Abstract
We have reported several aspects of a mouse sperm maturation-associated antigen, a 54, 000 dalton (54K) sialoglycoprotein, of which epitope can be masked by sialic acid residues against a monoclonal antibody T21 (35-38). However, the producing cell of the 54K sialoglycoprotein has long been unclear. This report documents the origin of the 54K sialoglycoprotein based on morphological evidence using avidin-biotin peroxidase complex (ABC) immunohistochemistry and indirect immunoperoxidase-electron microscopy with a recently established monoclonal antibody MC81. Immunoblots of Nonidet P-40 extract of cauda epididymidis sperm after one-dimensional, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that MC81 recognized a major protein of 54, 000 dalton (54K). The epitope for MC81 antibody was suggested to be present on a sugar chain of the 54K protein when assessed by periodate treatment. Immunoblots of sperm extracts separated by two-dimensional SDS-PAGE indicated that a major spot of 54K dalton was in acidic range less than 5 in the isoelectric focusing point. These results suggest that the antigen of MC81 is identical to that of T21. Immunohistochemistry with MC81 revealed that principal cells located at the distal caput to corpus epididymidis were immunoreactive. The region where the principal cells were first recognized by MC81 was more proximal than the region where luminal sperm were first stained. In addition, MC81 exclusively recognized the Golgi apparatus area at the supranuclear region of the immunoreactive principal cells. Sialidase-treatment prior to primary antibody (MC81) increased of the immunostaining intensity in all immunochemical studies examined. Together with the result of the previous part of this study, it is strongly suggested that the 54K sialoglycoprotein can be secreted by the principal cells at the distal caput to corpus epididymidis, with the epitope for MC81 masked by sialic acids.
