奶牛卵母细胞体外成熟与受精的研究

发布时间：2018-03-21 23:20

本文选题：奶牛　切入点：laminin　出处：《河南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：本试验主要探讨了laminin在奶牛体外受精及胚胎发育中的影响,同时对提高牛体外胚胎数量与质量的方法进行了研究,旨在建立一套比较完善的IVP技术体系。主要研究内容为:卵母细胞的体外成熟、体外受精及整合素β1在精卵融合中的作用、评价胚胎质量技术的优化和早期胚胎的体外培养。研究结果如下:1.卵丘细胞对牛卵母细胞体外成熟的影响试验结果显示:添加离散卵丘细胞能够显著促进DOs的体外成熟(57.98±14.27 vs35.53±14.00,P0.05),对COCs具有促进趋势但差异不显著(76.66±5.77 vs 66.76±9.46,P0.05);卵母细胞胞外卵丘细胞对与之共培养的DOs的体外成熟促进作用效果不明显(35.83±18.32 vs 35.53±14.00,P0.05)。分析结果显示:卵母细胞胞外卵丘细胞主要通过间隙连接促进卵母细胞的体外成熟,添加的离散卵丘细胞主要以旁分泌途径促进卵母细胞的体外成熟;3层及其以上胞外卵丘细胞能够提高其包裹的卵母细胞平均体外成熟能力的85.34%,添加的离散卵丘细胞能够提高COC平均体外成熟能力的16.18%,能够提高DO平均体外成熟能力的60.61%。2.不同受精液及有无卵丘细胞对牛体外成熟卵母细胞体外发育的影响试验研究了体外成熟卵母细胞在IVF-100、BO液和TALP液三种体外受精液中的受精效果,同时比较了裸卵(NOs)与卵丘卵母细胞复合体(COCs)对受精效果的影响。结果显示体外成熟卵母细胞在受精液IVF-100中的卵裂率显著高于BO液的(0.7055±0.0745 VS0.5794±0.0956,P0.05),而TALP液中的卵裂率与IVF-100和BO液的差异都不显著。NOs的卵裂率虽然低于COCs的,但差异不显著;NOs能够发育到囊胚阶段,但囊胚率极显著低于COCs的。说明BO液和TALP液作为体外受精液还有很大的改进空间,NOs的体外发育能力显著低于COCs的。3.laminin在牛早期胚胎中的分布及添加层粘连蛋白对体外胚胎发育的影响将经IVM-IVF的牛卵子移入含不同浓度laminin(0、5、10 and 20μg/ml)的体外培养基中进行培养,并对不同阶段胚胎的发育情况进行检测。结果显示10μg/ml组卵裂率及囊胚率显著高于对照组(0.8031±0.0667 vs 0.7039±0.0688,P0.05;0.4835±0.0167 vs0.3765±0.0257,P0.05)。虽然受精48h后各试验组4-8cell数与对照组之间差异不显著,但5、10μg/ml组的囊胚细胞团总数显著高于对照组(97.00±8.24、98.71±13.59 vs87.86±6.22,P0.05)。在滋养层细胞数上试验组与对照组之间无差异,各组内细胞团数与囊胚细胞团总数的比值均接近0.33。在对0-8天牛体外胚胎层粘连蛋白的分布检测试验显示,最早可于8细胞胚胎检测到laminin的表达分布,在16细胞到囊胚阶段层laminin的表达呈递增趋势,并于桑囊胚阶段大量表达。在囊胚及扩张囊胚阶段同时还能检测到滋养层细胞laminin部分的表达分布情况。结果表明:laminin随胚龄的增加需求量不断加大,添加一定量的的外源性laminin能够在不同程度上提高体外胚胎的卵裂率、囊胚率以及囊胚细胞数;运用免疫荧光技术最早可于8细胞阶段检测到奶牛胚胎laminin的表达分布,而且在桑囊胚阶段能够检测到laminin的大量表达。4.牛体外胚胎质量评定技术的优化为建立快速、可靠的评估牛囊胚质量的双重染色方法,本试验对4种双重染色方案进行了改进。结果显示改进的方案一、方案三及方案四都能达到牛囊胚双重染色的效果。改进的方案三、方案四囊胚细胞总数与Hoechst33342染色结果无差异(84.33±9.98 VS83.63±9.74,P0.05;93.00±10.50 VS83.63±9.74,P0.05),而且囊胚内细胞团(ICM)数与囊胚细胞总数的比值分别为0.3382与0.3356,均接近0.33。改进的方案四染色最为稳定,效果较好,整个染色时间不到3分钟,极大的提高了染色效率。试验结果表明改进的方案三、方案四都可以作为一种快速简洁有效的牛囊胚质量鉴别方法。5.整合素β1在精卵融合中的作用此试验通过不同条件下laminin与anti-β1对整合素β1的阻断来探索整合素β1在精卵融合中的作用,同时对laminin特意消失于受精阶段的原因进行分析。结果显示当分别用laminin与anti-β1对卵子上的整合素β1进行阻断时,laminin能极显著的影响受精(10μg/ml:67.38±5.41,20μg/ml:71.51±4.96 VS 86.04±3.73,P0.01)及卵裂(10μg/ml:41.43±8.33VS 59.30±7.85,P0.01),而anti-β1对受精及卵裂的影响不大。当分别将laminin与anti-β1添加到受精液中,对laminin肝素受体进行封闭时,结果显示随laminin浓度的升高,对精卵融合的影响显著增加(10μg/ml:80.36±1.92 VS 86.04±3.73,P0.05;20μg/ml:64.95±7.61 VS86.04±3.73,P0.01),但对卵裂的影响不大;而anti-β1能极显著的影响受精(50μg/ml:29.49±11.39,100μg/ml:34.98±7.54 VS 86.04±3.73,P0.01)及卵裂(50μg/ml:5.44±7.37,100μg/ml:3.65±6.84 VS 59.30±7.85,P0.01)。当分别用laminin与anti-β1对精子表面上的整合素β1进行阻断时,结果显示两者都显著的影响受精及卵裂(P0.05),而且laminin浓度越大,显著性越强。同时,当分别用laminin与anti-β1对精子表面和卵子表面整合素β1进行阻断再进行体外受精时,结果显示两者都极显著的影响精卵的融合(P0.01),卵裂率上除50μg/ml anti-β1组外都极显著的低于对照组(P0.01)。结果表明,整合素β1参于了奶牛体外精卵融合的过程;阻断精子表面上的整合素β1能极显著的阻碍精卵的融合,而阻断卵子表面上的整合素β1对受精影响不大;laminin在受精过程中的消失可能是由于对整合素β1及肝素的阻断而阻碍了精卵的正常融合。
[Abstract]:This experiment mainly discusses the influence of laminin in dairy cows in vitro fertilization and embryo development, and the methods to improve the quantity and quality of bovine embryo in vitro were studied to establish a perfect IVP system. The main contents are as follows: in vitro maturation of oocytes, in vitro fertilization and integrin beta 1 in sperm egg fusion and optimization of cultivation and early embryo quality evaluation technology of embryo in vitro. The results are as follows: 1. effects of cumulus cells on in vitro maturation of bovine oocytes showed that the addition of discrete cumulus cells could significantly promote DOs maturation in vitro (57.98 + 14.27 vs35.53 + 14, P0.05), but the difference is to promote the trend there was no significant difference on the COCs (76.66 + 5.77 vs 66.76 + 9.46, P0.05); in vitro oocyte cumulus cells on extracellular co cultured with DOs maturation promoting effect is not obvious (35.83 + 18.32 vs 35. 53 + 14, P0.05). The analysis results show that the oocyte cumulus cell extracellular mainly through gap junctions promote oocyte maturation in vitro, add the discrete cumulus cells mainly by paracrine pathway promote oocyte maturation in vitro; the 3 layer and above extracellular cumulus cells can improve the inclusion of oocytes the average maturation capacity of 85.34%, add the discrete cumulus cells can improve the average COC in vitro maturation capacity of 16.18%, affected by semen and without cumulus cells of bovine oocytes matured in vitro experimental study on the in vitro development of oocytes matured in vitro in IVF-100 can enhance the DO average in vitro maturation ability of 60.61%.2., BO and TALP solution three kinds of liquid in vitro fertilization effect in semen, and made a comparison of naked oocytes (NOs) and cumulus oocyte complexes (COCs) effect on fertilization effect. The results showed that the in vitro maturation of oocytes Cells in the cleavage of semen IVF-100 was significantly higher than those in the BO solution (0.7055 + 0.0745 VS0.5794 + 0.0956, P0.05), while the difference of TALP in liquid IVF-100 and liquid BO and cleavage rate were not significant.NOs although the cleavage rate of less than COCs, but the difference was not significant; NOs can develop to the blastocyst stage, but the blastocyst rate was significantly lower than that of COCs. BO and TALP solution as in vitro semen, there is still much room for improvement, and add the laminin effects on embryonic development in vitro by IVM-IVF in bovine oocytes with different concentration of laminin, the distribution of NOs in vitro development ability was significantly lower than that of COCs.3.laminin in bovine embryos in (0,5,10 and 20 g/ml) in the medium were cultured in vitro, and the development of different embryonic stages were detected. The results showed that 10 g/ml group, the cleavage rate and blastocyst rate was significantly higher than the control group (0.8031 + 0.0667 vs 0.7039 + 0.068 8, P0.05; 0.4835 + 0.0167 vs0.3765 + 0.0257, P0.05). Although 48h after fertilization in each experimental group and control group the difference between the 4-8cell number is not significant, but the total number of blastocysts group 5,10 g/ml group was significantly higher than the control group (97 + 8.24,98.71 + 13.59 vs87.86 + 6.22, P0.05). The number of cells in trophoblastic layer between the experimental group and the control group had no difference. The ratio of the total number of groups and the number of blastocyst inner cell mass cells were close to 0.33. in the display of the detection of 0-8 beetles in vitro embryo distribution of laminin expression distribution test, the earliest in 8 cell embryos detected by laminin, in the 16 cell to blastocyst stage presentation layer laminin a trend of increasing, and a large number of expressions. At the same time on the blastocyst stage but also in the expansion of the blastocyst stage to blastocyst and expression of trophoblast cell laminin detection part of the distribution. The results show that the laminin increase with the increasing demand of embryo age, add A certain amount of exogenous laminin can improve the in vitro embryo cleavage rate in different degrees, and the rate of blastocyst cell number of blastocyst; distribution of expression by immunofluorescence technique was first detected in 8 cell stage laminin of bovine embryos, but also in mulberry to blastocyst stage expression quality assessment technique of.4. optimization detection of bovine embryo in vitro to laminin in order to establish a rapid, reliable method of double staining evaluation of bovine blastocyst quality, this experiment was improved. 4 kinds of scheme of double staining results show that the improved scheme, scheme three and scheme four can reach the bovine blastocyst double staining results. The improved scheme three, and the total number of Hoechst33342 staining showed no difference scheme four blastocysts (84.33 + 9.98 VS83.63 + 9.74, P0.05; 93 + 10.50 VS83.63 + 9.74, P0.05), and the inner cell mass (ICM) and the ratio of number of total cells were blastocysts 0.3382 and 0.3356 are close to the 0.33. improvement plan four was the most stable, the effect is good, the dyeing time less than 3 minutes, which greatly improves the efficiency of dyeing. The experimental results show that the improved scheme three, scheme four can be used as a rapid method for identification of bovine blastocyst quality effective.5. integrin beta 1 the role of the sperm egg fusion test by laminin and anti- under different conditions of beta 1 integrin beta 1 integrin beta 1 to explore the blocking effect on sperm egg fusion, to analyze the causes of laminin specially disappeared from the stage. The results showed fertilization when using laminin and anti- beta 1 on the egg integrin beta 1 blocking, laminin can significantly affect the fertilization (10 g/ml:67.38 + 5.41,20 g/ml:71.51 + 4.96 VS 86.04 + 3.73, P0.01) and cleavage (10 g/ml:41.43 + 59.30 8.33VS + 7.85, P0.01), and anti- beta 1 on fertilization and egg Little effect on crack. When laminin and anti- were added to the beta 1 in semen of laminin, heparin receptor blocking, results show that with the increase of laminin concentration, the effect of sperm egg fusion increased significantly (10 g/ml:80.36 + 1.92 VS 86.04 + 3.73, P0.05; 20 g/ml:64.95 + 7.61 VS86.04 + 3.73 P0.01),, but the effect on the cleavage of the little; anti- beta 1 can affect the fertilization significantly (50 g/ml:29.49 + 11.39100 g/ml:34.98 + 7.54 VS 86.04 + 3.73, P0.01) and cleavage (50 g/ml:5.44 + 7.37100 g/ml:3.65 + 6.84 VS 59.30 + 7.85 P0.01, respectively). When using laminin and anti- beta 1 on sperm on the surface of the integrin beta 1 block, the result shows that the influence of both fertilization and cleavage significantly (P0.05), and higher laminin concentration, significantly stronger. At the same time, when using laminin and anti- beta 1 on the surface of the egg and sperm surface integrin beta 1 blocked again In vitro fertilization, sperm egg fusion results show that both are affected significantly the cleavage rate (P0.01), except 50 g/ml anti- beta 1 group were significantly lower than the control group (P0.01). The results showed that integrin beta 1 participate in the process of cattle sperm egg fusion in vitro; integrin blocking fusion sperm on the surface of beta 1 can significantly hinder the sperm egg, and the egg on the surface of the block is not 1 integrin on fertilization effect; Laminin in the process of fertilization was probably due to the blocking of the disappearance of integrin beta 1 and heparin and hinder the normal sperm egg fusion.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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