静电场对小鼠体外受精胚胎质量的影响

利用静电场处理体外受精（ＩＶＦ）小鼠原核期胚胎，并在体外进行发育培养，在此过程中通过观察ＩＶＦ胚胎的发育速度及冷冻－解冻后囊胚孵化率，探讨了静电场对小鼠ＩＶＦ胚胎质量的影响，实验结果显示，授精处理后８４ｈ，处理组的囊邳经（４６．５％）显高于对照组（２５．５％），并且到１０８ｈ时，处理组的囊胚孵化率（９４．４％） 于对照组（６０．０％），表明静电场处理与对照组无差异，然后囊胚孵化率却高于组１８．４     

克服昆明小鼠早胚2-细胞阻滞的研究

目的：评价两种培养液对昆明小鼠胚胎２－细胞阻滞的克服效果。方法：用添加１５％ＦＣＳ或ＢＳＡ的Ｍ１６和ＣＺＢ培养液培养昆明小鼠２－细胞胚胎和体外受精卵,以观察这两种培养液对昆明小鼠早胚发育的影响及在克服昆明小鼠２－细胞阻滞中的作用,并通过ＣＺＢ培养液中葡萄糖成份的增减,了解其作用及效应的时间。结果：添加ＦＣＳ的Ｍ１６和ＣＺＢ培养注均能较好地支持２－细胞胚胎发育到囊胚（７８．９％,７２．７％）,当进行     

山羊胚胎的体外发育及冷冻保存研究

采用体外培养的方法，对山羊２～８细胞胚进行培养以后将体外发育至囊胚的胚胎进行冷冻保存，以便探讨山羊早期胚的体外培养方法和冷冻方法对体外发育胚冷冻效果的影响。将胚胎分别放入Ｍ１９９、Ｍ１９９＋输卵管上皮细胞和Ｍ１９９＋输卵管上皮细胞＋ＥＧＦ的培养液中进行培养，选取发育至囊胚期的胚胎分别以甘油和乙二醇为冷冻保护剂进行冷冻保存。结果体外培养胚的囊胚发育率在三个处理组中分别为１２．５％、７０．６％和７６．９％。使用Ｍ１９９处理组与添加输卵管上皮细胞、添加输卵管上皮细胞和ＥＧＦ的处理组之间具有极显著性差异（Ｐ＜０．０１）。冷冻解冻胚的形态正常率和发育率在以甘油和乙二醇为防冻剂的处理组中分别为６８．７、５８．３％和７８．６％、７２．８％。两组之间无明显差异，结果表明，采用与输卵管上皮细胞共同培养的方法能够提高山羊胚胎体外培养的发育率，以甘油和乙二醇为冷冻防冻剂均适合于山羊体外发育胚的冷冻保存，两者相比乙二醇的效果略优于甘油。     

不同体外培养条件对牛体外受精胚胎质量影响的研究

研究探讨了不同体外培养条件下牛体外受精胚胎的发育速度及囊胚细胞数，从而了解不同体外培养系统对牛体外受精胚胎质量的影响，实验一中伴体外受精卵分别在ＳＯＦ＋牛输卵管上皮细胞（ＢＯＥＣ）和ＴＣＭ１９９＋ＢＯＥＣ两种培养系统内培养，在这两种培养条件下囊胚发育率分别为２２．２％和２１．２％，囊胚细胞数分别为１１３．３±５．０和９７．９±８．３，受精后第６～９ｄ的囊胚出现率分别为４０．１％，３７．０％，１９．     

The Effects of Different Culture Systems on the Quality of Bovine In Vitro Fertilized Embryos

研究探讨了不同体外培养条件下牛体外受精胚胎的发育速度及囊胚细胞数，从而了解不同体外培养系统对牛体外受精胚胎质量的影响．实验一中将体外受精卵分别在ＳＯＦ＋牛输卵管上皮细胞（ＢＯＥＣ）和ＴＣＭ１９９＋ＢＯＥＣ两种培养系统内培养，在这两种培养条件下囊胚发育率分别为２２．２％和２１．２％，囊胚细胞数分别为１１３．３±５．０和９７．９±８．３，受精后第６～９ｄ的囊胚出现率分别为４０．１％ａ、３７．０％、１９．２％ｂ、２．７％和１３．５％ｃ、３２．７％、３６．５％ｄ、１７．３％（ａ＞ｃ，Ｐ＜０．０１，ｄ＞ｂ，Ｐ＜０．０５），表明牛体外受精卵在ＳＯＦ＋ＢＯＥＣ中的发育速度快于在ＴＣＭ１９９＋ＢＯＥＣ中．实验二中将体外受精卵分别培养于ＳＯＦ＋ＢＯＥＣ、ＳＯＦ＋牛卵丘细胞、ＳＯＦ＋ＢＳＡ三种培养系统中，结果囊胚发育率分别为３５．５％ｅ、２９．４％和２２．４％ｆ（ｅ＞ｆ，Ｐ＜０．０１），囊胚细胞数分别为１１７．３±８．０ｇ、９４．２±９．３和９０．２±９．４ｈ（ｇ＞ｈ，Ｐ＜０．０５）．实验三观察了体外受精胚胎在ＳＯＦ＋ＢＯＥＣ培养条件下发育速度与囊胚细胞数的关系，结果第６～９ｄ的囊胚细胞数分别为１１０．８±１０．２ｉ、１２８．     

绒山羊胚胎的体外培养,冷冻保存和移植的研究

采用体外培养和冷冻保存的方法将起排处理后的绒山羊２～８细胞期胚胎经体外在Ｍ１９９＋ＦＣＳ（对照组）、Ｍ１９９＋ＯＥＣ＋ＦＣＳ、Ｍ１９９＋ＯＥＣ＋ＥＮＧＳ和，Ｍ１９９＋ＯＥＣ＋ＮＳＳ的培养基中分别进行培养以后，选取发育为桑椹胚和早期囊胚期胚胎分别以１０％Ｇｌｙ、１０％ＥＧ为防冻剂和玻璃化方法进行冷冻保存，并将用常规和玻璃化法冷冻保存的胚胎解冻后进行移植．结果表明，体外培养胚胎的囊胚的发育率在四个培养处理组中分别为１２．５％、７０．６％、８１．８％和７３．９％，对照组与添加ＯＥＣ处理组之间具有极显著性差异（Ｐ＜０．０１），囊胚发育率在添加ＦＣＳ、ＥＮＧＳ和ＮＳＳ的实验组之间无明显差异．经冷冻保存的桑椹胚和早期囊胚的冷冻解冻胚的形态正常率在甘油、乙二醇和玻璃化三个冷冻处理组中分别为７５．０％、８６．７％和７５．０％，发育率分别为５８．３％、７８．８％和８０．０％．冷冻解冻胚的移植妊娠率在三个处理组中分别为５０％、７５％和５０％，产羔率分别为３０％、６８．８％和５０％．采用乙二醇冷冻绒山羊胚胎产生的效果优于甘油和玻璃化方法的冷冻效果。     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎．在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％,P〈0．05）,1．8％的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

冷冻中甘油对小鼠胚胎保护效果的研究

将小鼠２－细胞、４－细胞、８－细胞以及桑棋期的胚胎快速冷冻,以甘油作低温保护剂,浓度分别为０．５ｍｏｌ／Ｌ,１．５ｍｏｌ／Ｌ,２．５ｍｏｌ／Ｌ,３．５ｍｏｌＬ培养到囊胚阶段。结果表明：随着保护剂浓度的升高,胚胎发育率增加;在囊胚之前,发育程度越高,抗低温的能力越强,胚胎的发育率也就越高,各阶段组发育到囊胚期的胚胎经移植后均能得到正常发育的胎儿。     

久效磷对小鼠早期胚胎体外发育的影响

采用小鼠早期胚胎的体外培养技术,研究了久效磷对小鼠着床前胚胎发育的影响。结果表明,剂量低于0.22mmol/L对小鼠早期胚胎无明显作用,剂量为0.34mmol/L时,70.6％的8细胞胚胎能发育至囊胚,剂量达0.45mmol/L,对小鼠早期胚胎有显著的毒害作用,只有5.7％可以发育至囊胚。在经久效磷处理的小鼠早期胚胎电镜照片上可以看到,细胞内网架结构保持完好,线粒体被破坏,数量变少,空泡变大、增多,细胞内不同结构对久效磷的敏感性有一定差异。     

Smad2基因敲除小鼠冷冻胚胎库的建立

目的 建立Smad2基因敲除小鼠胚胎库。方法 利用OPS法对Smad2基因敲除小鼠胚胎进行玻璃化冷冻保存,并比较不同杂交组合小鼠的超数排卵数、解冻胚胎的复苏率及发育率。结果 两组不同杂交组合（Smad2^＋/－♂×Smad2^＋/－♀和Smad2^＋／－×Smad2^＋／－♀）小鼠平均超排卵数分别为14．13枚和24．60枚;复苏率分别为90．16％和91．67％;囊胚发育率分别为73．08％和77．05％。这些结果表明Smad2基因敲除杂合子母鼠的超排数量明显低于野生型母鼠的超排数量,而二者胚胎解冻后的复苏率和囊胚发育率没有显著差异。因此我们主要通过对野生型小鼠超数排卵,然后与Smad2基因敲除杂合子雄鼠交配的方法获取胚胎,进行玻璃化冷冻保存,现已冻存胚胎1256枚。结论 成功建立了Smad2基因敲除小鼠胚胎库。     

Aberrant DNA methylation patterns in cultured mouse embryos

Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofluorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.     

牛体外受精卵发育培养条件的研究

将采自屠宰母牛卵巢的未成熟卵子在含有促进性腺激素和胎牛血清的TCM199培养基内培养成熟后,使用以钙离子载体Ionophore A23187诱导获能的牛精子进行授精处理.在受精卵发育用培养基中添加了谷氨酰胺,并以此与无添对照进行了对比,探索了培养基内谷氨酰胺的存在与否对胚胎发育的影响.另外,在发育用培养基内还分别添加了胎牛血清(FCS),阉牛血清(NSS)以及发情母牛血清(OCS)以观察其培养效果,结果表明,谷氨酰胺对胚胎的发育有极大的促进作用,添加谷氨酰胺的处理组与对照组中发育为桑椹胚、囊胚的胚胎比例分别为23.4%和6.6%,具有极显著的整异.而在含有FCS、NSS和OCS的TCM199培养基内培养的卵子中分别有81.4%,70.7%和82.9%发育为二细胞期胚胎,将胚胎继续培养至授精后第8天时,三个处理组中发育为桑椹胚、囊胚的胚胎比例分别为30.0%,40.2%和47.1%,以OCS的培养效果为最佳.本研究的结果证明,谷氨酰胺和发情母牛血清对牛体外受精卵的体外发育具有良好的促进作用.     

不同体外培养系统对牛体外受精胚胎冷冻解冻后孵化能力的影响

采用ＳＯＦ和ＴＣＭ１９９与输卵管上皮细胞共同培养系统对牛体外受精卵进行培养,观察了在这两种培养条件下得到的囊胚经冷冻—解冻后的孵化能力．在ＳＯＦ＋输卵管上皮细胞的培养条件下,囊胚的发育率为２１．３％,囊胚经冷冻—解冻后的孵化率为３７．５％;在ＴＣＭ１９９＋输卵管上皮细胞的培养条件下,囊胚的发育率为２６．０％,囊胚经冷冻—解冻后的孵化率为４７．７％．研究结果表明这两种体外培养系统的培养效果无显著差异（Ｐ＞０．０５）．     

The first cleavage of the mouse zygote predicts the blastocyst axis

One of the unanswered questions in mammalian development is how the embryonic-abembryonic axis of the blastocyst is first established. It is possible that the first cleavage division contributes to this process, because in most mouse embryos the progeny of one two-cell blastomere primarily populate the embryonic part of the blastocyst and the progeny of its sister populate the abembryonic part. However, it is not known whether the embryonic-abembryonic axis is set up by the first cleavage itself, by polarity in the oocyte that then sets the first cleavage plane with respect to the animal pole, or indeed whether it can be divorced entirely from the first cleavage and established in relation to the animal pole. Here we test the importance of the orientation of the first cleavage by imposing an elongated shape on the zygote so that the division no longer passes close to the animal pole, marked by the second polar body. Non-invasive lineage tracing shows that even when the first cleavage occurs along the short axis imposed by this experimental treatment, the progeny of the resulting two-cell blastomeres tend to populate the respective embryonic and abembryonic parts of the blastocyst. Thus, the first cleavage contributes to breaking the symmetry of the embryo, generating blastomeres with different developmental characteristics.     

Regulation of mouse blastocyst adhesion,outgrowth and secretion of matrix metalloproteinase-2 by cGMP and nitric oxide <Emphasis Type="Italic">in vitro</Emphasis>

Nitric oxide (NO) is a multifunctional messenger molecule produced through oxidation of L-arginine to L-citrulline by enzyme NO synthase (NOS). In the current study, mouse blastocysts were cultured in the different media, and the implantation capacity of blastocyst was evaluated by evaluating the percentage of embryos adhesion and outgrowth after culture for 12, 24 or 48 h. Matrix metalloproteinase-2 (MMP-2) mRNA was detected by RT-PCR, and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst adhesion and outgrowth was observed in embryo cultured with 500 μmol/L NOS inhibitor N^G-mono-methyI-L-arginine (L-NMMA) alone; however, 100 μmol/L S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, and 20μmol/L cGMP analogue, 8-Br-cGMP could block this inhibition. The expression and production of MMP-2 in the blastocysts were suppressed by L-NMMA, and SNAP or 8-br-cGMP could reverse this suppression. These results suggest that NO induces embryo implantation by cGMP signaling pathway.     

牛体外受精胚胎在成份确定培养液内的发育

将牛体外受精胚胎培养于化学成份确定的 SOF+PVA的培养系统内 ,观察了不同PVA浓度对胚胎体外发育的影响 .结果在 1 mg/ml、3mg/ml和 5mg/ml的 PVA浓度条件下 ,囊胚的发育率分别为 2 7.5% a、1 9.1 % a和 4 .3% b( a>b,P<0 .0 1 ) .将体外受精胚胎分别培养于 M1 99+输卵管上皮细胞、SOF+BSA和 SOF+PVA的三种培养系统内 ,囊胚的发育率分别为 2 0 .4 %、2 9.0 %和 2 7.5% ,结果表明本研究确立的化学成份确定的体外培养系统可以用于牛体外受精胚胎的发育培养 .     

超声波对4种海水鱼类受精卵孵化的影响

采用复合式纵向压电换能器,谐振频率为21．5kHz,谐振阻抗为120Ω,施加电压值为80V的超声波辐射海水鱼类受精卵,研究不同的超声辐射时间和辐射次数对大弹涂鱼、中华乌幅鳢、真鲷和大黄鱼受精卵孵化的影响,结果表明,超声处理大弹涂鱼胚胎发育后期的受精卵,呆提高其孵化率,但对大弹涂鱼和中华乌塘鳢胚胎发育早期受精卵的孵化影响,每次辐射时间为1min、总幅射次数为4次的辐射剂量有利于真鲷受精卵的孵化;每次辐射时间超过1min,孵化率降低,以每次辐射时间为10s或30s处理大黄鱼受精卵,其孵化率分别比对照组高出10％和12％。     

小鼠胚胎移植研究

目的研究不同时期胚胎对胚胎移植成功率的影响以提高胚胎移植率。方法采用经超量排卵的KM雌鼠与正常615雄鼠交配,取受精卵,分别将单胚卵、双胚卵、桑椹胚和囊胚、移入发情的假孕KM雌鼠的输卵管和子宫中妊娠产仔。结果移入单胚卵、双胚卵、桑椹胚和囊胚的假孕母鼠妊娠率分别为36.36%、42.86%、10%、27.27%,产仔率分别为46.58%、38.46%、58.33%、60%。结论移植双胚卵可提高小鼠的胚胎移植成功率。     

Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse acti-vation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.     

牛体外受精胚胎成份明确培养系统的建立

以 SOF为基本培养液 ,分别添加血清、PVA以及同时添加 PVA、肌醇和柠檬酸钠 ,进行牛体外受精卵的发育培养 ,三个处理组的囊胚发育率分别为 :3 2 .6%、1 1 .9%和 1 5 .9% ,血清处理组极显著高于两个 PVA处理组 (p<0 .0 1 ) .在此基础上 ,将受精处理后的卵子去掉卵丘细胞后 ,培养于 SOF PVA 肌醇 柠檬酸钠培养液内 ,以未去掉卵丘细胞的卵子为对照 ,两个处理组的囊胚发育率分别为 1 0 .8%和 2 1 .1 % .二者间没有显著差异 .结果表明没有血清和卵丘细胞的成份明确的培养系统能够支持牛体外受精卵发育至囊胚阶段 .