Modulation of membrane fluidity in the primate (Macaca mulatta) corpus luteum: correlation with changes in gonadotropin binding

Abstract

Addition of alcohols to particulate or cellular preparations of the monkey corpus luteum unmasks gonadotropin-binding sites via a temperature-sensitive process. Since alcohols and temperature are known modulators of membrane fluidity, we measured the fluidity of luteal membranes and determined whether the effects of ethanol and temperature on gonadotropin binding correlated with changes in the fluid state of the membrane. The fluidity of membranes from the macaque and rat corpus luteum was estimated from the fluorescence polarization of the lipophilic membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The absorption and emission spectra of DPH incorporated into luteal membranes were typical of those in other systems. Fluorescence intensity increased rapidly during the first 60 min of incubation and reached steady state conditions within 3 h. In contrast, polarization was constant within minutes and was insensitive to pH, ionic strength, tissue concentration, or DPH levels over the ranges tested. Fluorescence polarization was acutely sensitive to the temperature of the assay medium; polarization decreased as temperature increased from 4-50 C, and no phase transitions were observed. Addition of 4-20% and 8-20% ethanol to monkey and rat membranes, respectively, decreased (P less than 0.05) polarization relative to control values. However, ethanol was less effective on rat membranes, such that 20% ethanol was required to elicit a similar change in polarization as 8% ethanol in macaque membranes. The decrease in polarization was reversed to control levels when ethanol was removed from the incubation medium. Changes in fluorescence polarization of DPH-labeled macaque membranes elicited by ethanol and temperature correlated significantly (r = -0.97) with changes in specific [125I]iodohuman LH binding. In contrast, pretreatment of luteal membranes from the monkey and rat with neuraminidase, which unmasks another population of LH-binding sites in both species, did not alter polarization. We conclude that the fluorescence polarization of DPH is a useful tool for estimating membrane fluidity in the corpus luteum. Furthermore, changes in membrane fluidity may play an important role in the masking/unmasking of alcohol-sensitive (but not neuraminidase-sensitive) gonadotropin-binding sites in the macaque corpus luteum. Finally, the lesser effects of ethanol in the rat suggest important species differences in the receptor milieu and composition of luteal membranes.