异源精子诱导栉孔扇贝(Chlamys farreri)雌核发育二倍体早期胚胎的细胞学研究及GISH鉴定

CYTOLOGY OF GYNOGENTIC DIPLOIDS INDUCED BY HETEROLOGOUS SPERMIN SCALLOP CHLAMYS FARRERI AND GYNOGENETIC IDENTIFICATION WITH GISH

采用紫外线遗传失活的太平洋牡蛎精子作激活源,经6-DMAP加倍诱导栉孔扇贝第二极体抑制型雌核发育二倍体;运用石蜡切片显微技术,对雌核发育二倍体卵子早期胚胎发育过程中的核相变化进行观察;采用荧光原位杂交(GISH)技术对担轮幼虫期胚胎进行检测。结果显示,紫外线处理过的精子入卵后发生一次轻微膨胀,形成雄性原核,但不形成染色体,而是浓缩为致密的染色质小体(DCB),DCB或滞留于两卵裂球的分裂沟上或进入其一的细胞质中,不与雌原核融合;GISH检测结果显示,在早期胚胎中没有检测到外源太平洋牡蛎精子的遗传物质。实验结果可为研究异精诱导栉孔扇贝雌核发育二倍体提供细胞学依据。

Artificial diploid allo-gynogenesis of Chlamys farreri was induced using ultraviolet （UV）-irradiated heterogenous sperm of closely-related evolution species （Crassostrea gigas） to activate meiotic division of the eggs by blocking extrusion of the second polar body from fertilized eggs with 6-dimethylaminopurine （6-DMAP）; the chromosome was doubled by physical or chemical methods, and then all-female inheritance was obtained. Nuclear behavior of its gynogenetic embryos was observed on paraffin section. Maternal hereditary material and its ploidity were checked with GISH technique. The results showed that the UV-irradiated sperm could expand slightly after entering the egg and developed into a male pronucleus, but could not form chromosomes and became a dense chromatin body （DCB）. No paternal signals were detected by both C. farreri and C. gigas probes, indicating that there was no paternal chromosomes participate in embryo. The results showed that the UV-irradiated sperms of C. gigas could enter the eggs of C. farreri and activate the eggs, but failed to develop into zygocyte. 6-DMAP could prohibit the excursion of the second polar body to produce diploids. Cytological evidence of gynogenesis in C. farreri induced by heterologous sperm was therefore demonstrated.