兔卵巢组织冷冻移植后卯母细胞成熟及受精发育能力的研究

目的 探讨兔卵巢组织冷冻复苏后移植对卵母细胞生长、成熟、受精发育潜能的影响. 方法 25只性成熟新西兰雌兔随机分为对照组(5只)、新鲜移植组(10只)及冷冻复苏移植组(10只),卵巢组织均自体多点移植于子宫系膜内.卵巢移植90d后对兔联合使用促排卵、体外成熟培养和卵泡浆内单精子注射技术,观察卵子的成熟情况以及体内和体外成熟卵子的受精率、卵裂率和囊胚形成率. 结果新鲜移植组和冷冻复苏移植组获卵数均低于对照组(P<0.05),而两移植组间获卵数差异无显著性;各组末成熟卵子所占比率以及未成熟卵子体外成熟后的成熟率间均无显著性差异;各组间体内成熟卵子、体外培养成熟卵子的受精率、卵裂率和囊胚形成率的差异不明显;各组内体内成熟卵子与体外培养成熟卵子相比受精率、卵裂率间无显著性差异,但体外培养成熟卵子囊胚形成率明显低于体内成熟卵子(P<0.05). 结论兔卵巢经冷冻复苏自体移植后,其卵泡能够重新生长发育;通过体外成熟和受精技术,体内和体外成熟卵子均可产生正常的胚胎.提示卵巢组织冻融移植结合辅助生殖技术可作为一种生育力保存的方法.     

兔卵巢组织冷冻移植后卵母细胞成熟及受精发育能力的研究

目的 探讨兔卵巢组织冷冻复苏后移植对卵母细胞生长、成熟、受精发育潜能的影响。 方法 25只性成熟新西兰雌兔随机分为对照组（5只）、新鲜移植组（10只）及冷冻复苏移植组（10只），卵巢组织均自体多点移植于子宫系膜内。卵巢移植90d后对兔联合使用促排卵、体外成熟培养和卵泡浆内单精子注射技术，观察卵子的成熟情况以及体内和体外成熟卵子的受精率、卵裂率和囊胚形成率。 结果 新鲜移植组和冷冻复苏移植组获卵数均低于对照组(P＜0.05），而两移植组间获卵数差异无显著性；各组未成熟卵子所占比率以及未成熟卵子体外成熟后的成熟率间均无显著性差异；各组间体内成熟卵子、体外培养成熟卵子的受精率、卵裂率和囊胚形成率的差异不明显；各组内体内成熟卵子与体外培养成熟卵子相比受精率、卵裂率间无显著性差异，但体外培养成熟卵子囊胚形成率明显低于体内成熟卵子(P＜0.05）。 结论 兔卵巢经冷冻复苏自体移植后，其卵泡能够重新生长发育；通过体外成熟和受精技术，体内和体外成熟卵子均可产生正常的胚胎。提示卵巢组织冻融移植结合辅助生殖技术可作为一种生育力保存的方法。     

Ü卵子体外成熟对胞质线粒体DNA拷贝和功能的影响

目的 研究卵子体外成熟（IVM）过程对胞质内线粒体的数目、功能及卵子质量的影响,进而探讨IVM卵子低发育潜能的可能机制。方法 实验小鼠随机分为IVM组和体内成熟（IVO）组;应用Real-time-PCR、免疫荧光和荧光-荧光素酶测定法,分别检测IVM和IVO来源卵子的线粒体DNA（mtDNA）拷贝数、线粒体膜电位强度、ATP含量、线粒体分布、胞质ROS水平、卵子骨架和染色体结构。 结果 相对于IVO卵子,IVM卵子的mtDNA拷贝数显著降低;而胞质ROS生成和纺锤体及染色体结构异常率则显著增加。胞质线粒体分布模型和氧化磷酸化活性在IVM和IVO卵子间差异无显著意义。结论 非生理性IVM过程显著抑制了胞质mtDNA的复制,并增加了ROS生成和纺锤体和染色体结构异常,继而可能影响卵子细胞质的成熟进程,这可部分解释IVM卵子低发育潜能的机制。     

姜黄素对小鼠卵母细胞体外成熟及早期胚胎发育的影响

目的探讨姜黄素对小鼠卵母细胞体外成熟质量及早期胚胎发育能力的影响。方法获取小鼠卵丘卵母细胞复合体(COC),分别用含(0、5、10、15)μmol/L姜黄素的体外成熟培养液培养18 h。根据卵母细胞极体的排出计算成熟率;体外受精(IVF)后根据受精率、卵裂率、囊胚形成率及囊胚细胞数判断胚胎发育情况;线粒体荧光探针Mito-Tracker Green标记法检测线粒体在卵母细胞内的分布;实时定量PCR检测线粒体合成相关基因线粒体转录因子A(TFAM)和解偶联蛋白2(UCP2)的mRNA水平;免疫荧光细胞化学染色法检测TFAM、UCP2的蛋白表达;2′, 7′-二氯二氢荧光素二乙酸酯(DCFH-DA)负载法检测卵母细胞内活性氧(ROS)水平。结果卵母细胞体外成熟率和早期胚胎发育情况各组无显著性差异;姜黄素浓度与卵母细胞内线粒体成簇分布呈正相关;5μmol/L姜黄素处理组TFAM、UCP2 mRNA表达显著高于其他各组,TFAM、UCP2的蛋白表达水平与对照组相比显著升高;5μmol/L姜黄素处理组卵母细胞内ROS水平显著低于对照组。结论姜黄素处理显著改变小鼠体外成熟卵母细胞中线粒体的分布,增加线粒体合成相关基因的表达,降低卵母细胞内ROS水平,但并未对体外成熟率及早期胚胎发育能力造成显著的影响。     

玻璃化溶液EG5.5对小鼠未成熟卵发育能力的影响

目的研究玻璃化溶液EG5.5对未成熟卵母细胞成熟、受精和发育能力的影响.方法小鼠成熟卵母细胞经玻璃化溶液EG5.5暴露处理但不予以冷冻保存,然后行体外成熟、体外受精和胚胎培养;新鲜的未成熟卵直接行体外成熟和受精作为对照组,比较两组卵母细胞的成熟率、受精、卵裂率和囊胚形成率.结果EG5.5暴露组卵母细胞的成熟率、受精率、卵裂率和囊胚形成率与对照组均无显著差异.结论EG5.5不影响未成熟卵的成熟、受精和进一步发育能力.     

卵子成熟度与受精时间

目的 探讨卵子的成熟度与受精时间的关系,从而提高体外受精-胚胎移植(IVF-ET)治疗时卵子的受精率、卵裂率和妊娠率。方法 采用OCCS的形态学特征将卵子分为1-4级,然后对不同级别的卵子在不同时间受精,对接受IVF-ET治疗的110周期1004个卵子的临床资料进行回顾性分析。结果 Ⅱ、Ⅲ级在取卯后5h受精率最高,Ⅰ级在取卵后6-8h受精较好,Ⅳ级卵子受精率最低直至不受精。受精时间不是一成不变的,在同一级卵中也有不同的受精时间,这要根据卵子的形态及颗粒细胞的多少来确定,也就是说各个人的受精时间可不同,同一个人不同的卵也可用不同的受精时间。结论 卵子成熟度越低,体外培养成熟时间越长,受精时间越晚,而卵子成熟度越高,体外培养成熟时间越短,受精时间越早,体外成熟培养的时间依据原来成熟卵子成熟度越高,外培养成熟时间越短,受精时间越早,体外成熟培养的时间越短,受精时间越早,体外成熟培养的时间依据原来成熟卵的成熟程度而定。由于卵子的成熟不是同步的,故受精的时间也就不能是固定的,只有正确判断卵子的成熟度,也才能适时受精。     

骨桥蛋白对小鼠体外受精及早期胚胎发育的影响

目的 探讨骨桥蛋白（OPN）对小鼠体外受精及早期胚胎发育的影响。方法 120只昆明雌鼠和30只雄鼠,采用体外受精、胚胎培养方法,将小鼠精子、卵子、原核期胚胎和2-细胞期胚胎分别用不同浓度的OPN抗体预处理,观察小鼠受精、卵裂以及早期胚胎发育情况。结果 不同浓度的OPN抗体分别预处理精子和卵子后,与对照组比较,受精率显著降低(P＜0.01)。OPN抗体预处理原核期胚胎后,0.01mg/L OPN抗体组的卵裂率较对照组低,但差异无显著性 (P =0.052),1.00mg/L OPN抗体组的卵裂率低于0.01mg/L 组（P ＜0.01）及0.10mg/L组（P ＜0.05）。不同浓度的OPN抗体预处理2-细胞胚胎后可抑制胚胎发育。1.00mg/L OPN抗体组的4-细胞率和8-细胞率与0.10mg/L OPN抗体组相比差异无显著性(P ＞0.05),但前者的囊胚形成率显著低于后者（P ＜0.01）。1.00mg/L OPN抗体组的4-细胞率、8-细胞率以及囊胚形成率均显著低于0.01mg/L OPN抗体组（P ＜0.01）。结论 OPN可促进小鼠的受精和早期胚胎发育。     

鱼藤素对斑马鱼胚胎毒性的作用*

背景：以模式生物斑马鱼构建生物模型,易于开展活体实验。 目的：探讨了不同鱼藤素浓度对斑马鱼胚胎发育的影响。 方法：不同鱼藤素浓度作用于斑马鱼胚胎,分别在受精后48,72 h观测其表型变化,受精后72 h统计其胚胎孵化率。 结果与结论：鱼藤素在0.1 μmol/L浓度以下对斑马鱼胚胎发育无影响,0.2 μmol/L浓度延缓斑马鱼胚胎发育,0.3 μmol/L浓度以上抑制了斑马鱼胚胎发育,使胚胎致死。结果表明,鱼藤素高浓度使胚胎致死,低浓度延缓胚胎发育。       

精子浓度及受精时间对于受精结局和胚胎发育潜能的影响

目的通过对比分析体外受精(IVF)过程中不同受精浓度、不同受精时间多精受精、胚胎质量及发育潜能的差异,探讨体外受精过程中合适的受精浓度和精卵孵育的时间。方法选择首次行IVF并且获卵数大于12个的周期,将卵子随机分为数目相当的两组,分别采用低、高两种浓度的精子受精,低浓度采用0.15×10~6/ml,高浓度采用0.5×10~6/ml的精子浓度进行受精。根据受精时间的不同,分为短时受精低浓度组(A1组)、短时受精高浓度组(B1组),过夜受精低浓度组(A2组)和过夜受精高浓度组(B2组)。分别比较不同受精方式下各组间正常受精率、多精受精率、优质胚胎率、囊胚形成率之间的差异。结果各组间正常受精率、多精受精率、优质胚胎率、囊胚形成率均无统计学差异(P0.05)。结论受精浓度在一个比较大的范围内,无论短时受精还是过夜受精,多精受精率及胚胎发育潜能都不会随着受精浓度的增加而发生变化,体外受精多精受精率的增加可能更多的来源于非精子浓度的因素。     

鱼藤酮致多巴胺能神经细胞早期凋亡模型的研究

目的 :探讨鱼藤酮引起多巴胺能神经细胞早期凋亡的时间和浓度规律 ,为进一步研究其机制建立平台。方法 :不同浓度鱼藤酮作用多巴胺能神经细胞系SH SY5Y细胞不同时间 ,用四唑盐比色法检测细胞活性 ,用流式细胞术检测细胞的线粒体膜电位。结果 :低剂量 ( 5nmol/L和 2 0nmol/L)鱼藤酮处理 4 8h内对细胞活性无明显影响 (P >0 .0 5) ;超过 10 0nmol/L的鱼藤酮明显抑制细胞活性 (P <0 .0 5) ,并且抑制的程度呈剂量依赖性。 2 0nmol/L及更高浓度的鱼藤酮作用 2 4h可以引起细胞线粒体膜电位明显下降 (P <0 .0 5)。结论 :采用2 0nmol/L鱼藤酮作用 2 4h可以建立鱼藤酮多巴胺能神经细胞早期凋亡模型。     

Effects of griseofulvin on in vitro porcine oocyte maturation and embryo development

Griseofulvin is an orally administered antifungal drug that affects microtubule formation in vitro and interferes with microtubule dynamics in vivo as clearly shown for mitotic cells in several cell systems. This article reports the effects of griseofulvin on in vitro maturation of porcine oocytes and subsequent effects on embryo development. Our results revealed a concentration‐dependent effect on meiotic spindles with 20–40 μM griseofulvin affecting oocyte maturation, and 40 μM affecting fertilization and embryo development. These concentrations of griseofulvin did not affect mitochondrial and cortical granule distribution that also depend on microtubule and cytoskeletal functions during oocyte maturation. Specific effects on the meiotic spindle included spindle disorganization and aberrant chromosome separation displayed as prominent chromosome clusters in oocytes treated with 40 μM griseofulvin. These results strongly suggested that griseofulvin affected porcine oocyte in vitro maturation and following embryo development by disturbing microtubule dynamics. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence

It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization.     

Mitochondrial distribution and function in oocytes and early embryos

Active mitochondria relocate during oocyte maturation or fertilization in several species. Detailed studies with hamster oocytes and early embryos reveal a pattern of active mitochondria migrating to surround the pronuclei to form a pattern that persists through the early cleavage stages. Although the functional significance of this relocation is unknown, it appears to be an important part of normal development in hamsters. Treatments that disrupt embryo development in vitro (such as the presence of inorganic phosphate or alteration of intracellular pH) also disrupt the normal pattern of mitochondrial distribution. Active mitochondria also reorganize during maturation in bovine oocytes and during fertilization in rhesus monkey oocytes. Examination of these changes in mitochondrial organization may provide insights into the regulation of normal embryo development and might serve as predictors of oocyte or embryo developmental competence.     

Low oocyte mitochondrial DNA content in ovarian insufficiency

BACKGROUND: Mitochondrial biogenesis and bioenergetics play an important role in oocyte maturation and embryo development. We have investigated the relationship between defective mitochondrial biogenesis and the lack of oocyte maturity observed during IVF procedures with patients suffering from ovarian dystrophy and ovarian insufficiency. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in 116 oocytes obtained from 47 women undergoing the ICSI procedure. We compared the mtDNA content of oocytes from women with a normal ovarian profile with that of oocytes from women with ovarian dystrophy and ovarian insufficiency. RESULTS: We found an average of 256,000 +/- 213,000 mitochondrial genomes per cell. The mean mtDNA copy number was not significantly different in ovarian dystrophy compared with controls, but it was significantly lower in oocytes from women with ovarian insufficiency (100,000 +/- 99,000, P < 0.0001). CONCLUSIONS: Our results suggest that low mtDNA content is associated with the impaired oocyte quality observed in ovarian insufficiency.     

Mitochondrial DNA content affects the fertilizability of human oocytes

Mitochondrial DNA content varies considerably in oocytes, even when collected from the same patient. In the present study, real-time quantitative polymerase chain reaction analysis of 113 unfertilized oocytes obtained from 43 patients revealed an average of 193,000 (range: 20,000 to 598,000) mitochondrial genomes per cell. We compared several groups of oocytes to investigate the relationship between mitochondrial DNA content and fertilizability. The average mitochondrial DNA copy number was significantly lower in cohorts suffering from fertilization failure compared to cohorts with a normal rate of fertilization. In addition, the mitochondrial copy number of oocytes from patients with fertilization failure due to unknown causes was significantly lower than that of oocytes from patients in which IVF failure was due mainly to a severe sperm defect. The lower mtDNA copy number could be due to defective cytoplasmic maturation of oocytes. We conclude that low mitochondrial DNA content, due to inadequate mitochondrial biogenesis or cytoplasmic maturation, may adversely affect oocyte fertilizability.     

Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle imaged with PolScope in in vitro matured human oocytes

BACKGROUND: Birefrigent meiotic spindle in live human oocytes can be visualized by the PolScope. This study investigated the relationship between birefrigent meiotic spindle and cytoplasmic mitochondrial DNA (mtDNA) and ATP contents in in vitro matured human oocytes. METHODS: Oocytes at germinal vesicle stage were collected and cultured for 24-48 h with or without the metabolic inhibitor, carbonyl cyanide p-(tri-fluromethoxy) phenyl-hydrazone (FCCP). All in vitro matured oocytes were examined by PolScope for the presence of meiotic spindle, then the oocytes were used for either intracytoplasmic sperm injection or the measurement of mitochondrial quantity and ATP content. RESULTS: Meiotic spindles were observed in 51.3% (60/117) of the in vitro matured oocytes. Oocytes with detectable meiotic spindle contained significantly higher mtDNA copies (637 250 +/- 237 606 versus 491 454 +/- 153 406, P = 0.027) and ATP content (1.97 +/- 0.38 versus 1.65 +/- 0.32 pmol, P = 0.028) when compared with those without detectable meiotic spindle. However, in vitro matured oocytes showed a significantly reduced rate of positive meiotic spindle and a lower ATP content when cultured with FCCP. A lower incidence of normal fertilization and good quality embryos were observed if meiotic spindles were not detected. CONCLUSIONS: Low mtDNA and ATP content might contribute to the absence of birefringent spindle imaged with the PolScope in human in vitro matured oocytes.     

Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos

Mitochondria play a vital role in the metabolism of energy-containing compounds in the oocyte cytoplasm to provide adenosine trisphosphate for fertilization and preimplantation embryo development. In this study, ratiometric confocal microscopy with the mitochondrion-specific membrane potential-sensitive fluorescence dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) was used to measure the activity of mitochondria in human oocytes and developing preimplantation embryos. Mitochondria in oocytes and embryos were characterized by distinct localized aggregation patterns. These patterns however did not determine localized regions of heterogeneity in mitochondrial activity. Mitochondrial activity was analysed during oocyte maturation and after fertilization. The activity of mitochondria in fresh metaphase II oocytes was negatively correlated with maternal age. This trend continued when the activity of developing embryos was analysed. Mitochondrial activity was strongly correlated with the rate of embryo development on day 3 after fertilization, but not on day 2. Partial regression analysis showed that the rate of cleavage of preimplantation embryos was more highly correlated with embryo mitochondrial activity than maternal age. These data suggest that the efficiency of mitochondrial respiration in oocytes and preimplantation embryos is closely correlated with the programmed rate of embryo development, and suggest that maternal age further influences this factor. The loss of mitochondrial activity in oocytes obtained from ageing couples may therefore contribute to lower embryo development and pregnancy rates observed during cycles of IVF.     

Identification of possible mediators of embryonic mortality caused by mastitis: actions of lipopolysaccharide, prostaglandin F2alpha, and the nitric oxide generator, sodium nitroprusside dihydrate, on oocyte maturation and embryonic development in cattle

PROBLEM: Mastitis and immunization against constituents of organisms causing mastitis can reduce fertility of cattle and sheep, respectively. For the current experiments, it was hypothesized that these effects are mediated via actions of lipopolysaccharide (LPS), prostaglandin F2alpha (PGF2), and nitric oxide on oocyte maturation and embryonic development. METHOD OF STUDY: To evaluate effects on oocyte maturation, oocytes were matured with various concentrations of LPS, PGF2alpha, or the nitric oxide (NO) generator, sodium nitroprusside (SNP). Following maturation, oocytes were fertilized and cultured until day 8 after fertilization. To test effects on embryo growth, oocytes were matured and fertilized and cultured after fertilization with LPS, PGF2alpha, or SNP. RESULTS: Addition of 100 and 1000 ng/mL LPS and 50 and 100 ng/mL PGF2alpha to oocyte maturation medium reduced the proportion of oocytes that became blastocysts at day 8 after fertilization. When added after fertilization, in contrast, neither LPS nor PGF2alpha reduced development to the blastocyst stage. Unlike for LPS and PGF2alpha, addition of SNP during oocyte maturation was without effect on the proportion of oocytes that became blastocysts at day 8 after fertilization. However, addition of 10 microM SNP to culture medium after fertilization completely prevented development to the blastocyst stage while 0.1 and 1 microM SNP did not affect development. CONCLUSIONS: Results indicate that increased local concentrations of LPS, PGF2alpha, and NO can have deleterious consequences on oocyte function (LPS, PGF2alpha) and embryonic development (NO). Thus, these molecules are putative mediators of effects of infectious disease or inflammation, including mastitis, on fertility of cattle.