超排卵周期未成熟卵体外培养的研究

目的研究来源于超排卵周期中的未成熟卵母细胞，观察其体外培养成熟、受精及胚胎发育能力，探讨IVM技术在超排卵周期中的未成熟卵母细胞体外成熟的临床应用。方法比较同源体内成熟卵和体外成熟卵进行IVF／ICSI后的正常受精、卵裂、优质胚胎和囊胚形成情况。结果体外培养中72．41％的GV期卵和76．08％的MI期卵在36h内达到成熟，两组成熟率无明显差异（P〉0．05）。体内成熟与体外成熟卵相比较，受精率无明显差异（P〉0．05），但卵裂、优质胚胎形成率低，差异有显著性（P〈0．05）。未成熟卵母细胞组共有13个囊胚培养形成。结论超排卵周期中的未成熟卵，能够继续在体外发育成熟。虽然优质胚胎的形成率低于体内成熟卵，但在同一促排周期增加了可移植胚胎数。同一促排周期所获的未成熟卵行体外成熟培养是可行的。     

IGF-Ⅱ对小鼠2-细胞胚胎体外发育的影响

目的研究胰岛素生长因子-Ⅱ对小鼠2-细胞胚胎体外发育的影响。方法在mKSOM培养液中添加不同浓度的IGF—Ⅱ（insulin—like growth factor—Ⅱ）对小鼠2-细胞胚胎进行体外培养,观察囊胚发育率、孵化率和囊胚细胞数的变化。结果（1）培养到72h,实验组囊胚率显著高于对照组。（2）培养到96h,0．1、1、10ng／ml IGF-Ⅱ添加组的孵化率显著高于对照组和100ng／ml IGF—Ⅱ添加组。（3）进行囊胚细胞计数,实验组与对照组比较,差异无显著性;四个实验组之间比较,差异亦无显著性。结论IGF-Ⅱ浓度为0．1、1、10ng／ml时,能促进小鼠2-细胞胚胎的体外发育。     

雌激素受体抑制剂对小鼠囊胚形成的影响

目的观察雌激素受体（estrogen receptor,ER）抑制剂对昆明（Kunming,KM）小鼠体外囊胚形成的影响,为进一步探讨雌激素受体在小鼠囊胚形成中的作用和机制提供实验依据。方法以空白KSOM培养液为对照组,KSOM中添加浓度为1、2、3、4、5p~mol／L的ER仪抑制剂MPP,以及浓度为1、10、50、100μmol／L的ERB抑制剂PHTPP为实验组,收集KM小鼠8．细胞胚进行体外连续培养,计数各组发育至桑葚胚和囊胚两个阶段的数目,比较各组发育到囊胚的比率。结果添加浓度为1~mol／L、2Ixmol／L的MPP实验组囊胚发育率分别为86．36％、86．40％,与对照组（82．26％）相比无明显差别（P〉O．05）,而添加浓度为3μmol／L、4μmol／L、5μmol／L的MPP实验组囊胚发育率分别为46．58％、7．50％、0,发育率显著下降（P〈0．01）。添加浓度为1μmol／L、10μmol／L、50~mol／L的PHTPP实验组囊胚发育率分别为82．61％、87．04％、83．98％,与对照组（82．09％）比较没有明显差异（P〉O．05）,而添加高浓度（100μmol／L）的PHrrPP实验组囊胚发育率（51．39％）较明显下降。结论低浓度（5μmol／L）的ERo的抑制剂MPP可完全抑制KM小鼠8一细胞胚体外发育到囊胚,而50~mol／L的ERB抑制剂PHTPP对KM小鼠囊胚形成无明显影响,高浓度（100μmol／L）的PHTPP才可部分抑制囊胚形成。提示在小鼠囊胚形成中,ERα起着更为主要的作用。     

8型解脲支原体对小鼠早期胚胎发育的致病力研究

目的观察不同浓度的8型解脲支原体对小鼠早期胚胎发育的影响,以探讨解脲支原体对早期胚胎发育的致病作用。方法取小鼠2-细胞期胚胎随机分为实验组与对照组,对照组：组1,2-细胞期鼠胚在不含8型UU的HTF培养液中培养;实验组：组2、组3、组4、组5,2-细胞期鼠胚分别在含102CCU/ml～105CCU/ml浓度的8型UU的HTF培养液中培养,在37℃、5%CO2的培养箱中培养72h,每24h在Nikon倒置显微镜下观察记录,通过统计各组的D2评分、4-细胞期胚胎形成率、桑椹期胚胎形成率、囊胚形成率,评价早期胚胎的发育情况。以观察不同浓度8型UU对小鼠早期胚胎发育的影响。结果 8型UU在培养液中的终浓度为102CCU/ml组2-细胞胚胎以后的D2评分、4-细胞期胚胎形成率、桑椹期胚胎形成率、囊胚形成率等各阶段的胚胎发育指标与对照组比较差异均无统计学意义（P〉0.05）,终浓度分别为104CCU/ml、105CCU/ml组2-细胞胚胎以后的各项胚胎发育指标与对照组比较均有显著性差异（P〈0.05）,终浓度为103CCU/ml组的4-细胞期胚胎形成率、囊胚形成率与对照组比较无显著性差异〉0.05,而D2评分、桑椹期胚胎形成率与对照组比较有显著性差异（P〈0.05）,103CCU/ml组的P值均接近0.05显著性差异临界值。结论 UU能够影响小鼠早期胚胎的发育,8型UU≥104CCU/ml时就可阻碍小鼠早期胚胎的发育。随着UU浓度的增高,对早期胚胎发育的影响也增大。     

两种小鼠胚胎体外发育系统对内毒素敏感性的比较研究

目的 :探讨小鼠 1细胞和 2细胞胚胎体外发育系统对内毒素的敏感性。方法 :获取小鼠 1细胞胚胎和 2细胞胚胎 ,分别与 3个内毒素剂量 (0 .7,7,70EU/ml)组共培养 ,计算各阶段胚胎的发育率。结果 :小鼠 1细胞和 2细胞胚胎的内毒素0 .7EU/ml组 ,从 2细胞至囊胚各期发育率与对照组比较没有显著性差异 (P >0 .0 5 )。内毒素在 7EU/ml水平显著降低小鼠 1细胞胚胎的体外发育率 (P <0 .0 1) ,但对 2细胞胚胎未见明显影响。内毒素在 70EU/ml水平则完全抑制 1细胞和 2细胞胚胎的体外发育 (P <0 .0 1)。培养液中含有内毒素引起碎裂胚胎增多 ,卵裂球退化和轮廓不清。结论 :内毒素对体外培养的胚胎有明显毒性 ,小鼠 1细胞胚胎体外发育试验对内毒素较 2细胞胚胎试验敏感     

不同激素剂量和组合对不同品系小鼠超数排卵的影响

背景：超数排卵效果受动物品系、营养水平、年龄、发情周期阶段、光照、超排方法、超排所用激素种类和剂量等诸多因素影响,其中激素剂量和动物品种是关键因素。 目的：探讨不同剂量孕马血清促性腺激素(pregnant mare serum gonadotropin,PMSG)和人绒毛膜促性腺激素(human chorionic gonadotropin, HCG)组合,对不同品系小鼠超数排卵效果的影响。 方法：分别用不同剂量的PMSG和HCG对ICR鼠、KM鼠和BALB/c鼠进行超数排卵处理,比较激素处理后各品系小鼠超数排卵的胚胎总数、平均胚胎率、正常胚胎及平均可用胚率。 结果与结论：5 IU PMSG+7 IU HCG剂量组合对ICR鼠和KM鼠超排处理效果较好,BALB/c鼠超排的最适激素剂量为3 IU PMSG+5 IU HCG;使用5 IU PMSG+7 IU HCG剂量组合分别处理3种品系小鼠时,ICR鼠和KM鼠平均胚胎率和平均可用胚率显著高于 BALB/c鼠(P < 0.05)。为获得较多的胚胎进行相关实验,应当选择ICR和KM等小鼠进行超排,特别是选择国际通用的ICR鼠。     

输卵管积水对小鼠胚胎体外发育能力的影响

目的探讨输卵管积水对小鼠胚胎体外发育能力的影响。方法收集人输卵管积水，小鼠促超排卵，收集2细胞胚胎和囊胚，随机分配到含有不同浓度输卵管积水的培养液中，观察胚胎的发育，计算成囊率，囊胚孵出率。结果输卵管积水组的胚胎成囊率和囊胚孵出率低于无积水组，并呈剂量依赖性。结论输卵管积水能显著影响胚胎的早期发育潜能。     

小鼠2-细胞胚胎卵裂球后代在囊胚中随机分布

目的 用异硫氰荧光素（FITC）-右旋糖苷和四甲基罗丹明（TMR）-右旋糖苷标记小鼠2-细胞胚胎的两个卵裂球,观察其发育,以探讨囊胚Em-Ab极性的形成。方法 向受精卵注射FITC-右旋糖苷确定标记物对胚胎的伤害性,向2-细胞胚胎两个卵裂球分别FITC-右旋糖苷分别和TMR-右旋糖苷,将标记后的胚胎体外培养发育至囊胚,观测两个卵裂球后代在囊胚中的分布情况。 结果 2-细胞胚胎两个卵裂球的后代在囊胚中分布并无规律。 结论 2-细胞胚胎卵裂球随机分布在囊胚的胚胎部分和胚外部分。     

内毒素对去卵透明带2-细胞小鼠胚胎体外发育的影响

目的 观察不同剂量浓度内毒素对去卵透明带2—细胞小鼠胚胎体外发育的影响。方法 获取小鼠2—细胞胚胎，用胰蛋白酶法去除卵透明带后，分别置于含有0，1pg/ml，5pg/ml和10pg/ml内毒素的CZB液滴中培养，此外，用蛋白酶法去除卵透明带，在不含内毒素的CZB液滴中培养，观察胚胎体外发育情况。结果 用胰蛋白酶法去卵透明带的2—细胞小鼠胚胎，其囊胚率随着培养液中内毒素剂量的增加而降低，且5pg/ml和10pg/ml时与对照组(不含内毒素)相比差异显著(P＜0．001)。在有内毒素存在时，停滞在2—细胞或4细胞期的胚胎彼此分离排成列，不再呈球形。部分卵裂球出现空泡或碎裂。另外，用蛋白酶法去除透明带的2—细胞鼠胚的囊胚率为61．7％，显著低于胰蛋白酶法的73．8％(P＜0．001)。结论 微量内毒素即明显抑制去卵透明带2—细胞小鼠胚胎的体外发育，并表现出剂量效应；去卵透明带2—细胞小鼠胚胎试验可应用于培养液的微量内毒素检测；胰酶法去除卵透明带后胚胎的囊胚率优于蛋白酶法。     

恶性肿瘤细胞对小鼠2-细胞胚胎体外发育的影响

目的： 探讨生命早期形式与恶性肿瘤细胞在相同微环境下的早期胚胎发育情况和肿瘤细胞的生物学行为。方法： 建立小鼠2-细胞胚胎-恶性肿瘤细胞(HepG2,SKOV3,HNE1,Hepa1-6,B16,CHO)体外共培养模型，观察小鼠胚胎的发育状况和恶性肿瘤细胞的生物学行为。结果： 处于不同种属源性和胚层来源恶性肿瘤微环境的小鼠胚胎4-细胞形成率，桑葚胚形成率和囊胚形成率及其形成的囊胚细胞数目与对照组比较无显著差异（P>0.05）。小鼠的2-细胞胚胎能在人源性和鼠源性不同胚层来源的恶性肿瘤细胞体外培养环境中按一定的时间顺序发生卵裂、卵裂球紧密化、分化、囊胚腔形成，而肿瘤细胞的形态、增殖及核分裂相与对照组比较没有明显差异。结论： 在共培养环境中，恶性肿瘤细胞对小鼠2-细胞胚胎的发育时程没有阻滞和破坏作用，且与恶性肿瘤细胞的种属和胚层来源无关。小鼠早期胚胎能在不同种属和胚层的恶性肿瘤环境中按正常体外培养发育时程发育，这可能与早期胚胎发育的自我组织、高适应性和肿瘤细胞分泌的某些因子有关。     

EGCG对昆明小鼠早胚体外发育的影响

目的观察表没食子儿茶素没食子酸酯(-)(EGCG)对昆明(kunming,KM)小鼠早胚体外发育的影响,为改善小鼠早胚体外培养体系奠定实验基础。方法以空白M16培养液为对照组,M16中添加浓度为0.1μg/mL、1μg/mL、10μg/mL、20μg/mL EGCG为实验组,收集KM小鼠1-细胞胚进行体外连续培养,计数各组发育至2-、4-细胞胚、桑椹胚和囊胚等各个阶段的数目,并以2-细胞胚为基数,计算各组至不同阶段的发育率。结果添加EGCG实验组发育到桑椹胚和囊胚的比率明显高于对照组,其中以添加浓度为10μg/mL和20μg/mL的EGCG实验组的效果最显著,其桑椹胚发育率分别为77.3%和78.7%,而对照组只有43.2%(P0.01)。10μg/mL和20μg/mL的EGCG实验组的囊胚发育率分别为53.0%和47.3%,也明显高于对照组(19.2%,P0.01)。结论EGCG可以显著提高KM小鼠1-细胞胚体外发育到桑椹胚及囊胚的比率,以10μg/mL和20μg/mL浓度的EGCG效果最显著。     

Preimplantation exposure to high insulin-like growth factor I concentrations results in increased resorption rates in vivo

BACKGROUND: Women with polycystic ovarian syndrome suffer increased rates of miscarriage. Elevated insulin and insulin-like growth factor I (IGF-I) concentrations have been implicated. Here, we hypothesize that the high concentrations of IGF-I result in miscarriage, represented by decreased normal pregnancy rates and increased resorption rates in a mouse model. METHODS: In-vitro studies: 2-cell embryos were cultured in either 1.3 or 130 nmol/l IGF-I; or 500 nmol/l IGF-I receptor (IGF-IR) sense and antisense oligoprobes for 72 h. Embryos were then transferred into pseudo-pregnant ICR females. In-vivo studies: IGF-I-containing slow-release pellets or mock pellets were implanted within the uterine horn in ICR female mice. For both studies, the recipient females were killed on day 14.5 and the numbers of normal implantation sites versus resorption sites were recorded. RESULTS: In-vitro studies: blastocysts cultured in low IGF-I exhibited significantly higher normal implantation rates than blastocysts cultured in high IGF-I concentrations (P < 0.01). Blastocysts cultured in IGF-IR sense oligoprobes exhibited a significantly higher normal implantation rate than blastocysts cultured in antisense oligoprobes. In-vivo studies: mice implanted with IGF-I-containing pellets exhibited significantly lower normal implantation rates as compared with mock-pellet controls (P < 0.01). CONCLUSIONS: High preimplantation IGF-I concentrations in vitro or in vivo lead to increased resorption rates in the mouse.     

Temporal effects of taurine on mouse preimplantation development in vitro.

Previously it has been shown that significantly more 2-cell mouse embryos reach the blastocyst stage when cultured in medium supplemented with taurine. In this study, in-vitro fertilized zygotes from a hybrid mouse strain were used to examine the temporal effects of 10 mM taurine on embryonic development in vitro during the preimplantation period. Taurine exerted its beneficial effect exclusively during the first 2 days post-insemination. The effect of taurine on blastocyst formation appeared to be restricted mostly to the period 20-48 h after fertilization, during which time mouse embryos are at the two-cell stage. Although more blastocysts were found when embryos were cultured in taurine-containing medium from 5 to 20 h post-insemination, this difference was not significant compared to the number of blastocysts when embryos were cultured without taurine. Taurine did not appear to affect the two-cell block of mouse embryos from random-bred strains.     

血管内皮生长因子受体-2在植入前小鼠胚胎发育中的表达变化

目的：探讨植入前小鼠胚胎血管内皮生长因子受体-2（VEGFR-2/Flk-1）的表达规律及其在小鼠胚胎发育中的作用。方法:对0.5－3.5 d小鼠胚胎行全胚免疫荧光染色及反转录－聚合酶链反应，观察胚胎发育过程中Flk-1表达时相及其变化；取8细胞期胚胎行体外培养，加入5 mg/L Flk-1中和抗体，分析36 h囊胚形成率。结果:在植入前小鼠胚胎中Flk-1 mRNA于4细胞期开始表达，8细胞期达高峰，桑椹胚期和囊胚期无表达；Flk-1蛋白表达定位于胚胎外缘和细胞交界处，于4细胞期和8细胞期表达呈阳性，桑椹胚期、囊胚期表达呈阴性；于体外培养的8细胞期培养液中加入5 mg/L中和抗体后，36 h囊胚形成率降低。结论:在小鼠植入前胚胎发育不同阶段Flk-1表达不尽相同，其作用可能与Flk-1促进植入前胚胎发育有关。     

Evidence for the involvement of a species-specific embryonic protease in zona escape of hamster blastocysts

The source and nature of zona lytic factors during zona escape of hamster blastocyts were investigated. When cultured in hamster embryo culture medium (HECM)-2h, all 8-cell embryos (n = 135) developed to zona escaped-blastocysts with complete zona lysis. In addition, 2-cell embryos, when co-cultured with zona escaping-blastocysts (at a ratio of 1:10), exhibited zona lysis. Various other embryos at the 1-8-cell stages also showed zona lysis when cultured with zona-escaping blastocysts. However, zonae from mice, rats, sheep and humans were resistant to lysis under these conditions. Pronase treatment resulted in rapid zona lysis in hamsters (7 +/- 1 s), whereas in other species zona lysis was much slower: mouse (662 +/- 27 s), rat (532 +/- 16 s), sheep (120 +/- 12 s) and human (104 +/- 8 s). When cysteine protease inhibitors (antipain, leupeptin, E-64 and p-hydromercuricbenzoate) were tested, they completely inhibited zona escape, while trypsin inhibitors (TLCK and SBTI) did not. Uterine zona lysin contribution in zona escape was discounted since: (i) uterine luminal flushing and endometrial extract from day 4 (the time of zona escape in vivo) pregnant females failed to lyse zonae and (ii) endogenous oocytes and transferred 2-cell embryos (to day 3 pseudopregnant recipients) were all zona-intact, while 71% of transferred blastocysts exhibited zona escape, following their recovery after 24 h. These observations suggest that a species-specific, embryonic proteolytic factor, with a cysteine protease-like activity, is involved in the zona escape of blastocysts in hamsters.     

猪体外成熟卵母细胞卵质膜电场行为的研究

目的 研究体外成熟不同时间猪犷母细胞卵质膜的抗电击能力和电融合能力。方法 利用电刺激,显微操作与电融合方法分别对体外成熟时同时间猪卵母细胞的抗电击和电融合能力进行了研究。结果 １．培养４８ｈ卵母细胞质膜的抗电击能力显著高于培养２４ｈ和３６ｈ的未成熟卵以及培养６０ｈ和７２ｈ的老化卵;２．体外成熟４８ｈ卵母细胞,在进行同卵或异卵核体移植时,其融合能力最强（融合率９０％以上）;３．将小鼠２、４、８细胞和     

The effect of Vero cell coculture on the development of mouse embryos exposed to monoclonal antibodies specific for mammalian heat shock protein 60

Heat shock proteins (HSP) have been identified as an important factor of a very complex and highly conserved cellular defense mechanism to preserve cell survival under adverse environmental conditions. HSP 60 are immunodominant antigens of microbe such as Chlamydia trachomatis and have a potentiality to become a target antigen due to antigenic similarity between chlamydial and human HSP. This study was conducted to investigate the effects of Vero cell coculture to anti-HSP 60 on the early mouse embryo development in vitro. The 2-cell mouse embryos (ICR) were cultured and mouse embryo development was observed every 24 hr for 3 days. 45% and 22.1% of the embryos cultured in Ham's F-10 plus anti HSP 60 with Vero cells developed to the 4- to 8- cell stage (day 1) and morular stage (day 2) as compared with 29.2% and 2.7% of those cultured without Vero cells respectively. But at day 3, the beneficial effect of Vero cells was not noted. These findings suggest that Vero cells have some roles to overcome the detrimental effect of anti-HSP 60 to some degree. These results suggest that Vero cells coculture will promote reproductive outcome in patient previously sensitized to microbial (e.g. Chlamydia trachomatis) HSP 60.     

Effects of myo-inositol on the in-vitro maturation and subsequent development of mouse oocytes

BACKGROUND: The study aim was to assess whether the incorporation of myo-inositol (MI) into culture medium could improve oocyte maturation in vitro. METHODS AND RESULTS: We performed a controlled prospective study using female ICR strain mice superovulated with pregnant mare's serum gonadotrophins. Cumulus-enclosed germinal vesicle (GV) oocytes were randomly cultured in medium with or without MI supplementation. The kinetics of GV breakdown after 4 h of incubation was significantly higher in oocytes incubated with 30 mmol/l of MI than in controls (P < 0.001). Accordingly, this concentration of MI was used for subsequent experiments. The proportion of metaphase II oocytes achieved after 24 h of culture, their fertilization and cleavage rates were significantly higher in the MI-treated group (P < 0.01, P < 0.05, P < 0.05 respectively). This group also demonstrated significant improvement in postimplantation development after transferring the 2-cell embryos to pseudopregnant mice. Confocal microscopy revealed spontaneous intracellular Ca(2+) oscillations within competent GV oocytes and treatment with MI caused an earlier onset of these Ca(2+) signals. CONCLUSIONS: Our results suggest that MI may affect meiotic progression of mouse GV oocytes possibly by enhancing the intracellular Ca(2+) oscillations. Supplementation of MI in culture medium may be useful for human oocyte maturation.     

Prostacyclin enhances the implantation and live birth potentials of mouse embryos

BACKGROUND: Recently we reported that iloprost, a stable analogue of prostacyclin, enhanced mouse embryo hatching. Here we present a follow-up study to determine whether exposure to iloprost augments the implantation and live birth potentials of mouse embryos. METHODS: Two-cell embryos (C3B6F1) were harvested 42 h after HCG injection and cultured in medium supplemented with iloprost. After 48 h, the embryos were transferred to 2.5 day pseudopregnant gestational carriers. The number of gestation sacs was counted 72 h later; the number of live pups and the weight of pups and placentae were determined 14 days later. The implantation rate was defined as gestation sac per embryo transferred; the live birth rate was defined as live pup per embryo transferred. RESULTS: The prostacyclin analogue enhanced the implantation rate from 42 to 76% [relative risk 1.84, 95% confidence interval (CI) 1.38-2.43]. The rate of live pups also increased from 28 to 36% (relative risk 1.28, 95% CI 1.04-1.56). The weights of the pups and of the placentae of the two groups were comparable. CONCLUSION: Prostacyclin enhances the potentials of implantation and live birth of mouse embryos.