Oocyte mitochondria: Strategies to improve embryogenesis

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.     

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae)

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.     

Chromosomal mapping of the host resistance locus to rodent malaria (Plasmodium yoelii) infection in mice

The disease outcome in malaria caused by the protozoan parasite Plasmodium is influenced by host genetic factors. To identify host genes conferring resistance to infection with the malaria parasite, we undertook chromosomal mapping using a whole-genome scanning approach in cross-bred mice. NC/Jic mice all died with high parasitemia within 8 days of infection with 1 x 10(5) parasitized erythrocytes. In contrast, 129/SvJ mice all completely excluded malaria parasites from the circulation and remained alive 21 days after infection. We performed linkage analysis in backcross [(NC/Jic x 129/SvJ)xNC/Jic] mice. The Pymr ( Plasmodium yoelii malaria resistance) locus was mapped to the telomeric portion of mouse Chromosome (Chr) 9. This locus controls host survival and parasitemia after infection. The Char1 locus ( P. chabaudi resistance locus 1), controlling host survival and peak parasitemia in P. chabaudi infection, was previously mapped to the same region. This host resistance locus mapping to Chr 9 may represent a ubiquitous locus controlling susceptibility to rodent malaria. Elucidation of the function of this gene will provide valuable insights into the mechanism of host defense against malaria parasite infection.     

Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells

The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.     

Developmental abnormalities in mouse embryos tetrasomic for chromosome 11: apparent similarity to embryos functionally disomic for the x chromosome

Adopting a mating system involving two different Robertsonian translocations with monobrachial homology, we studied the early development of mouse embryos trisomic or tetrasomic for chromosome 11. A developmental delay of 12-24 hours was evident in trisomic embryos at embryonic day (E)7.5, whereas tetrasomic embryos apparently had stopped growth by E6.5 without formation of extraembryonic structures. This extremely severe developmental abnormality found in tetrasomic embryos is similar to that reported in embryos having two active X chromosomes in extraembryonic cell lineages. Autosomal tetrasomy, but not autosomal trisomy, can lead to such early developmental errors. Thus, a reasonable inference would be that the X chromosome is twice as active as the autosome. Probably, the X chromosome became upregulated in response to the evolutionary necessity of minimizing haplo-insufficiency brought about by miniaturization of the Y chromosome.     

Active-dimeric form of lipoprotein lipase increases in the adipose tissue of patients with rheumatoid arthritis treated with prednisolone

A thermodynamic analysis of the interaction of 125I-labeled human chorionic gonadotropin (IhCG) with two of its monoclonal antibodies (MAbs) was carried out. The dissociation profile of IhCG-MAb complex conforms to a two-step model. vant Hoff enthalpies were calculated with the K(A) (equilibrium constant) values obtained from dissociation at different temperatures. Free energy and entropy changes were calculated using the standard equations. DeltaH values for one of the MAbs, viz. VM7 were favorable at temperatures beyond 30 degrees C. Interestingly, the DeltaS values were also favorable at all temperatures. In the case of MAb VM4a, however, the interaction throughout the temperature range was driven by large favorable entropic contributions, indicating the importance of hydrophobic interaction in the binding of this MAb to hCG. The energetics of the interaction of these two monoclonals with hCG is discussed.     

An improved method for estimating sequence divergence between related DNAs from changes in restriction endonuclease cleavage sites

Summary We have developed a theory to estimate the degree of sequence divergence between related DNAs from the comparison of restriction endonuclease recognition sites. Two major improvements have been made upon a similar method reported by Upholt (1977). First, the most probable value is calculated by the collective use of all available data. This reduces intrinsic statistical error and extends the analyzable range of sequence divergence. Second, all variables are redefined so that they have strict mathematical implications. This corrects a serious error arising from the misinterpretation of the meaning of the fraction of conserved cleavage sites. With this refined method, sequence divergence between rat and mouse mitochondrial DNAs (mtDNAs) was calculated to be about 25% substitutions/nucleotide, which is in good agreement with the DNA-DNA hybridization data obtained by Jakovcic et al. (1975). It was also estimated that the three types of rat mtDNAs differ from one another by 0.3 ~1% of total base pairs. These values are 2 ~5 times smaller than those obtained with the conventional method.     

Tolerance to, and uptake and degradation of 2,4,6-trinitrotoluene (TNT) are enhanced by the expression of a bacterial nitroreductase gene in Arabidopsis thaliana

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C.1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The obtained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the non-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mM TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when theywere floated on TNT solution. HPLC analysis showed that the peak due to TNT taken upinto plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indicates that the expression of bacterial NTR improved the capability of plants to degrade TNT.     

Cellular and Vacuolar Fusion of Protoplasts Electrofused Using Platinum Microelectrodes

Protoplasts isolated from cultured cells of Coptis japonicaand Euphorbia millii were electrically fused using platinummicroelectrodes. The process involved two stages, cellular andvacuolar fusion, which are characterized respectively by transientwrinkling of the membrane and the formation of a dark-red precipitate. (Received June 12, 1987; Accepted October 13, 1987)     

River to river: First evidence of eel movement between distant rivers via the sea

Environmental Biology of Fishes - Eel movement patterns have been frequently studied to learn about their movements within the fresh- and brackish waters of the same river before their spawning...