Freeze-fracture alterations in guinea pig sperm membranes preceding gamete fusion

Normal fertilization in mammals depends on several events involving membrane fusion. One, a secretory phenomenon, is the fusion of the spermatozoan acrosomal granule membrane with the plasmalemma, and another concerns the union of this nascent hybrid membrane with that of the egg. Using freeze-fracture and ancillary cytochemical procedures, we can identify three general membrane constituents: proteins, apparent as intramembranous particles; phospholipids, visible as smooth, particle-free areas; and sterols, observed as 25-nm ellipsoid protuberances when complexed in situ with the polyene antibiotic filipin. Freshly removed from the epididymis, guinea pig sperm displays distinctive patterns of intramembranous particles and a nonhomogeneous distribution of sterols. When incubated in a Ca2+-free medium that permits processing spermatozoan membranes for fusion, these membrane components become altered. In the circular foci where union occurs, the particle pattern is modified from an orderly, geometric quilted design to a random distribution; filipin-reactive sterols are deleted, followed by the loss of particles from the nonesterified sterol-deficient patches. With the addition of Ca2+, the residual smooth areas meld as the number of particles equilibrates in the plane of the hybrid acrosomal-plasma membrane. Sterols appearing in the postacrosomal segment remain reduced in quantity. After acrosomal-plasma-membrane fusion, a second line of particle-cleared circles emerges behind the margin of the new membrane suture. These clearings appear to be identical to the circles observed in the acrosomal and plasma membranes preceding the acrosome reaction. We interpret this second series of clearings as the preparation of the spermatozoan membrane for fusion with the egg. In sperm, the focal alterations antecedent to organelle (acrosome) plasma membrane union are evidently comparable to those preceding plasma membrane (sperm) plasma membrane (egg) fusion. It will be of great interest to us to discover whether this type of protein and sterol/phospholipid juncture proves to be a unifying feature of fusions in all the various biological systems discussed in this volume.