【作者】 卢庆；

【导师】 芮荣；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 在猪上,体外生产胚胎一直为囊胚发育率低、胚胎质量低的问题所困扰。早期胚胎发育过程中伴随着大量发育异常的细胞,尤其在体外培养过程中更为普遍。研究表明,早期胚胎中普遍存在凋亡现象,即使是形态正常的胚胎当中也存在凋亡细胞。胚胎细胞中出现高比例的凋亡对胚胎的生长是不利的,而早期胚胎的体外死亡又是制约哺乳动物胚胎生物技术发展的重要因素。随着胚胎生物技术的发展,囊胚前死亡越来越引起人们的关注,早期胚胎死亡的增加也是相关动物繁殖新技术应用于生产的最大障碍。通过对牛和猪胚胎的观察发现,在早期胚胎的培养过程中,随着培养时间的延长,胚胎会出现发育阻滞、卵裂球破碎、胞质不均匀、透明带软化等现象,有相当一部分胚胎发生退化,进而死亡。本实验通过对猪体外受精胚胎早期发育过程中的形态学变化、核相变化及DNA损伤等情况的研究,以期建立其与卵裂、发育能力间的联系;通过分析胚胎形态异常与退化的关系,了解胚胎退化死亡的实质;通过猪胚胎早期发育过程中正常与异常胚胎发生的比率,凋亡胚胎与形态异常胚胎的比例,建立凋亡与猪胚胎发育不良间的关系,为克服早期胚胎退化死亡,提高胚胎体外发育率提供实验依据。本实验所获结果如下:1、早期胚胎在体外培养过程中会发生退化。退化死亡胚胎的比例随培养时间延长逐渐上升,但总体上表现为培养早期相对平稳,第3 d、第4 d、第5 d的死亡率分别为3.6%,5.7%和9.6%,培养6、7 d时死亡率似乎突然增加,分别达45.6%(21/46)和55.7%(44/79)。2、早期胚胎在体外发育过程中,呈现退化的形态学变化,包括发育速度减慢,胞质碎片增多等等。从胚胎发育速率上看,培养3 d的胚胎正常发育的比例较高,为59.1%(39/66),培养5、6 d的胚胎普遍发生发育延迟,其正常发育的胚胎比例仅为5.3%(5/94)和7.7%(5/65)。随着体外培养时间的延长,无胞质碎片的胚胎比例逐渐减少,并且胚胎的碎片含量也有所增加,培养3 d的胚胎碎片含量都＜20%,而培养6 d时碎片含量＞20%的胚胎数可占到13.8%。3、胚胎第3 d的发育速率、胞质碎片含量、碎片类型与胚胎的后期发育能力关系紧密。本实验参照人胚胎评分标准,首次建立猪胚胎质量评分标准(3项指标各20分,满分60分)。按此标准,高分值胚胎的发育潜能高于低分值胚胎。综合评分中分值46-60的胚胎桑椹胚、囊胚发育率为32.8%,明显高于分值30-45的胚胎(10.4%,P＜0.05)。4、IVF胚胎发生退化时,胚细胞核发生凝聚、断裂,DNA产生损伤、断裂,彗星电泳表现出不同程度的凋亡形态。核相分析表明,培养3 d的胚胎有多个细胞核,形态较完整;培养6 d后部分胚细胞核发生凝集、浓染;培养至9 d,大多数胚胎的细胞核染色均为阴性,仅偶而可见胚胎边缘有浓密核物质存在。彗星试验表明,培养时间越长则彗尾长度越短,尾部DNA含量越高,表明DNA损伤程度随着培养时间的增加而加重,胚胎出现细胞凋亡的比例也增高。胚胎的形态异常与否和胚胎DNA损伤的比例之间关系不显著。更多还原

【Abstract】 The low development rate and quality of blastocysts have been the main obstacle to produce porcine embryos in vitro.Huge number of abnormal development of embryonic cells underwent during early stage embryo development,particularly to great extent existing during culture in vitro.Apoptosis is prevalent in preimplantation embryos,even the embryo is morphologically normal.The large ratio of apoptosis happened in embryos may influence the viability of embryos.The death of preimplantation embryos was paid more and more attention.Observation of bovine and porcine embryos had been conducted and the fragmented blastomere, developmental arrest,nonhomogeneous cytoplasm together with soften zona pellucida were found in embryos with the prolonged culture time during early development. Some embryos were degenerate or died.This research studied on the anaplasia of early porcine embryos,discussed the relationship between morphologic index and development potential,in order to set up a scoring system to predict the late development.We use the PI staining and comet assay,to analysis the change of nuclear and the damage of DNA,and to discuss the apoptosis rate of normal and abnormal embryos.In addition,the outcomes of the study could be regarded as one part of research and application basics in exploiting and obtaining high quality of blastocyst.The results derived from present experiments were showed as follows:1.During development of early porcine embryos derived from fertilization in vitro, we found the death rate increased gradually on Day 3,Day 4 and Day 5（Respectively 3.6%, 5.7%and 9.6%）,and then on Day 6 and Day 7,the death rate increased suddenly（Possiblely exceeding 50%）,because most of arrest embryos didn’t died until Day 7,while before Day 5, senescence of the embryos without cleavage.2.During development of early porcine embryo,we found the morphologic change of embryos occurred,including decreased development velocity,increased the number of cytoplasmic fragmentation etc.On Day 3,the number of embryos of normal development rate of 59.1%（39/66） was higher when compared with embryos on Day 5 and Day 6(5.3%（5/94） and 7.7%（5/65）.In addition,the ratio of embryos without fragmentation decreased gradually. The ratio of fragmentation in embryos on Day 3 was less than 20%,but on Day 6,the embryos increased（over 13.8%） in which the ratio of fragmentation was more than 20%.3.The development velocity,the ratio and type of cytoplasmic fragmentation could influence the late embryo development potential.Accordaing to the scoring criteria of human embryo,we set up a scoring system with these three indexes to predict the late porcine embryo development potential.The result showed that compared to embryos with lower score,the quality of the embryo who obtained higher score was much better.The developmental rate to morula of those embryos scored 46-60 was 32.8%,while other embryos scored 30-45 only yielded 10.4%of developmental rate to morula.4.The cellular nucleus of embryos when degenerated would condense and disrupt. Their DNA were damaged and fragmented,showing different extent apoptosic morphology by comet assay.The nuclear morphological analysis showed that there were number of cellular nucleus with relatively normal structure in embryos cultured for 3 days. When embryos were cultured for 6 days,partial cellular nucleus of embryos became to be condensed and behaved dark staining.To the 9^th day of culture,most embryos appeared to be negative for the nuclear staining,occasionally some condensed nucleus could be found on the edge of the embryo.The commet assay showed that the longer embryos were cultured the shorter commet tail be together with the higher content of DNA.It indicated that degree of the damage of DNA went worse with the longer culture and the apoptosic percentage would increase also.There existed no significant relationship between the embryonic morphology and the damage of embryonic DNA.更多还原