In order to isolate genes whose expression is up-regulated after the initiation of meiosis, we screened a cDNA expression library of newt testes with antiserum against homogenates of testes derived from the spermatogonial and spermatocyte stages. We report the isolation of spermatocyte-specific cDNA clones encoding a newt homologue of the calcium-dependent phospholipid-binding protein, annexin V. Northern blot analysis showed that newt annexin V mRNA was 1.7 kb in length and was expressed strongly in testes, but weakly in other organs. In situ hybridization revealed that the expression of newt annexin mRNA was barely observed in spermatogonia, but increased significantly in leptotene-zygotene primary spermatocytes and reached a maximum level in pachytene spermatocytes and round spermatids. The newt annexin V cDNA predicted a 323-amino acid protein and had a 68% homology to human annexin V. The predicted amino acid sequence contained a conserved 4-fold internal repeat of approximately 70 residues like other annexin proteins. Immunoblot analysis using the monoclonal antibody against newt annexin V showed that the protein was expressed scarcely in spermatogonia but was abundantly expressed in stages from primary spermatocytes to spermatids; this pattern was consistent to that of the mRNA. Immunohistochemical analysis revealed that newt annexin V was localized in the cytoplasm of the spermatogenic cells, but not in somatic cells such as Sertoli cells or pericystic cells. These results indicate that the expression of newt annexin V is up-regulated in the spermatogenic cells after the initiation of meiosis and suggest that newt annexin V plays an important role in spermatogenesis.

Animals and reagentsAdult male newts (C. pyrrhogaster) collected during winter and early spring, were purchased from Hamamatsu Seibutsu Kyozai Ltd, Hamamatsu, Japan. Adult female newts were collected during autumn. All chemicals were obtained from Nacalai, Kyoto, Japan, unless otherwise stated. A cDNA encoding a newt homolog of Escherichia coli RecA and yeast RAD51 from a testis cDNA library was isolated. The newt RAD51 (nRAD51) cDNA predicted a 337 amino acid protein with a 95-96% amino acid identity to Xenopus and mammalian RAD51. Northern blot analysis showed that nRAD51 mRNA, 1.7 kb in length, was expressed strongly in the testis and ovary, but weakly in the liver, kidney and brain. In situ hybridization revealed that expression of nRAD51 mRNA was barely observed in primary spermatogonia (one cell in a cyst) and early secondary spermatogonia (two to four cells in a cyst), but increased in late secondary spermatogonia (≥ eight cells in a cyst), reaching a maximum level in leptotene-zygotene spermatocytes, and thereafter declined. These results suggest that nRAD51 is involved in mitotic recombination in spermatogonia as well as in meiotic recombination in spermatocytes.

To isolate genes whose expression is up-regulated after initiation of meiosis, we employed an mRNA differential display method using RNA extracted from newt testis fragments in the spermatogonial and spermatocyte stages. We report here isolation of a spermatocyte stage-specific cDNA clone encoding a newt homologue of dynein intermediate chain (IC). The newt dynein IC cDNA was found to encode a polypeptide consisting of 694 amino acid residues with 66.8% and 45.8% amino acid sequence similarity to sea urchin dynein IC3 and Chlamydomonas IC69, respectively. The predicted protein contains five WD repeats and a novel repeated motif in the C-terminal region. Northern blot analysis revealed that newt dynein IC mRNA was expressed in the spermatocyte and round spermatid stages, suggesting that dynein IC plays a role in formation of flagella as well as in meiotic events.