Studies On Induced Gynogenesis And Its Mechanism In Paddlefish (Polyodon Spathula)

The American paddlefish Polyodon spathula (Walbaum) is a large freshwater fish belonging to the family Polyodontidae of order Acipenseriformes. The paddlefish of the Mississippi River basin and its tributaries and adjacent Gulf Slope drainagesand and the Chinese paddlefish Psephurus gladius (Martens) of the Yangtze River are the only two species in the family Polyodontidae. The American paddlefish has characteristics such as fast growth, high quality, wide adaptation, etc. which has become increasingly important as a high-value species in aquaculture. Chinese sturgeon aquaculture companies introduced the fertilized eggs of paddlefish and successfully culture in the 1990s. At present, the species has become more and more popular and famous.Paddlefish is an important cultured freshwater fish as well as a promising model fish for the study of conservation of endangered sturgeons and paddlefishes species. In the present study, a protocol for artificial gynogenesis of paddlefish was developed in order to produce a pure line and to obtain a technique routing of conservation of endangered the Chinese paddlefish. On the other hand, the paddlefish aquaculture industry has appeared in China. Therefore, it is necessary to breed new stains that have a good economic characteristic which is grow fast, strong disease tolerance and so on. Artificial induction of gynogenesis is an effective method of constructing homozygote quickly, which is used to select choiceness breed.Artificial induction of gynogenesis is one of the main methods of genome engineering, which can help to solve problems of fish genetics and selection. In this study, we report the methods of inducting meiotic gynogenesis in paddlefish using Amur sturgeon (Acipenser schrenckii) sperm. And apply four methods (the morphometric and chromosome counting analysis, flow cytometry and microsatellite analysis) to confirm the success of the offspring of the gynogenesis. At the same time, electron microscopies (Scanning electron micrograph and Transmission electron micrograph) were used to investigate the changes in morphology and ultrastructure of UV-irradiated Amur sturgeon sperm. And comet assay was also used to detect DNA integrity of Amur sturgeon sperm following UV irradiation. Cytological studies on artificially gynogenetic paddlefish and the sex determinination mechanism in paddlefish were discussed.The following results were received:1. Paddlefish eggs were fertilized with normal Amur sturgeon sperm, the embryo were haploid (n=60) which were identified by the chromosome counting analysis. Based on this result, apply hybridization-heatshock to induce gynogenesis in paddlefish was made. The optimal iniation time for heat shock of gynogenetic eggs was determined, the optimal temperature and treatment time were determined. The gynogenetic diploid were obtained by eggs fertilized with normal Amur sturgeon sperm then heatshocked at 37℃for 2 min, at 18 min post-fertilization. With this treatment the fertilization rate and hatching rate was 76.4±9.2%,7.8±1.4%, respectively. On the other hand, we also obtained gynogenesis diploid in paddlefish using ultraviolet (UV)-irradiated Amur sturgeon sperm for 5-min and consequent heat shock treatment, and UV irradiation of sperm for 5 min at a UV intensity of 863μw/cm2 was the optimum dose to achieve diploid gynogenesis on the basis of observations on hatching rate of eggs. Morphologically examination, chromosome testing, flow cytometry, andmicrosatellite DNA analysis were involved in the identification of offspring of gynogenes and confirmed that all offspring were gynogenetic diploid paddlefish. Thus both distant hybridization-heatshock and irradiatation-heatshock were effective methods at producing gynogenetic paddlefish.2. In the present study, the effect of UV irradiation on sperm morphology and ultrastructure was examined by scanning electron microscopy (SEM) and transmission electron micrograph (TEM). The result shows that the mature spermatozoa of Amur sturgeon consistes of an elongated head with a distinct acrosome and nudeus region, a midpiece and a flagellum. The mean length of the head and midpiece, the flagellum and total length of spermatozoon were 8.95,36.80 and 45.75μm, respectively. The acrosome contains posterolateral projection, subacrosome and granular material and actin filament found within the anterior acrosome and etc. The flagellum was thin and long with a typical "9+2" microtubular structure. Scanning electron microscopy(SEM) observation showed that after iradiated by UV, the acrosome membranes of some of sperm were cracked,and acrosome substances were released to form acrosome filament. Transmission electron micrograph (TEM) observation showed that after iradiated by UV, the plasma membrane swelled and broke off from the nuclear membrane; the acrosome disrupted and its contents lost; some mitochondria lost external membrane, accompanied by the disaggregation of mitochondria or the deformation of mitochondria cristae; swelling of flagellum membrane or loss of flagellum. UV irradiation damage on plasma membrane, acrosome, mitochondria and flagellum probably caused the decrease in motility and fertility of the UV-irradiated sperm. It was estimated that UV irradiation on plasma membrane, acrosome, mitochondria and flagellum probably caused the decrease in motility and fertility of the UV-irradiated sperm.In this paper, detection of DNA damage in response to a UV irradiation process in Amur sturgeon spermatozoa was carried out. The results demonstrated that there were no significant differences The results demonstrated that there were no significant differences(P>0.05) between sperm by UV-irradiated 1,3min and fresh sperm. However, a significant drop in sperm motility and fertilization rate was observed in sperm by UV irradiated 5,7,9 min. The comet rate and damage coefficient of UV irradiated sperm with 1,3 min was similar to fresh sperm, but UV irradiated 5,7,9min were significantly differed to fresh sperm. In fact, there was a positive correlation between comet rate of UV irradiated sperm and intensity and time of UV irradiation in protocol. The majority of sperm with DNA damage within the nucleus were slightly and mildly damaged, while minorities were heavily damaged. Few were totally damaged, and only occurred under the conditions of 7min and 9min UV irradiaton. Our analysis suggests that high intensity of UV irradiation is the main factor that causes the DNA damage in UV irradiated sperm nucleus.3. This paper reports the cytological process on Amur sturgeon sperm induced gynogenesis in paddlefish. It was observed that after being inseminated, Amur sturgeon sperm can enter the egg, spermatozoon nucleus beside female pronucleus was condensed and cannot fuse with female pronucleus.At the same time, whe fry growth over 1 year, the sex of the fry from gynogenesis diploid group and normal paddlefish group were identified. We successfully checked the sex ratios in 3 families from gynogenesis diploid group and 2 families from normal fish. The results showed the male to female ratios in the two normal fish group were not significantly different from theoretical 1:1 ratio (P>0.05), while there were all female in gynogenesis diploid group. Based on this result, We suggest these sex determination mechanism in paddlefish is XX/XY type.