Gamete Biology

The Macaque Sperm Actin Cytoskeleton Reorganizes in Response to Osmotic Stress and Contributes to Morphological Defects and Decreased Motility

Abstract
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 biological 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, stained with phalloidin-FITC and processed for microscopy. The majority of sperm incubated in medium osmolality media exhibited post acrosomal stain while the minority displayed banding patterns of F-actin stain in the head. High osmolality media, and 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 sub-lethal sperm flagellar and motility defects.

Key words: Gamete Biology • Sperm • Sperm motility and transport • Stress