Development of avian embryo manipulation techniques and their application to germ cell manipulation

The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.     

Mos and the mitogen-activated protein kinase do not show cytostatic factor activity in early mouse embryos

Mos and the mitogen-activated protein kinase (MAPK) cascade have been established as crucial regulators of second meiotic metaphase arrest, the so-called CSF arrest, in mammalian oocytes. They are also thought to play a role in regulating mitotic metaphase arrest of early mammalian embryos. In the present study, we examined whether mitotic arrest is induced in early mouse embryos by activation of extracellular signal-regulated kinases (ERKs), which are major MAPKs in mouse eggs, and their substrate, p90Ribosomal S6 kinase (RSK), as reported in Xenopus embryos. Wild-type Mos (wt-Mos), degradation-resistant Mos mutant (P2G-Mos) or constitutive active mutant of MAPK/ERK kinase, MEK (SDSE-MEK), was expressed in early mouse embryos by injecting the respective expression vectors into the pronucleus of fertilized eggs, and the developmental rates were then examined up to 72 h after insemination. Expression of P2G-Mos and SDSE-MEK succeeded in activating ERKs and RSK in developing mouse embryos, while wt-Mos failed to activate them in spite of expression of mos mRNA, indicating that the wt-Mos protein is unstable in early mouse embryos. Although the activated levels of ERKs and RSK in the vector-injected embryos were comparable to those of meiotically arrested mouse oocytes, their developmental rates were identical to those of the control embryos. These results suggest that activation of MAPK and RSK does not induce mitotic arrest in early mouse embryos. The present study indicates that there are large physiological differences between early mouse embryos and mouse oocytes and that CSF arrest of mouse eggs in mitosis should be discussed separately from that in meiosis.     

Aluminum-tolerance of carrot (daucus carota L.) plants regenerated from selected cell cultures

Abstract

Aluminum-tolerant variants from cell cultures of carrot (Daucus carota L. cv. MS Yonsun) were selected by exposing them to excess ionic aluminum. The medium contained A1CI₃ and 0.1 mM phosphate at pH 4.0. Under ionic aluminum stress (0.5-1.0 mM), 282 calli were selected. Twenty-eight fertile plants were regenerated from 7 selected calli. Based on the root elongation tests of the seedlings, the seeds displayed aluminum-tolerance. In the case of the cell line H7-08, the selection occurred at a frequency of ca. 6 × 10^-7 when the materials were plated on agar medium containing 0.5 mM aluminum.     

ATP depletion inhibits retraction of chromatophores induced by GABA in cephalopod Todarodes pacificus

Color changes in cephalopods are generated by the expansion or retraction of chromatophores located under the dermis. The behavior of the chromatophores is regulated by neurotransmitters; l-glutamate (l-Glu) is an excitatory transmitter that causes the chromatophores to expand. To date, serotonin [5-hydroxytryptamine (5-HT)] is the only neurotransmitter known to stimulate retraction of chromatophores. We found that the chromatophores in the Japanese squid Todarodes pacificus were regulated by γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and that GABA caused expanded chromatophores to retract. We also found that adenosine 5′-triphosphate (ATP) concentrations in skin samples remained stable at their initial values for more than 24 h after the death of each squid; therefore, the chromatophores could respond to both l-Glu and GABA during that period. Furthermore, we attempted to reduce the levels of ATP by storing skin sample in sodium azide solution. The chromatophores in sodium azide-treated skin samples were induced to expand by l-Glu, but these expanded chromatophores could not be induced to retract by GABA. Based on these observations, we conclude that ATP is essential for retraction, but not expansion, of chromatophores.     

Structure of thermal convection development based on inhomogeneous water surface cooling

The generation and development of thermal convection based on inhomogeneous water surface cooling were examined by hydraulic and numerical experiments to examine the characteristics of thermal convection in a closed water body with aquatic plants. A visualization experiment revealed the structural characteristics of a whirlpool when thermal convection was generated quantitatively by using PIV analysis. Then, a water temperature measurement experiment demonstrated that a steady cold water mass generated based on the heat flux transport from the water surface increases. This explained each of the three stages in the convection development process. Moreover, aquatic plants, which grow thickly on the water surface, cause not only vertical but also horizontal flows based on the density difference with the water surface that is not covered by plants, and thus change the development process of the convection cell.     

Deformation and blemishing of pearls caused by bacteria

Bacteria cause deformation and blemishing in pearls, and we investigate this relationship. We examined pearls derived from Pinctada margaritifera, Pinctada maxima, and Pinctada fucata, and determined the location of bacteria using histopathological and immunohistochemical techniques and 16S ribosomal RNA (rRNA) sequence analyses. The most remarkable change was the inflammatory reaction located between the pearl nucleus and the nacreous layer, composed of hemocytic infiltration with melanization, periostracum, and fibrous aragonite-like structures. These anomalous changes were limited to abnormal sites, and such inflammatory reaction sites are a major factor in the formation of pearl abnormalities. Bacteria were detected from the inflammatory sites and are suspected as the causative agent. Most of these bacteria were anaerobic.     

QTLs for phosphorus deficiency tolerance detected in upland NERICA varieties

Phosphorous (P) deficiency is a major yield limiting factor in rice (Oryza sativa L.) production. The interspecific New Rice for Africa (NERICA) varieties combine general stress tolerance from African cultivated rice (Oryza glaberrima Steud) with characteristics associated with high yield from O. sativa. However, little is known about their ability to tolerate P deficiency. Here, we examined the variation for tolerance to P deficiency among the 18 upland NERICAs and their parents in multi‐year field experiments. The good performance under P deficiency of the O. glaberrima parent CG 14 and some NERICAs suggested that these tolerant NERICAs contain loci associated with P deficiency tolerance inherited from CG 14. Additionally, four QTL clusters for P deficiency tolerance were detected on chromosomes 4, 6 and 11 using F₃ lines derived from the cross between the P deficiency tolerant variety NERICA10 and a Japonica‐type sensitive variety ‘Hitomebore’. These QTLs represent the first step in identifying stress tolerance genes from O. glaberrima that could subsequently be used to enhance P deficiency tolerance in O. sativa.