F-Actin live cell staining with SiR-actin reveals specific dynamic changes during mouse sperm acrosomal exocitosis.

VELASCO FÉLIX AG; BALESTRINI, PAULA; LUQUE, GUILLERMINA; KRAPF, DARÍO; DARSZON A; RAMÍREZ GÓMEZ HV; GILIO, NICOLÁS; GERVASI MG; VISCONTI PE; BUFFONE, MARIANO G; ROMAROWSKI, ANA; TORRES RODRÍGUEZ P; PUGA MOLINA, LIS; XU X; GUERRERO AO

Congreso; Gordon Conference on Fertilization and the Activation of Development.; 2017

Acrosomal exocytosis (AE) is an absolute requisite for fertilization in mammals. This complexexocytic process is controlled by several players including membrane potential, proteins fromthe fusion machinery system, ion channels, calcium and other ions and changes in the actincytoskeleton. Most studies to evaluate actin polymerization (formation of filamentous actin orF-actin) in mammalian sperm were performed using phalloidins, which are toxic and notcapable to cross the plasma membrane of the cell. As a result, it is not possible to use thisapproach to study this dynamic process in real time using single cells. SiR-actin is a novelmembrane permeable fluorescent probe that binds to actin filaments in vivo. Using this newtool, we aimed to describe actin polymerization dynamics in live mouse sperm. A comparisonbetween cells labeled with Alexa Fluor 488 phalloidin and SiR-actin revealed that whilephalloidin displayed a homogeneous staining over the whole sperm, SiR-actin stained specificregions within the sperm head. We also observed by image-based flow cytometry acapacitation-induced increase in polymerized actin levels. A detailed analysis of the structuresstained by SiR-actin revealed 6 patterns of F-actin localization within the sperm head:perforatorium, lower acrosome, upper acrosome, ventral, septum and neck. The same patternswere also observed when we used sperm from transgenic mice expressing LifeAct-EGFP,supporting our in vivo observations using SiR-actin. The proportion of these patterns withinginthe sample changed over the course of capacitation. We were able to observe in great detailthese structures of F-actin by using a combination of super resolution techniques includingBayesian blinking and bleaching (3B), Super-Resolution Radiality Fluctuations (SRRF)and Stochastic Optical Reconstruction Microscopy (STORM). To evaluate for the first time, thedynamic actin cytoskeletal changes at the onset of the AE, we loaded sperm with SiR-actin andFM4-64 (a marker for the AE). We also obtained recordings in TIRFM, which allowed us toevaluate the cortical actin cytoskeleton and the sperm membrane in the same cell in vivo.Interestingly, we found that all sperm that possessed the septum or the lower acrosome F-actinpattern, the actin cytoskeleton was depolymerized in those regions prior to the AE induced withionomycin, while the rest of the F-actin patterns remained unchanged. Thus, we havesimultaneously visualized actin dynamics and the AE by super resolution in vivo anddemonstrated for the first time that actin depolymerization occur in specific regions of thesperm head during the AE.