Cholesterol-enriched nanodomains analysis by single molecule localization of UnaG-D4 cholesterol probe and transient formation and detection of BODIPY-cholesterol red-shifted dimers in the plasma membrane of mouse sperm.

Abstract

Capacitation is an essential process of sperm maturation that occurs in the uterus. A key event for this process is the cholesterol efflux from the cell membrane. Evidence suggest that cholesterol is a component of lipid nanodomains (lipid rafts) whose organization and distribution change due to its efflux during the capacitation, however until now it has not been possible to observe these nanodomains directly in the cell. Our objective is to visualize and characterize the organization of cholesterol enriched nanodomains during the mouse sperm capacitation. To achieve this, we propose the use of TIRF microscopy and single molecule localization microscopy of the two separate cholesterol probes, the ligand activatable fluorescent protein UnaG and the red-shifted emission of the transiently formed DII dimer of BODIPY-cholesterol. Preliminary results show that in the plasma membrane of mouse sperms cholesterol is organized in nanodomains, but differences in size and distributions along the sperm head are observed depending on whether they were incubated in medium to induce capacitation. Experiments are currently underway to determine if these nanodomains features are characteristic of the capacitated-sperm physiology state. This research is supported by DGAPA-PAPIIT 211216.