Human embryonic stem cell lines derived from single blastomeres

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos.     

Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo

The widespread use of clomiphene citrate and exogenous gonadotrophins for in vitro fertilization (IVF) in human frequently results in the production of multiple embryos. Replacement of more than two embryos increases pregnancy rate but may result in multiple pregnancies with increased pre- and post-natal abnormality. Preservation of embryos for a limited time allows fewer embryos to be replaced on several different occasions and thus the problems of multiple pregnancy can be minimized, the effectiveness of a single IVF procedure increased and embryo replacement in adverse maternal conditions avoided. Preimplantation embryos have been successfully cryopreserved in many animal species. The sensitivity of embryos to cooling and freezing varies between species and stages of embryo development. We report here the cryopreservation procedures that allow a high survival rate of four- and eight-cell human embryos and the establishment of a pregnancy following the freezing and storage of an eight-cell embryo for 4 months in liquid nitrogen. The pregnancy terminated at 24 weeks' gestation due to development of a septic Streptomyces agalactiae chorion amnionitis after premature membrane rupture.     

Rapid diagnosis with FISH for chromosomal abnormality of fetal pyelectasia

Fluorescence in situ Hybridization （FISH） was used to investigate whether the chromosome of the fetus prenatally diagnosed as pyelectasis was normal or not. Amniotic fluid was taken from the pregnant woman whose fetus was detected with pyelectasia by prenatal examination. The chromosome of the amniotic fluid cell without culture was examined with FISH. The result shows that compared with the traditional amniotic fluid cell culture, FISH has the advantages of more rapid, higher sensitivity and specificity, and was 10-12 days earlier to complete the diagnosing than the traditional method. The fetuses detected chromosomal abnormality in each groups were induced during the middle and late trimester, while those fetuses with normal chromosome continued pregnancy, the rate of spontaneous disappearance of pyelectasia decreased as the severity of pyelectasia increased. FISH can satisfy the urgent need in the clinical prenatal diagnosis due to its rapidity to determine whether fetus with pyelectasia was accompanied with chromosomal.     

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     

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。     

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.     

微注射人TCP10L基因对斑马鱼形态的影响

利用模式生物斑马鱼,通过微注射的方法,研究了人肝脏特异性转录因子TCP10L对斑马鱼早期胚胎发育的影响.结果显示:人TCP10L基因对斑马鱼胚胎发育有一定的毒性.表现为早期的脊索发育异常,个体严重畸形;当血液循环出现后,表现为血流延缓和围心腔水肿等症状.该项研究表明:人肝脏特异性转录因子TCP10L在斑马鱼胚胎中的过表达会引起显著的形态异常和心血管障碍,并且这种影响与其是否在斑马鱼肝内的特异表达有关,为进一步探明该转录因子在人肝脏内发挥的功能奠定了基础.     

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.     

Extra-embryonic function of Rb is essential for embryonic development and viability

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5 (refs 2-4). However, the actual cause of the embryonic lethality has not been fully investigated. Here we show that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. We used two complementary techniques-tetraploid aggregation and conditional knockout strategies-to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals were virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development.     

促排卵周期血清雌二醇比值及孕酮／雌二醇比值与体外受精-胚胎移植妊娠率的关系

目的探讨促排卵周期血清雌二醇（E2）比值及孕酮／雌二醇（P/E2）比值与体外受精-胚胎移植（IVF-ET）妊娠率的关系。方法接受常规体外受精胚胎移植术的不孕患者695例,于HCG日、移植前1d、移植第7d检测血清E2水平;移植第7d同时检测血清P水平。分别比较HCG日E2与移植前1dE2的比值、HCG日E2与移植第7dE2的比值、移植第7dP的绝对值及P/E2的比值与妊娠率的关系。结果HCG日E2与移植前1d E2的比值与妊娠率无相关性;HCG日E2与移植第7dE2的比值与妊娠率呈负相关性,比值越高妊娠率越低,有显著性差异;移植第7dP/E2的比值与妊娠率呈负相关性,比值越高妊娠率越低,有显著性差异。结论在IVF周期中,HCG日E2与移植第7dE2的比值、移植第7d孕雌激素比值可以作为预测IVF妊娠结局的指标之一。     

锯缘青蟹胚胎发育过程中几种水解酶活力的比较研究

测定了锯缘青蟹胚胎发育过程中九个发育期的四种水解酶比活力。结果表明，蛋白酶、淀粉酶、脂肪酶在青蟹的整个胚胎发育过程中均有活力，１－８期活力低但较稳定，纤维素酶则到胚胎的第八期才表现出活力。临孵化前的第九期，胚胎的掾酶活力有所提高，而其余三种水解酶活力则有显著提高，表现为开口摄食饵料作准备。胚胎水解酶活力的高低可作为幼体能否培育成功的一项良好指标。     

氯化锂对泥鳅胚胎发育的影响

研究了ＬｉＣｌ对泥鳅早期胚胎发育的影响。结果表明，ＬｉＣｌ对卵裂早期至神经胚期的形态发育有明显的致畸作用，不同发育时期的胚胎对ＬｉＣｌ的敏感性不同。ＬｉＣｌ对原肠期胚胎的致畸效应相对较强。     

776例畸形、死胎、习惯性流产患者的染色体分析及临床意义

回顾性总结患者核型资料，为产前诊断提供依据。对776例分娩畸形、死胎、习惯性流产患者进行染色体检查，共检出异常核型25例，占受检人数的3．2％，其中平衡易位携带者13例，臂间倒位5例，性染色体异常7例。这表明染色体异常是导致畸形、死胎、习惯性流产的重要原因。在遗传咨询门诊中，应对患者进行染色体检查，若再次妊娠，则应进行生育指导。     

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 .     

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.     

Expression of LIF,VEGF, CD57 and CD68 after the transfer of rat embryos to mouse uteri

The high failure rate of interspecific preganacy is a major obstacle to the successful interspectific cloning of mammals,To in vestigate the reasons for the failure of inter-specfic pregnancy between rats and mice,we transferred rat blastocysts into mouse uteri on the third day of pseudopreg -nancy (D3),oure previous study showed that intact rat embryos could still be observed in mouse uteri on D9.In the present study ,we found that expression of CD57 and CD68 increased significantly at the maternal -fetal interface fol-lowing the transfer of rat embryos,Similarly ,Leukaemia inhibitory factor(LIF) expression increased ,but vascular endothelial growth factor(VEGF) expession decreased,In a co-culture system ,the percentage of rat ectoplacental cones (EPCs) with adhesion and outgrowth and outgrowth area on mouse uterine decidual cells were less than that of mouse EPCs,These results indicate that an increase in the immunological rejection response and a decrease in the in vasiveness of rat embryos may be important reasons for the failure of interspecific pregnancy between rat and mouse.     

EMS对枇杷花药胚状体细胞有丝分裂的影响

采用不同浓度的EMS对枇杷花药胚状体进行离体诱变,并以未诱变的材料作对照,对其引起的细胞学效应进行了研究．结果表明：在EMS诱变过的材料中均观察到一定数量的染色体畸变;球胚细胞中随着EMS浓度的升高有丝分裂指数降低,而染色体畸变率升高．胚性细胞与球胚细胞中有相同的变化趋势,但各自的分裂指数和染色体畸变率并不相同．     

用荧光原位杂交技术检测性染色体异常

用性染色体探针分别对９例性染色体异常患者和１名性别不明患儿进行了荧光原位杂交，结果表明，２个病列的杂交结果与原诊断核型不符，性别不明患儿的核型为４６，ＸＹ，结合中期及间期杂交结果，ＦＩＳＨ的应用价值进行了评估。