Regionalization and Redistribution of Membrane Phospholipids and Cholesterol in Mouse Spermatozoa during in Vitro Capacitation1

Abstract

Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.

Reproductive Pathology and Sperm Physiology in Acid Sphingomyelinase-Deficient Mice

“…27 In our ASM-deficient mice, lipid accumulation was histologically evident in somatic cells of the gonads (Figure 1), as well as in fully formed caudal spermatozoa ( Figure 2). Cholesterol has been localized to the acrosome region of the sperm head and the tail midpiece, 29,30 and our data showed that spermatozoa from ASMKO mice had an increase in filipin-sterol complexes over the acrosome (Figure 3, A and B).…”

Chromosome aberrations in mouse embryos and fetuses produced by assisted reproductive technology

“…Moreover the cholesterol distinctively distributes in the plasma membrane over the acrosomal region [19−22]. In conventional IVF, these components never enter the oocyte because the cholesterol is dissociated from the acrosomal plasma membrane during sperm capacitation [20,21,23] and the acrosome breaks down immediately before oocyte penetration [24].…”

Sterols in spermatogenesis and sperm maturation

“…Cholesterol effl ux is promoted by albumin ( Fig. 2 ) and high bicarbonate levels, which facilitate lipoprotein-mediated cholesterol effl ux (77)(78)(79). Cholesterol removal was restricted only to the nonraft membrane fraction of the sperm plasma membrane, whereas the content of cholesterol in the raft membrane fraction remained unchanged ( 75 ).…”