Super-Resolution Imaging of Live Sperm Reveals Specific Dynamic Changes of The Actin Cytoskeleton During Acrosomal Exocytosis

GUERRERO AO; XU X; PUGA MOLINA LC; DARSZON A; TORRES RODRIGUEZ P; KRAPF D; ROMAROWSKI A; LUQUE GM; BALESTRINI PA; BUFFONE MG; VELASCO FELIZ AG; VISCONTI PE; GERVASI MG; GILIO N; RAMIREZ GOMEZ HV

New Orleans

Congreso; 51st Annual meeting of the Society for Study of Reproduction (SSR); 2018

In spermatozoa, acrosomal exocytosis (aka acrosome reaction; AR), a special type of controlled secretion, is an absolute requisite for fertilization in mammals. This complex exocytic process is controlled by several players including the actin cytoskeleton. However, how this dynamic change of the actin cytoskeleton occurs in live cells remain completely unknown in part, due to the fact that most studies were performed using phalloidins, which are toxic and not capable of crossing the cell plasma membrane. Here, we used the powerful abilities of SiR-actin, a novel membrane permeable fluorescent probe that binds to actin filaments in vivo, in combination with super-resolution microscopy to examine actin dynamics in live mouse sperm at the onset of AR. A detailed localization analysis of sperm loaded with SiR-actin or from transgenic mouse containing Lifeact-EGFP revealed six filamentous actin (F-actin) structures within the sperm head: perforatorium, lower acrosome, upper acrosome, ventral, septum and neck. The proportion of sperm possessing these structures statistically changed with capacitation. We were able to observe in great detail these F-actin structures using a combination of super-resolution techniques including Bayesian analysis of Blinking and Bleaching (3B), Super-Resolution Radiality Fluctuations (SRRF) and Stochastic Optical Reconstruction Microscopy (STORM). We have registered for the first time, depolymerization of the actin cytoskeleton in specific regions of the sperm head prior to the initiation of the AR. In contrast, we have also demonstrated that the cortical F-actin network is not actively depolymerized at the initiation of the AR suggesting that the loss of SiR-actin florescence may result from shedding through the space formed between the outer acrosomal membrane (OAM) and plasma membrane hybrid vesicles. Finally, our work emphasizes the utility of live-cell nanoscopy for the study of sperm ultra-structures, given that these imaging capabilities will undoubtedly impact the search for mechanisms that underlie basic sperm functions.