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Biomedical subjects

T Wakayama

Publications and source records attributed to T Wakayama.

At least 55 records · Page 3Linked to original sources
PubMed1999

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules.

The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I-III and IX-XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell.

Animals↗
PubMed1999

Simulation of the daily sunlight illumination pattern for bacterial photo-hydrogen production.

Methods of illumination to simulate the daily sunlight irradiation pattern were studied in relation to photohydrogen production using the photosynthetic bacterium Rhodobacter sphaeroides RV. Three illumination patterns were compared, in which the light intensity was changed in 1, 3, or 6 steps. As a control, outdoor experiments were also carried out over a 3-d period in Tsukuba, August 1996. Outdoors, hydrogen production by Rba. sphaeroides RV was dependent on the sunlight intensity: the total volume of hydrogen produced per day varied from 14 to 28 l.m(-2), while the total light energy ranged from 5.5 to 6.4 kWh.m(-2). d(-1). The maximum hydrogen production rate was 2.8 l.m(-2).h(-1) under a 4.5-cm light path and the average light energy conversion efficiency was 1.1%. Indoors, the hydrogen production rate was found to be independent of the mode of illumination among the three patterns employed. The maximum hydrogen production rate was 3.3 l.m(-2).h(-1) with a light energy conversion efficiency of 1.0%, and it was concluded that the single-step illumination method provides an appropriate simulation of sunlight. Saturation of hydrogen production occurs during high light intensity around noon and this plays a key role in the simulation.

Journal Article↗
PubMed1999

Entrapment of Rhodobacter sphaeroides RV in cationic polymer/agar gels for hydrogen production in the presence of NH4+.

Cationic polyelectrolytes (chitosan, poly-L-lysine (PLL), polyethyleneimine (PEI) and trimethylammonium glycol chitosan iodide (TGCI)) were used to entrap anoxygenic phototrophic bacteria in order to prevent the inhibitory effect of NH4+ on hydrogen production. When combined with agar gel, chitosan and PLL demonstrated no obvious repressive effect on hydrogen production by Rhodobacter sphaeroides under light-anaerobic conditions with lactate and glutamate as the carbon and nitrogen sources, respectively. On the other hand, both PEI and TGCI exerted a detrimental effect on hydrogen production under these conditions. Hydrogen production in the presence of NH4+ by the bacteria entrapped in the complex gel containing chitosan and agar improved considerably compared to that in the control containing only agar. Evidence shows that chitosan improves the hydrogen production via various effects. Diffusion tests demonstrated that the addition of chitosan increased to some extent the resistance to the diffusion of positively charged NH4+, but had no effect on negatively charged lactate. A buffer effect in the complex gels was also revealed.

Journal Article↗
PubMed1999

Enhanced hydrogen production by a mutant of Rhodobacter sphaeroides having an altered light-harvesting system.

A stable mutant of the photosynthetic bacterium Rhodobacter sphaeroides with an altered light-harvesting (LH) system (P3 mutant) was obtained by UV irradiation and characterized. The mutant exhibited a 2.7-fold decrease in the core antennal (LH1) content and 1.6-fold increase in peripheral antennal (LH2) content compared to the wild-type strain. The H2 evolution rates in the P3 mutant under 800- and 850-nm light, corresponding to the absorption maxima of LH2, were 1.5 times higher than in the wild-type strain. The wild-type absorption spectrum was restored in the P3 mutant when a 1.1-kb PCR-amplified fragment containing the puf promoter and pufQBA genes was ligated into a pRK-415 derivative and introduced into it. The transformant showed lower H2 production rates at 800 and 850 nm than the P3 strain carrying the control plasmid, indicating that the accelerated H2 production in the P3 mutant was a result of alterations in the LH system.

Journal Article↗
PubMed1999

Comparison of oocyte-activating agents for mouse cloning.

Since somatic cell components are unable to activate oocytes following injection or fusion, enucleated oocytes receiving adult somatic cells during the cloning process must be activated artificially for their development. We compared the efficiency of four types of oocyte-activating agents: strontium, ethanol, single electric pulse, and spermatozoa. Although strontium was the best in supporting preimplantation development of reconstructed mouse oocytes, there was no significant difference among the four agents with respect to the rate of postimplantation embryo development and the birth of live offspring.

Animals↗
PubMed1999

A novel trans-complementation assay suggests full mammalian oocyte activation is coordinately initiated by multiple, submembrane sperm components.

To initiate normal embryonic development, an egg must receive a signal to become activated at fertilization. We here report that the ability of demembranated sperm heads to activate is abolished after incubation over the range 20-44 degreesC and is sensitive to reducing agents. On the basis of this observation, we have developed a microinjection-based, trans-complementation assay in order to dissect the heat-inactivated sperm-borne oocyte-activating factor(s) (SOAF). We demonstrate that the failure of heat-inactivated sperm heads to activate an egg is rescued by coinjection with dithiothreitol-solubilized SOAF from demembranated sperm heads. The solubilized SOAF (SOAFs) is trypsin sensitive and is liberated from demembranated heads in a temperature-dependent manner that inversely correlates with the ability of sperm heads to activate. This argues that SOAFs is a proteinaceous molecular species required to initiate activation. Injection of oocytes with mouse or hamster sperm cytosolic factors, but not SOAFs alone, induced resumption of meiosis, further suggesting that these cytosolic factors and SOAF are distinct. Collectively, these data strongly suggest that full mammalian oocyte activation is initiated by the coordinated action of one or more heat-sensitive protein constituents of the perinuclear matrix and at least one heat-stable submembrane component.

Animals↗
PubMed1999

Anti-herpesvirus activities and cytotoxicities of 2-thiopyrimidine nucleoside analogues in vitro.

Twenty 2-thiopyrimidine nucleoside analogues were synthesized and examined for inhibitory activity against herpes simplex virus (HSV) type 1 and 2, varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and thymidine kinase-deficient HSV (HSV-TK-) replication in vitro. 2-thiouracil (thymine) arabinoside, 2'-deoxy-2-thiouridine (or 2-thiothymidine) and their 5-halogenated derivatives showed anti-HSV activity in both RPM18226 (human B-lymphoblastoid cells) and MRC-5 (human embryo lung cells). 2'-deoxy-5-halogenated-2-thiocytidines were also inhibitory against HSV, whereas 2-thiocytosine arabinoside and its derivatives were not inhibitory against HSV replication, except 5-bromo and 5-iodo congeners (TN-31, TN-32). Substitution of the halogen atom at the 5-position of the pyrimidine rings to an atom with a higher molecular weight increased anti-HSV and VZV activities, except for the anti-HSV activity of 2-thiouracil arabinosides. 2'-deoxy-5-methyl-, and 2'-deoxy-5-iodo-2-thiouridines (TN-17, TN-44) showed the most potent anti-HSV activity, and 2'-deoxy-5-chloro- and 2'-deoxy-5-bromo-2-thiocytidines were potent inhibitors of VZV replication. However, none of the compounds inhibited HCMV and HSV-TK- replication. TN-31 and TN-32 were shown to inhibited HCMV and HSV-TK- as well as HSV and VZV replication. The cytotoxicity of the 2-thio-pyrimidine nucleoside analogues was less than that of the 2-oxy-congeners of the compounds (5-iodo-2'-deoxyuridine, 5-iodo-2'-deoxycytidine, thymine arabinoside and cytosine arabinoside). The selectivity index of 2'-deoxy-5-iodo-2-thiouridine (TN-44) was higher than that of 5-iodo-deoxyuridine. TN-17 and TN-44 were not cytotoxic to resting or stimulated human peripheral blood mononuclear cells at 400 microM, although TN-32 was cytotoxic at a concentration of 20 microM.

Antiviral Agents↗
PubMed1999

Cytotoxicity and pharmacokinetics of 1-beta-D-arabinofuranosyl-2-thiocytosine, a 2-sulphur substituted derivative of cytarabine.

1-(beta-D-Arabinofuranosyl)-2-thiocytosine (araSC), a 2-substituted derivative of cytarabine (araC), has been investigated for its cytotoxicity, enzymatic stability, plasma concentration-time profile in mice, and cytokinetics. This derivative showed strong cytotoxicity in several mammalian cell lines, although activity (IC50s) was weaker than araC. Greater stability to mouse cytidine deaminase was observed; the half-life in the presence of the enzyme was about 4-times longer than that of araC. The plasma concentration-time profile in mice in vivo showed prolonged retention of araSC when compared with araC. Cytokinetic study using flow cytometry indicated a non-S-phase specific effect of this compound.

Animals↗
PubMed1998

Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.

Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.

Animals↗
PubMed1998

Expression and cellular localization of the mRNA for the 25-kDa heat-shock protein in the mouse.

The 25-kDa heat-shock protein (Hsp25) is a member of the small heat-shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot and in situ hybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin,eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.

Adrenal Glands↗
PubMed1998

Flavor deterioration in yogurt.

Volatile components of flavored yogurt preserved at 5 degrees C in the dark for 0 day, 3 days and 10 days were recovered by simultaneous distillation extraction (SDE) and headspace (HS) procedures. Gas chromatography (GC) and GC-mass spectrometry (GC-MS) analyses of those samples showed remarkable changes in some compounds. Aldehydes which contribute to expressing citrusy notes were reduced to alcohols during fermentation process and storage. As a result, the strength of flavors which expressed well-balanced citrusy notes in yogurt were weakened, and fatty or oily notes mainly caused from alcohols were strengthened reversely. Hydrocarbons were also digested by bacteria during a fermentation process. A small amount of other compounds, such as esters and terpene alcohols changed. Fewer effects of sorption into a package material and chemical reactions, such as hydrolysis esters, hydration or oxidation of hydrocarbons, were observed.

Adsorption↗
PubMed1998

Fate of microinjected spermatid mitochondria in the mouse oocyte and embryo.

Mouse round spermatids labelled with MitoTracker were microinjected into Sr(2+)-activated mouse oocytes. The labelled mitochondria were tracked up to the morula/blastocyst stage using fluorescence microscopy. The overall incidence of embryos with labelled mitochondria fell from 80% in the 1-cell zygote to 25% in 2-cell, 9% in 4-cell and approximately 1% in 8-cell or later stages. Thus it appears that almost all round spermatid mitochondria finally disappear from embryos during the 4-cell to 8-cell transition, as happens for mature spermatozoa (Cummins et al. Zygote 1997, 5:301-8). The spermatid mitochondria remained tightly bound together during this process. In contrast, labelled primary spermatocyte and cumulus mitochondria dispersed rapidly throughout the oocyte cytoplasm within 3 h. We hypothesise that spermatid mitochondria may be bound together by cytoskeletal elements produced in the early haploid spermatid. These elements, together with terminal differentiation of the sperm mitochondria, may be central to the processes by which the embryo 'recognises' the sperm mitochondria and inhibits inheritance of paternal mitochondrial DNA. These results suggest that round spermatid injection for infertile men will not pose a significant risk to offspring by transmitting abnormal mitochondrial genomes.

Animals↗
PubMed1998

Can alcohol retain the reproductive and genetic potential of sperm nuclei? Chromosome analysis of mouse spermatozoa stored in alcohol.

Alcohol is known to preserve genomic DNA and the primary structure of sperm protamines. To determine whether alcohol can retain the genetic and reproductive potential of mammalian sperm nuclei, mature mouse spermatozoa were stored in 70% ethanol or propanol for up to 2 months before injection into oocytes. Live offspring were obtained after injection of spermatozoa stored in 70% ethanol for 1 day at -20 degrees C. About 20% of the spermatozoa stored under this condition had normal chromosomes. The remaining 80% of spermatozoa and all the spermatozoa stored in 70% ethanol for 2 months had structurally aberrant chromosomes, and none could support the development of normal embryos. High concentrations of alcohol do not alter the primary structure of either DNA or small-molecular-weight protamines. However, alcohol may modify protamine-protamine or protamine-DNA interactions in a manner that results in the induction of DNA strand breaks during sperm chromatin decondensation within the oocyte. The limited success in obtaining normal offspring with ethanol-stored spermatozoa is encouraging. It may be possible to overcome these problems and develop a simple method for preserving mammalian spermatozoa without freezing.

Animals↗
PubMed1998

Fertilisability and developmental ability of mouse oocytes with reduced amounts of cytoplasm.

To determine the minimal amount of cytoplasm necessary to support normal development of mouse oocytes, mature unfertilised oocytes were reduced in size into approximately 1/2, 1/4 and 1/8 by removing the cytoplasm, then inseminated. More than 80% of 1/2-size oocyte were fertilised normally and almost all fertilised eggs developed to blastocysts. When transferred to foster females, 31% of the blastocysts developed into normal offspring. In contrast, 1/4- and 1/8-size oocytes, although they were penetrable by spermatozoa and extruded the second polar bodies, could not reach the 2-cell stage. In these oocytes, sperm nuclei did not develop into full-sized pronuclei. These results suggest that an oocyte can develop to full term after losing about half its cytoplasm, but not more.

Animals↗
PubMed1998

Development of normal mice from oocytes injected with freeze-dried spermatozoa.

Freeze-dried mouse spermatozoa are all motionless and dead in the conventional sense. When injected into oocytes, however, their nuclei can support normal embryonic development even after three month preservation in a dried state. Although the freeze-drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genomes at room temperature.

Animal Husbandry↗
PubMed1998

The first polar body can be used for the production of normal offspring in mice.

The purpose of this study was to determine whether chromosomes in the first polar body can participate in normal embryonic development. In the mouse the majority of first polar bodies degenerate soon after ovulation, but a few remain viable for 10 h or more. When the contents of a live polar body were injected into an enucleated mature oocyte and examined 2 h later, the polar body chromosomes were arranged on a metaphase plate as seen prior to the secondary meiotic division. Such oocytes were fertilized normally by sperm injection. When 2-cell embryos were transferred to foster females, 30-57% developed into fertile offspring. This outcome supports a longstanding belief that chromosomes ejected into the first polar body have the same genetic potential as those remaining in the oocyte after the first meiotic division. As the chromosomes in the second polar body are known to have full potential to participate in normal embryonic development, it is theoretically possible to reproduce four offspring by using chromosomes in one oocyte.

Animals↗
PubMed1998

Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection.

Epididymal mouse spermatozoa were suspended in various physiological solutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffinose before cooling to -20 degrees, -50 degrees or -196 degrees C and storage for 1-28 days. After thawing, a few spermatozoa frozen with raffinose were partially motile (about 2%) but in all other treatments they were immotile and diagnosed as 'dead' by staining that differentiates between live and dead spermatozoa. Almost all oocytes injected with sperm heads (nuclei) from spermatozoa frozen with and without raffinose were fertilized normally (95-100%) and developed to the two-cell stage (89-100%). No differences were found between the physiological media. The majority of oocytes fertilized with spermatozoa frozen in CZB medium developed to blastocysts (80-94%) but development was significantly reduced after fertilization with spermatozoa frozen in PBS and isotonic saline especially in the absence of raffinose (69 and 70% versus 51 and 50%). Normal fertile offspring were obtained in all treatments but there were significantly fewer offspring with spermatozoa stored at -196 degrees C in isotonic saline with or without raffinose and CZB with raffinose. Testicular spermatozoa were extremely sensitive to cryodamage: about 50% frozen to -196 degrees C in CZB with or without raffinose disintegrated after thawing. Almost 100% of oocytes injected with sperm heads from intact (at light microscope level) testicular spermatozoa developed to the two-cell stage but development to blastocysts was reduced significantly compared with that of controls especially those without raffinose. The data indicate that cryopreservation of sperm nuclei requires less stringent conditions than those for the retention of normal physiological function of intact spermatozoa. Motility and plasma membrane integrity are not essential for fertilization and the production of live offspring when nuclei of nonviable spermatozoa are injected into oocytes.

Animals↗