玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体的影响_cropped

玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体的影响_cropped 关键词玻璃化冷冻;卵母细胞;纺锤体;染色体;猪 哺乳动物卵母细胞冷冻保存不仅成为保护品种1.1 材料 资源和拯救濒危动物不可缺少的技术保障，而且也 除特别说明外，本实验所用化学药品与试剂均 是人类辅助生育技术(ART)的重要研究内容。玻璃 购自 Sigma 公司。 化保存方法具有许多优点，其操作程序简单，耗 体外成熟培养液: TCMI99(Gibco，12340-030) [1]时少、更安全有效，所以在卵母细胞低温保存的 +10%卵泡液(PFF)+0.57 mmol/L半胱氨酸+10 IU/ml 胚胎生物技术研究中已成为不可缺少的技术手段。 PMSG +10 IU/ml hCG+10 mg/ml EGF+200 IU/ml 青 [2][3]利用玻璃化方法冷冻胚胎和卵母细胞在多种哺乳 霉素 +10% 胎牛血清(NCS) 。 动物中获得成功。家畜卵母细胞的冷冻保存主要集 受精卵培养液: NCSU-23液+0.4%BSA(fraction 中在牛上，目前已经获得来自冷冻 - 解冻的牛体外 V, A-9647)+5 g/ml胰岛素+50 mol -基乙成熟和未成熟卵母细胞的牛犊，而猪卵母细胞冷冻 醇。 精子洗涤液: BTS 液 +3 mg/ml BSA+0.85 保存的研究进展缓慢，尚未建立起成熟的保存方 mg/L 法，目前仍处于实验室探索阶段，且解冻后卵母 亚牛磺酸。 [4]细胞的存活率、受精率及胚胎发育率仍然很低。 精子受精液: TALP液+2 mmol/L咖啡因+3 mg/ml 要提高卵母细胞的冷冻效果，就必须清楚低温保存 BSA+0.85 mg/L 亚牛磺酸 +1 mg/ml 乙烯乙醇 +0.17 前后猪卵母细胞超微结构的变化以及冷冻过程引起 mmol/L 卡那霉素 +20 mg/L 肝素。 卵母细胞发育潜力下降的原因。本研究从保存体外 玻璃化平衡液与冷冻液: 依照冷冻保护剂及降温 培养成熟的猪 MII 卵母细胞入手，探讨不同低温冷 速度不同有所差异。EG40 组[单用乙二醇(ethylene 冻保护剂和降温速率对卵母细胞形态、纺锤体微管 glycol, EG)组]的冷冻平衡液为 20%EG+TCM199+ 和染色体形态结构影响及其与体外受精后续发育能 20%NCS，冷冻液为40%EG+10 g/L ficoll+0.65 mol/L 力的关系，为研究和提高猪冷冻后卵母细胞体外生 海藻糖;ED20(-)组[EG 和二甲基亚砜(dimethyl 产效率打下基础。 收稿日期:2006-03-20接受日期:2006-09-01 Fig.1 Immunofluorescentmicrographsofporcineoocytesmaturedinvitro chromosomes (blue) stained Hoechst 33342 group spindle microtubules (green) labeled Thearewithina,b,candd.Thearewithantior tubulin antibodies and FITC-conjugated secondary antibody in group a', b', c' and d'. a: a normal MII oocyte prior to vitrification (control had a polar body (pb) and chromosome alignment at equatorial plane of the spindle; a': A normal spindle morphology (control group); group) b: chromosomes disperse (group EG40); b': spindle structures severely distort ( group EG40); c: chromosomes completely disperse in the cytoplasm [group ED20(-)]; c': microtubules of spindle become dissembled in the cytoplasm [group ED20(-)]; d: chromosomes return to MII phase[group ED20(+); d': spindle morphology basically maintain, while microtubules disperse at the top of the structures. Table 1Normalrateofspindleorganizationandchromosomesmorphologyofporcineoocytesaftervitrification GroupsTotal No. of oocytesNormal chromosomes, %(n)Normal spindle organization, %(n) aa Control11182.9(92)84.7(94)bcbEG40 93 19.4(18) 20.4(19) Influence of Vitrification on the Chromosomes and Spindles of Porcine Oocytes Matured in Vitro 11Zhi-Feng Xiang*, Jin-Zhou Zhang, Pei-Qing Li, De-Fu Zhang, Shi-Qing Zhang 1(Department of Animal Science, Henan Sci-tech College, Xinxiang 453003, China; Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China) Abstract To investigate the influence of vitrification freezing on the chromosomes and spindles of porcine oocyte matured in vitro by using nylon cryoloop. Porcine oocytes were verified with ethylene glycol (EG) singly or EG combined with dimethyl sulphoxide (DMSO) as cryoprotectants , and cooled by taking oocytes directly into liquid nitrogen or by vitrification machine. After frozen porcine oocytes thawed, the microtubulin of the spindles and chromosomes were fixation and stained by immunofluorescent method. Normal morphological oocytes in every groups were used to fertilize in vitro. Comparatively, in protocol of EG combined with DMSO and at ultra-rapid cooling rate, the normal configuration of spindle and chromosomes rate of thawed porcine oocytes was remarkably higher than that of the two protocol of single EG used and EG combined with DMSO, As the same in the rate of oocytes activated. The protocol of EG combined with DM SO as cryoprotectants and with extremely high cooling rate can produce better effect on conservation of chromosomes and spindle configuration in vitrification of porcine oocytes, while the development ability of thawed oocytes are severely affected. Key words vitrification; oocyte; spindle; chromosome; porcine Re cei ved : Ma rch 20 , 2 006 Acc ept ed: Se pte mber 1, 20 06 Th is work wa s su ppo rted by th e Yo uth Fun d o f Henan Sc i-t ech Col lege (No.0 4010 6) *Cor res po ndi ng au th or. Tel : 8 6- 373 -3 040 83 3, Fa x: 86 -3 73- 30 407 18 , E-mail : zhi fe ng7 80 9@ yah oo .co m.cn