Dynamics of satellite binding protein CENP-B and telomere binding protein TRF2/MTBP in the nuclei of mouse spermatogenic line.

The location of centromeric protein CENP-B and telomeric protein TRF2/MTBP in the mouse spermatogenic line has been studied using indirect immunofluorescent and immunoelectron microscopy. CENP-B localized to the heterochromatic parts of the nuclei at meiotic stages. A clearly distinct chromocenter forms in the nucleus at stages 3-4 of spermatid maturation; CENP-B localizes in it and in the area adjacent to the future acrosome. CENP-B localization in the subacrosomal area and in the chromocenters' periphery demonstrates that centromeres are organized in two groups in mouse spermatozoa, unlike human centromeres. TRF2/MTBP concentrates around the forming chromocenter at spermiogenesis early stages. The TRF2/MTBP main signal migrates into the area of acrosomal membrane at the course of spermatozoon maturation. TRF2/MTBP never localizes inside the synaptonemal complex but can be found in the areas where the synaptonemal complex attaches to the nuclear envelope. At the pachytene and diplotene stages when chromosomes separate from the nuclear envelope, some amount of the protein remains bound to the nuclear membrane while the other part reveals itself in chromosomes. TRF2/MTBP accumulates in the future acrosome from the very beginning of its formation. In the mature spermatozoon TRF2/MTBP decorates the acrosomal membrane as well as spreads in condensed chromatin.

Telomere-binding TRF2/MTBP localization during mouse spermatogenesis and cell cycle of the mouse cells L929.

Observations of the organization and distribution of telomeres (Tel) in somatic tissues still remain controversial. The Tel topography revealed by modern microscopy shows them to be associated with the nuclear envelope (NE) in a wide variety of eukaryotic cells, although not at the Rabl orientation (peripheral position at one pole of the nucleus at prophase). We used two cell types that have different nuclear architectures. The cell line L929 shows lack of any rigid Tel architecture in the nucleus. In contrast, spermatozoa have a precise architecture established during spermiogenesis. We observed Tel and membrane Tel binding protein (MTBP/TRF2) position by immunoFISH in L929 cells and by immunofluorescence and immunogold electron microscopy, using antibodies against Membrane Tel Binding Protein (MTBP/TRF2), during different stages of spermiogenesis. At all stages of the L929 cell cycle, MTBP/TRF2 is co-localized with Tel. The only Tel order found in this cell type is similar to the Rabl-orientation, probably due to fast divisions. In the mouse pachytene spermatocytes, the membrane structures abut on the synaptonemal complex (SC) attachment sites contain MTBP/TRF2. In fully formed spermatozoa and during spermiogenesis, apart from the expected MTBP/TRF2 position at the nuclear periphery, MTBP/TRF2 unexpectedly localized at the acrosomal membrane that is adjacent to the nucleus. The difference in the MTBP/TRF2 distribution in the oocyte and spermatozoa leads to the suggestion that the MTBP/TRF2 location might reflect preparation for fertilization events. The Tel distribution is not static in cultured cells throughout the cell cycle or during spermatogenesis. When the Tel are attached to the NE, as during SC formation, MTBP/TRF2 is the member of the protein complex, which appears to be responsible for this attachment.