The Molecular Cytological Mechanism Of Mitogynogenetic Diploid Induction In Olive Flounder (Paralichthys Olivaceus)

Suitable methods of egg fixation and membrane removal were selected and modified by a serial of experiments and microtubule skeletal observation of egg using immunofluorescence microscopy were established in marine flatfish. The developmental process of olive flounder (Paralichthys olivaceus) and turbot (Scophthamus maximus) fertilized eggs from blastodisc formation to 4-cell stage were observed dynamically with these methods. The results showed that the proper egg fixation method of microtubule skeletal immunofluorescent micro-observation in turbot (also in flatfish) was to be fixed with FGP-fix fixation solution under room temperature, then stored in PBST at 4℃.The best method of egg membrane removal was to be removed manually with sharp forceps under stereo-microscope directly. A serial of images of centrosomes, spindle and nuclear in olive flounder and turbot fertilized eggs from blastodisc formation to 4-cell stage were observed. The dynamic process and the morphologic configuration of the microtubule skeletal structure—spindles and centrosomes in the developmental eggs of olive flounder were similar to those of turbot eggs. The time of blastodisc formation of turbot eggs was about 10 min later than that of olive flounder eggs, and this was consistent with the results obtained by stereo-microscope. At anaphase stage, the phenomenon of the regenerated nuclear assembled the microtubules as the microtubule organizing center in olive flounder eggs was not distinct as that of turbot eggs. Above results would provide basic information for the fertilizing cytological study and offer cytological data for chromosome manipulation in flatfish. The present study was a preliminary investigation for examining cytological changes and subsequent mitotic processes when the haploid eggs of olive flounder were submitted to cold-shock treatment around the time of early metaphase of the first cell cycle. Indirect immunofluorescence staining was applied to the isolated blastodisks of the fixed eggs. Changes on microtubule structure and the expected suppression of the second cleavage had been observed during the period from blastodisc formation to 8-cell stage. Obvious differences among controls and treated eggs had been detected during early egg cleavage. The developmental process of gynogenetic haploid control eggs was similar to that of the diploid control eggs except several minutes (about 7-9 minutes) delayed. The microtubule-depolymerizing resulted from cold-shock treatment did not change much the pronuclear migration. Spindles disassembled by the cold-shock treatment regenerated after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole constituted by tubulin molecules was easily depolymerized by cold shock treatment, thus resulting in the disassembly of the daughter centriole, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle. The monopolar spindle was assembled in each blastomere in the second cell cycle, which led to the failure of the anaphase disjunction of duplicated chromosomes and of the second cleavage, eventually resulting in the chromosome set doubling. We had also found few 2-cell stage embryos, which had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These embryos had a high potentiality developing into haploid-diploid mosaics. This study was also a preliminary investigation for examining cytological changes and subsequent mitotic processes when the gynogenetic haploid eggs of olive flounder were submitted to hydrostatic pressure treatment around the time of early metaphase of the first cell cycle using indirect immunofluorescence staining method and system. Changes on microtubule structure and the expected suppression of the second cleavage had been observed during the period from blastodisk formation to the thirdcell cycle. Similarily, obvious differences among controls and treated eggs had been detected. The developmental process of haploid control eggs was also similar to that of the diploid control eggs except several minutes (about 7-9 minutes) delayed. Most of the microtubules were disassembled and the nucleation capacity of the centrosomes was just temporarily inhibited after the pressure treatment, but the pair of centrosomes in some eggs was not disassembled because of its resistance to hydrostatic pressure. Within 15 min after treatment, the nucleation capacity of the centrosomes began to gradually recovered, and regenerated microtubules slowly. About 20 min after treatment, the nucleation capacity of the centrosomes recovered completely, regenerated distinct bipolar spindles and resulted in the occurrence of the first mitosis. As the fertilized eggs developed asynchronously, centrosomes generated different responses to pressure shock treatment and formed different spindles subsequently or in the second cell cycle. During the second cell cycle, most 2-cell embryos had a monopolar spindle in each blastomere, part of the 2-cell embryos had a bipolar spindle in each blastomere, few 2-cell embryos had a bipolar spindle in one of the two blastomeres and a monopolar spindle in the other, some eggs still remained 1-cell stage and some of them had a monopolar spindle. The induction rate of mitotic gynogenetic diploid by hydrostatic pressure treatment was higher and the haploid-diploid mosaics rate was lower than those of the cold-shock treatment obviously. To the best of our knowledge, the new information obtained was the first time to adapt immunofluorescence staining clarifying the mechanism of mitotic gynogenetic diploid induction by cold-shock treatment and hydrostatic pressure treatment in marine fishes, and provide a preliminary cytological basis for developing a reliable and efficient mitotic gynogenesis induction in olive flounder.