摘要
【目的】明确黄鳝卵原细胞是否能迁移、整合到斑马鱼生殖嵴上,为建立黄鳝异体生殖途径打下研究基础。【方法】通过酶消化法制备黄鳝卵原细胞悬液,经PKH26标记卵原细胞后显微注射到受体斑马鱼胚胎中,在荧光显微镜下追踪受体斑马鱼胚胎中供体黄鳝卵原细胞的迁移、整合及增殖情况;采用组织形态学方法检测移植受体斑马鱼胚胎的发育情况,并以半定量PCR和荧光原位杂交检测受体斑马鱼胚胎中供体黄鳝卵原细胞标记基因nanos3的表达情况。【结果】黄鳝卵巢经蛋白酶消化后,分离得到的卵原细胞悬液浓度为2.4×10^(6)个/mL,以PKH26染色后在红色荧光下呈橙黄色。荧光追踪观察结果显示,在移植后0、12、24和48 hpt的受体斑马鱼胚胎中均能检测到供体黄鳝卵原细胞,且黄鳝卵原细胞迁移至受体斑马鱼胚胎的生殖嵴上进行整合及增殖,但72 hpt受体斑马鱼出膜后荧光信号丢失。组织形态学观察结果表明,黄鳝卵原细胞注入受体斑马鱼胚胎中48 hpt后其细胞质结构松散,生殖细胞由动物极向两端迁移而分化形成组织,72 hpt和30 dpt的移植受体斑马鱼与对照组斑马鱼无明显差别。半定量PCR检测结果显示,在48 hpt受体斑马鱼胚胎中能检测黄鳝nanos3基因表达,而在72 hpt和30 hpt受体斑马鱼中表达微弱。荧光原位杂交检测结果显示,48 hpt、72 hpt和30 dpt的移植受体斑马鱼均有黄鳝nanos3基因表达。【结论】黄鳝卵原细胞在48 hpt后仍存在于受体斑马鱼胚胎内,但72 hpt后在受体斑马鱼胚胎未检测到黄鳝卵原细胞,即黄鳝卵原细胞在斑马鱼受体中迁移并最终整合到生殖嵴上,但在斑马鱼出膜后无法继续存活。因此,为了提高黄鳝生殖细胞移植成功率应选择亲缘关系近的鱼种或构建性腺不育种质作为受体。
【Objective】To determine whether the oocyte of Monopterus albus could migrate and integrate into the reproductive ridge of zebrafish,and lay a foundation for the study of the allogenic reproductive pathway of M.albus.【Method】Oocyte of M.albus was prepared by enzyme digestion method.Reproductive cells were labeled with PKH26 and microinjected into recipient zebrafish embryos.The migration,integration and proliferation of donor reproductive cells in the recipient were tracked under fluorescence microscope.The development of transplant recipients was detected by histomorphology;semi-quantitative PCR and fluorescence in situ hybridization detection techniques were used to detect the expression of the marker gene nanos3 mRNA in the oogonium of the recipient zebrafish embryos.【Result】After the ovaries of M.albus were digested with protease,the concentration of cell suspension isolated was 2.4×10^(6) cells/mL,and was yellow-orange under red fluorescence after PKH26 staining.Fluorescence tracing PKH26 labeled donor oocytes showed that donor oocytes were detected at 0,12,24 and 48 hpt in zebrafish embryos,and migrated to the recipient reproductive crest for integration and proliferation.At 72 hpt,the fluorescence signal of the recipient zebrafish was lost.The results of histomorphology showed that the cytoplasm structure of 48 hpt was loose when the eel oocytes were injected into the recipient zebrafish embryos,and the germ cells migrated from the animal pole to both ends,and differentiated into tissues.There was no great difference between the recipient zebrafish transplanted with 72 hpt and 30 dpt and the control zebrafish.Semi-quantitative PCR results showed that donor germ cell marker gene nanos3 was expressed in recipient fish at 48 hpt,and donor germ cell marker gene nanos3 was weakly expressed in recipient fish at 72 hpt and 30 hpt.Fluorescence in situ hybridization technique indicated that gene nanos3 was expressed in zebrafish transplanted at 48 hpt,72 hpt and 30 dpt.【Conclusion】The oogonia ofM.albus still exists in the recipient zebrafish embryo at 48 hpt,but no oogonia ofM.albus was detected in the recipient zebrafish embryo at 72 hpt,that is,the oogonia ofM.albus migrates in the zebrafish receptor and finally integrates into the reproductive ridge,but can not survive after the zebrafish membrane is formed.In order to improve the survival and differentiation rate of transplantated oogonia of M.albusgerm,fishes with closer relatives or germplasm with gonadal sterility should be selected as recipients.
作者
何坤
吴华东
张士林
刘铮铮
阮记明
孙艺文
李福贵
HE Kun;WU Hua-dong;ZHANG Shi-lin;LIU Zheng-zheng;RUAN Ji-ming;SUN Yi-wen;LI Fu-gui(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang,Jiangxi 330045,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2023年第1期315-324,共10页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31860735)
江西省科学计划重点研发项目(20192ACB60009)
江西省教育厅科技计划项目(GJJ200401,GJJ180168)。
关键词
黄鳝卵原细胞
斑马鱼胚胎
移植
nanos3基因
荧光追踪
荧光原位杂交
半定量PCR
oogonium ofMonopterus albus
zebrafish embryos
transplantation
Nanos3 gene
fluorescent tracking
fluorescence in situ hybridization
semi-quantitative PCR
