静电场对小鼠胚胎发育能力的影响

首次报道哺乳动物胚胎经静电场处理后对＂细胞阻滞＂和后期发育能力的影响．结果表明，用静电场处理小鼠体外受精卵和２细胞胚胎时，对克服小鼠＂２细胞阻滞＂无显著影响．处理发育于Ｍ１６＋１００μＭＥＤＴＡ＋输卵管上皮的２细胞胚胎时，显著提高了胚胎的发育能力，最佳处理剂量的囊胚率从４２％提高到６４％，囊胚孵化率从２０％提高到４５％．     

Identification of embryonic chromosomal abnormality using FISH-based preimplantation genetic diagnosis

Objective: Embryonic chromosomal abnormality is one of the main reasons for in vitro fertilization (IVF) failure. This study aimed at evaluating the value of Fluorescence in-situ Hybridization (FISH)-based Preimplantation Genetic Diagnosis (PGD) in screening for embryonic chromosomal abnormality to increase the successful rate of IVF. Method: Ten couples, four with high risk of chromosomal abnormality and six infertile couples, underwent FISH-based PGD during IVF procedure. At day 3, one or two blastomeres were aspirated from each embryo. Biopsied blastomeres were examined using FISH analysis to screen out embryos with chromosomal abnormalities. At day 4, embryos without detectable chromosomal abnormality were transferred to the mother bodies as in regular IVF. Results: Among 54 embryos screened using FISH-based PGD, 30 embryos were detected to have chromosomal abnormalities. The 24 healthy embryos were implanted, resulting in four clinical pregnancies, two of which led to successful normal birth of two healthy babies; one to ongoing pregnancy during the writing of this article; and one to ectopic pregnancy. Conclusion: FISH-based PGD is an effective method for detecting embryonic chromosomal abnormality, which is one of the common causes of spontaneous miscarriages and chromosomally unbalanced offsprings.     

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.     

牛体外受精技术研究的现状与展望

系统综述了牛体外受精技术研究的现状及存在的问题．并对今后的研究方向和前景提出了展望。牛卵母细胞可在不含血清的成熟培养液中获得受精及胚胎发育能力，促卵泡素、促黄体素及上皮细胞生长因子均对这一过程具促进作用。肝素是迄今最为有效的精子获能处理方法，但在受精过程中对精子获能起决定作用的是卵丘细胞和卵母细胞本身。提高受精质量可明显提高受精卵的胚胎发育能力。体外受精卵可通过改善培养条件或与体细胞进行复合培养发育到囊胚阶段．但后者更为稳定可靠。目前，牛卵母细胞的体外受精分裂率可达８０％，囊胚发育率达４０％左右．两枚鲜胚的移植妊娠率可达５０％～６０％．而两枚冻胚的移植妊娠率仅３０％～５０％。因此，尚需进一步提高体外受精胚胎的活力和改进胚胎的冷冻保存技术。     

牛体外受精胚胎移植的应用研究

探讨了利用牛ＩＶＦ技术,在国外生产纯种肉牛胚胎运用国内进行大批量移植后受体母牛的妊娠率及产犊情况,共移植受体母牛１５８头,妊娠率（９０ｄ）为２１．４％￣４８．５％,共计产纯种犊牛５５头,５￣８日龄胚移植给发情第７ｄ的母牛能够得到较高的妊娠率（４３．８％￣５０．０％）,受体母牛在前两交伯移植妊娠率（３０．０％￣３５．０％）,明显高于第三次（１５．４％）以后的妊娠高,研究结果证实,在国外生产的的占ＩＶ     

Unborn children (protection) bill

Enoch Powell's Unborn Children (Protection) Bill passed a second reading in the British House of Commons on 22 February 1985. The authors contend that most informed scientists are dismayed because, if enacted into law, the bill would criminalize the in vitro fertilization of any human oocytes except for purposes of "embryo insertion," and many promising avenues of research would be blocked. While the pro-life lobby maintains that the development of possible methods to cure genetic diseases does not require research on human embryos, Evans and McLaren cite scientific data to argue that such research is necessary to insure that embryos chosen for implantation are chromosomally normal, to study the etiology of chromosomal disorders and eventually develop therapies for them, to improve IVF procedures, and to develop new contraceptive techniques.     

LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing

Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies. Here we report a newly discovered molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA3 (refs 2-4). Targeted deletion of LPA3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase 2 (COX2) (ref. 5), was downregulated in LPA3-deficient uteri during pre-implantation. Downregulation of COX2 led to reduced levels of prostaglandins E2 and I2 (PGE2 and PGI2), which are critical for implantation. Exogenous administration of PGE2 or carbaprostacyclin (a stable analogue of PGI2) into LPA3-deficient female mice rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA3 receptor-mediated signalling as having an influence on implantation, and further indicate linkage between LPA signalling and prostaglandin biosynthesis.     

Experimental chimaeras--removal of reproductive barrier between sheep and goat

Following reciprocal embryo transfer between sheep and goats, the embryo of the foreign species is able to induce early pregnancy but the embryos do not survive beyond the first weeks of gestation. Similar results have been obtained from hybridization experiments on sheep and goats. While the causes for early embryonic death of hybrid eggs from donor sheep are unknown, the reciprocal event has been associated with immunological implications. As a means of overcoming the reproductive barrier between sheep and goats, we have transferred interspecific chimaeric embryos. We report here the birth of a goat kid from a sheep mother.     

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 .     

原代和传代培养的人输卵管上皮细胞对小鼠胚胎体外发育的影响

为了检测人输卵管上皮细胞对早期胚胎体外发育的影响，将小鼠２细胞胚胎与人输卵管上皮细胞进行体外共培养．结果显示：无论原代培养还是传代培养的人输卵管上皮细胞都可使胚胎发育率提高，发育速度加快．经传代４次以后的细胞对胚胎发育的促进作用有下降的趋势，所以传代第１至第４代是应用于共培养的最佳选择．同一代细胞中已贴壁稳定生长的细胞对胚胎发育的促进作用比刚经传代尚未贴壁的细胞大．     

Interfamily pregnancy and expression of CD57, CD68 in deciduas between golden hamster and mouse

Pregnancy between different species is one of the key steps to interspecific somatic cell cloning. Although interspecific clone embryos have been constructed, they could not develop to birth after being transferred to recipi-ents. In order to clarify the mechanism of this phenomenon, interfamily pregnancy between golden hamste (Mesocricetus auratus) and mouse (Mus musculus) was studied. Co-culture results indicated that the adhesion ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 12, 24, 48 and 72 h after co-culture were all significantly lower than those of mouse blastocysts. The outgrowth ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 48, 72 h after co-culture were both significantly lower than those of mouse blastocysts (P < 0.01). Golden hamster抯 blastula could be implanted and develop to D 11 of pregnancy after being transferred to mouse uterus (the 7th day after embryo transfer). Compared to the transfer of mouse embryo to mouse uterus, the successful ratio of interfamily embryo transfer was lower and the bulk of fetus was smaller than that of intraspecific fetus. Compared to intraspecific preg-nancy of mouse, the remote decidual tissue of interfamily pregnancy on D8 is looser. At the same time, expressions of CD57 and CD 68 in remote deciduas were both higher than those in the secondary deciduas in both intraspecific and interfamily pregnancy. However, expressions of the two molecules in interfamily pregnancy were lower than those in intraspecific pregnancy. These results showed that interfam-ily pregnancy could be established between golden hamster and mouse. But the development of fetus in interfamily pregnancy was slower than that in intraspecific pregnancy. The expression difference of CD57 and CD68 indicates the difference of immunoreaction between interfamily and in-traspecific pregnancy, which may be one of the reasons lead-ing to interfamily pregnancy termination.     

小鼠胚胎移植技术优化及对巨细胞病毒净化研究

目的 建立小鼠巨细胞病毒净化方法,以获得无MCMV感染的小鼠。方法 利用胚胎移植技术,通过使用不同激素、不同发情周期注射激素、受体鼠品系、不同的移植方法、不同时期胚胎移植,以及移植胚胎数量等对比试验,优化了净化条件。利用优化的胚胎移植净化方法,对供体鼠进行了净化。结果 SIGMA生产的激素,在10 IU剂量下超排得到的可用胚胎数量约为17枚/只;在小鼠发情间期超排得到的可用胚胎最多,超排的可用胚胎数约为23枚/只;选取C57雄性小鼠与ICR雌性小鼠交配的子一代作为受体鼠,产仔率达44.5%;输卵管移植较子宫移植效果好,产仔率为40.64%;移植2细胞胚胎的妊娠率为80%,明显优于单细胞和8细胞;受体鼠移植24枚胚胎时,产仔率达到了43.75%。利用优化的小鼠巨细胞病毒胚胎移植净化方法,净化得到了无MCMV感染的小鼠。结论 建立了小鼠巨细胞病毒净化方法,为获得无MCMV感染的小鼠种群提供了可靠的保障。     

无核白葡萄胚挽救机理的研究

选用无核白葡萄在开花后35￣60d,取其胚接种在含有GA3+IAA+6-BA的不同激素水平MS培养基上,进行胚挽救,以探讨研究无核葡萄的胚挽救机理.结果表明无核白在开花后50d胚挽救效果最好.在附加GA30.5mg.L-1+IAA1.5mg.L-1+6-BA 0.5mg.L-1的MS培养基上得到了84%的发育率和31.9%的成苗率(50d);开花后60d的发育率最高,得到了100%的发育率,却未得到成苗,说明在胚挽救中幼胚的不同部位其发育与败育过程不同步.对无核葡萄胚挽救适期的确定依据提出了新的见解.     

Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification

Over 200 recessive X chromosome-linked diseases, typically affecting only hemizygous males, have been identified. In many of these, prenatal diagnosis is possible by chorion villus sampling (CVS) or amniocentesis, followed by cytogenetic, biochemical or molecular analysis of the cells recovered from the conceptus. In others, the only alternative is to determine the sex of the fetus. If the fetus is affected by the defect or is male, abortion can be offered. Diagnosis of genetic defects in preimplantation embryos would allow those unaffected to be identified and transferred to the uterus. Here we report the first established pregnancies using this procedure, in two couples known to be at risk of transmitting adrenoleukodystrophy and X-linked mental retardation. Two female embryos were transferred after in vitro fertilization (IVF), biopsy of a single cell at the six- to eight-cell stage, and sexing by DNA amplification of a Y chromosome-specific repeat sequence. Both women are confirmed as carrying normal female twins.     

Effect of leptin on oocyte maturation and subsequent pregnancy rate of cloned embryos reconstructed by somatic cell nuclear transfer in pigs

Cloning pigs by somatic cell nuclear transfer （SCNT） has wide applications in basic research, human medicine and agricultural production. To improve cloning efficiency, the effect of two basic maturation media, NCSU-23 and TCM199, was compared, and TCM199 was selected for the following experiments with leptin. We systematically studied the effects of leptin supplementation on oocytes in vitro maturation （IVM）, in vitro development of parthenogenetically activated （PA） and SCNT embryos and in vivo development of SCNT embryos after embryo transfer （ET）. The results showed that supplementation of 100 or 200 ng/ml leptin into the maturation medium did not greatly affect nuclear maturation of oocytes, or cleavage rates of PA and SCNT （P 〉 0.05）. Blastocyst rates of PA and SCNT embryos were significantly improved when 100 or 200 ng/ml leptin was added to maturation medium, and the number of cells in PA blastocysts was also improved （P 〈 0.05）. The number of cells in blastocyst of SCNT was improved, when 100 ng/ml leptin was added （P 〈 0.05）. Furthermore, supplementation of 100 or 200 ng/ml leptin to the IVM medium may improve pregnancy rate and the delivery rate inpig cloning.     

实验小鼠胚胎冷冻技术实验研究

本文在建立和引进实验小鼠超排卵技术、卵采集技术、胚胎输卵管移植技术的基础上 ,应用小鼠初期胚的快速冷冻复苏法对实验小鼠C5 7BL 6J、BDF1 、A wy及昆明种小鼠进行了初期胚的冷冻保存与仔鼠存活率的研究 ,并将冷冻复苏胚与非冷冻胚经移植后的生存率作了比较 ,均取得了重复性的结果 ,可望为实验小鼠胚胎种子库提供参考依据。     

人输卯管组织与小鼠胚胎共培养的初步观察

为了检测人输卵管组织共培养系统对胚胎早期体外发育的影响,将超排得来的小鼠2细胞期胚胎与人输卵管组织共培养,或以人输卵管组织培养后的条件培养液进行培养。培养液为Ham's F10+10％FCS,并以其作为对照培养液。实验结果显示:无论是组织共培养或是条件培养液共培养,胚胎体外发育达囊胚率,胚胎逸出透明带率及胚胎的发育速度都较F10对照组高。其中输卵管壶腹部组织共培养及其条件培养液共培养对胚胎发育的促进作用较峡部大,而且其条件培养液共培养是以组织培养一周内所得的条件培养液效果最好。组织共培养和条件培养液共培养发育的胚胎正常分裂,没有差别,形态也均正常。实验提示人类输卵管上皮可能分泌某种因子能促进胚胎发育。     

茉莉酸甲酯对杂交鹅掌楸体胚发育的影响

【目的】探讨作为植物生长调节剂的茉莉酸甲酯(MeJA)对杂交鹅掌楸体胚发育的影响,为进一步提高杂交鹅掌楸体胚发生效率奠定基础。【方法】在已建立的杂交鹅掌楸未成熟胚体细胞发生体系的基础上,选用杂交鹅掌楸3种基因型C138、233012、253010为材料,分别添加5种浓度的茉莉酸甲酯,测定杂交鹅掌楸在添加不同浓度茉莉酸甲酯条件下的体胚发生率、成熟率、畸形胚发生率,以及再生植株率。【结果】在体胚诱导培养基中添加不同浓度的MeJA可以显著影响杂交鹅掌楸体胚发生效率,且随着MeJA浓度的增加,体胚发生率和成熟率逐渐升高,畸形胚发生率逐渐降低,而当MeJA浓度超过一定值后,体胚发生率和成熟率呈现下降趋势,畸形胚发生率也逐渐升高。杂交鹅掌楸3种基因型体胚发生率和成熟率从高到低的次序为233012、C138、253010。脱落酸(ABA)能够增强MeJA的上述效应。在体胚苗的生长阶段,浓度逐渐升高的MeJA限制了根的生长,但ABA不能改变该效应。【结论】添加1 μmol/L MeJA和2 mg/L ABA时杂交鹅掌楸体胚诱导效果最佳,此时体胚发生率和体胚成熟率相对较高,体胚畸形率最低。     

乙二醇玻璃化冷冻液二步法对长爪沙鼠 2-细胞胚胎的冷冻保存

摘要: 目的 评价用乙二醇玻璃化冷冻液二步法对长爪沙鼠 2-细胞胚胎冷冻保存的效果。方法 通过超排卵处理 后与雄鼠交配获得长爪沙鼠 2-细胞胚胎,胚胎在冷冻液 EFS20 中平衡 3 min,然后移入冷冻液 EFS40 中平衡 1 min, 之后将细胞冻存管直接浸入液氮中保存。采用二步法复苏胚胎,复苏后移入改良的胚胎培养液( mKSOM) 中体外 培养。结果 采用该方法冷冻的胚胎复苏后胚胎回收率为 86. 73% ,存活率为 82. 94% ,复苏后存活的胚胎中约 1 / 3 胚胎可以在体外发育到囊胚。结论 乙二醇玻璃化冷冻液适于冷冻保存长爪沙鼠 2-细胞胚胎。     

基因工程小鼠制备和保种、育种技术研究

摘要: 本文从小鼠的超数排卵、采集胚胎、原核受精卵显微注射、胚胎移植、基因型鉴定等多个环节,详细阐述制备基因工程小鼠的技术操作要领; 通过实施辅助生殖技术IVF( 体外受精) 、精子和胚胎的冷冻、复苏技术实现了基因工程小鼠的保种和育种,涉及相关技术的要点改进和操作体会,成功建成了基因工程小鼠的制备和保种、育种平台,提供了良好的技术服务。