异源精子诱导栉孔扇贝雌核发育二倍体的微管动态变化

Dynamic status of microtubule in early developmental stages of gynogenetic diploid induced by heterologous sperm in Chlamys farreri

采用紫外线遗传灭活的长牡蛎(Crassostrea gigas)精子激活栉孔扇贝(Chlamys farreri)卵子,并用6-DMAP诱导染色体加倍的方法,获得第二极体抑制型雌核发育二倍体早期胚胎。多聚甲醛固定、免疫荧光染色后,运用荧光显微镜观察栉孔扇贝正常发育卵子和异源精子诱导的雌核发育卵子,对其受精过程中微管的动态变化过程进行观察和分析。结果表明:(1)正常发育组受精卵内以微管为基础的纺锤体能够顺利地组装并引导卵细胞进行减数分裂和第一、第二极体的排放,以及雌、雄原核融合和第一次卵裂;(2)异源精子诱导雌核发育的卵子经6-DMAP处理后,部分微管变得模糊或消失,纺锤体受到破坏导致染色体的分离无法进行,第二极体的形成受到抑制并使雌核染色体二倍化;去除6-DMAP作用后,微管重新组装,雌核分裂重新启动继而进行卵裂;精核保持固浓缩状态或轻微膨胀形成雄性原核,但卵裂后期则以致密的染色质体(DCB)形式存在于分裂沟上或进入一个卵裂球中。结果证实,长牡蛎精子经紫外线遗传灭活后可顺利进入并激活栉孔扇贝成熟卵子但不参与合子核的形成,6-DMAP也可有效抑制第二极体的排放而获得雌核发育二倍体胚胎。本实验结果为研究异源精子诱导栉孔扇贝雌核发育的可行性提供了细胞学依据。

Artificial diploid gynogenesis techniques in shellfish have developed rapidly in recent years, however, few studies on the dynamic status of microtubule in this field were reported. This study depicted the microtubule status in early developmental stages of normal fertilized diploids and gynogenetic diploids of scallop Chlamys farreri, and, provided cytological bases for cytoengineering breeding and related researches. The meiotic division of the eggs of Chlamysfarreri was activated by ultraviolet （UV） -irradiated heterogenous sperm of Pacific oyster Crassostrea gigas and the gynogenetic diploids were induced by blocking extrusion of the second polar body from fertilized eggs with 6-dimethylaminopurine （6-DMAP）. The dynamic status of microtubule was studied by direct immunofluorescence with FITC-anti-α-tubulin. The results showed that the spindle assembled periodically towed the chromosomes to accomplish the extrusion of the first and second polar bodies, and the female and male pronuclei fused into zygotonucleus in normal fertilized eggs. However, the behavior of microtubule in eggs of gynogenetic diploids was considerably complicated. The microtubule in 6-DMAP treated eggs was destroyed badly and the spindle became anomalistic shape or disappeared, the microtubule became shorter and unconspicuous. The chromosomes arranged irregularly and couldn＇ t move to form the second polar bodies. After relieving the treatment of 6-DMAP, the microtubule reassembled and conduced the diploid female pronuclei＇ s movement to activate the first cleavage. In this process, the sperm nucleus of gynogenesis kept dense or expanded again to become a dense chromatin body （DCB） which didn＇ t join into the karyokinesis. The results of this study suggested that the ultraviolet （UV） -irradiated sperms of Crassostrea gigas could enter into the eggs of Chlamys farreri and activate the eggs while they did not form the zygocyte. And the 6-DMAP could prohibit the excursion of the second polar body to produce diploids. The feasibility of gynogenesis in Chlamys farreri induced by heterologous sperm was demonstrated.