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

青鳉属生殖隔离的分子机制:种间杂交种胚胎发育异常和不育的分子和细胞生物学分析

• 批准号: 11236201
• 负责人: YAMASHITA Masakane
• 金额: $ 41.92万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11236201/
• 关键词: Abnormal embryonic development; Genome; Germ cell formation; Interspecific hybrid; Medaka; MPF; Reproductive isolation; Sterility; 遺伝子組み換え

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

在这个项目中调查了以下两个主题。l)种间杂交水稻胚胎发育异常的分子基础。种间杂交的麻麻与爪哇麻麻具有胚胎致死性。m期促进因子（MPF，一种Cdc2和Cyclin B的复合物）是细胞分裂的关键调节因子。除了同种亚基组成的野生型强积金外，种间杂交种细胞还具有不同种亚基组成的杂种强积金。因此，我们研究了一种可能性，即杂种MPF催化的异常磷酸化诱导了异常细胞分裂，从而使种间杂种离开。利用重组Cdc2和在昆虫细胞中产生的Cyclin B，我们证实了杂交MPF磷酸化的蛋白与野生型MPF磷酸化的蛋白不同。在此基础上，我们进一步研究了杂交强积金的产生是否会引起细胞的异常分裂。注射了O. latipes Cyclin B的O. lanpes胚胎在m期染色体正常，而注射了O. javanicus突变体Cyclin B的O. lanpes胚胎细胞分裂异常，细胞发生染色体消除。这些结果强烈提示，种间杂交种的胚胎发育异常是由于来自不同物种的亚基杂交MPF所致。我们还发现，在杂交胚胎的早期阶段，来自javanicus的染色体被选择性地从杂交胚胎中剔除，这表明除了杂交MPF之外，还有其他机制参与了这种异常发育。2)种间杂交水稻生殖细胞形成异常导致不育的分子机制。大纹稻与弯纹稻的F_1杂交表现出性别特异性异常。我们发现，在雄性中，精子发生继续进行，每个具有4C DNA的精子样细胞在没有事先减数分裂的情况下产生。在雌性中，二倍体卵是通过正常的减数分裂产生的，在减数分裂前DNA复制之前，卵原细胞内有丝分裂会产生额外的DNA复制。为了确定与生殖细胞产生异常有关的分子，我们研究了SMC1α、SMC1β、SMC3、Rad21、SCP3、Mad2、Cdc20、Separin、Securin和p53等参与姐妹染色单体和同源染色体内聚和细胞周期控制的蛋白的表达模式。免疫细胞学分析显示：1)杂交睾丸中黏结素成分SMC3的表达模式不同于野生型睾丸；2)在杂交睾丸中，与野生型睾丸一样，SCP3（突触复合体的一个组成部分）开始在瘦素精细胞中表达；然而，在杂交中没有检测到串状表达（代表突触复合体）；3)其他蛋白的表达模式在杂交和野生型睾丸之间没有差异。这些结果提示，杂种睾丸中产生的缺乏SCP3的不完整的突触复合体是导致精子发生异常的原因。少

项目成果

Takashi Iwamatsu: "Studies on fertilization in the teleost IV : Effects of aphidicolinand camptothecin on chromosome formation in fertilized medaka eggs"Development, Growth and Differentiation. 44. 293-302 (2002)

Takashi Iwamatsu：“硬骨鱼受精研究 IV：阿菲迪林和喜树碱对青鳉受精卵染色体形成的影响”发育、生长和分化。

Jibak Lee: "Analyses of mRNA expression patterns of cohesin subunits Rad21andRec8 in mice : Germ cell-specific expression of rec8 mRNA in both male and female mice"Zoological Science. 19. 539-544 (2002)

Jibak Lee：“小鼠中粘连蛋白亚基 Rad21 和 Rec8 mRNA 表达模式的分析：雄性和雌性小鼠中生殖细胞特异性的 rec8 mRNA 表达”《动物学科学》。

Matsui, H., Ogiwara, K., Ohkura, R., Yamashita, M., Takahashi, T.: "Expression of gelatinases A and B in the ovary of the medaka fish Oryzias latipes."Eur. J. Biochem.. 267. 4658-4668 (2000)

Matsui, H.、Ogiwara, K.、Ohkura, R.、Yamashita, M.、Takahashi, T.：“明胶酶 A 和 B 在青鳉鱼 Oryzias latipes 卵巢中的表达。”Eur。

Lee, J., Yokoyta, T., Yamashita, M.: "Analyses of mRNA expression patterns of cohesin subunits Rad21 and Rec8 in mice : Germ cell-specific expression of rec8 mRNA in both male and female female mice."Zool. Sci.. 19. 539-544 (2002)

Lee, J.、Yokoyta, T.、Yamashita, M.：“小鼠中粘连蛋白亚基 Rad21 和 Rec8 mRNA 表达模式的分析：雄性和雌性雌性小鼠中生殖细胞特异性的 rec8 mRNA 表达。”Zool。

Koichi Mita: "Differential expression of cyclins B1 and B2 during medaka (Oryzias latipes) spermatogenesis"Zoological Science. 17(in press). (2000)

Koichi Mita：“青鳉 (Oryzias latipes) 精子发生过程中细胞周期蛋白 B1 和 B2 的差异表达”《动物学科学》。