体外发育

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cytoplasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methylation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribution of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明：利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程，在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样，呈现高度甲基化状态；在细胞核开始融合的时候，甲基化水平急速下降，在细胞核完全融合的时候甲基化水平达到最低点；随着胚胎继续分裂，胚胎甲基化水平逐渐增加，在桑葚胚期甲基化水平最高；但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别，这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cyto-plasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methy-lation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribu-tion of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明：利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程，在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样，呈现高度甲基化状态；在细胞核开始融合的时候，甲基化水平急速下降，在细胞核完全融合的时候甲基化水平达到最低点；随着胚胎继续分裂，胚胎甲基化水平逐渐增加，在桑葚胚期甲基化水平最高；但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别，这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastcysts fellowed by fusion of cytoplasm and nucleus and cleavage in vitro.After fusion of cytoplasm, the DNA methylation levels of the fused embryos was very high as well as two-cell diploid embryos in vivo.Then the embryos was rapiddly demethylated when the nucleus begin to fuse, resulting the lowest DNA methylation levels when the nucleus fused completely.After that, the DNA methylation levels of fused embryos were gradually increased until the blastocysts stage.However, whereas an asymmetric distribution of DNA methylation was established in an vivo-derived blastocysts with a higher methylation level in the inner cell mass than in the trophectoderm, in most vitro-derived tetraploid blastocysts, we can not detect the asymmetric distribution.

结果表明：利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程，在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样，呈现高度甲基化状态；在细胞核开始融合的时候，甲基化水平急速下降，在细胞核完全融合的时候甲基化水平达到最低点；随着胚胎继续分裂，胚胎甲基化水平逐渐增加，在囊胚期甲基化水平最高；但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别，这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Until now, only within the mouse model, the birth of live young have been reported.

在目前的技术条件下，啮齿类的腔前卵泡已能经体外培养发育至有腔卵泡期，而且从有腔卵泡分离的卵母细胞经体外成熟、体外受精后已能产生新的个体。

The rate of Flow cytometry to analyse retinoic acid haploid is 19.3%.The sperm-like cells achieved could activate bovine oocytes by sperm injection, and the development rate of blastosphere was 20.36%. Conclusions SSCs of newly -born mice can be induced and differentiated into sperm-like cells and are also capable of activating oocytes.

本实验通过体外诱导新生小鼠SSCs分化为精子样细胞，探讨不同诱导方法的诱导效率；采用显微注射法将精子样细胞注射到卵母细胞和去核卵母细胞，探讨其体外重构胚的发育能力，为SSCs体外分化研究打下基础。

Objective To observe the development of mouse kidney in vitro at embryonic day 12、14、16 and postnatal day 1, and compare with the development of homeochronous kidney in vivo. And to select eligible the embryonic age for kidney culture in vitro.

目的 观察胚龄12、14、16、以及生后1天的肾组织体外培养的发育情况，与同期在体发育进行比较，探讨肾组织体外培养的胚龄选择。

Ovine immatured oocytes, matured oocytes and embryos from 2-cell stage to blastocyst produced in vitro were recovered, and RT-PCR was used to semi-quantify mRNA expression. Proteins of Cx43 and Cx45 were detected by using a combination of immunocytochemistry and Laser Scanning Confocal Microscope.

收集绵羊未成熟和成熟卵母细胞以及体外受精早期胚胎各发育阶段的卵裂球细胞，通过RT-PCR半定量检测Cx43和Cx45基因的mRNA表达量；通过免疫组织化学方法结合激光扫描共聚焦显微镜检测Cx43和Cx45蛋白在绵羊体外受精的早期胚胎发育过程中的表达情况及表达区域。

The mice were used as experimental animals in this study,through the experiment in vitro and in vivo,systemic invested the dynamic changes in expression and localization of FGF7,FGF10 and its receptor in mammals development,lactation and involution;revealed the relationships between the expression of FGF7,FGF10 and its receptor and the function;clarified the function of FGF7 and FGF10 in mammary gland development,lactation and involution,and the effect of mammogenic hormones on expression of FGF7 and FGF-10 and its receptor in different periods in vitro.

本研究主要以小鼠为实验材料，通过体外和体内实验，系统研究FGF7、FGF10及KGFR在哺乳动物乳腺发育、泌乳及退化过程中表达定位的动态变化，揭示FGF7、FGF10及KGFR表达变化与乳腺发育及泌乳功能间的对应关系，阐明FGF7、FGF10在乳腺发育、泌乳及退化过程中的功能以及乳腺发育激素对不同时期乳腺FGF7、FGF10及KGFR表达的影响。

Our study includes four aspects. In the first aspect we study several important conditions of porcine oocytes maturation in vitro and oocytes cleavage after parthenogenetic activation and found mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15% PFF+0.57mMcysteine is a good culture condition .When the Cocs are cultured in it ,the maturation rate and oocytes cleavage rate are higher than those of foreign covered. Our result are (86.7±3.35)% and (86.3±4.16)% and the highest report of foreign is(85.7±4.1)%.In the second aspect we study the effect of different chemical activations on development of porcine parthennogenetic embryo and found two best activation method. The first one is that putting the maturation MII oocytes in the 20μmol/L ionomycin for 30 minutes and then putting them in the NCSU-23 condition containing 5μg/mICB and 5mM/L6-DMAP for 3.5 hours, the oocytes cleavage rate and morulae/blastocysts development rate are (76.7±7.6)% and (37.1±6.4)%.The second one is that putting the maturation MII oocytes in the 200μM/L Thimerosal for 20 minutes and then putting them in the NCSU-23 condition containing 8mM DTT for 30 minutes

本研究分为4个部分，第一部分对影响猪卵母细胞体外成熟和孤雌激活后胚胎分裂的几个重要条件进行了比较研究，确立了一种较好的培养方法：与颗粒细胞共培养，找到了一种适合猪卵母细胞体外成熟的培养基：mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15%PFF+0.57mM半胱氨酸，成熟率和分裂率分别为(86.7±3.35)%和(86.3±4.16)%，国外报道的最高成熟率为(85.7±4.1)%；第二部分对猪卵母细胞孤雌激活的化学方法进行了研究，确立了化学激活猪卵母细胞的两种最佳方法：1将成熟的去卵丘颗粒细胞的MII期卵母细胞用20μmol/Lionomycin作用30min，再将卵母细胞培养于含5μg/mlCB和5mM/L 6-DMAP(6-二甲基氨基嘌呤)的NCSU-23培养液中，卵裂率和桑囊胚发育率达到(76.7±7.6)%和(37.1±6.4)%2将成熟的去卵丘颗粒细胞的MII期卵母细胞在200μM/L的Thimerosal中处理20min，再与8mM的DTT共孵育30min，卵裂率和桑/囊胚形成率为(81.0±2.8)%和(39.6±2.7)%；第三部分对孤雌激活胚胎的培养条件进行了研究，确立了一种最佳的胚胎培养条件：在SOF简单培养基中添加颗粒细胞进行前3天的培养，然后转入添加胎牛血清的NCSU—23培养基并和输卵管上皮细胞进行后期的培养，其桑椹胚和囊胚的发育率为(59.5±3.2)%；第四部分研究了IGF-I

The results show that: 1 supplementation of protein to maturation media improves cumulus expansion in vitro compared to the protein-free control, but cumulus expansion is not necessarily related to oocyte nuclear maturation in pigs, and cumulus expansion is not the criterae for determination of nuclear maturation of pig oocytes, but only the exclusion of the first polar body; 2 exposure of pig COCs to hormone supplements for 23-24 hours improved cumulus expansion but had no significant effect on nuclear maturation compared to that for 46-48 hours; 3 under our research conditions, supplementation of different proteins into different maturation media has different effects on porcine oocyte nuclear maturation, but has no significant effect on subsequent embryonic development after IVF; 4 the nuclear maturation rates of pig oocytes matured in mTCM+pFF and mNCSU+pFF are superior than that in mNCSU+FCS; 5 different maturation media have no effect on pig oocyte cumulus expansion and subsequent embryonic development after IVF.

结果显示：(1)在成熟液中添加蛋白质可以加强卵丘细胞的扩散，但猪卵母细胞的核成熟与其周围的卵丘细胞扩展没有必然的联系，卵丘细胞扩散或成放射状不宜作为猪卵母细胞核成熟的标准，只有排出第一极体才能作为猪卵母细胞核成熟的标志；(2)在猪COCs的46-48小时成熟培养的后23-24小时阶段去除成熟液中的激素不但可以保证卵母细胞的核成熟率，而且可加强卵丘细胞的扩散；(3)在现有实验条件下，在mTCM和mNCSU中添加10％pFF与在mNCSU中添加10％FCS相比可获得较高的猪卵母细胞核成熟率；(4)在不同的成熟液中添加不同的蛋白质对猪卵母细胞核成熟率的影响效果不一样，但对体外受精后的早期胚胎发育影响不明显；(5)成熟液种类对猪卵母细胞的卵丘细胞扩散和体外受精后的早期胚胎发育无显著影响。

Cynanchum Lingtai apricot production in the average weight 65 grams, the brightly-colored fruit, juicy rich, sweet-sour taste, sweet from the nucleolus, when the late Qing Dynasty famous Shaanxi, Gansu provinces, the Qing imperial court Tongzhi tribute for years.

灵台生产的牛心杏平均单果重65克，果实色泽鲜艳，汁多味浓，甜酸适口，离核仁甜，清末时就驰名陕、甘两省，清同治年间曾为朝廷贡品。

Chenopodium album,Solanum nigrum, and Amaranthus retroflexus were very susceptible to the herbicides. Polygonum persicaria and Abutilon theophrasti were relatively less susceptible to the herbicides, and Lycopersicon esculentum was not susceptible to it. The relationship between reduction rates of weed biomass and PPM values of weed leaves 2,4, and 6 days after treatment was established.

供试的6种杂草对该混剂的敏感性存在显著差异：红心藜Chenopodium album、龙葵Solanum nigrum和反枝苋Amaranthus retroflexus对该混剂最敏感，ED90值分别为47.65、71.67和29.17g/hm2；春蓼Polygonum persicaria和苘麻Abutilon theophrasti敏感，ED90值分别为96.91、114.20g/hm2；而番茄不敏感。