【摘要】 用1500μW/cm2的紫外线照射长牡蛎(Crassostrea gigas)精子60 s以进行灭活处理,并使之与栉孔扇贝(Chlamys farreri)卵子受精,在卵子受精后排出第一极体前用6-DMAP(50 mg/L)处理受精卵,持续处理35 min,抑制第一极体和第二极体的排放,诱导异精雌核发育四倍体。采用二脒基苯基吲哚(DAPI)荧光染色显微观察法,对灭活的长牡蛎精子诱导的栉孔扇贝雌核发育四倍体早期胚胎发育过程进行细胞学观察。结果表明:经紫外线灭活过的长牡蛎精子进入栉孔扇贝卵子后发生轻微膨胀;在第一次卵裂中期,精核形成一致密的染色质小体(DCB),位于两组分开的母本染色体之间,不参与核分裂;第一次卵裂结束时DCB滞留于两卵裂球的分裂沟上或进入其中一分裂球中;第二次卵裂过程中,DCB的去向与第一次卵裂时基本一致。6-DMAP处理有效地抑制了第一极体和第二极体的排出,从而使雌核四倍化。对担轮幼虫染色体倍性分析结果表明,通过本方法可以获得6.25%的四倍体幼虫。本研究还对灭活的异源长牡蛎精子诱导栉孔扇贝雌核发育四倍体过程中产生的复杂倍性、核物质分离紊乱及多精附卵现象进行了观察和分析。更多还原

【Abstract】 Zhikong scallop plays an important role in aquaculture in north of China because of their economic benefits.However,in recent years,culturable scallop sometime dies abnormally in different sea areas,and its yield and quality per unit reduce.Artificial polyploid provides a new way for us to improve the serious situation.Triploid shellfish are of great interest in aquaculture because of their sterility,improved meat quality,and superior growth.But the triploid population is hard to be used in production abroad because they can’t reproduce and must be induced each year.Tetraploid breeding can solve the problem because that induced tetraploids provide an alternative method for mass production of triploids by mating them with normal diploids.The method is the best one for producing triploids in factory.Then study on the technique of tetraploid breeding is meaningful for triploid industrialization.The work results of our lab before showed that sperm of Crassostrea gigas could attach Chlamys farreri egg rapidly and penetrate into it after insemination,and sperm nucleus diffused and formed male pronucleus.Egg of Chlamys farreri resumed meiosis,released polar body and formed female pronucleus when it was activated by the heterogenous sperms.At last,male and female pronuclei fused and fertilized egg began the first cleavage.The chromosome could not be distributed equally into two cells in most of the zygote during the first cleavage,which resulted in that the hybrids between Zhikong scallop and Pacific oyster are not viable.In order to prevent any genetic paternal contamination,sperm of Crassostrea gigas must be genetically inactivated.This study aims to induce allogynogenesis tetraploid with 6-DMAP by inhibiting the release of the first and the second polar bodies,and observe cytological process of fertilization and the early embro development of allogynogenetic tetraploid,which can provide us some evidence for inducing allogynogenetic tetraploid and finding its mechanism.Allogynogenetic tetraploidy of scallop Chlamys farreri was induced by UV-irradiated heterogenous sperms of Pacific oyster Crassostrea gigas.The induction was attempted by blocking release of the first and second polar bodies from fertilized eggs with 6-dimethylaminopurine(6-DMAP,50 mg/L,35 min),which was applied just before the first polar body was going to be expelled from the fertilized eggs.Heterogenous sperms were ultraviolet-irradiated for 60 s at an intensity of 1 500 μW/cm2.The cytological process of fertilization and development of early embryos of gynogenetic tetraploidy were investigated under fluorescent microscope with DAPI stained.Cytological observation revealed that nucleus of ultraviolet-irradiated sperm expanded after penetrating into eggs of Chlamys farreri.At metophase of the first cleavage,male pronucleus became a dense chromatin body(DCB),which did not participated in karyokinesis and located between the two maternal chromosomes.At completion of the first cleavage,DCB was observed either in region of the first cleavage furrow or in one of the two blastomeres.During the second cleavage,the experience of DCB was fundamentally identical to that the first cleavage’s.Treatment with 6-DMAP effectively blocked release of the first and second polar bodies,and resulted in big tetraploid female pronucleus.Percentage of tetraploids produced in the study was much lower than that produced with other methods though we did get the allogynogenetic tetraploids.Levels of allogynogenetic tetraploids in eggs treated with 6-DMAP may be due to four factors.The first factor was inactivation of heterogenous sperm which involved intensity of irradiation,concentration of sperm and irradiation time.The second one was treatment of reduplication which included the duration of 6-DMAP treatment,treatment opportunity and concentration of 6-DMAP.The third one was condition of cultivation which was also very important.And the fourth factor was whether development of treated eggs was synchronized or not.Synchronization of egg development in zhikong scallop was greatly affected by female condition and other environmental factors.The results may suggest that a longer duration of 6-DMAP may produce more tetraploids than a shorter duration.Further studies are needed to define optimum condition for the induction of allogynogenetic tetraploids in the future.Many results of gynogenetic cytological observation proved that sperm nucleus was in condensation after penetrating into egg and did not disperse.Results of cytological observation documented that during the second cleavage,experience of DCB was fundamentally identical to the first cleavage’s in which DCB was observed either in the region of the first cleavage furrow or in one of the two blastomeres.Fate of the irradiation male pronucleus after 4-cell stage was unknown which need a new way.In addition,the embryos with varied polyploid,irregular division of nuclear materials and multi heterogenous sperms attaching to the eggs were observed and discussed in the paper.更多还原