| Japanese flounder Paralichthys olivaceus (Temminck et Schlegel) is a commercially important marine finfish, widely cultured along the coastlines of China, Japan and Korea. Owing to the low level of metabolism of this bottom-dwelling species, it is possible to culture them in terrestrial tanks supplied with relatively small amounts of seawater or in semi-closed recirculating tanks. At present, Japanese flounder has become a major cultivated marine finfish species in China, and its aquaculture is now considered economically very important. Difference in growth rate between male and female animals is frequently observed in nature. The growth rate of Japanese flounder is significantly faster in females than that in male. So the effective methods for gynogenetic inducement in this species are important for all-female population construction.Here an effective method to induce meiogynogenesis for all female population construction in Japanese flounder was provided and the cytological processes of induced meio- and mitogynogenesis were studied.Meiogynogenesis was induced in Japanese flounder by using UV irradiated sperm of red sea bream Pagrus major. The sperm of red sea bream exhibited a significant Hertwig effect after irradiating by UV. The embryo had the lowest hatching rate(2.0±0.1%) when UV dosage was 3.4mJ/cm2, and recovered with the increase of UV dosage. It reached the highest hatching rate (57.7±3.1%) when UV dosage was 73mJ/cm2, and ploidy analysis by flow cytometer showed that the embryos were all haploid. After a series of tests for initiation and duration at water temperature of 0±0.5℃, the best parameters for meiogynogenesis inducing in Japanese flounder were determined that cold shock begun at 3 min after insemination and continued for 45min. The fertilization rate and hatching rate at this scenario were 80.7±1.1% and 57.3±2.3%, respectively, which were significantly higher than all the other treat groups(p<0.05).Mature eggs were at the metaphase of the second meiosis when inseminated with UV-irradiated sperm of red sea bream. After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into female-like pronucleus. Then, it fused with the female pronucleus to generate diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs.In mitogynogenesis group, before hydrostatics pressure treatment, the eggs were at the metaphase of the first mitosis. The spindle was disassembled by the treatment and then resembled in its pretreatment position, and the chromosomes were rearranged, i.e., the first mitosis was not blocked. During the second mitotic cycle, only a monopolar spindle was assembled in each blastomere and the chromosomes doubled, but cell cleavage was blocked. In the third cycle, mitosis proceeded normally with a bipolar spindle in each blastomere.From the time of the insemination to the cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed, formed a dense chromatin body, and randomly entered one blastomere.The results of flow cytometric analysis indicated that the relative DNA content of the diploid control was 51.0, while for the haploid it was 27.0, which was almost half that of the diploid control. The relative DNA content of the meio- and mitogynogenetic diploid was 50.0, which were equal to that of the diploid control. These results demonstrated that both meio- and mitogynogenetic fries were all diploid.
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