Molecular Mechanisms of Reproductive Isolation in the Genus Medaka : Molecular and Cellular Biological Analyses of the Abnormal Embryonic Development and the Sterility in Interspecific Hybrids

本课题主要研究了以下两个方面的问题：1）青（Oryzias latipes）与野生稻（Oryzias latipes）种间杂种胚胎发育异常的分子基础;爪哇人。O. latipes和O.爪哇鳄是胚胎致死的。M期促进因子（MPF，Cdc 2和Cyclin B的复合物）是细胞分裂的关键调节因子。除了由来自相同物种的亚基组成的野生型MPF之外，种间杂种的细胞配备有由来自不同物种的亚基组成的杂种MPF。因此，我们研究了杂种MPF催化的异常磷酸化诱导异常细胞分裂，从而使种间杂种Lethat的可能性。使用重组Cdc 2和细胞周期蛋白B在昆虫细胞中产生的，我们证实，杂交MPF磷酸化的蛋白质不同于那些由野生型MPF磷酸化。然后，我们研究了在地中海中混合MPF的产生是否 ...更多信息 也就是胚胎诱导异常细胞分裂。O. latipes胚胎注射O. Cyclin B的M期染色体正常，而O. lanpes胚胎注射O. javanicus突变体细胞周期蛋白B显示异常细胞分裂，发生细胞染色体消除。这些结果表明，种间杂种胚胎发育异常是由于杂种MPF亚基来自不同物种。我们还发现，染色体起源于O。爪哇野生稻在杂种早期被选择性地从杂种胚中清除，表明在杂种胚的异常发育过程中还存在着其他机制的参与。2）导致Oryzias latipes与O. curvinotus的F_1代。latipes和O.弯背龙表现出性别特异性异常。我们发现，在男性，精子发生的进展和精子样细胞，每个具有4C DNA的产生没有事先减数分裂。在雌性中，二倍体卵子通过正常的减数分裂产生，减数分裂前的DNA复制之前，卵原细胞中发生一次核内有丝分裂进行额外的DNA复制。为了鉴定导致杂种中异常生殖细胞产生的分子，我们研究了参与姐妹染色单体和同源染色体的凝聚以及细胞周期控制的蛋白质的表达模式，包括SMC 1 α，SMC 1 β，SMC 3，Rad 21，SCP 3，Mad 2，Cdc 20，Separin，Securin和p53。免疫细胞学分析显示：1）SMC 3在杂种睾丸中，SCP 3（粘着蛋白的一种成分）显示出与野生型睾丸不同的模式; 2）在杂种睾丸中，（联会复合体的一个组成部分）开始在细线期精母细胞中表达，与野生型睾丸一样;然而，在杂种中未检测到串状表达（代表联会复合体）; 3）所检测的其他蛋白质的表达模式在杂种和野生型睾丸之间没有差异。这些结果表明，杂种精巢中产生的缺乏SCP 3的不完全联会复合体是精子发生异常的原因。少

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 … More 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