38. Immunochemical Localization of Novel Nuclear Protein NP95 in Testis

Masahiro Muto, Yasuyoshi Kanari, Eiko Kubo, Kouichi Tatsumi, Takatoshi Uemura^* and Toshimichi Ikemura^*
(^*Dept. Evolutionary Genetics, National Institute of Genetics, Mishima, Japan)

Keywords: nuclear protein NP95, PCNA, DNA replication, cell cycle, spermatogenesis

NP95 (nuclear protein, 95 kDa) is expressed during S phase progression but is suppressed during G1 and G2/M phases in normal mouse thymocytes. Cloning and sequencing revealed that NP95 is a novel nuclear protein of 782 amino acids and contains a leucine zipper motif, a zinc finger motif, a putative cyclin A/E-CDK2 phosphorylation site, and RB-binding motifs. We also found abundant expression of Np95 mRNA in the testis. In this organ, the unique organization of the seminiferous epithelium allows precise determination of the temporal expression of specific genes during spermatogenesis. Spermatogenesis has been divided into three phases based on functional considerations: (a) the proliferative phase (spermatogonia) in which cells undergo rapid successive divisions; (b) the meiotic phase (spermatocytes) in which genetic material is recombined and segregated; and (c) the spermiogenic phase (spermatids) in which haploid spermatids differentiate into elongated ones. We studied localization of NP95 in testis sections. Immunostaining using anti-NP95 antibody revealed that NP95 was expressed in almost all cells except those of the lumenal surface of the epithelium, where elongated spermatids exist. When anti-PCNA antibody was used, the stained regions were restricted to the proliferating spermatogonial layers. To study the spatial and temporal localizations of NP95 in meiotic nuclei, we isolated spermatogenic cells and carried out double immunostaining of NP95 and RAD51 which were detected as discrete foci along the synaptonemal complex in early prophase I. The results indicated that RAD51 was detected as foci in spermatocytes, but little staining was observed in round spermatids. In contrast, NP95 was detected as discrete foci in the nuclei of both spermatocytes and round spermatids; co-localization with RAD51 was very rare. Results of the immunohistochemical analysis in testis were consistent with the expression pattern of NP95 that was observed from spermatogonia to round spermatids. RAD51 is involved in recombination and is associated with synaptonemal complex formation in meiotic prophase cells. Since NP95 was not colocalized with RAD51 in meiotic prophase cells, NP95 may not be associated with the synaptonemal complex or involved directly in the recombination process itself. The finding that NP95 was expressed in spermatids where replication does not occur is consistent with the prediction that NP95 might play some role in the mismatch-repair mechanism. As a consequence of homologous recombination and heteroduplex formation, a small amount of DNA is synthesized through mismatch DNA repair. We propose that NP95 is involved in both mitotic and meiotic progression, presumably in a post-replication fashion and is a novel cell-cycle regulator that is associated with DNA fidelity maintenance.

Publications:
1) Fujimori, A., Matsuda, Y., Takemoto, Y., Hashimoto, Y., Kubo, E., Araki, R., Fukumura, R., Mita, K., and Muto, M. : Mammalian Genome , 9, 1032-1035, 1998.
2) Kawai, H., Kitamura, Y., Nikaido, O., Tatsuka, M., Hama-Inaba, H., Muto, M., Ohyama, H. and Suzuki, F. Radiat. Res. 149, 41-51, 1998.
3) Hama-Inaba, H., Wang, B., Mori, M., Matsushima, T., Saitoh, T., Takusagawa, M., Yamada, T., Muto, M., and Ohyama, H. Mutation Research , 403, 85-94, 1998.