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

BUFFONE MG; GUERRERO AO; VISCONTI PE; GILIO N; XU X; BALESTRINI P; GERVASI MG; TORRES RODRÍGUEZ P; RAMÍREZ GÓMEZ HV; ROMAROWSKI A; DARSZON A; KRAPF D; PUGA MOLINA L; LUQUE GM; VELASCO FÉLIX AG

Conferencia; Gordon Research Conference and Seminars: Fertilization and Activation of Development; 2017

Acrosomal exocytosis (AE) is an absolute requisite for fertilization in mammals. This complex exocytic process is controlled by several players including membrane potential, proteins from the fusion machinery system, ion channels, calcium and other ions, and changes in the actin cytoskeleton. Most studies to evaluate actin polymerization (formation of filamentous actin or F-actin) in mammalian sperm were performed using phalloidins, which are toxic and not capable of crossing the cell plasma membrane. As a result, it is not possible to use this approach to study this dynamic process in real time using single cells. SiR-actin is a novel membrane permeable fluorescent probe that binds to actin filaments in vivo. Using this new tool, we aimed to examine actin polymerization dynamics in live mouse sperm. A comparison between cells labeled with Alexa Fluor 488 phalloidin and SiR-actin revealed that while phalloidin displayed a homogeneous staining over the whole sperm, SiR-actin stained specific regions within the sperm head. We also observed by image-based flow cytometry a capacitation-induced increase in polymerized actin levels. A detailed analysis of the structures stained 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 patterns were 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 in the sample changed over the course of capacitation. We were able to observe in great detail these F-actin structures using a combination of super resolution techniques including Bayesian blinking and bleaching (3B), Super-Resolution Radiality Fluctuations (SRRF) and Stochastic Optical Reconstruction Microscopy (STORM). We evaluated the dynamic actin cytoskeletal changes at the onset of AE, by labelling sperm with SiR-actin and FM4-64 (a marker of AE). We also obtained recordings in TIRFM, which allowed us to evaluate the cortical actin cytoskeleton and the sperm membrane in the same cell in vivo. Interestingly, we found that in all sperm that possessed the septum or the lower acrosome F-actin pattern, the actin cytoskeleton depolymerized in those regions prior to ionomycin induced AE, while the rest of the F-actin patterns remained unchanged. Thus, we have simultaneously visualized actin dynamics and AE by super resolution in vivo and demonstrated for the first time that actin depolymerization occurs in specific regions of the sperm head during the AE.