Differential subcellular distribution of tubulin epitopes in boar spermatozoa: Recognition of class III β-tubulin epitope in sperm tail

Abstract

The exposure of tubulin epitopes was studied in ejaculated boar spermatozoa using a panel of four monoclonal antibodies specific to the N-terminal or C-terminal structural domains of tubulin and three monoclonal antibodies against class III β-tubulin, The specificity of the antibodies was confirmed by immunoblotting. Immunocytochemical staining showed that antibodies discriminated between various parts of a spermatozoon, and that epitopes of class III β-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of β-tubulin was detected in the triangular segment of the postacrosomal part of the sperm head. Its distribution changed after an A23187 ionophore-induced acrosome reaction, indicating that tubulin participates in the early stages of fertilization, Three monoclonal antibodies, IU-20, SDL.3D10, and TuJ1 directed against epitopes on the C-terminal end of neuron-specific class III β-tubulin that is widely used as a neuronal marker, stained the flagella. The reactivity of TU-20 was further confirmed by absorbing the antibody with the immunizing peptide and by immunoelectron microscopy. Immunoblotting after two-dimensional electrophoresis revealed that the corresponding epitope was not present on all β-tubulin isoforms, These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.