Acrosome Biogenesis in the Hamster: RESULTS(5)
Immunoelectron microscopy was employed to define precisely the intra-acrosomal distribution of AM22 and AM29. Post-embedding immunolabeled cauda epididymal spermatozoa exhibited specific labeling throughout the ml and m2 matrix regions of the acrosome (Fig. 7, A-С), and no labeling was noted in the equatorial segment (Fig. 1C).
Other structures of the head, including the perinuclear material, the nucleus, and the postacrosomal sheath, as well as all flagellar organelles, exhibited no specific labeling. For pre-embedding immunolabeling, spermatozoa were permeabilized using nitrogen cavitation, before treatment with antibody probes. Specific labeling of both the ml and m2 matrix elements of the acrosomal cap was noted (Fig. 8, A and B), but neither the outer nor the inner acrosomal membranes were specifically labeled (Fig. 8, A and B). Gold particles were located at the surface of both the ml and m2 acrosomal matrix with the pre-embedding labeling protocol, while no gold particles were noted within the matrix; this probably reflects the inability of the primary and/ or secondary antibodies to penetrate the interior of the intact matrix (Fig. 8, A and B). birth control pills
FIG. 7. Post-embedding immunoelectron microscopy of epididymal spermatozoa using polyclonal anti-AM22. A) Cross section through anterior region of sperm head. Note the specific localization of gold particles to the regions of the acrosome corresponding to the location of the ml and m2 matrix elements. No specific labeling of the nucleus (n) or perinuclear material (p) is noted. B) Cross section through anterior region of sperm head again showing specific intra-acrosomal labeling at the sites occupied by ml and m2 matrix assemblies. C) Section through sperm head at level of the equatorial segment. Labeling is apparent over regions occupied by the ml and m2 matrix, but the equatorial segment (es) is not labeled. A and В, X78 ООО; С, X60 000 (reproduced at 69%). n, Nucleus; p, perinuclear material; ia, inner acrosomal membrane; ps, postacrosomal segment.
FIG. 8. Pre-embedding immunoelectron microscopy of epididymal spermatozoa permeabilized by nitrogen cavitation and then labeled with anti-AM29/22. A) Portion of released acrosomal cap showing specific labeling of ml and m2 matrix elements. Gold particles are confined to surface of the matrix assemblies and appear unable to penetrate its interior. Note the absence of gold particles on the outer acrosomal membrane (oa), which remains associated with the acrosomal matrix. B) Cross section through anterior tip of sperm head showing the inner acrosomal membrane (ia), the perinuclear material (p), the outer acrosomal membrane (oa), and m2 matrix. Only the m2 matrix exhibits specific labeling. A, X46 ООО; В, X75 000 (reproduced at 91%).