The Effect Of HIRA Knockdown On Embryogenesis In Gibel Carp

Gibel carp is a gynogenetic crucian carp that have unique reproductive characteristics, which play a important position in fish developmental biology. The Meiosis mechanism is different to traditional system in the oogenesis of the gibel carp. The main performance mainly include abnormal spindle, and the first polar body is not expelled from the oocyte. And In the process of fertilization, the responses are also different between homologous and allogenetic sperm. The homologous sperm from gibel carp males can undergo decondensation following insemination, and there are a percentage of male progenies. However, the heterologous sperm nuclei can not decondense when incorporated in the egg of gibel carp. The genetic information of sperm nuclei can not transfer to offspring, which results in all of progenies are female. Because of the typical reproductive mode, this fish has been regarded as a model to study cytogenesis of germ cell and mechanisms of fertilization. In recent years, the research in histone chaperone HIRA are mainly focus on yeast, Drosophila and mouse, but less common in fishes. Through previous studies, we know that HIRA is an important factor in development process. Mutation of Hira gene cause female infertility but not affected the survival in Drosophila. The research in mouse indicated that deletion of Hira gene lead to the postnatal lethality. We had cloned full-length sequence of Hira gene in gibel carp, detected expression pattern of HIRA transcript at different stages in embryos development. And we prepared the polyclonal antibodies of gibel carp. Whereafter, we examined the dynamic variation characteristics of Hira gene in oogenesis by means of hybridization in situ. Further experiments about sperm decondensation in vitro and immunodepletion revealed that HIRA may have an important role in sperm decondensation of gibel carp and colored crucian carp.On the basis of previous studies, in order to initiate the role of Hira gene in oogenesis and early embryonic development, we first investigated the expression of Hira gene during oogenesis of gibel carp. Subsequently, we knocked down HIRA in the development of embryos using morpholino. We observed the phenotype of the embryos to get more information after HIRA knocked down. The paraffin section and hybridization in situ experiments were also contribute to reveal the effect after HIRA knocked down in embryos. The study will further enrich research on fish development biology. And it hopes to provide services on revealing the function of HIRA in animal development.The results of this study indicate that the expression of Hira transcript gradually increase from stageâ… to stage IV in the oogenesis of the gibel carp, in addition, the expression level of stageâ…£is significantly higher than that stageâ… (p<0.05). The result of western blot on expression of HIRA protein shows the same feature. With the development of oocyte, the expression level of HIRA protein gradually increase, the expression level of stageâ…¢is significantly higher than that stageâ… (p<0.05) The results indicate that no significant difference come up between transcription and translation of HIRA in oogenesis of the gibel carp, which also showed that maternal gene(Hira) may play an important role in oogenesis and early embryonic development of the gibel carp.Knock down of HIRA results in the generation of fish with dramatically phenotype deficiencies, including the slowing of epiboly in gastrulation, abnormal cardiovascular system, the inhibition of yolk utility and short body axis, curly tail. One day after hatching larvae stage, the tail of HIRA-MO embryos atrophied, bad abdominal swelling, blocked development of digestive organ, and many embryos died in two days. There was no swim bladder emerged in the HIRA-MO embryos six days after hatching larvae stage. The larvas made harder lives in the water. In addition, a number of embryos died. In situ hybridization of shh gene expression suggests that the HIRA morphant embryos have defective notochord development. In paraffin section of the embryos suggest that the sarcomere ball and neural tube are notobvious, and we couldn't observe notochord clearly. Unlike Drosophila in invertebrate, these results suggest that HIRA may affect the foundation process of the body axis, development of nervous system, digestion and circulation system, directly or indirectly. Combining the research results in mouse, we can say that HIRA play an important role during development of vertebrate.