Survival rate to term of chimeric morulae produced by aggregation of five to nine embryos in the mouse, Mus musculus

Chimeric morulae from five to nine embryos were produced by aggregation after removal of the zonae pellucidae and brief incubation in phytohemagglutinin-P. After 28 hr of in vitro culture, these aggregates were transferred to the left uterine horn of pseudopregnant recipient female mice. Each recipient also received control embryos in the right uterine horn. Genetic markers were included so that aggregate-derived offspring could be distinguished from control offspring. Aggregate-derived embryos survived to term, but at a much reduced rate. In the 25 recipients that produced litters, 6.2% (N=211) of the aggregated embryos developed into liveborn young. Survival-to-term for control embryos was 38.8% (N=387). Survival-to-term was independent of the number of embryos aggregated. The aggregate-derived embryos were apparently undergoing size regulation following implantation.     

Production of transgenic mice

A “transgenic” mouse is identified by the integration of a foreign DNA into its genome. Such animals serve as experimental systems for the study of gene expression and are often generated as models for human diseases. Direct microinjection of DNA into the male pronucleus of a mouse zygote has been the method most extensively used in the production of transgenic mice. Our method is subdivided into three sections: First, Preinjection, where the animals, the donor eggs, and the injection tools are presented. Second, Injection, where the egg handling and micromanipulation is described. Third, Postinjection, where the surgical transfer into pseudopregnant females completes the procedural sequence. Additional sections are provided to include the materials we use and to offer a series of technical tips that cover various aspects of the transgenic process.     

Reduced survival in utero from transferred mouse blastocysts compared with morulae

Studies on the survival of mouse embryos revealed that fewer offspring were produced when blastocysts, rather than morulae, were transferred to foster mothers. Approximately 8–10 h after fertilization F1 hybrid eggs (C57BL/6J × LT/Sv) were collected and cultured to morulae (day 4) or blastocysts (day 5 ) before transfer into uteri of day 3 foster mothers. A few recipients were killed on day 8 of gestation and deciduae were examined histologically. Embryos developing from transferred morulae were found to lie deep within the deciduae and were surrounded by numerous, large blood islands. Conversely, embryos developing from transferred blastocysts implanted more distally to the maternal blood vessels with only a few blood islands surrounding the embryos. These observations, suggesting abnormal implantation with insufficient embyro nourishment, were confirmed when uteri of foster mothers were examined on day 19 of gestation. Although the proportion of implantations from transferred morulae or blastocysts was similar (42 % and 47%, respectively), significantly more of the implantations were resorbed after transfer of blastocysts (78%) as compared with morulae (15%). These results demonstrate that transfer of day 5 cultured blastocysts into uteri of foster mothers increases embryonic mortality as a consequence of improper implantation.     

Production of monozygotic mouse twins from microsurgically bisected morulae

Mouse monozygotic twins were produced by bisection of the compacted morulae and transfer of the pairs of half-embryos after culture in vitro. The compacted morulae (about 16 cells) were microsurgically bisected, using a fine glass needle attached to a micromanipulator, without any supporting micro-instruments, after pretreatment for zona-softening and decompaction. About 80% of the morulae were bisected without visible cell damage. After 20 h in culture, the half-embryos were classified morphologically as eu-blastocysts, pseudo-blastocysts, or trophectodermal vesicles or non-integrated forms. After culture of 131 pairs of bisected morulae, 75 (57.3%) pairs of eu-blastocysts, 20 (15.3%) pairs comprising a eu-blastocyst and pseudo-blastocyst, and 9 (6.9%) pairs of pseudo-blastocysts, were obtained. The pseudo-blastocysts were considered to be derived from half-morulae in which some blastomeres were destroyed or dissociated as a result of micromanipulation. From 30 pairs of eu-blastocysts transferred to 21 recipients, 5 twin fetuses on Day 17 (18 pairs/9 recipients) and 3 twin male young (12 pairs/12 recipients) were obtained. Survival rate of the twin-embryo pairs was 27.8% at autopsy and 25.0% at term. None of the 20 pairs of pseudo-blastocysts transferred to 10 recipients gave rise to normal conceptuses.     

Testes of XX ↔ XY chimeric mice develop from fetal ovotestes

The majority of XX ? XY chimeric mice develop into fertile males. The sexual differentiation of the gonads in these animals has been examined on days 12–14 postcoitum to determine if their development parallels that of normal testes. It was found that 50% of chimeric fetuses, the proportion predicted to be XX ? XY, had neither normal testes nor ovaries. Instead, ovotestes were present, with varying proportions of presumptive ovarian and testicular tissue. On day 12 the ovotestes were organized with testicular tissue in the central region and ovarian tissue at the craniad and/or caudad poles. In the more advanced fetuses there was evidence of regression of the ovarian portion, which would account for the testes found in adults. These results are discussed in light of current theories of sex determination and differentiation and what was previously known about gonads of sex mosaics.     

Production of chimeric calves by aggregation of in vitro-fertilized bovine embryos without zonae pellucidae

Bovine embryos produced by in vitro maturation (IVM), fertilization (IVF) and culture (IVC) were used to produce aggregation chimeras. An aggregated chimera was produced by combining bovine IVF embryos (Holstein × Japanese Black and Japanese Brown × Limousin breeds) which were cultured in vitro without the zonae pellucidae. Forty-eight hours after IVF, embryos at the 8 cell-stage were used to produce aggregation chimeras. In Experiment I, the zonae pellucidae was removed by a microsurgical method using a microblade or by treatment with 0.25% pronase. Holstein × Japanese Black embryos were aggregated with Japanese Brown × Limousin embryos after zonae removal by hand manipulation in culture medium. In Experiment II, the viability of the aggregated embryos developing into blastocysts was examined by measuring the extent of development. The number of aggregated embryos and embryos developed into blastocysts was 34 (91.9%) and 24 (70.6%), respectively, when the zonae pellucidae was removed by the microsurgical method; and 12 (92.3%) and 6 (50.0%), respectively, when the zonae pellucidae was removed using the 0.25% pronase treatment. The size of the aggregated embryos was significantly different from that of the normal embryos when cultured in vitro until Day 10, but not different thereafter. Five aggregated embryos were transferred nonsurgically to the recipients, resulting in 1 pregnancy and the birth of 2 chimeric calves. Skin color was used as evidence of chimerism.     

Production of normal piglets from microsurgically split morulae and blastocysts

The embryo splitting technique was applied to pig embryos, and the developmental ability of the split embryos was examined by means of in vitro culture and transfer. Morulae, early blastocysts and blastocysts were collected from Landrace x Large White F(1) gilts which had been mated to Duroc boars. The embryos were bisected with a fine glass or alloy (PtIr) needle after the softening of zonae pellucidae. The halved-embryos, which had either been placed in zonae pellucidae or not, were transferred to recipient gilts immediately after the micromanipulation (Experiment 1) or after cultivation for 15 to 20 h (Experiment 2). In Experiment 1, two fetuses were obtained from one of three recipients which had received 12 half-embryos. In Experiment 2, three of five recipients became pregnant, and in one recipient, seven piglets of a litter were obtained from 12 zona-free half-embryos produced from the original seven blastocysts. The results obtained indicate that a simple method not requiring the encasing of split embryos into zonae pellucidae is satisfactory to produce viable half-embryos.     

Production of identical twins by separating two-cell rat embryos

Rat identical twins were produced from two-cell embryos. In the presence of cytochalasin B, rat two-cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one-half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two-cell embryos.     

Quantitative release of fatty acids from lipids by a simple hydrolysis procedure

Glycerolipids and sphingolipids are hydrolyzed with 0.5 M HCl in acetonitrile-water 9:1 (by vol) for 45 min at 100 degrees C or 4 hr at 70 degrees C. After hydrolysis, free fatty acids (FFA) are recovered in chloroform and separated by thin-layer chromatography. More than 95% of the radioactivity from labeled phospholipids is recovered as FFA, and more than 97% of the lipid phosphorus is recovered as water-soluble phosphate. The yields of FFA, including polyunsaturated acids, after hydrolysis are as good as or better than those obtained for methyl esters using methanolysis catalyzed by acid, alkali, or BF3. High recoveries of FFA from glycerophospholipids, sphingomyelin, and neutral glycerides are attained. The procedure is quantitative, simple, inexpensive, and produces no methyl esters as secondary products.     

The production of chimeric rats and their use in the analysis of the hooded pigmentation pattern

New, improved media and procedures for making rat chimeric embryos and culturing them in vitro have been developed. We have produced 27 rat chimeras: 20 males and 7 females. This ratio of males to females is consistent with that seen in mouse chimeras, suggesting that rat sex chimeras develop as phenotypic males. By aggregating embryos containing appropriate genetic markers for pigment cell differentiation, it is possible to produce chimeras that elucidate the site of action of the hooded gene. The coat color patterns of black ? black hooded chimeras display a white belly spot. In black ? albino hooded chimeras, small patches of white hair appear on the head and a large white spot occurs on the belly. Black ? agouti hooded chimeras display both agouti and nonagouti pigmentation over the entire surface of the chimera. These animals are fully pigmented with no white spots. In black ? albino non-hooded chimeras, rather small irregular patches of black and white hairs are distributed throughout the pelage. Histological examination of sections of hair follicles obtained from the white areas in the head of black ? albino hooded chimeras revealed amelanotic melanocytes. On the other hand, hair bulbs from the white belly spots do not contain any such melanocytes. Thus the white hairs of the head are due to the presence of albino melanocytes, but the white hairs of the belly are due to the total absence of melanocytes. All these observations are consistent with the conclusion that the hooded gene acts within melanoblasts, probably to retard their migration from the neural crest and/or to prevent their entrance into the hair follicles of the white areas of hooded rats.     

A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures

A simple method is presented for the isolation of nuclei from somatic embryos of carrot (Daucus carota L.), which is applicable to small amounts of material in synchronized culture. The method employs buffers containing a high concentration of glycerol to stabilize the structure of the nuclei. Purification was carried out by centrifugation using preformed Percoll gradients. Treatment with cell wall-degrading enzymes prior to homogenization improved the efficiency of isolation and permitted a reproducible yield of nuclei. The pure preparations were obtained with an efficiency of approximately 60%. The isolated nuclei retained their morphological characteristics as demonstrated by phase — contrast and electron microscopy. Nuclear proteins displayed the expected species of histones by two-dimensional gel electrophoresis. The isolated nuclei showed high RNA polymerase activity.     

Production of transgenic chimeric rabbits and transmission of the transgene through the germline

Here we report that improved reproductive technologies combined with an efficient microinjection method and in vitro cultivation medium enabled us to create germ line chimeric rabbits. To follow the fate of the chimeric embryo a blastomere marked with the human blood coagulation factor VIII (hFVIII) transgene was microinjected into a morula stage wild type embryo. The degree of chimerism in different tissues was estimated by real-time PCR and was found to be in the range of 0.1-42%. Among the four chimeric animals, one was identified as a chromosomal intersex and two were germline chimeras.     

Production of human erythropoietin by chimeric chickens

The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells.     

High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method.

Rabbit morulae were exposed to a vitrification solution-modified PBS [PB1] medium containing 40% ethylene glycol + 18% Ficoll + 0.3 M sucrose (EFS) for 2, 5, or 10 min at 20 degrees C and were vitrified in liquid nitrogen. When morulae were rapidly warmed, 96% had an intact zona pellucida. When embryos were cultured after removal of the mucin coat, high proportions of them formed blastocoel (79-100%), but the percentage of embryos developed to fully expanded blastocysts decreased with increased exposure time 87%, 40%, and 17%). The survival rate of morulae vitrified after removal of the mucin coat was lower than that of mucin-intact embryos. To assess the development potential in vivo, 131 embryos were vitrified after 2 min of exposure to EFS solution; all the embryos were recovered and 120 were transferred to recipients without removal of the mucin coat, resulting in 78 (65%) full-term fetuses or young. This simple method, which yields high survival both in vitro and in vivo, will be of practical use for vitrifying rabbit embryos.     

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor‐derived gametes

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES‐like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma‐irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor‐derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells. Mol. Reprod. Dev. 53:394–397, 1999. © 1999 Wiley‐Liss, Inc.     

Inhibition of L-threonine intestinal absorption in rabbits by cadmium

Cadmium compounds are widely spread in the environment. Animal exposure to cadmium compounds occurs mainly through foods or drinks contaminated by this metal. Cadmium has been shown to produce several negative effects on the gastrointestinal tract such as inhibition on sugars and amino acids absorption. The aim of the present work was to study the inhibitory characteristics of cadmium on L-threonine intestinal absorption in rabbits in order to understand about this malabsorption of nutrients. Our results show that L-thereonine tissue accumulation as well as mucosal to serosal transepithelial fluxes are decreased in a dose-dependent manner in rabbit jejunum. Amino acid diffusion across the intestinal epithelium was not affected by cadmium. A noncompetitive mechanism and a partial reversion by dithioerythritol (thiol groups protector) is described for this inhibition.     

电刺激家兔迷走神经中枢端诱导的黑-伯反射中的非联合型学习现象

本文旨在研究电刺激家兔迷走神经诱导的黑-伯（Hering-Breuer,HB）反射中的学习和记忆现象。选择性电刺激家兔迷走神经中枢端（频率10～100Hz,强度20～60μA,波宽0．3ms,持续60s）,观察对膈神经放电的影响。以不同频率电刺激家兔迷走神经可模拟HB反射的两种成分,即类似肺容积增大所致抑制吸气的肺扩张反射和类似肺容积缩小所致加强吸气的肺萎陷反射。（1）长时高频（≥40Hz,60s）电刺激迷走神经可模拟呼吸频率减慢,呼气时程延长的肺扩张反射。随着刺激时间的延长,膈神经放电抑制的程度逐渐衰减,表现为呼吸频率的减慢（主要由呼气时程延长所致）在刺激过程中逐渐减弱或消失,显示为适应性或“习惯化”的现象;刺激结束时呼吸运动呈现反跳性增强,表现为一过性的呼气时程缩短,呼吸频率加快,然后才逐渐恢复正常。长时低频（〈40Hz,60s）电刺激迷走神经可模拟呼吸频率加快、呼气时程缩短的肺萎陷反射。随着刺激时间的延长,膈神经放电增强的程度逐渐衰减,同样表现出“习惯化”现象;刺激结束后,膈神经放电不是突然降低,而是继续衰减,表现为呼气时程逐渐延长,呼吸频率逐渐减慢,直至恢复到前对照水平,表现了刺激后的短时增强效应。（2）HB反射的适应性或“习惯化”程度反向依赖于刺激强度和刺激频率,表现为随着刺激强度和频率的增加,膈神经放电越远离正常基线水平,即爿惯化程度减弱。结果表明,家兔HB反射具有“习惯化”这一非联合型学习现象,反映与其有关的呼吸神经元网络具有突触功能的可翅性,呼吸的中枢调控反射具有一定的适应性。     

Mitochondrial dysfunction in sepsis: evidence from bacteraemic baboons and endotoxaemic rabbits

Mitochondria, that provide most of the ATP needed for cell work, and that play numerous specific functions in biosyntheses and degradations, as well as contributing to Ca²⁺; signaling, also play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome, and by acute processes, are responsible for numerous diseases.The involvement of impaired mitochondria in the pathogenesis of sepsis is discussed. By means of the skinned fiber technique and high resolution respirometry, we have detected significantly reduced rates of mitochondrial respiration in heart and skeletal muscle of endotoxaemic rabbits. Mitochondria from heart were more affected than those from skeletal muscle. Decreased respiration rates were accompanied by reduced activities of complex I+III of the respiratory chain. Endotoxin-caused impairment was also detectable at the level of the Langendorff perfused heart, where the coronary vascular resistance was significantly increased.For an investigation of the influence of bacteraemia on the mitochondrial respiratory chain, baboons were made septic by infusion of high and low amounts of E. coli. For complex I+III and II+III, a clear dose-dependent decrease was detectable and in animals which died in septic shock, a further decrease of enzyme activities in comparison to the controls were found.These results are discussed in the light of current knowledge on the role of mitochondria in cell pathology in respect to sepsis.In conclusion, we present evidence that mitochondrial function is disturbed during sepsis. Besides ischaemic and poison-induced disturbances of mitochondrial function, sepsis is a further example of an acute disease where impaired mitochondria have to be taken into account.     

Production of chimeric loach by cell transplantation from genetically pigmented to orange embryos

To establish techniques for chimera formation and to obtain further knowledge of chimerism, chimeric loach were produced using the wild strain as the donor and the orange strain as the recipient by cell transplantation. Transplantation between embryos at two different stages was performed to achieve efficient chimera formation. In the combination of the early-mid-blastula as the donor and the late-blastula as the recipient, 100-150 blastomeres were injected into the blastoderm of the recipient and the rate of chimera formation was 46.2%. On the other hand, in the combination of early-mid-blastula and early-gastrula, only 30 blastomeres were injected and the rate of chimera formation was 80.0%. These results demonstrating the combination of embryonic stages may provide a key for efficient chimera formation. We also compared the number of melanophores on chimeric larvae with that on donor cells labelled with latex beads; it was found that the number of transplanted cells has a profound effect on chimerism, whereas the site of pigmentation is not always in agreement with the site of actual transplantation of donor cells.