Behavior of the Gamete Membranes during Sperm Entry into the Mammalian Egg

Techniques for fertilizing golden hamster eggs in vitro have recently been developed (4, 14). Consequently, sperm entry into the egg can be followed with the phase-contrast microscope, and after semination eggs can be fixed at desired intervals for subsequent examination with the electron microscope. The probability of obtaining thin sections that demonstrate sperm penetration of an egg is enhanced if the eggs are polyspermic. Phase-contrast microscope observations show that monospermic entry and polyspermic entry into eggs does not differ with respect to behavior of the spermatozoa. Compare Figs. 1, 6, and 7 to photographs in earlier studies (1, 2) of monospermic, in vivo fertilized eggs (also see reference 9 for discussion). Thus, in the present study use was made of polyspermic eggs obtained from a preparation set up for fertilization in vitro as described by Barros and Austin (4) and Barros (3). Eggs were fixed either with osmium tetroxide alone or with glutaraldehyde followed by osmium tetroxide. Recent evidence (5, 6) has shown that the acrosome reaction of mammalian spermatozoa involves extensive vesiculation between the sperm plasma membrane and the outer acrosomal membrane except in that portion covering the posterior region of the acrosome, the equatorial segment. In some specimens the acrosomal material of this segment has a septate appearance. Most of the equatorial segment persists on spermatozoa that have crossed the zona pellucida, whereas the rest of the acrosome remains outside. Some spermatozoa in the zona or in the perivitelline space show vesiculation (as described above) in the equatorial segment, but whether these sperm enter the egg is unknown. As previously indicated in a study of sperm penetration in the rat (11), the mid-lateral region of the sperm head enters the egg first (Figs. 1 and 2). Entry of the sperm head involves fusion of the plasma membranes of the sperm and egg (Fig. 3) as was first demonstrated in invertebrates by Colwin and Colwin (7, 8) and in mammals by Szollosi and Ris (12). However, as has not been previously recognized in mammals, incorporation of the sperm head progresses from the mid-region toward the anterior and posterior ends, the anterior end entering the egg last. Incorporation of the posterior region of the sperm head involves a degree of invagination of the fused cell membranes of the sperm and egg (Figs. 2 and 3), but apparently follows the pattern of sperm entry first described in the annelid, Hydroides hexagonus (7, 8). However, the anterior end of the sperm head is drawn below the egg surface within a channel. The channel may later become a true phagocytotic vesicle, but this has not yet been ascertained (Fig. 4). The vesicle or channel originates from the anterior limit of the equatorial segment of the sperm head (Fig. 5). Any portion of the sperm head which lies anterior to the equatorial segment appears to be enveloped by an intact egg cellmembrane. A large part of the equatorial segment is seen within the egg cortex (Figs. 4 and 5), and the structural organization of that region resembles that of a "septate" desmosome (see reference 13). The origin of the outer membrane (om, Fig. 5) of the desmosome-like region has not yet been determined. If the vesiculation observed at the equatorial segment is complete before sperm penetration, the outer membrane is of egg origin, but if the equatorial segment persists intact until after sperm entry the outer membrane is of sperm origin. This problem of membrane identification does not exist anterior to the equatorial segment (Fig. 5), the anterior limit of which is indicated by a ring of subacrosomal material (Fig. 5). The equatorial segment is a characteristic structure of mammalian spermatozoa (10) and probably plays a significant