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Differential Subcellular Distribution of Tubulin Epitopes in Boar Spermatozoa: Recognition of Class III ß-Tubulin Epitope in Sperm Tail¹

Jana Pknicová^2,a

Vladimír Viklick^b

^a Department of Biology and Biochemistry of Fertilization ^b Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic ^c Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic

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 ß-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 ß-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of ß-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 ß-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 ß-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.

FOOTNOTES

First decision: 5 January 2001.

¹ This work was supported by grant nos. 524/96/K162 and 204/98/1054 from the Grant Agency of the Czech Republic, grant no. NJ 5851-3 from the Grant Agency of the Ministry of Health of the Czech Republic, and Eureka 1985-NFDK-MOAB.

² Correspondence: Jana Pknicová, Department of Biology and Biochemistry of Fertilization, Academy of Sciences at the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. FAX: 420 2 44471707;jpeknic{at}biomed.cas.cz

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