Acrosome Biogenesis in the Hamster: RESULTS(7)
Immunoblotting was performed on Triton X-100-soluble and -insoluble fractions of round spermatids using both polyclonal anti-AM22 and monoclonal anti-AM29/22 (Fig. 11). The Triton Х-100-soluble fraction possessed a polypeptide of 40 kDa that reacted with polyclonal anti-AM22 (Fig. 11, lane 2) and no immunoreactive polypeptide was found in the pellet fraction (Fig. 11, lane 3). In addition, no AM22 or AM29 were noted in these fractions. cialis professional
Parallel immunoblots stained with monoclonal anti-AM29/22 exhibited no immunoreactive bands in either the supernatant or pellet fractions (Fig. 11, lanes 5 and 6). These data suggest that the 40-kDa polypeptide of round spermatids represents a precursor that is processed during late spermio-genesis into mature AM29 and AM22. Since the monoclonal anti-AM29/22 interacts only with the fully mature acrosomal matrix polypeptides and not the precursor form, this suggests that other posttranslational modifications, in addition to size modification, may occur during processing of the acrosomal matrix precursor polypeptide.
FIG. 11. Immunoblot of 12% SDS-PAGE minigel showing cauda sperm ALM fraction (lanes 1 and 4) and Triton X-100 supernatant (lanes 2 and 5) and pellet (lanes 3 and 6) fractions of round spermatids (RS) immunostained with polyclonal anti-AM22 (lanes 1-3) and monoclonal anti-AM29/22. Note that the polyclonal antibody recognizes a 40-kDa polypeptide in the supernatant (lane 2) but not the pellet fraction (lane 3) of round spermatids, and the monoclonal antibody (lanes 5 and 6) does not react with any round spermatid protein. Molecular weight markers (x 10~3) are at left.