| Research Abstract |
The following two subjects were investigated in this project.
l)Molecular basis of the abnormal embryonic development in the interspecific hybrid between Oryzias latipes and O. javanicus. The inter-specific hybrid between O. latipes and O. javanicus is embryonic lethal. The M-phase promoting factor (MPF, a complex of Cdc2 and Cyclin B) is a key regulator of the cell division. Besides the wild-type MPF consisting of subunits from the same species, the cells of inter-specific hybrids are equipped with the hybrid MPF consisting of subunits derived from different species. We therefore investigated a possibility that anomalous phosphorylation catalyzed by the hybrid MPF induces the abnormal cell division, thereby making the inter-specific hybrids lethat. Using recombinant Cdc2 and Cyclin B produced in insect cells, we confirmed that the hybrid MPF phosphorylates proteins different from those phosphorylated by the wild-type MPF. We then investigated whether the production of hybrid MPF in med
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aka embryos induces the abnormal cell division. O. latipes embryos injected with O. latipes Cyclin B have normal chromosomes at M-phase, whereas O. lanpes embryos injected with O. javanicus mutant Cyclin B shows abnormal cell divisions, with the occurrence of chromosome elimination from the cells. These results strongly suggest that the abnormal embryonic development in the inter-specific hybrid is due to the hybrid MPF with subunits from different species. We also found that chromosomes derived from O. javanicus are selectively eliminated from the hybrid embryos in the early stages, suggesting the involvement of other mechanisms independent of the hybrid MPF in the abnormal development.
2)Molecular mechanisms of the abnormal germ cell formation that leads to the sterility in the interspecific hybrid between Oryzias latipes and O. curvinotus.
The F_1 hybrid between O. latipes and O. curvinotus exhibits sex-specific abnormalities. We showed that in the males, spermiogenesis proceeds and sperm-like cells each having 4C DNA are produced without prior meiosis. In the females, diploid eggs are produced through normal meiosis that follows an extra DNA replication by endomitosis occurring once in oogonia just prior to the premeiotic DNA replication. To identify the molecules responsible for the abnormal germ cell production in the hybrid, we investigated the expression patterns of proteins involved in the cohesion of sister chromatids and homologous chromosomes and in the cell cycle control, including SMC1α, SMC1β, SMC3, Rad21, SCP3, Mad2, Cdc20, Separin, Securin and p53. Immunocytological analyses revealed the followings : l)SMC3 (a component of the cohesin) in the hybrid testis shows patterns different from those found in the wild-type testis ; 2)In the hybrid testis, SCP3 (a component of the synaptonemal complex) begins to be expressed in leptotene spermatocyties, as in the wild-type testis; however, the string-like expression (representing the synaptonemal complex) is not detected in the hybrid ; 3) The expression patterns of the other proteins examined show no difference between the hybrid and wild-type testes. These results suggest that the incomplete synaptonemal complex devoid of SCP3 produced in the hybrid testis is the cause of the abnormal spermatogenesis. Less
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