The Functional Analysus Of Hira Gene During Oogenesis And Embryogenesis In Fishes

HIRA, a conserved replication-independent chromatin assembly factor, is a conserved family of proteins present in various animals and plants. The function of Hira gene in the development still remains to be elucidated. It has been shown that mutation of Hira in Drosophila does not affect the viability, but results in sterility of the females due to the defect of sperm nucleus decondensation, which suggest that HIRA is essential for the assembly of paternal chromatin and for the formation of male pronucleus at fertilization. However, Hira is essential for proper embryonic development and survival in mouse. Gibel carp Carassius auratus gibelio is a unique triploid species that has two different reproduction modes: allogynogenetic reproduction and gonochoristic reproduction. The first polar body was not observed during oogenesis of gibel carp, which could be the strategy of maintaining original ploidy of chromosomes during oogenesis of this fish. Gibel carp has a similar phenotype of abnormal male pronucleus to that in Hira mutated Drosophila. In order to investigate the role of Hira gene in fish development and in gynogenesis, we have previously compared the expression patterns of Hira gene during embryogenesis between gynogenetic gibel carp and gonochoristic colored crucian carp and found that the Hira mRNA was consistently expressed during all embryonic development stages in gibel carp. However, in colored crucian carp, the abundance of Hira mRNA significantly decreased (p<0.05) shortly after fertilization and then increased again in gastrula stage and kept stable till hatching. In this study, in situ hybridization and immunofluresence experiments were performed on the ovaries of gibel carp and colored crucian carp to examine dynamic expression patterns of Hira mRNA and protein during oogenesis. The results show that Hira mRNA expression patterns during oogenesis of these two fishes are generally identical. In stage I oocytes, Hira transcript is localized in the nuclei. At stage II, the mRNA transfers to cytoplasm and is found uniformly distributed in the cytoplasm of the developing oocytes. By stages III, Hira mRNA gradually moves to the cortex. The positive hybridization signal is hardly observed within the fully grown oocytes (stage IV). The dynamics of HIRA protein during oogenesis in the two fishes are a little different. In gibel carp, HIRA protein is not appeared at stage I, and it is strongly expressed along the cortex of the oocytes at early stage III. While in colored crucian carp, the protein is observed as early as stage I, even the signal is weak. HIRA protein is strongly expressed at stage II. By the late period of stage III and stage IV, HIRA protein is barely to be seen in both of fish oocytes. The signals of Hira mRNA and protein are hardly to be observed in stage IV oocytes of these two fishes could be attributed to the enormously enlargement of the oocytes at the later stages and thus greatly diluting the gene products. The nearly identical dynamic patterns of Hira expression during oogenesis between these two fishes with different reproductive modes suggest that Hira gene might not affect meiosis during oogenesis but play a role at and after fertilization.We also analyzed the function of Hira gene during embryogenesis in gibel carp by knocking down the expression of HIRA after fertilization using morpholino anti-sence mRNA (Hira-MO). Western analysis showed that HIRA expression in the early embryos injected with Hira-MO and in the early embryos injected with control-MO did not display obvious difference. However, the expression of HIRA decreased evidently at heart-beat stage. This interference resulted in severe deficiencies of the phenotype during embryogenesis, including the slowing of epiboly in gastrulation, abnormal cardiovascular system, the inhibition of yolk utility and abnormality of the tail. In addition, the development of nerve system was aberrant and the distribution of the neural cells in the neural tube was changed. The malformation of digestive system was also observed in the Hira-MO interfered fries of gibel carp. These results suggest that HIRA is not only related to the decondensation of sperm nuclei and the formation of male pronuclei at fertilization, but also play an important role during embryogenesis.