Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.
The boundary layer on the wall surface of the nozzle, the wave suppressor and the channel wall itself affects strongly the velocity profile of the test section of a circulating water channel. The velocity near the free surface is always smaller than the uniform velocity which is normally determined by the mean value at fairly deep position. In order to obtain the uniform velocity near water surface, the rotor method, a jet-like flow generated by the rotor at the edge of the nozzle accelerated the velocity, is employed here. In this paper, it is shown that at first the 2-D wake theory represents the velocity defect near the free surface, and the 2-D jet flow theory can be used for representing the flow by the rotor itself. The uniformed velocity profile is obtained by superimposing these two profiles. The optimum rotation number of the rotor is theoretically determined as a function of the uniform velocity.
The authors have developed a measurement technique by means of statistical analysis of visualized images. The technique is based on the fact that the probability of the particle passing is proportional to the flow speed if the particle concentration is uniform through the measurement area. In this paper, the governing equation for the measurement is formed using Lagrange's differentiation. The process of forming the equation is similar to that of the so-called spatio-temporal derivative method. The stream line coordinate system has been introduced in order to obtain the general form so as to apply this technique to the flow field where the particle concentration is not uniform. The new governing equation also suggests the measurement of the velocity component. The image temporal derivative gives the velocity component normal to the particle edge. Therefore, the velocity vectors can be obtained through the ensemble average of the product of temporal and spatial derivatives. The principles of the measurement is explained in the case of both one- and two-dimensions. The result of a numerical simulation in the two-dimensional case shows that the time-mean velocity vector can be obtained directly from the visualized image data.
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We investigated the optical properties of ZnTe:O∕GaAs before and after atomic hydrogen irradiation. Oxygen incorporation gives rise to energy levels associated with single O atoms, O–O pairs, and O clusters, and to a blueshift of the energy gap of the material with respect to that of pure ZnTe∕GaAs. All of these effects disappear progressively after irradiation with H, which also leads to an increase in the tensile strain of the epilayer. These observations provide experimental evidence of H-induced passivation of an isoelectronic impurity in II–VI alloys.
Wind tunnel tests on an artificially roughened model of a slender ship-hull were carried out. The rough surface was made up of irregular, tightly packed sand grains and the corresponding roughness function was determined by the Liverpool pipe flow technique. A special three-hole Preston tube, suitable for the ship model, was constructed and calibrated to measure the wall friction on both rough and smooth surfaces. The results obtained were compared with those predicted by an integral method of calculation of boundary layer on rough ship hulls. In order to perceive the roughness effects on the hull surface pressure distribution, the latter was measured on the model fitted with special roughness strips distributed girthwise from the fore to the after peak. The results are compared with potential flow calculations by the Hess and Smith method.
Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required for these techniques to be successful, and patients incapable of producing spermatids cannot be helped. Male mice homozygous for the mutant juvenile spermatogonial depletion (jsd) gene show spermatogonial arrest and an elevated intratesticular testosterone level like many other experimental infertility models such as those with iradiation- or chemotherapy-induced testicular damage. In this category of infertile males, suppression of the testosterone level induces spermatogonial differentiation to the stage of spermatocytes but no further. In the present study with jsd mutant mice, we induced spermatogenesis first to spermatocytes and then to elongated spermatids by suppression of testosterone levels with a GnRH antagonist, Nal-Glu, at a dose of 2500 μg kg−1 day−1 for 4 wk and then withdrawal of Nal-Glu. Spermatids were seen in the cross-sections of seminiferous tubules in all mice treated by administration and subsequent withdrawal of Nal-Glu. Four weeks after withdrawal of Nal-Glu, some of the germ cells differentiated into elongated spermatids. Supplementation with testosterone and Nal-Glu after 4 wk of treatment with Nal-Glu alone also induced spermatogenesis similar to the induction by withdrawal of Nal-Glu. Thus, we ascribe the restoration of the differentiation of spermatocytes to spermatids to reelevation of the testosterone level. Furthermore, we successfully rescued male sterility in jsd mice by subsequent intracytoplasmic sperm injection using the elongated spermatids induced by the programmed hormone therapy.
