Acrosome Biogenesis in the Hamster: RESULTS(4)
Distribution of AM29 and AM22 in Epididymal Spermatozoa
The distribution of AM22 and AM29 between soluble and particulate sperm fractions was assessed by immuno-blotting. Total lysates of caput and cauda spermatozoa stained with polyclonal anti-AM22 exhibited the same pattern of immunoreactive polypeptides (Fig. 5, lanes 1 and 2). Triton X-100 lysates of cauda sperm were centrifuged in a microcentrifuge at 12 000 X g for 2 min, and the supernatant and pellet fractions were adjusted to the same volume. Immunoblot analyses revealed that immunoreactive polypeptides were absent in the supernatant fluid (Fig. 5, lane 3) but that they localized to the sperm pellet (Fig. 5, lane 4). This result demonstrates that the total pool of AM22 and AM29 is associated with a particulate sperm fraction.
To define the intracellular distribution of AM22 and AM29, cauda epididymal spermatozoa were immunostained with polyclonal anti-AM22 (Fig. 6, A-H) or with monoclonal anti-AM29/22 (Fig. 6, I and J). The two antibodies gave identical staining patterns. Sperm exhibited intense fluorescence of the acrosomal cap and no other structures under all conditions of permeabilization and fixation employed (Fig. 6). In formalin-fixed Triton Х-100-permeabil-ized spermatozoa, staining was restricted to the apical and principal segments of the acrosome, and no staining of the equatorial segment was noted (Fig. 6, A and B). Spermatozoa that were permeabilized by Triton X-100 extraction (Fig. 6, C, D, I, and J) or by nitrogen cavitation (Fig. 6, G and H) before formaldehyde fixation exhibited intense fluorescence of both partially or fully detached acrosomal caps, while the exposed principal segment and equatorial segment of the sperm head were negative. Control specimens immunostained with identical dilutions of preimmune serum or normal ascites fluids exhibited no fluorescence (Fig. 6, E and F). These data demonstrate that AM22 and AM29 are restricted to the apical and principal segments of the acrosome and are primarily associated with the acrosomal cap and not the inner acrosomal membrane. ventolin inhaler
FIG. 5. Immunoblot of 12% SDS-PAGE minigel showing total caput sperm lysate (lane 1), total cauda sperm lysate (lane 2), Triton X-100-soluble cauda sperm proteins (lane 3), and Triton Х-100-insoluble cauda sperm proteins (lane 4) stained with polyclonal anti-AM22. Each lane was loaded with extracts representing equivalent numbers of sperm. Note the similar patterns of immunoreactive polypeptides in lysates of caput and cauda spermatozoa. Also note that all immunoreactive cauda sperm polypeptides are present in the pellet fraction. Molecular weight markers (X 10~3) are at left.
FIG. 6. Matched phase contrast and fluorescence photographs of immunostained cauda epididymal spermatozoa. A, B) Formaldehyde-fixed Triton X-100-permea-bilized sperm stained with polyclonal anti-AM22 showing intense fluorescence of the acrosomal cap and absence of fluorescence of equatorial segment. C, D) Triton X-100-pretreated spermatozoa immunostained with polyclonal anti-AM22. Note the intense fluorescence of the partially detached acrosomal caps while the underlying anterior head and equatorial segment are negative. E, F) Triton X-1 OO-pretreated sperm immunostained with preimmune serum. No fluorescence is noted. G, H) Spermatozoa permeabilized by nitrogen cavitation and immunostained with polyclonal anti-AM22 show intense fluorescence of the partially detached acrosomal caps, whereas the underlying sperm head is negative. I, I) Triton X-1 OO-pretreated spermatozoa immunostained with monoclonal anti-AM29/22 show fluorescence of the acrosomal cap, whereas all other sperm structures are negative, ac, Acrosomal cap; es, equatorial segment; ah, anterior head; ps, postacrosomal segment; mp, midpiece. Bar = 10 ц,т.