从植入前遗传学诊断异常的胚胎中分离人胚胎干细胞系

在体外受精过程中,通过胚胎植入前遗传性诊断（PGD）对有遗传风险患者的胚胎进行植入前活检和遗传学分析,选择无遗传性疾病的胚胎植入子宫,而PGD诊断异常的胚胎则会被丢弃。本研究尝试将PGD异常胚胎用于分离人胚胎干细胞,以获得携带遗传缺陷的人胚胎干细胞系。利用荧光原位杂交技术对第3-5天胚胎进行PGD检测,结果异常的胚胎进一步用于分离获取胚胎干细胞系,然后对h ES细胞系进行核型及干细胞表面标记、多能性基因表达、端粒酶活性以及分化能力等特征性鉴定。总共从13个PGD异常胚胎中分离获得8个人胚胎干细胞系,建系效率为61.5%,其中1个核型正常,5个核型异常。说明利用PGD异常胚胎可以获得携带遗传缺陷的人胚胎干细胞系,不仅为评估PGD技术临床结论的准确性提供了一种新方法,更重要的是为研究各种遗传性疾病的发病机理提供了有效的细胞模型。     

Embryonic mortality and the uterine environment in first-service lactating dairy cows

Embryos were collected non-surgically from the tip of one uterine horn of 23 lactating dairy cows on Day 7 of pregnancy. Embryos were classified on the basis of morphological criteria as normal (n = 12) or abnormal (n = 13). Abnormal embryos were further classified as cleavage stage (n = 9) or morula/blastocyst (n = 4). Cows producing an abnormal embryo did not differ in days post partum at oestrus, age or parity from cows producing a normal embryo. Cows with an abnormal morula/blastocyst also did not differ with respect to days post partum at oestrus from cows with abnormal cleavage-stage embryos but cows with an abnormal morula/blastocyst were significantly older and of greater parity than cows with an abnormal cleavage-stage embryo. Hepes-saline-PVP solution (30 ml) was initially infused into the uterine tip, mixed and then withdrawn with a syringe. Analysis of this fluid revealed that the concentrations of glucose, total protein, calcium, magnesium and potassium were significantly higher in the flushings from the uterus of cows with abnormal embryos than from cows with normal embryos and zinc and phosphorus tended to be higher in the uterine flushings of cows with abnormal embryos. Phosphorus, total protein, calcium and magnesium tended to be higher in the flushings from cows with abnormal morulae/blastocysts than from cows with abnormal cleavage-stage embryos. Plasma progesterone did not differ between cows with normal or abnormal embryos or in cows with abnormal morulae/blastocysts or abnormal cleavage-stage embryos. Most embryonic mortality therefore occurred before Day 5 (during cleavage) in these cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degeneration pattern in somatic embryos of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.     

Aberrant convergence of the neural folds in the mouse mutant vl.

Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuration of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22-28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30-35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds.     

A high degree of aneuploidy in frozen-thawed human preimplantation embryos

We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34 patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest. Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed. Received: 19 November 1998 / Accepted: 4 March 1999     

Growth of 9.5-day rat embryos in folic-acid-deficient serum

Rat embryos (9.5-day-old) were cultured for 48 hours in serum from diet-induced folic-acid-deficient rats. Resultant embryos were frequently abnormal; many were growth retarded and exhibited a defect in the turning mechanism that inverts the embryo from ventrally to dorsally convex. Affected embryos displayed abnormal twisting or kinking of the neural tube. Gross anaemia was also frequently observed, and the protein content of the embryos was markedly less than that of embryos grown in normal rat serum. Supplementation of the deficient serum with folic acid improved growth and greatly reduced the occurrence of deformities. It virtually eliminated the incidence of gross anaemia but only partially restored the protein content of the embryos to the level observed in those grown in normal rat serum. The effects of the folate deficiency could be eliminated by supplementation with normal rat serum. The data indicate that embryos have a requirement for adequate folate in order for normal growth and differentiation to take place; they also suggest that some of the embryopathic effects of maternal folate deficiency are mediated by secondary effects on maternal metabolism. This may take the form of a disturbance in the production of maternally synthesised growth factors necessary for normal embryonic development.     

Ultrastructure of the neural basal lamina in loop-tail mice

Ultrastructural features of the neural basal lamina were studied by means of the tannic acid and ruthenium red techniques in normal and abnormal dysraphic loop-tail mice at 9-11 days of gestation. With ruthenium red, the configuration of the neural basal lamina is similar in both normal and abnormal embryos at 9-11 days. However, differences were detected in the abnormal 9-day embryos processed with tannic acid, as compared with normal littermates. These include irregularities in the lamina rara externa, as well as differences in the staining pattern of the neuroepithelial cell plasma membrane. By 11 days of gestation, the lamina rara externa of the normal embryos shows features similar to those observed in the 9-day abnormal embryos.     

The Hectd1 ubiquitin ligase is required for development of the head mesenchyme and neural tube closure

Closure of the cranial neural tube depends on normal development of the head mesenchyme. Homozygous-mutant embryos for the ENU-induced open mind (opm) mutation exhibit exencephaly associated with defects in head mesenchyme development and dorsal-lateral hinge point formation. The head mesenchyme in opm mutant embryos is denser than in wildtype embryos and displays an abnormal cellular organization. Since cells that originate from both the cephalic paraxial mesoderm and the neural crest populate the head mesenchyme, we explored the origin of the abnormal head mesenchyme. opm mutant embryos show apparently normal development of neural crest-derived structures. Furthermore, the abnormal head mesenchyme in opm mutant embryos is not derived from the neural crest, but instead expresses molecular markers of cephalic mesoderm. We also report the identification of the opm mutation in the ubiquitously expressed Hectd1 E3 ubiquitin ligase. Two different Hectd1 alleles cause incompletely penetrant neural tube defects in heterozygous animals, indicating that Hectd1 function is required at a critical threshold for neural tube closure. This low penetrance of neural tube defects in embryos heterozygous for Hectd1 mutations suggests that Hectd1 should be considered as candidate susceptibility gene in human neural tube defects.     

Immunofluorescent analysis of fibronectin and laminin distribution in the vl mutant mouse

Summary The distribution of fibronectin and laminin was determined in the basement membrane surrounding the caudal neural tube and at the site of initial apposition of the caudal neural folds by means of indirect immunofluorescence histochemistry on 9.0- to 10.5-day mouse embryos fixed in Carnoy's solution and serially sectioned in paraffin. At early phases of development of normal (+/+) and abnormal (vl/vl) embryos the dorsolateral neural basement membrane overlying putative neural crest cells caudal to the hindlimb shows a patchy fibronectin reaction, with laminin virtually absent. In older embryos, both components are present but are discontinuous overlying the neural crest. The results suggest that since discontinuities occur in the basement membrane of abnormal as well as normal embryos, the neural crest cells are not prevented from emigrating from the abnormal neural tube; thus the faulty neural fold fusion that characterizesvl/vl embryos does not appear to be due to a suppression of emigration by the basement membrane. The results also demonstrate the advantages and reliability of embedding in paraffin for analysis of serially sectioned pathological material by means of indirect immunofluorescence, provided that normal controls and abnormals are processed simultaneously.     

Karyotypically normal and abnormal human embryonic stem cell lines derived from PGD-analyzed embryos

Although a normal karyotype is generally a requirement for stem cell lines, new applications are likely to emerge for stem cells with defined chromosomal aneuploidies. We therefore investigated the use of embryos found to be aneuploid on biopsy followed by preimplantation genetic diagnosis (PGD) with fluorescent in situ hybridization (FISH), and developmentally arrested embryos for stem cell derivation. Eleven stem cell lines were obtained from 41 embryos in 36 cultures, with higher success rate achieved from PGD-analyzed, developmentally advanced embryos (45%) than from clinically unsuitable non-PGD embryos (13%). The resulting stem cell lines were karyotyped, and surprisingly, six of the nine lines from aneuploid embryos as well as both lines from non-PGD embryos were karyotypically normal. Three lines from PGD embryos were aneuploid exhibiting trisomy 5, trisomy 16, and an isochromosome 13, respectively. None of the aneuploid lines presented the same anomally as the original PGD analysis. Our study has three important implications. First, we confirm the ability to produce stem cell lines from PGD-tested embryos as well as developmentally abnormal embryos, offering specialty stem cell lines for research into the clinically important aneuploidies. Second, we observe that stem cell derivation from apparently aneuploid embryos is often thwarted by underlying mosaicism and emerging dominance of the stem cell line by karyotypically normal cells. The corollary, however, is that regular production of normal stem cell lines from developmentally abnormal embryos ordinarity discarded opens a new source of embryos for stem cells, whether for research or for eventual therapeutic use within the donating families.     

Left-right lineage analysis of the embryonic Xenopus heart reveals a novel framework linking congenital cardiac defects and laterality disease

The significant morbidity and mortality associated with laterality disease almost always are attributed to complex congenital heart defects (CHDs), reflecting the extreme susceptibility of the developing heart to disturbances in the left-right (LR) body plan. To determine how LR positional information becomes ;translated' into anatomical asymmetry, left versus right side cardiomyocyte cell lineages were traced in normal and laterality defective embryos of the frog, Xenopus laevis. In normal embryos, myocytes in some regions of the heart were derived consistently from a unilateral lineage, whereas other regions were derived consistently from both left and right side lineages. However, in heterotaxic embryos experimentally induced by ectopic activation or attenuation of ALK4 signaling, hearts contained variable LR cell composition, not only compared with controls but also compared with hearts from other heterotaxic embryos. In most cases, LR cell lineage defects were associated with abnormal cardiac morphology and were preceded by abnormal Pitx2c expression in the lateral plate mesoderm. In situs inversus embryos there was a mirror image reversal in Pitx2c expression and LR lineage composition. Surprisingly, most of the embryos that failed to develop heterotaxy or situs inversus in response to misregulated ALK4 signaling nevertheless had altered Pitx2c expression, abnormal cardiomyocyte LR lineage composition and abnormal heart structure, demonstrating that cardiac laterality defects can occur even in instances of otherwise normal body situs. These results indicate that: (1) different regions of the heart contain distinct LR myocyte compositions; (2) LR cardiomyocyte lineages and Pitx2c expression are altered in laterality defective embryos; and (3) abnormal LR cardiac lineage composition frequently is associated with cardiac malformations. We propose that proper LR cell composition is necessary for normal morphogenesis, and that misallocated LR cell lineages may be causatively linked with CHDs that are present in heterotaxic individuals, as well as some 'isolated' CHDs that are found in individuals lacking overt features of laterality disease.     

Stage Specific Effects of Zn2+ on Sea Urchin Embryogenesis

The treatment of sea urchin embryos by Zn²⁺ followed by culture with Zn²⁺-specific chelators such as ethylenediamine-N, N'-diacetic acid and N-hydroxyethylethylenediamine-N, N', N'-triacetic acid, was performed at various developmental stages to find out specific stages for Zn²⁺ to induce abnormal differentiation. The treatment with 1 mM ZnSO₄ at 20°C during a period including two spans of development between 0 and 8 hr and between 14 and 16 hr post fertilization yielded permanent blastulae. Zn²⁺-treatment during the former span produced abnormal prisms and plutei with small archenteron. The treatment for a period including only the latter span failed to produce abnormal ones. Zn²⁺-treatment during a period including the gastrula stage also produced abnormal spherical embryos. Without the culture with these chelators, abnormal embryos were produced by Zn²⁺-treatment performed at any stages before gastrulation. A high zinc amount in the embryos just after the treatment became as low as in normal embryos soon after the culture with these chelators and was maintained during the culture without them. These results indicate that zinc retention occurs in the Zn²⁺-treated embryos and causes abnormal differentiation when the treated embryos develop in normal sea water through the Zn²⁺-specific periods of development.     

低温预处理对水曲柳体细胞胚胎发生的影响

用4℃低温预处理未成熟的水曲柳种子0-30d,取出种子内的合子胚为外植体诱导体胚发生,研究低温预处理影响体胚发生的结果表明：低温预处理过的外植体其体胚发生总数和子叶胚发生数均低于未作低温预处理的;随着预处理时间的延长,畸形胚发生数和发生比率与总的体胚发生数和发生率的变化趋势基本相同;处理20d的正常胚发生数和发生比率的绝对数虽然很低,但远高于不作低温预处理的。说明4℃低温预处理对水曲柳体胚发生没有促进作用,对畸形胚的发生也不能控制,总的来讲,适当的低温处理有一定的改善正常体胚发生的潜力。     

Morphogenetic routes of long-term embryogenic callus culture of <Emphasis Type="Italic">Areca catechu</Emphasis>

Early morphogenetic events and repetitive embryogenesis from callus culture of betel nut palm (Areca catechu L.) were studied using scanning electron microscopy. On Murashige and Skoog (MS) medium supplemented with 2 mg dm⁻³ dicamba, callus culture has capacity to form plantlets via somatic embryogenesis and to form secondary embryos for about 4 years. However, various abnormal embryos without differentiation of the leaf sheath and shoot apical meristem were observed, which showed bell-shaped and then cup-shaped or mushroom-shaped structures. These abnormal embryos contained distinctive structures, including a disk-shape interior region, surfaces with grooves and a stalk-like posterior region. During subculture, these abnormal embryos enlarged, became deformed and gradually lose their shape and then converted into nodular, compact embryogenic callus. It was also found that secondary embryos originated from interior surfaces or posterior regions of abnormal embryos, and gave rise to the next cycle of normal and abnormal embryos.     

The fine structure of ventricular cells in the brains of mouse embryos homozygous for the loop-tail gene

The neural tube in normal (+/+), heterozygous (Lp/+), and abnormal (Lp/Lp) mutant mouse embryos ranging in age from 10 to 12 days of gestation was studied by means of transmission electron microscopy. In the abnormal embryos, ventricular cells in defective regions of the brain show distortions and crowding together of internal cellular processes and a decrease in blebs and bulbous projections, as compared with their normal counterparts. At 12 days' gestation the abnormal brains show a scarcity of the T-shaped internal cellular processes characteristic of normal brains. The abnormal brains also show increased amounts of intercellular space and extensive gaps between the cells, particularly in basal regions. There are no striking differences between the normal and abnormal brains at 10 to 12 days' gestation with respect to the appearance and distribution of cilia, microfilaments, microtubules, tight junctions, and ribosomes.     

第二极体数目与受精结局及胚胎发育潜能的相关性

目的 探讨第二极体数目与卵母细胞受精结局和胚胎发育潜能之间的关系.方法 根据受精后5 h内卵母细胞极体数目的 不同分为1个极体组（1PB组）、2个极体组（2PB组）、3个极体组（3PB组）和4个以上极体组（≥4PB组）.分别统计各组的正常/异常受精率（2PN率,1PN和3PN率）、优质胚胎率（优胚率）、移植胚胎所占比例以及相应着床率.采用χ2检验对数据进行统计学处理.结果 ① 2PB组的2PN率显著高于其它组,而异常受精率显著低于其它组;随着第二极体数目的 增加,异常受精比例逐渐增加;② 2PB组和3PB组的胚胎着床率显著高于1PB组,以2PB组为最;③ 2PB组和3PB组用于移植的胚胎比例无显著差异.结论① 短时受精后显示有两个极体的卵母细胞其受精结局和发育潜能优于其它极体数目的 胚胎;② 随着第二极体数目的 增加,异常受精比例逐渐增加,可能与卵母细胞减数分裂或基因调控异常有关;③ 1PB组的总受精率高达48.9 %.因此,短时受精后对于仅显示一个极体的卵母细胞需要延长观察时间,谨慎确定受精与否,以防止过度早补救ICSI;④ 1PB组的着床率显著低于2PB组,也不建议首选用于移植.     

Histochemical analysis of the neural basal lamina in the hindbrain region of exencephalic mutant mice

The neural basal lamina in hindbrain regions of exencephalic loop-tail (Lp/Lp) mice and of their normal (+/+; Lp/+) littermates was analyzed histochemically at the electron microscopic level by means of enzyme digestion and alcian blue staining with critical electrolyte concentrations (CEC) of MgCl2. At 9 days of gestation, the normal and abnormal embryos showed a similar pattern of alcian blue staining with a CEC of 0.00 M or 0.05 M MgCl2. However, with a CEC of 0.30 M MgCl2, the basal lamina in the abnormals stained more prominently, particularly the lamina rara externa, suggesting the presence of more sulfated glycosaminoglycans (GAG) in the abnormals. Moreover, predigestion of the tissues with Streptomyces hyaluronidase, which removes hyaluronic acid (HA), indicated that the abnormal basal lamina contained relatively less HA than in the normal embryos. By 10 days of gestation the normal basal lamina contained relatively more sulfated GAG and less HA and was thus more similar in appearance to that in the abnormal embryos. This apparently premature shift from HA predominance to sulfated GAG predominance in the abnormal basal lamina may be of significance in the etiology of dysraphism in this mutant.     

Chromosomal abnormalities in Day-6, in vitro-produced pig embryos

A cytogenetic study was undertaken to quantify, by chromosomal karyotyping, the incidence and type of chromosomal abnormalities present in Day-6 in vitro-produced (IVP) porcine embryos. Morphologically normal Day-6 blastocysts (n=318) were fixed and grouped into six classes according to the number of total cells (from < or =20 to 61-70). Of 248 embryos suitable for analysis, 97 (39.1%) displayed chromosomal abnormalities. The abnormalities included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%). Within polyploid embryos, triploidy and tetraploidy showed the highest incidence (56.5 and 27.5%, respectively); among mixoploid embryos, diploid-triploid embryos (2n/3n) were prevalent (36.8%). Overall, the mean cell number was 34.3 +/- 12.1 and the mitotic index was 8.6 +/- 6.1. Chromosomally abnormal embryos had fewer (P<0.01) total cells compared to normal (2n) embryos (31.8 +/- 1.3 versus 35.9 +/- 1.0). In addition, the incidence of polyploidy decreased as the number of cells increased, while that of mixoploidy did not differ. These data indicate that polyploidy affects a large percentage of IVP porcine embryos capable of developing to blastocysts and the incidence of chromosomal abnormalities is much higher than that reported previously in in vivo embryos in this species. Given the ability of morphologically normal embryos with an abnormal chromosome complement to undergo preimplantation development in vitro, and the inability to identify blastocysts with abnormal karyotype without cytogenetic analysis, careful consideration should be given to factors affecting ploidy of IVP embryos, especially the incidence of polyspermic fertilization, when evaluating criteria of a porcine in vitro embryo production scheme.