猪孤雌激活早期胚胎线粒体分布及mtDNA拷贝数变化的研究

本研究通过线粒体分子探针标记技术检测孤雌激活早期胚胎线粒体的分布变化,运用实时荧光定量PCR技术检测mtDNA拷贝数的变化,揭示早期胚胎发育过程中线粒体分布、mtDNA拷贝数变化趋势。结果表明,成熟卵母细胞电激活后,由2-细胞胚胎开始,卵裂球内线粒体分布均匀且密集,每个细胞均有分布,卵裂球之外的空隙未见线粒体分布,直到囊胚形成,线粒体均有分布。孤雌激活4-细胞胚胎mtDNA拷贝数显著高于8-细胞胚胎mtDNA拷贝数(907210.77±145520.77,186224.33±103308.00,P<0.05),但显著低于2-细胞胚胎、桑椹胚、囊胚的mtDNA拷贝数(1563422.54±224666.51、1697626.25±176999.53和1752301.29±101146.64,P<0.05)。孤雌激活扩张囊胚mtDNA拷贝数最高,为2812545.67±156819.31,显著高于其他发育时期胚胎的mtDNA拷贝数(P<0.05)。由此可见,孤雌激活早期胚胎发育进程中线粒体分布及mtDNA拷贝数会发生变化。     

线粒体移植对水牛卵母细胞发育潜能的影响

本研究以水牛卵泡颗粒细胞作为线粒体的来源细胞,初步探讨水牛卵母细胞进行线粒体移植(MIT)后对其发育潜能的影响。试验比较了不同级别水牛卵母细胞mtDNA的拷贝数,并研究了水牛卵母细胞进行MIT后,其后续早期胚胎发育及胚胎线粒体膜电位(ΔΨm)的变化情况。结果显示:一级卵母细胞组的平均mtDNA拷贝数极显著高于二、三级卵母细胞组((202 101±74 432)vs(118 483±17 028),(39 177±7 938),P0.01),二级卵母细胞组的平均mtDNA拷贝数极显著高于三级卵母细胞组((118 483±17 028)vs(39 177±7 938),P0.01);孤雌激活处理后发现:一级卵母细胞组的卵裂率和囊胚率也都极显著高于二、三级卵母细胞组(P0.01),而二级卵母细胞组激活后胚胎的卵裂率和囊胚率亦极显著高于三级卵母细胞组(P0.01)。用Mito-Tracker探针标记的外源线粒体经移植后,会随着胚胎的发育而发生移动,分布到各个卵裂球中;且发现二级卵母细胞MIT组孤雌激活后的囊胚率显著高于对照组和空注组(27.3%vs 17.4%,7.84%,P0.05),而三级卵母细胞MIT组激活后的囊胚率与对照组和空注组差异不显著(P0.05);对胚胎ΔΨm检测发现,水牛植入前各时期胚胎ΔΨm总体呈上升趋势,线粒体移植后胚胎各时期的ΔΨm均显著高于对照组(P0.05)。综上表明:不同质量的水牛卵母细胞其mtDNA拷贝数存在显著差异,且mtDNA拷贝数与卵母细胞质量和发育能力成正相关;通过移植外源线粒体可以提高二级水牛卵母细胞的发育潜能。     

表儿茶素对小鼠卵母细胞mtDNA拷贝数和胚胎体外发育能力的影响

本研究旨在探究表儿茶素(epicatechin,EC)对小鼠体外成熟培养卵母细胞线粒体DNA(mtDNA)拷贝数及其随后孤雌激活胚胎发育能力的影响。小鼠卵丘-卵母细胞复合体(COCs)在添加不同浓度EC(0、5、10、15、20μmol/L)的成熟液中体外成熟培养16h后,采用实时荧光定量PCR的方法检测卵母细胞mtDNA拷贝数;同时,通过对卵母细胞进行孤雌激活处理,探讨其后续胚胎的体外发育能力。实时荧光定量PCR分析结果显示,添加EC各处理组的卵母细胞mtDNA拷贝数均有所增加,其中,10、15μmol/L组的mtDNA拷贝数均显著高于0μmol/L对照组(P0.05);但10μmol/L组mtDNA拷贝数更接近自然排卵周期合子的mtDNA含量(P0.05)。体外培养观察结果发现,成熟液中添加10μmol/L EC能提高卵母细胞第一极体排出率,与对照组相比差异不显著(P0.05),但能显著提高卵母细胞孤雌激活后胚胎的囊胚发育率(P0.05)。综上表明,小鼠卵母细胞体外成熟液中添加10μmol/L EC可提高卵母细胞的mtDNA拷贝数,有利于促进卵母细胞后续的发育能力。     

2,2'-偶氮二(2-脒基丙烷)二盐酸盐诱导的氧化应激对猪肠上皮细胞活力及线粒体功能的影响

本试验旨在评估2,2’-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)诱导的氧化应激对猪肠上皮细胞(IPEC-J2细胞)活力及线粒体功能的影响,以期建立一种稳定的体外研究肠道氧化应激的模型。以不同浓度(0、30、32、34 mmol/L) AAPH处理IPEC-J2细胞24 h后,检测细胞活力、细胞增殖、细胞内总活性氧(ROS)含量、线粒体膜电位、线粒体DNA(mtDNA)拷贝数以及线粒体功能相关基因[线粒体转录因子A(TFAM)、线粒体转录因子B1(TFB1M)、线粒体转录因子B2(TFB2M)、解耦连蛋白1(UCP1)、解耦连蛋白2(UCP2)、解耦连蛋白3(UCP3)]表达的变化。结果表明:AAPH对IPEC-J2细胞活力的影响呈剂量及作用时间依赖关系。与0 mmol/L AAPH处理相比,不同浓度(30、32、34 mmol/L) AAPH处理均显著抑制了IPEC-J2细胞增殖(P0.05),细胞活力显著下降(P0.05),细胞内总ROS含量显著升高(P0.05),细胞线粒体功能相关基因(TFAM、TFB1M、TFB2M、UCP1、UCP3)表达显著下调(P0.05),mtDNA拷贝数显著降低(P0.05)。综上所述,AAPH诱导的氧化应激通过增加IPEC-J2细胞内总ROS含量引起细胞线粒体功能损伤,进而导致细胞活力下降,抑制细胞增殖。     

绵羊卵母细胞体外成熟过程中的线粒体分布变化

为了检测绵羊卵母细体外成熟前后的线粒体分布变化,将成熟过程不同阶段的卵母细胞用活体细胞线粒体跟踪试剂盒(GENMED kit)对线粒体进行染色,之后用碘化丙啶(Propidium Iodide,PI)对细胞核进行染色,最后用激光共聚焦显微镜观察线粒体分布情况以及相应的细胞核所处的发育时期.结果:生发泡期(Germinal Vesicle,GV)的卵母细胞线粒体呈周边分布;生发泡破裂期(Germinal Vesicle Breakdown,GVBD)、第一次减数分裂中期(Metaphase Ⅰ,MⅠ中期)的卵母细胞呈半周边分布;第一次减数分裂末期(M Ⅰ Anaphase,MⅠ后期)的卵母细胞线粒体基本呈均匀分布,但是线粒体簇较小,着色较浅;第二次减数分裂中期(Metaphase Ⅱ,MⅡ期)的卵母细胞线粒体呈均匀分布,而且细胞质中央的线粒体簇变大,着色变深.结论:线粒体分布随着绵羊卵母细胞体外成熟阶段的进展,由细胞周边向均匀、由疏松向致密呈波动变化,这种变化有可能作为一种判定绵羊卵母细胞胞质成熟的有效参考指标.     

3种冷冻方法对猪MⅡ期卵母细胞线粒体分布和损伤的影响

本研究旨在探讨3种冷冻方法对猪MⅡ期卵母细胞玻璃化冷冻后线粒体的分布和超微结构变化的影响.通过透射电镜、Rhodamine-123 (R-123)荧光染色和体外发育观察,结果显示,(1)无论是FDA-DAPI复染存活率,还是孤雌激活卵裂率,CLV (Cryoloop vitrification)法(72.00%,7.22％)效果最好,OPS(Open Pulled Straw)法(60.00％,4.85％)次之,straw法(42.22％,0％)最低；(2)CLV法经R-123染色后,卵母细胞线粒体的正常分布率(52.24％)要比其它2组要高(OPS,48.65％；straw,37.68%),但三者之间没有显著差异(P＞0.05)；(3)透射电镜超微结构表明,冻后卵母细胞线粒体变得粗糙和模糊,有的线粒体嵴减少甚至消失.结果表明,冷冻过程中卵母细胞线粒体的分布和形态损伤严重,CLV法可提高冷冻速率,增强冻后卵母细胞的发育能力,降低冷冻造成的卵母细胞线粒体异常分布比例.     

高海拔地区牦牛心肌骨骼肌ATP酶活性的测定

为了探讨高原动物对高原低氧的适应机制,采用标准ATP酶测试盒对青海省(平均海拔4 000 m)16头成年牦牛心肌、骨骼肌ATP酶活性进行了测定和分析.结果:Na+ -K+ -ATP,Mg2+ -ATP,Ca2+ ATP和Ca2+-Mg2+-ATP 4种酶活性在骨骼肌线粒体中分别为3.65±1.31(μmolP/mg蛋白·h,下同),2.70士1.36,2.87±1.23和4.08士1.30;在心肌线粒体中分别为4.44±1.19,2.42士0.87,2.29±0.57和3.62±0.92;在骨骼肌组织中分别为2.02±0.80,1.54士0.36,1.69士0.91和2.02±0.96;在心肌组织中分别为1.41士0.67,1.04士0.51,1.24±0.54和1.42±0.60;骨骼肌、心肌线粒体中4种ATP酶活性均显著高于骨骼肌、心肌组织(P＜0.05).     

猪季节性高低温的分子适应性研究

旨在探讨环境温度变化对猪能量代谢的影响,为阐明猪适应性进化的分子调控机制奠定理论基础。选取6月龄荣昌猪为研究对象,分别在季节性高温和低温时期,采集12头猪的大脑、心、肝、肺、肾、眼肌和腰肌,检测其mtDNA拷贝数及能量代谢相关基因表达水平。结果表明:猪体重增长率与环境温度呈现极显著相关性(|r|≥0.77,P0.01);不同组织mtDNA拷贝数在低温组和高温组之间表达模式高度一致(r=0.92,P0.01);与低温组相比,高温组除了心,其它组织的mtDNA拷贝数均显著(P0.05)或极显著(P0.01)降低,同时各组织中调控mtDNA生物合成的TWINKLE基因表达水平也显著(P0.05)或极显著(P0.01)降低;能量代谢相关基因(ND1、COX1、ATP6和CYTB)在组织中的表达水平基本上均是低温组显著(P0.05)或极显著(P0.01)高于高温组,而眼肌中ND1和COX1基因,肝和心中CYTB基因在低、高温组间无显著差异(P0.05),大脑中CYTB基因在高温组显著高于低温组(P0.05);除了肺组织(P0.01),其余组织中HSP70基因表达水平在低、高温组间无显著差异(P0.05)。综上表明,猪可通过调节基因转录水平及线粒体DNA拷贝数来重塑代谢,进而适应环境温度的变化。     

甘氨酸对水貂GV期卵母细胞冷冻保存效率的影响

试验旨在探究玻璃化冷冻及培养过程中添加甘氨酸(glycine,Gly)对水貂GV期卵母细胞冷冻解冻后存活率、核发育、线粒体和皮质颗粒分布的影响。试验分为3组:对照组(没有进行冷冻处理)、冷冻组和Gly添加处理组(1 mmol/L Gly)。对玻璃化冷冻解冻后的水貂GV期卵母细胞分别进行平衡恢复3 h和体外成熟培养,采用免疫荧光标记法检测各组GV期卵母细胞线粒体分布的差异及MⅡ期皮质颗粒分布的变化。结果显示,Gly添加处理组卵母细胞在解冻后3 h的存活率与冷冻组相比差异不显著(P0.05),但显著低于对照组(P0.05);Gly添加处理组卵母细胞的减数分裂恢复率显著高于冷冻组(P0.05),但与对照组相比差异不显著(P0.05)。免疫荧光结果显示,Gly添加处理组的GV期卵母细胞线粒体正常分布率显著高于冷冻组(P0.05),但Gly添加处理组和冷冻组的GV期卵母细胞线粒体正常分布率均显著低于对照组(P0.05)。皮质颗粒分布结果显示,水貂GV期卵母细胞在冷冻后体外成熟培养至MⅡ期时,Gly添加处理组皮质颗粒的正常皮质区分布比例显著高于冷冻组(P0.05),但Gly添加处理组与冷冻组的正常皮质区分布比例均显著低于对照组(P0.05)。结果表明,添加Gly可以提高冻融后水貂卵母细胞的减数分裂恢复率,降低冷冻对其线粒体及皮质颗粒的损失。     

不同促性腺激素添加方式对猪卵母细胞生发泡染色质构型和卵母细胞成熟能力的影响

在卵母细胞体外成熟培养过程中,培养基中添加激素与否及其激素添加的先后顺序是影响猪卵母细胞核成熟和质成熟的一个重要因素.本试验将猪卵母细胞分别在FSH→不含激素、FSH→LH、FSH LH不含激素中培养48 h(培养第20～22 h后换液),并于成熟培养的第24 h(未换液)、48 h将卵母细胞进行荧光染色,观察其生发泡内染色质构型及卵母细胞核成熟情况.实验表明:(1)在IVM的前24 h,添加FSH LH组的GVIV期卵母细胞比例低于只添加FSH组,但差异不显著(8.99%比17.19%,P＞0.05);(2)在FSH存在的情况下,IVM的前期和后期添加LH能促进卵母细胞发生GVBD;(3)FSH LH培养24 h后转入不含激素培养基组,卵母细胞的核成熟比率显著高于添加FSH组和先添加FSH培养24 h后转入添加LH组(P＜0.05).     

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization.     

毛喉素对猪卵母细胞降脂及冷冻保护效果研究

猪卵母细胞脂质含量高被认为是其冷冻效率低下的重要因素之一。本文通过在猪卵母细胞体外成熟过程中添加化学降脂剂毛喉素(forskolin),检测冷冻前后成熟卵母细胞的脂滴含量、脂滴超微结构、线粒体膜电位、活性氧水平、早期凋亡指标、冻后存活率及发育潜能变化等,研究其对猪卵母细胞降脂和冷冻保护的效果。结果显示,成熟过程中毛喉素处理可部分提高卵母细胞成熟率,但差异不显著(P>0.05)。尼罗红荧光染色显示,毛喉素处理后卵母细胞内脂滴数量及面积在冷冻前后均极显著低于未处理组(P<0.01);超微结构观察发现,经毛喉素处理的卵母细胞中非均质脂滴数量与均质脂滴数量的比例扩大,且比均质脂滴面积更大;冷冻后,卵母细胞中以非均质脂滴为主,脂滴变小变少,分布不均匀,毛喉素处理后非均质与均质间的脂滴数量比例进一步增加。毛喉素处理极显著上调冻后卵母细胞线粒体膜电位(1.04 vs. 0.51,P<0.01),减轻氧化应激,降低早期凋亡率(68.30%vs. 86.03%,P<0.01),从而有效提高了冷冻后卵母细胞的存活率(71.17%vs. 51.47%,P<0.01)和孤雌激活卵...     

Mitochondrial activity and morphology in developing porcine oocytes and pre-implantation non-cultured and cultured embryos

Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.     

线粒体动力学平衡对心脏结构与功能的影响

心脏是一个高耗能、高耗氧的器官,其正常收缩及电信号的正常传导也需大量能量供应,因此心肌细胞含有大量线粒体。正常生理状态下,线粒体在细胞内的数量、形态和功能是相对稳定的。当机体内细胞能量产生不足或两个独立的线粒体存在不同的缺陷时,线粒体会受到Mfn1/2和OPA1的调控而发生融合,发生融合后线粒体基质含量相互混合形成一个功能完善的新的线粒体,此过程即为线粒体融合。为维持线粒体DNA及线粒体膜电位的稳定,线粒体受到Drp1及其受体的调控而发生分裂,分裂可使受损的线粒体DNA及去极化的线粒体膜在分裂时聚集到一个子线粒体中,并通过泛素化-蛋白酶系统或自噬作用消除,从而维持线粒体的正常功能。线粒体融合与分裂是一个连续波动的过程,被认为是维持线粒体和细胞正常功能和形态的关键过程。近年来研究发现,心肌细胞线粒体的融合与分裂失衡会引起自身形态和功能的紊乱,进而损害心脏结构和功能。因此,维持心肌细胞的稳态需要线粒体分裂和融合之间的动态平衡,而维持线粒体的动态平衡则需要介导线粒体融合与分裂相关的动力蛋白。作者对参与线粒体融合及分裂过程的关键蛋白的功能进行了综述,同时讨论了线粒体动力学平衡对心脏结构与功能的影响,以期为后期的研究提供一定理论参考。     

Effect of Maternal Age on the Ratio of Cleavage and Mitochondrial DNA Copy Number in Early Developmental Stage Bovine Embryos

Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease.     

线粒体DNA与山羊进化

线粒体DNA由于其母系遗传、高拷贝、高进化速率以及缺乏重组等特点而被广泛的应用于山羊的起源进化研究。本文就山羊的多母系起源、弱的地理结构、群体扩张及分化时间等方面的mtDNA研究概况作了说明,并对线粒体DNA数据分析方法作了简要的介绍。