Hira基因产物在银鲫和彩鲫卵子发生过程中的动态变化
EXPRESSION ANALYSIS OF HIRA mRNA AND PROTEIN DURING OOGENESIS IN GYNOGENETIC AND GONOCHORISTIC CRUCIAN CARPS
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摘要: 为进一步研究Hira基因在卵子发生和雌核发育过程中的作用,通过原位杂交和免疫荧光定位的方法检测了Hira mRNA和蛋白质在雌核发育银鲫和两性生殖彩鲫卵子发生过程中的动态变化。结果表明,银鲫和彩鲫卵子发生过程中Hira基因转录产物的变化基本一致,在Ⅰ期卵母细胞的细胞核中大量表达,至Ⅱ期卵母细胞时转至细胞质中均匀分布,在Ⅲ期卵母细胞中,杂交信号逐渐移向细胞的周边,到Ⅳ期时随着卵黄物质大量积累,杂交信号几乎不见。HIRA蛋白在银鲫和彩鲫卵子发生过程中的变化略有差别。HIRA蛋白在银鲫Ⅰ期卵母细胞中没有表达,在Ⅱ期卵母细胞的细胞质中有弱表达,在Ⅲ期早期卵母细胞的周边有强烈表达;而在彩鲫Ⅰ期卵母细胞中就有HIRA蛋白的弱表达,至Ⅱ期时HIRA蛋白在细胞质中大量表达,Ⅲ期早期卵母细胞的细胞质中有弱表达。在银鲫和彩鲫Ⅲ期末和Ⅳ期卵母细胞中都很难观察到荧光信号。Hira mRNA和蛋白质在银鲫和彩鲫早期卵母细胞中有较强表达,且在银鲫和彩鲫卵子发生过程中没有显著差异,说明其可能对于脊椎动物卵子发生和减数分裂没有显著影响,而是在受精和/或胚胎发育过程中起作用。Abstract: 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 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 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 showed that Hira mRNA expression patterns during oogenesis of these two fishes were generally identical. In stage Ⅰ oocytes, Hira transcript was localized in the nuclei. At stage Ⅱ, the mRNA transferred to cytoplasm and uniformly distributed in the cytoplasm of the developing oocytes. By stages Ⅲ, Hira mRNA gradually moved to the cortex. The positive hybridization signal was hardly observed within the fully grown oocytes (stage Ⅳ). The dynamics of HIRA protein during oogenesis in the two fishes showed a little different. In gibel carp, HIRA protein was not appeared at stage Ⅰ, and it was strongly expressed along the cortex of the oocytes at early stage Ⅲ. While in colored crucian carp, the protein was observed as early as stage Ⅰ, though the signal was weak. HIRA protein was strongly expressed at stage Ⅱ. By the late period of stage Ⅲ and stage Ⅳ, HIRA protein was barely to be seen in both of fish oocytes. The signals of Hira mRNA and protein were hardly to be observed in stage Ⅳ oocytes of these two fishes. This 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.
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