Differential subcellular distribution of tubulin epitopes in boar spermatozoa: recognition of class III beta-tubulin epitope in sperm tail.
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 beta-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 beta-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of beta-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, TU-20, SDL.3D10, and TUJ1 directed against epitopes on the C-terminal end of neuron-specific class III beta-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 beta-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.
Pĕknicová
J
J
Department of Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. jpeknic@biomed.cas.cz
Kubátová
A
A
Sulimenko
V
V
Dráberová
E
E
Viklický
V
V
Hozák
P
P
Dráber
P
P
D020101:Acrosome Reaction; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D008297:Male; D016253:Microscopy, Immunoelectron; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D013082:Sperm Tail / Q000737:chemistry*; D013094:Spermatozoa / Q000648:ultrastructure*; D013552:Swine; D014404:Tubulin / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D014404:Tubulin