Topographical distribution of phospholipids in boar sperm plasma and intracellular membranes as revealed by freeze-fracture cytochemistry

We used fracture-label and label-fracture cytochemistry in conjunction with the phospholipase A(2)-colloidal gold (PLA(2)-CG) technique to study the distribution of phospholipids in ejaculated boar spermatozoa, These techniques provide visualization of the topographical distribution of phospholipids in freeze-fractured sperm membranes in a three-dimensional view, In various freeze-fractured boar sperm membranes and crossfractured cytoplasmic structures, quantitative analysis revealed that the nuclear envelope membranes and the nuclear content possessed the highest labeling density of PLA(2)-CG. Moderate labeling was detected over acrosomal membranes, especially the inner acrosomal membrane, Replicas of both protoplasmic and exoplasmic fracture faces of the plasma membrane of boar sperm head showed a relatively low density of PLA(2)-CG labeling. Moreover, a differential distribution of phospholipids was seen over the protoplasmic face of the plasma membrane domains of the sperm head, which showed the highest concentration of gold particles in the postacrosomal region, followed by the equatorial segment and the anterior acrosome region. The PLA(2)-CG labeling densities over the postacrosomal region and the equatorial segment were significantly higher than that over the anterior acrosome region, In the flagellum, an intense labeling was also seen over cross-fractured mitochondria, dense fibers, and fibrous sheath, The protoplasmic fracture face of the plasma membrane over the middle piece, the annulus, and the principal piece was moderately labeled by PLA(2)-CG, No significant difference in mean labeling density of PLA(2)-CG was detected among the three membrane domains. In label-fracture preparations, exoplasmic halves of the plasma membrane of the head and the middle piece of the tail were uniformly labeled with PLA(2)-CG, However, the annulus and principal piece were devoid of PLA(2)-CG binding sites. These results indicate that differential distribution of phospholipids associated with the boar sperm membranes may reflect phospholipid composition of membrane domains characteristic of special physiological functions.