Sperm undergo extreme variations in temperature and osmolality during cryopreservation, resulting in cell damage that includes plasma membrane defects, changes in cell volume, decreased motility, and flagellar defects. However, the fundamental biologic mechanisms underlying these events are poorly understood. We investigated the effects of osmotic stress and cytochalasins b (CB) and d (CD), naturally occurring toxins that disrupt actin organization, on the actin cytoskeleton and motility of Rhesus macaque sperm (Macaca mulatta). Sperm were diluted in media of low, medium, or high osmolality, or medium-osmolality media containing CB or CD, were stained with phalloidin-fluorescein isothiocyanate, and were processed for microscopy. The majority of sperm incubated in medium-osmolality media exhibited postacrosomal stain, whereas the minority displayed banding patterns of F-actin stain in the head. High-osmolality media, as well as CB and CD incubation, resulted in reorganization of F-actin into bands of stain in the majority of sperm heads. Cytochalasin b treatment also resulted in curled and looped tails, a phenomenon of hyposmotic stress, and CB and CD caused significant, dose-dependent decreases in motility determined by computer-assisted sperm assessment. Rho A cell populations were determined using flow cytometry, and immunocytochemistry analysis demonstrated that Rho A localization was altered after osmotic stress. Together, our results support a mechanism in which reorganization of the actin cytoskeleton induced by osmotic stress and potentially mediated by a Rho A signaling pathway contributes to sublethal sperm flagellar and motility defects.
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Vol. 77 • No. 6