinositol and treatment of the pu rified [H-3]GPI with phosphatidylinositol-specific phospholipase C. La belling equilibrium of the GPI-lipid was achieved after 24 h ([H-3]gal actose and [H-3]myoinositol) or 72 h ([H-3]glucosamine) incubation, wh ereas incorporation of other labelled carbohydrates tested ([H-3]galac tosamine, [H-3]mannose, and [H-3]sorbitol) was negligible throughout t he time period studied. The glucosamine C-1 appears to be linked throu gh a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitr ous acid deamination of dual labelled ([H-3]glucosamine/[C-14]palmitat e or [H-3]glucosamine/[C-14]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5-72 hr) with [C-14]linoleate, [H-3]myristate, [H-3}-oleate, [H-3]palmitate, or [H- 3]stearate and the radioactivity associated with the purified glycosyl -phosphatidylinositol determined. Incorporation of [H-3]palmitate and [H-3]myristate into the GPI-lipid peaked after 8 h and 24 h of labelli ng, respectively, and both fatty acids were partially released after P LA, treatment of the dual labelled ([H-3]glucosamine/[C-14]palmitate o r [H-3]glucosamine/[C-14]myristate) GPI. In parallel experiments no si gnificant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosyl-phosphatidylinositol could be d etected. Granulosa cells were also labelled with [H-3]glucosamine in t he presence of FSH (30 ng/ml), cholera toxin (1 mug/ml), or the membra ne permeable cAMP analog (bUt)2cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3- , 5-, and 7-fold for FSH, CT, and (bUt)2cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were label led for 72 h with [H-3]glucosamine in the presence of (but)2cAMP (1 mM ), TPA (10(-7) M), or combination thereof. The effect of treatment wit h the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [H-3]GPI content could be observed in untreated granulosa cells or cells cultured in the pre sence of the protein kinase C-activating phorbol ester alone. In cells differentiated with FSH (30 ng/ml for 3 days) to induce LH receptors, treatment with hCG (100 ng/ml) induced a rapid (60 sec) and transient (5 min) decrease in the GPI content, whereas no effect of the hormone on undifferentiated granulosa cells could be observed. The rapid effe ct elicited by hCG on GPI content and turnover may be an early transdu ction mechanism involved in the biological effects of LH/hCG in differ entiated granulosa cells.