Role of inhibitor of growth 2 in the development of mouse preimplantation embryo

目的 探讨生长抑制因子2 (Ing2)在小鼠植入前胚胎中的表达及生物学功能.方法 使用ICR小鼠进行促进排卵以获得植入前胚胎.采用免疫荧光方法对Ing2蛋白在小鼠植入前胚胎中的定位进行研究,采用显微注射Ing2-esiRNA实验对其在早期胚胎中的功能进行研究.抽提小鼠囊胚期合子的RNA反转录为cDNA,进行实时PCR分析.结果 免疫荧光结果显示Ing2蛋白在小鼠植入前胚胎中定位于胞浆及胞核.从4-cell期胚胎开始,胞核中的表达量明显增高.Ing2-esiRNA显微注射实验显示在小鼠合子中干扰Ing2后,各期胚胎形成率均显著低于对照组(4-cell期胚胎比率:82.7％ vs.94.7％;桑葚胚比率:67.3％vs.89.4％；囊胚形成率:48.7％ vs.75.8％)(P＜0.01).结论 Ing2的下调,引起植入前小鼠胚胎发育阻滞.     

囊胚期与卵裂期胚胎培养与移植比较

目的分析早期卵裂期与囊胚期胚胎培养与移植结局。方法选取因单纯输卵管因素及男性因素接受体外受精-胚胎移植患者,其中选择卵裂期胚胎移植者110例,选择囊胚期胚胎培养与移植者57例。受精后早期胚胎培养采用G1,囊胚培养采用G2。比较两组的获卵数、受精数、胚胎植入率、妊娠率以及囊胚组囊胚形成率、移植胚胎数与多胎率、妊娠率的关系。结果囊胚组获卵率、受精率分别比早期胚胎组多4.98及3.98个;采用G1/G2序贯培养,囊胚形成率为32.9%;两组胚胎植入率、临床妊娠率分别为31.2%、42.1%及11.5%、25.5%,囊胚组明显高,差异有显著性;移植2个囊胚与移植3个囊胚比较,妊娠率无显著差异,但可减少双胎妊娠例数。结论囊胚期胚胎移植可明显提高胚胎植入率及妊娠率。移植两个囊胚为IVF-ET的最好选择。     

1658例玻璃化冻融周期的临床结局分析

目的：探索应用玻璃化冻融技术冻融卵裂期胚胎及囊胚的临床效果。方法来源于江西省妇幼保健院生殖中心2012年9月至2014年1月1658例玻璃化冻融移植周期（其中卵裂期胚胎1426周期共2907枚胚胎,囊胚232周期共341枚胚胎）,用自制麦管作为胚胎载体及相同的玻璃化方法进行冷冻、复苏,及胚胎移植,观察其复苏存活率、全胚融解率、临床妊娠率、植入率和流产率等。结果卵裂期胚胎复苏率为98.5%,全胚融解率是0.35%。囊胚复苏率为99.7%,全胚融解率是0.43%。卵裂期胚胎的胚胎植入率显著低于囊胚（35.4%vs 49.3%）（P〈0.05）,平均每周期移植胚胎数显著高于囊胚（2.0±0.3 vs 1.5±0.5）（P〈0.05）,临床妊娠率低于囊胚（50.7%vs 55.4%）,差异无显著性意义,P〉0.05,双胎率卵裂期胚胎显著高于囊胚（38.3%vs 28.9%）（P〈0.05）,早期流产率无显著性差异（6.8%vs 7.8%,P〉0.05）。结论使用相同的玻璃化冷冻方法对人类卵裂期胚胎和囊胚可获得理想的冷冻效果。     

用实时荧光定量RT-PCR检测单个小鼠种植前胚胎中特异性基因表达

目的用单细胞实时荧光定量RT-PCR,检测单个小鼠种植前胚胎中特异性基因表达。方法采集小鼠卵母细胞和原核期胚胎,后者经体外培养至囊胚。采用实时荧光定量RT-PCR对多个及单个卵母细胞和2-细胞期胚胎中Tcl1基因的表达进行定量检测;用同样方法对小鼠种植前各发育阶段的胚胎进行Tcl1基因的表达检测。结果在多个及单个卵母细胞和2-细胞期胚胎中,均检测出Tcl1的表达;多个卵母细胞、2-细胞期胚胎中Tcl1的表达量显著高于单个卵母细胞、2-细胞期胚胎;此方法也适用于胚胎早期发育的各个时期,Tcl1在单个卵母细胞、原核期胚胎和2-细胞期胚胎中表达量最高,在4-细胞期胚胎、8-细胞期胚胎、桑葚胚和囊胚中表达量极低。结论应用单细胞实时荧光定量RT-PCR可对单个小鼠种植前胚胎中基因的表达进行定量检测。     

生长激素在无优质囊胚形成患者中的应用研究

目的:评估生长激素在无优质囊胚形成患者中的应用效果。方法:研究纳入2019年1月前于某院就诊的无优质囊胚形成患者,其中对照组204例,实验组135例。实验组在促排卵周期启动前一次月经期第3～5d开始使用生长激素至取卵前注射HCG日,对照组则不使用生长激素。结果:两组之间的胚胎植入率、临床妊娠率、卵泡液IGF1和IGF2无统计学差异;实验组囊胚数大于对照组,实验组的促性腺激素总量少于对照组(P0.05)。两组卵泡液IGF1和IGF2与促性腺激素总量、囊胚数、优质囊胚数之间无明显相关性。结论:生长激素预治疗提高胚胎质量,但没有提高胚胎植入率以及妊娠率。     

囊胚培养的相关因素分析

目的探讨体外受精(IVF)和精子卵胞浆内注射技术(ICSI)治疗周期中体外培养囊胚形成的影响因素。方法将卵裂期第3d(D3)冷冻后剩余胚胎进行体外延长培养至囊胚期,观察囊胚形成情况。结果共收集7438个冷冻后剩余胚胎,经序贯培养,形成1382个优质囊胚(18.58%)。D3胚胎中6个细胞的优质囊胚形成率(10.34%)显著高于4-5个细胞的优质囊胚形成率(5.45%),差异有统计学意义(P=0.011);7-9个细胞的优质囊胚形成率(19.95%)显著高于6个细胞的优质囊胚形成率(10.34%),≥10个细胞的优质囊胚形成率(27.47%)显著高于7-9个细胞的优质囊胚形成率(19.95%),差异均有统计学意义(P<0.001)。D2胚胎中4个细胞的优质囊胚形成率(25.40%)显著高于3个细胞、5个细胞的优质囊胚形成率(10.26%,15.41%),差异有统计学意义(P<0.001)。碎片评级I-II级D2、D3胚胎的囊胚形成率均显著高于III-IV级的胚胎,差异有统计学意义(P<0.001)。结论体外延长培养能有效筛选具有发育潜力的胚胎,优质囊胚形成与D2、D3胚胎的卵裂球数及碎片评级密切相关。     

SARI基因真核表达载体的构建及稳定转染HeLa细胞系的建立

目的构建人SARI基因真核表达载体,并建立稳定转染的HeLa细胞系。方法采用RT-PCR技术从正常人外周血单个核细胞cDNA文库中扩增SARI基因序列,连接插入至pCMV-N-Flag真核表达载体,并对重组质粒进行酶切及测序鉴定;阳离子聚合物介导重组质粒转染HeLa细胞后,用G418抗生素筛选稳定转染的细胞系。间接免疫荧光检测Flag-SARI融合蛋白在HeLa细胞系中的表达定位。RT-PCR及Western blot检测SARI mRNA及蛋白的表达变化。结果双酶切和DNA测序表明pCMV-Flag-SARI真核表达质粒构建成功。经G418筛选后,RT-PCR和Western blot表明,转染重组质粒的HeLa细胞系中SARI mRNA及蛋白的表达明显升高。间接免疫荧光结果显示,Flag-SARI融合蛋白在HeLa细胞系的胞质和胞核中均有表达,且胞质荧光信号强于胞核。结论成功构建pCMV-Flag-SARI真核表达载体,表达的FlagSARI蛋白主要定位于HeLa细胞系的胞质中。     

不同质量胚胎囊胚培养比较以及对妊娠结局的影响

目的:探讨不同质量胚胎进行囊胚培养的情况以及移植后对妊娠结局的影响。方法:收集2018年1月—2019年12月太原市中心医院生殖中心行常规体外受精/卵胞浆内单精子显微注射-胚胎移植(IVF/ICSI-ET)助孕且进行囊胚培养的患者资料,根据囊胚培养前胚胎质量分为优质胚胎囊胚培养组(A组)与非优质胚胎囊胚培养组(B组),比较两组患者一般情况、囊胚培养情况以及妊娠结局。结果:共156个周期患者资料,两组年龄、体质量指数(BMI)、不孕年限、基础雌二醇(E₂)、基础促卵泡激素(FSH)、子宫内膜厚度等一般资料比较,差异无统计学意义(P>0.05)。囊胚培养结果显示,A组囊胚形成率和可利用囊胚形成率都高于B组(P<0.05)。妊娠结局显示,两组移植胚胎数、临床妊娠率比较,差异无统计学意义(P>0.05),但A组多胎率高于B组(P<0.05)。结论:优质胚胎囊胚培养形成率及可利用囊胚率优于非优质胚胎,但临床妊娠率无差异,只是优质胚胎囊胚培养移植后多胎率较高,临床可对非优质胚胎进行囊胚培养以提高临床妊娠率。     

氯化锰诱导脉络丛上皮细胞系Z310中PHB1上调表达的亚细胞分布和定位特征

目的测定氯化锰(MnCl_2)处理脉络丛上皮细胞质Z310后升高的抑制素蛋白1(PHB1)在亚细胞中的分布及重定位,以及在细胞胞浆中的主要定位亚细胞器,并观察PHB1与细胞生长调控蛋白P53在细胞内是否有共定位关系。方法采用免疫蛋白印迹法(Western Blot)测定MnCl_2(0,100,200μmol/L)处理24 h后Z310细胞胞浆和胞核内的PHB1表达的变化及其重定位;采用免疫荧光法(Immunofluorescence,IF)结合激光共聚焦显微镜,检测并观察MnCl_2处理后PHB1的亚细胞空间分布,及其与P53的亚细胞共定位特征。结果 Western Blot检测表明,与对照组比较,MnCl_2处理后Z310细胞内PHB1的表达在胞浆中升高,同时在胞核内降低(P0.05);免疫荧光法显示MnCl_2处理后PHB1在胞浆和胞核均存在,且在胞浆中主要分布于线粒体上;在胞浆和胞核内发现PHB1与P53有共定位(Co-localization)。结论MnCl_2处理后,上调表达的PHB1蛋白主要位于胞浆,同时发现胞核内PHB1降低,提示MnCl_2能够引起Z310细胞中PHB1蛋白升高并从胞核向胞浆移动,这种亚细胞水平的PHB1空间重定位(translocation)改变是锰毒性依赖性的;胞浆和胞核中的PHB1均与P53有共定位关系;胞浆中的PHB1主要存在于线粒体上。     

免疫荧光技术中不同固定剂对细胞p65核移位观察效果的影响

目的观察免疫荧光技术中不同固定剂以及Triton X-100不同渗透时间对小鼠腹腔巨噬细胞p65蛋白核移位的影响,找出最适合观察p65蛋白移位的固定方法及Triton X-100渗透时间。方法采用免疫荧光的方法观察使用不同固定剂(甲醇,1%～4%甲醛)固定,0.1%Triton X-100渗透3～15min的条件下,观察脂多糖刺激2h后,p65蛋白细胞内定位的情况。结果 p65蛋白在对照细胞主要定位于细胞质,脂多糖(LPS)刺激后p65蛋白移入胞核。甲醇固定后细胞荧光染色显色很弱且核区不明显,染色弥散;1%、2%甲醛固定不良可引起细胞收缩;4%甲醛固定后,核区与胞质p65蛋白荧光染色对比明显。0.1%Triton X-100作用3～5min效果最佳,而作用10～15min对照细胞核内也有较强荧光。结论本实验表明,在免疫荧光技术中采用4%甲醛固定20min,0.1%Triton X-100渗透3～5min可更好的观察LPS诱导的小鼠腹腔巨噬细胞p65核移位。     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on preimplantation mouse embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental contaminant that can exert developmental toxicity. To investigate the stage-specific effects of TCDD on preimplantation embryos, we exposed mouse embryos to TCDD at different stages (1-, 2-, and 8-cell) and collected them at different stages of development (the 1- or 2-, 8-cell, and blastocyst stage, respectively). Semiquantitative RT-PCR revealed increased constitutive gene expression of the arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) at the 1-cell stage, decreased expression at the 2- to 8-cell stage, and increased expression again at the blastocyst stage, and addition of TCDD to media did not affect their mRNA levels. Interestingly, no cytochrome P4501A1 (CYP1A1) mRNA was detected in embryos at the 1-, 2-, and 8-cell stages after exposure to 10 nM TCDD for 12 or 24 h, whereas CYP1A1 mRNA was significantly increased at the blastocyst stage in response to TCDD, and its induction was found to be concentration-dependent on TCDD exposure from 0.01 to 10 nM for 24 h. In addition, no significant differences in development rate of preimplantation embryos, cell number of blastocyst embryos, or apoptotic indices, such as TUNEL-positive cell number or Bax/Bcl-2 expression ratios were observed at the blastocyst stage between TCDD-exposed groups and non-exposed group. These results suggest that the sensitivity to TCDD differs with the embryonic stage, which may reflect an ability of embryos to adapt to environmental stressors, such as dioxins.     

Effects of 3H-thymidine on preimplantation mouse embryos in vitro

The effect of 3H-thymidine on in vitro development of preimplantation mouse embryos was studied. Two-cell and 4-8-cell embryos from B6CBA/F1 mice were continuously exposed to 3H-thymidine in medium containing 3H-thymidine in concentrations ranging from 10-500 nCi/ml. The effect of the radioactive precursor on embryo development to the blastocyst stage was studied by morphological observation, counting the blastocyst cell number and measuring 3H-thymidine incorporation. The continuous presence of 3H-thymidine significantly inhibited development of 2-cell and 4-8-cell embryos to the blastocyst stage. Embryos cultured from the 2-cell stage were more sensitive to 3H-thymidine than those exposed from the 4-8-cell stage. Even in morphologically normal blastocysts the cell number was significantly reduced. A 2 hr pulse of 100 nCi/ml 3H-thymidine at the blastocyst stage, did not affect the blastocyst formation or the blastocyst cell number and the amount of incorporated 3H-thymidine was sufficient to provide a reliable quantitation of DNA synthesis during the culture of preimplantation embryos in vitro. Continuous incubation with 3H-thymidine in order to measure DNA synthesis of preimplantation mouse embryos should be avoided when DNA synthesis is used as a means of evaluating toxic effect of an agent. Adverse radiation effects by 3H-thymidine on preimplantation mouse embryos during toxicity testing can be avoided by pulse labelling.     

Development of preimplantation mouse embryos after exposure to a 50 Hz magnetic field in vitro.

Effect of sinusoidal 50 Hz magnetic field (MF) on development of preimplantation CBA/S mouse embryos in vitro was studied. Superovulated and in vivo fertilized preimplantation embryos were collected at one cell stage and divided to control and MF-exposed groups. Sinusoidal 50 Hz MF with field strength of 10 A/m r.m.s., corresponding a flux density of 13 microT r.m.s., was used to expose the embryos in culture at 37 degrees C in a CO2-incubator. The developmental stage and abnormalities were recorded twice daily except once daily during weekends. The vitality and developmental stages of the embryos were similar in both groups although slightly more dead embryos were found during the 1st day in MF-exposed group (P<0.05) and the development of MF-exposed embryos was slightly impaired. In conclusion, the exposure to sinusoidal 50 Hz MF at field strength of 10 A/m did not significantly disturb the development of the mouse embryos in vitro up to the blastocyst stage.     

Embryotoxicity in vitro with extract of Indigofera suffruticosa leaves

Aqueous extract of leaves of Indigofera suffruticosa (AELIs) were studied for adverse effects in preimplantation mouse embryos. Two-cell mouse embryos were cultured for 94 h in human tubal fluid medium (HTF), and AELIs at a concentration of 5 or 10 mg/ml. On Day 4 of culture, morulae and blastocysts were collected for morphological analysis of blastomeres. We found that embryos exposed to the higher concentration of AELIs (10 mg/ml) did not develop and all embryos persisted at the two-cell stage. Those embryos exposed to the lower concentration (5 mg/ml) showed development until morula, blastocyst and hatched blastocyst stages that were similar to the controls. These results suggest that use of AELIs may be hazardous to humans who make use of it in folk medicine.     

Cleavage and development in cultured preimplantation mouse embryos exposed to lidocaine

Two-cell preimplantation mouse embryos were exposed in vitro to lidocaine (0 to 1,000 μg/mL) for 72 h to determine the effects of this anesthetic on subsequent cleavage and development during prolonged exposures. Embryonic development was monitored each 24 h for 3 d. Lidocaine adversely affected the in vitro development of the mouse embryos, altering the distribution of the development stages at the evaluated culture tunes. The percentage of two-cell embryos that cleaved and developed to more advanced stages was decreased by the exposure to lidocaine. After 24 h of culture, two-cell embryos were arrested before completion of cellular division; this occured in 30% of the embryos at concentrations of 10 to 100 μg/mL and in 73.2% of the embryos with 1,000 μg/mL. After 48 h the blastomeres of the arrested embryos began to degenerate, showing lysis or fragmentation. At the lowest concentration, 14.9% of the arrested embryos exhibited the capacity to recover. These embryos continued their cleavage and normal development towards blastocyst formation. The cytotoxic effect and arrest at the two-cell stage were observed in a dose-dependent manner after 72 h of culture. We conclude that sensitivity to lidocaine embryotoxicity occurs during a window at the two-cell stage.     

Knockdown of p66Shc by siRNA injection rescues arsenite-induced developmental retardation in mouse preimplantation embryos

Two-cell arrest plays a principal role in the elevated levels of embryo loss during the first week of development in mouse. Previously, we have shown that arsenic can apparently induce 2-cell arrest in mouse preimplantation embryo and the expression of oxidative stress adaptor protein p66^Shc is up-regulated in this process. In the present study, we demonstrated that microinjection of p66^Shc siRNA into the pronucleus of zygotes resulted in a markedly decrease in both mRNA and protein levels of p66^Shc. The arsenite-induced 2-cell arrests, along with a reduction in the levels of reactive oxygen species (ROS), were significantly inhibited and the number of embryos developing to morula stage concurrently increased upon p66^shc siRNA microinjection. These findings indicate that knockdown of p66^shc improves the developmental competence of arsenite-exposed embryos in vitro by increasing the resistance to oxidative stress. In addition, we highlight the utility of single-embryo analysis in preimplantation embryos.     

Sensitivity of preimplantation mouse embryos to abrin

The effect of the plant toxin abrin on preimplantation mouse embryos in vitro was studied. Two-cell embryos from C57BL/6J or B6CBA/F1/Bom female X B6CBA/F1/Bom male mice were exposed to abrin for 72 hrs in vitro, in medium containing abrin in concentrations ranging from 0.1 to 10,000 pg/ml. Two-cell mouse embryos were also exposed to corresponding concentrations of ricin in vitro. The effect of abrin was evaluated by daily morphological observation of the embryos up to the blastocyst stage and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. The effect of ricin was evaluated by morphological observation of the embryos up to the blastocyst stage. The results showed that 2-cell mouse embryos were highly sensitive to abrin, 1 pg/ml being the lowest concentration of the toxin that significantly inhibited the development of 2-cell embryos to the blastocyst stage. With ricin statistically significant inhibition of blastocyst formation was obtained at a concentration of 10 pg/ml. The ID50 for abrin was calculated to be 24 pg/ml and for ricin 47 pg/ml. A dose-dependent lag period was observed before the effect of abrin or ricin on the formation of blastocysts became evident. Incorporation of 3H-thymidine, 3H-uridine and 14C-leucine into DNA, RNA and protein, respectively, was also inhibited by abrin after different time intervals in culture.     

胰岛素与葡萄糖对ICR小鼠早期胚胎体外培养的影响

目的探讨胰岛素与葡萄糖对ICR小鼠早期胚胎体外发育的影响。方法同时添加胰岛素＋葡萄糖的CZB培养液对ICR小鼠胚胎进行体外培养,观察囊胚发育率、孵化胚率和囊胚细胞数的变化,并研究两者最佳的浓度搭配。结果在CZB培养液中同时加入葡萄糖与胰岛素可促进小鼠1-细胞胚胎体外培养,且0．05μg·ml^-1胰岛素与5mmol·L^-1葡萄糖添加组囊胚率、孵化胚率和囊胚细胞数均显著高于其他各浓度添加组。结论联合添加胰岛素和葡萄糖的培养液对小鼠胚胎体外发育具有促进作用,且胰岛素浓度为0．05μg／ml,葡萄糖浓度为5mmol／L时促进作用最大。     

Genotoxic and embryotoxic effects of gonadotropin-hyperstimulated ovulation of murine oocytes, preimplantation embryos, and term fetuses.

Compared to spontaneous ovulation, gonadotropin-hyperstimulated ovulation (superovulation) in mice resulted in a fourfold increase in the number of preimplantation embryos 3 days post coitum, 50% of which died before term. Both in vitro development of embryos during the preimplantation period and transfer of morulae from superovulated females to pseudopregnant untreated foster mothers indicate that the prenatal loss occurring shortly before implantation up to term is due to maternal factors rather than to direct hormonal effects on oocytes or early embryos. Indeed, no genotoxic events could be observed in 4-cell to blastocyst stage embryos from superovulated female mice as revealed by the chromosomal aberration test and the sister chromatid exchange assay. Chromosome analysis of the pronuclei from mouse zygotes showed an increased rate of aberrations in oocyte-derived nuclei after superovulation in comparison to spontaneous ovulation. The present data suggest that aberrant murine oocytes may be fertilized, but they do not survive the first cleavage stages. The result is discussed with respect to the high incidence of chromosomal abnormalities found in human oocytes after gonadotropin-hyperstimulated ovulation.