肿瘤转移抑制因子CD82/KAI1在假孕小鼠子宫的表达及其对小鼠胚胎着床的影响

目的:研究CD82/KAI1mRNA及蛋白在假孕d1-8小鼠子宫中的表达规律,以及CD82/KAI1抗体对小鼠胚胎体外发育及着床的影响。方法:用RT-PCR及免疫组织化学技术观察CD82/KAI1mRNA及蛋白在假孕小鼠子宫的表达规律。将8-细胞小鼠胚胎培养于含不同浓度CD82/KAI1抗体的培养液中,观察囊胚形成及脱透明带的情况。妊娠d4小鼠宫角注射CD82/KAI1抗体,于妊娠d8观察小鼠胚胎植入数量。结果:①CD82/KAI1mRNA在假孕d1-8小鼠子宫中均有表达,于d4、d5表达最丰富。CD82/KAI1蛋白在假孕d1-8的子宫基质细胞及腺上皮细胞均有表达,假孕d1-4腔上皮细胞无表达,假孕d5子宫腔上皮细胞出现弱阳性表达,从d6开始子宫腔上皮细胞表达逐渐增强。②一定浓度的CD82/KAI1抗体可明显抑制囊胚的形成率(1∶400,P<0.05)和脱带率(1∶800,P<0.05)。③宫角注射一定量的CD82抗体可以显著提高小鼠胚胎的着床数(8.35±0.17vs4.52±0.24,P<0.05)。结论:CD82/KAI1在妊娠小鼠子宫的表达是非胚胎依赖性的。CD82/KAI1在小鼠胚胎发育和着床过程中发挥重要作用。     

肿瘤转移抑制因子CD82/KAI1基因在小鼠子宫内膜的动态表达

目的：探讨肿瘤转移抑制因子CD82/KAI1在小鼠胚胎着床过程中的功能。方法：应用RT-PCR和免疫组织化学技术分别观察CD82/KAI1mRNA和蛋白在小鼠动情周期和早孕子宫中的表达。结果：在动情周期中,CD82/KAI1mRNA除在动情期较低外,其余三期的表达量基本相同,但蛋白质却是在动情期表达最丰富,子宫内膜上皮和基质均呈阳性。在早孕子宫中,CD82/ KAI1mRNA的表达逐渐增多,蛋白质表达的量和范围也逐渐增强。结论：CD82/KAI1在小鼠子宫中呈动态表达,提示它在胚胎精确侵袭子宫内膜的调节中发挥作用。     

玻璃化法冻融小鼠桑葚胚期和囊胚期胚胎的效果观察

目的 比较玻璃化法冻融小鼠桑葚胚期、囊胚早期和囊胚期胚胎后,胚胎存活及继续发育的能力。方法应用6 mol/L乙二醇和1 mol/L蔗糖的玻璃化冷冻液,冻融小鼠142个桑葚胚期、135个囊胚早期和148个囊胚期胚胎,观察冻融后胚胎存活及继续发育的情况。结果 小鼠桑葚胚期、囊胚早期和囊胚期胚胎的存活率分别为88.0％、73.3％和60.1％,囊胚孵出率分别为73.9%、61.5％和49.3％。桑葚胚期的胚胎存活率及囊胚孵出率均高于囊胚早期,囊胚早期高于囊胚期,各期胚胎存活率、囊胚孵出率比较,差异均有显著性(P<0.05)。结论 玻璃化法冻融桑葚胚期的效果好于囊胚早期及囊胚期,桑葚胚期是卵裂期后胚胎玻璃化冻融的最佳阶段。     

胎盘异铁蛋白对小鼠胚胎生长发育影响的研究

目的:探讨胎盘异铁蛋白对小鼠胚胎生长发育的影响。方法:将人早孕蜕膜细胞和小鼠2-细胞期胚胎置于含不同浓度胎盘异铁蛋白的培养液中共培养,倒置显微镜下观察记录胚胎数目并分级。结果:①发育至4-细胞期、8-细胞期及桑葚胚阶段鼠胚数的百分率比较,差异无显著性(P>0.05)。②在10 U/mL和100 U/mL浓度的胎盘异铁蛋白作用下,发育至囊胚期和孵出期鼠胚数的百分率明显提高(P<0.05)。③在1 000 U/mL浓度的胎盘异铁蛋白作用下,从2-细胞期发育至孵出期鼠胚数的百分率有所下降,部分细胞发生变性、退化和不规则分裂等现象,但无统计学意义(P>0.05)。结论:10～100 U/mL浓度的的胎盘异铁蛋白对鼠胚的早期发育没有显著影响,但能促进晚期鼠胚的生长、分化和孵出。     

表皮生长因子(EGF)和胰岛素样生长因子(IGF-Ⅱ)对小鼠1-细胞胚胎体外发育的影响

目的:研究表皮生长因子(epidermalgrowthfactor,EGF)在小鼠早期胚胎体外发育中的作用,以及EGF联合胰岛素样生长因子-Ⅱ(insulin-likegrowthfactor-II,IGF-Ⅱ)对小鼠早期胚胎体外发育是否有协同促进作用。方法:①在mKSOM培养液中添加0ng/ml(对照组)、0.1ng/ml、1ng/ml、10ng/ml、100ng/ml的EGF、10ng/mlIGF-Ⅱ、1ng/mlEGF+1ng/mlIGF-Ⅱ、1ng/mlEGF+10ng/mlIGF-Ⅱ、10ng/mlEGF+1ng/mlIGF-Ⅱ、10ng/mlEGF+10ng/mlIGF-Ⅱ培养小鼠1-细胞胚胎,每组胚胎在培养箱中连续培养120h,每24h观察胚胎发育情况,分别计算2-细胞率、4-细胞率、桑椹胚率、囊胚率和孵化率,并进行囊胚细胞计数。结果:添加1ng/ml、10ng/mlEGF组的囊胚率及孵化率显著增加;EGF+IGF-Ⅱ组较单一添加组、对照组囊胚率及孵化率显著增加(P<0.05),且EGF+IGF-Ⅱ组囊胚细胞计数显著高于其他组(P<0.05),其中10ng/mlEGF+10ng/mlIGF-Ⅱ组的孵化率及囊胚细胞数最高(P<0.05)。结论:EGF浓度在1￣10ng/ml范围内可以提高体外培养鼠胚的囊胚率及孵化率;EGF及IGF-Ⅱ对体外培养鼠胚的发育有协同促进作用;本实验范围内10ng/mlEGF+10ng/mlIGF-Ⅱ为最优组合。     

盐酸克仑特罗对小鼠胚胎体外发育的影响

目的:探讨盐酸克仑特罗对小鼠1-细胞胚胎和2-细胞胚胎体外发育的影响。方法:获取小鼠1-细胞和2-细胞胚胎,分别与盐酸克仑特罗10 ng/mL,3 ng/mL和1 ng/mL的3个剂量组共培养,观察胚胎各阶段的发育情况并计算胚胎的发育率。结果:1ng/mL和3ng/mL组的1-细胞小鼠胚胎,从4-细胞期到囊胚阶段的发育率与对照组相比差异显著(P<0.05),3 ng/mL组显现出比1ng/mL组更强的抑制作用(P<0.01),10 ng/mL组,2-细胞期就与对照组有差异(P<0.05,其中 4-细胞至囊胚阶段P<0.01),囊胚率只有2.4％。10 g/mL组及3 ng/mL组的2-细胞小鼠胚胎,分别从4-细胞期和8-细胞期与对照组相比差异显著(P<0.05),1 ng/mL组的各个阶段胚胎发育率与对照组相比未见统计学差异(P>0.05)。培养液中含有盐酸克仑特罗使胚胎的粗颗粒增多,部分印裂球碎裂、退化。结论:盐酸克仑特罗对小鼠胚胎的体外发育有毒性作用并呈一定的剂量效应。盐酸克仑特罗使1-细胞小鼠胚胎被抑制在2-细胞期,对处于晚2-细胞期胚胎的影响明显降低。     

人子宫内膜细胞共培养体系对早期鼠胚体外发育的影响

目的:研究人子宫内膜共培养体系对早期鼠胚体外发育的影响及移植后的妊娠情况。方法:将2-细胞小鼠胚胎与人子宫内膜细胞进行体外共培养,对照组为无营养细胞的单纯培养液,每日在显微镜下观察胚胎的发育情况。将培养到囊胚期的胚胎移植回小鼠的子宫腔,观察着床情况。结果:共培养体系中68.3％的2-细胞胚胎发育至桑椹胚期,50.8％发育至囊胚期,囊胚的孵化率为36.7％,胚胎的着床率为25.0％。而对照组只有24.8％的2-细胞胚胎发育至桑椹胚期,11.4％到达囊胚期,且其中大部分为早期囊胚即停止发育。另外对照组细胞碎片出现早且多,卵裂球不均匀,胚形态差,移植后胚胎的着床率仅为3.1％。结论:人子宫内膜细胞共培养体系可以促进小鼠胚胎的体外发育,改善胚胎的质量,提高着床率。     

肿瘤转移抑制因子CD9/MRP-1基因在小鼠围着床期子宫内膜的动态表达

目的:探讨肿瘤转移抑制基因(CD9)/运动相关蛋白(MRP-1)mRNA和蛋白在胚胎着床过程中的作用。方法:应用RT-PCR和免疫组化技术观察CD9/MRP-1mRNA和蛋白在早孕和假孕小鼠子宫中的表达规律。结果:早孕d1-4小鼠子宫组织均有CD9/MRP-1mRNA表达,且d4表达最多;其蛋白主要表达在d1-4的子宫内膜上皮细胞,且早孕d2-4CD9/MRP-1在子宫基质细胞散在阳性表达。假孕d1-8小鼠子宫组织均有CD9/MRP-1mRNA表达,假孕d5表达开始增加,至d6达到峰值。而CD9/MRP-1蛋白在假孕d1-8子宫内膜腺上皮均有表达,而子宫内膜腔上皮均无表达。假孕d2-5,子宫基质细胞出现散在阳性表达。结论:①CD9/MRP-1在早孕小鼠子宫中呈动态表达,提示它在胚胎精确侵袭子宫内膜的调节中发挥作用。②CD9/MRP-1在妊娠小鼠子宫的表达是非胚胎依赖性的。     

废弃胚胎继续囊胚培养研究

目的:探讨体外受精治疗周期中废弃胚胎的体外发育潜能。方法:通过囊胚序贯培养法将无原核(0PN)、单个原核(1PN)、多个原核(≥3PN)和卵裂期发育延缓的2原核(2PN)废弃胚胎培养至囊胚期。比较不同来源胚胎囊胚形成情况和d3胚胎的卵裂球数、质量分级等;并利用废弃胚胎囊胚形成情况对体外受精妊娠结局进行预测。结果:共收集801个废弃胚胎,经序贯培养,形成209个囊胚(26.09%),其中58个为优质囊胚(27.75%)。1PN胚胎、0PN胚胎、d3卵裂球数为7-9-细胞胚胎、d3评分为Ⅰ-Ⅱ级胚胎以及卵裂球数为偶数的胚胎囊胚形成率较高(P均<0.05)。废弃胚胎中有囊胚形成者的临床妊娠率明显高于无囊胚形成者(P<0.05)。结论:废弃胚胎有不同程度的发育潜能,部分可发育为囊胚;特别是:①0PN和1PN胚胎;②d3的4-9-细胞胚胎,偶数卵裂球者更佳;③I级和Ⅱ级胚胎。     

人类白细胞抗原G及其异构体mRNA在人类着床前胚胎的表达

目的研究人类白细胞抗原G（human leukocyte antigen，HLA-G）及其异构体mRNA在人类着床前胚胎的表达，探讨HLA-G在人类胚胎早期发育中的作用和意义。方法第四军医大学唐都医院妇产科生殖医学中心2003-01-2005-12期间，以体外受精-胚胎移植技术助孕过程中剩余的患者自愿捐赠的人类早期胚胎作为研究对象，采用巢式RT-PCR检测早期胚胎HLA-G及其异构体mRNA的表达。结果检测20个受精后2—3d的人类卵裂期胚胎，2、4、6、8细胞期胚胎各5个，有7个胚胎表达HLA-G及其部分的异构体mRNA。检测5个受精后4d的人类桑葚胚，全部表达HLA-G及其部分的异构体mRNA。检测25个受精后6d的扩张期囊胚，全部受检囊胚均表达HLA-G mRNA，但异构体表达不同。在受检的25个扩张期囊胚中，20个表达HLA-G1（表达率80．0％），4个表达G2（表达率16．0％），25个表达G3（表达率100％）、24个表达G4（表达率96．0％），5个表达G5（表达率20．0％），8个表达G6（表达率32．0％）。结论着床前的人类胚胎表达HLA-G及其异构体mRNA，其阳性表达胚胎的比例，随着胚胎发育的进程而增加。HLA-G异构体在人类着床前胚胎差异表达。     

卵裂期活检对胚胎体外发育能力的影响

目的　探讨不同的活检方法、活检时机和活检细胞数对胚胎体外发育能力的影响。方法　选取体外受精 胚胎移植剩余的形态学分级为Ⅰ级的胚胎 1 54个 ,对其中第 1阶段的 66个胚胎分别行化学法 (2 6个 )、机械法 (2 0个 )取出 1个卵裂球 ,并设对照 (2 0个 ) ;对第 2阶段的 88个胚胎分为用化学法取出 2个卵裂球 (44个 )和对照 (44个 )。观察记录活检时胚胎细胞数、活检时间、活检后卵裂球是否退化、活检后胚胎体外发育情况及囊胚总细胞数。结果　 (1 )化学法的活检时间为 (2 31±2 0 )s,明显短于机械法的 (2 62± 2 3)s(P <0 0 1 ) ;而囊胚形成率为 65 % ,高于机械法的 35 % (P<0 0 5)。(2 ) 6 细胞胚胎的囊胚总细胞数为 (44± 4)个 ,低于 7～ 8 细胞胚胎的 (49± 5)个和≥ 9 细胞胚胎的 (50± 6)个 (P <0 0 5) ;细胞融合的≥ 9 细胞胚胎活检后不仅囊胚形成率 (2 0 % )低于对照 (67% ,P<0 0 5) ,且活检后卵裂球退化率 (50 % )高于无细胞融合的胚胎 (1 7% ,P <0 0 5)。 (3)取出 1个或 2个卵裂球胚胎的囊胚形成率和囊胚总细胞数与对照比较 ,差异均无显著性 (P >0 0 5) ,而 6 细胞胚胎取出 2个卵裂球后囊胚形成率 (1 /8)低于对照 (5/8,P <0 0 5)。结论　化学法活检比机械法更为快速、安全 ;合适的活检时     

Effect of Different Concentrations of Recombinant Leukemia Inhibitory Factor on Different Development Stage of Mouse Embryo In Vitro

Purpose: To assess the influence of different concentrationsof recombinant human leukemia inhibitory factor (LIF) onthe in vitro development of mouse embryos. Methods: The 2- to 4-cell embryos of CB6F1 mice werecultured in the human tubal fluid (HTF) media containingdifferent concentrations of LIF. Mouse embryos were dividedinto seven groups: (1) HTF; (2) 1500 IU/ml LIF; (3) 1000IU/ml LIF; (4) 750 IU/ml LIF; (5) 500 IU/ml LIF; (6) 250IU/ml LIF; (7) 125 IU/ml LIF. The embryonic numbers ofdifferent stages including 5–8 cell, 9–16 cell, morula, blastocyst,and hatching blastocyst were recorded. Results: The percentage of early embryo stage (2-cellembryos to 6- to 16-cell stages) in all groups were nonsignificantlydifferent. There were higher formation rates of preimplantationembryos (morula to hatching blastocyst) ingroups 2, 3, 4, and 5 than in groups 1, 6 and 7. Conclusions: LIF has positive effects on preimplantationembryo development and has nonsignificant influence onthe early embryo development. The lowest concentration ofLIF which could provide the optimal embryo developmentis 500 IU/ml.     

Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching

Purpose

To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development.

Methods

Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy.

Results

PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos’ outer surface.

Conclusions

PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.     

Human papilloma virus DNA exposure and embryo survival is stage-specific

Purpose: Human papillomavirus (HPV) has been shown to disrupt late-stage implanting embryos. The objectives were (a) to assess the development of early embryos exposed to HPV DNA and (b) to analyze the blastocyst hatching process after HPV exposure. Methods: The study involved exposing two-cell and 4–8-cell mouse embryos to DNA fragments from either HPV type 16, type 18 or DQA1 (control). The embryos were incubated for 120 h and assessed. Results: HPV 16 and 18 inhibited two-cell embryo development. In contrast, delaying the exposure of HPV DNA until the 4–8-cell stage resulted in further embryonic development. There was 25.9% less blastocyst formed with HPV 16 exposure. Additionally, there were 25.9–31.8% more degenerated embryos with HPV 16 exposure. Conclusions: The study demonstrated embryo stage-specific effects of HPV on early development. The results suggested HPV exposure was linked to two-cell embryo demise and delaying the exposure of HPV until later embryo stages permitted embryo development. HPV 16 was shown to decrease blastocyst formation while HPV 18 inhibited the blastocyst hatching process.     

Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method

Purpose

The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos.

Methods

Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups.

Results

Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P?>?0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P?<?0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P?<?0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P?<?0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P?>?0.05).

Conclusions

Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.