Biochemistry & Analytical Biochemistry

Abstract

Remodelling of Lipid Rafts during In vitro Capacitation and Acrosome Reaction of Ram Spermatozoa

Carmen Colas, Rosaura Perez-Pe, Adriana Casao, Mario Ollero, Lucia Calleja, Margarita Gallego, Teresa Muiño-Blanco and Jose A Cebrian-Perez

Background: Lipid rafts are often known as Detergent-Resistant Microdomains (DRMs). We report for the first time the presence of two lipid raft markers, caveolin-1 and ganglioside GM1, on the ram sperm surface, and the effect of in vitro capacitation and acrosome reaction on these marker distributions, the protein content and lipid composition of DRM and non-DRM fractions.

Methods: Caveolin-1 and ganglioside GM1 were evidenced by immunocytochemical and fluorescence analysis, respectively. DRM and non-DRM fractions were separated by an OptiPrepTM density gradient. Cholesterol by fluorometry, GM1 by peroxidase reaction, protein content by spectrophotometry, and fatty acid profiling by gas chromatography were determined.

Results: Caveolin-1 was evidenced at the acrosome of 59.2 ± 4.3% fresh spermatozoa, and the proportion of stained cells increased (P<0.05) after capacitation. GM1 was detected at the post-acrosome and tail of all spermatozoa, and no change was found after capacitation. Cholesterol and GM1 were distributed all along the gradient, with a peak in DRM fractions. A higher proportion (P<0.001) of saturated fatty acids was found in DRM fractions, confirmed by the unsaturation index and a higher lipid/protein ratio. In vitro capacitation induced a decrease in the content of saturated fatty acids in both DRM (P<0.001) and non-DRM (P<0.01) fractions. Polyunsaturated fatty acids increased in DRMs after the acrosome reaction. All treatments resulted in lower content of cholesterol and proteins in DRM (P<0.01) and non-DRM fractions (P<0.001), and a higherGM1 content in DRMs (P < 0.05). Conclusions: Lipid raft-like microdomains were isolated in a discrete region of the gradient. Their high content of saturated fatty acids confers a highly ordered environment. Their composition is modified during in vitro capacitation and acrosome reaction.
General significance: These results represent the first characterization of ram sperm DRM, and may contribute to a better understanding of the sperm fertilizing potential acquisition mechanism.