Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: an electron microscope study

Affinity purified antibodies prepared against proteins isolated from fibrous sheath (FS) and outer dense fibers (ODF) were utilized in an immunocytochemical study of spermatids at various steps of spermiogenesis. This study, using the immunogold technique, was performed on sections of Epon or Lowicryl embedded tissues examined with the electron microscope. In the case of FS antibodies there was a selective immunoreactivity of the FS itself from step 10 onwards, but no reactivity over the plasma membrane associated FS anlagen. In addition there was a diffuse immunoreaction over the cytoplasmic matrix from step 9 until step 18 of spermiogenesis but no reactivity over the various types of dense bodies (e.g., granulated bodies, reticulated body, etc.) seen in the cytoplasm of these spermatids. In the case of ODF antibodies the ODF were immunolabeled throughout their development from step 11 onward. In addition to a diffuse immunoreactivity of the cytoplasmic matrix of spermatids from step 9 until step 18 of spermiogenesis, there was an immunolabeling of "granulated bodies." These bodies appeared in relation to ER cisternae during steps 10-14, increased in number and size during steps 15-17 and decreased in number thereafter leaving only a few coarsely granulated bodies in the residual cytoplasm which detached from late step 19 spermatids. No other cytoplasmic structures were labeled with the ODF antibody-gold complexes. Thus the granulated bodies appeared to serve as a transitory storage site for some proteins destined to form ODF, a major cytoskeletal element of the tail of rat spermatozoa.