Biomedical subjects

Michiaki Yamashita

Publications and source records attributed to Michiaki Yamashita.

12 recordsLinked to original sources

PubMed2005

Dynamics and histological characteristics of gonadal sex differentiation in pejerrey (Odontesthes bonariensis) at feminizing and masculinizing temperatures.

This study evaluated the effects of different temperatures on the histological process of sex differentiation in the pejerrey Odontesthes bonariensis, a fish with marked temperature-dependent sex determination (TSD), at feminizing, neutral, and masculinizing temperatures. Fish reared at three temperatures (17 degrees C, 24 degrees C, and 29 degrees C) from hatching were sampled weekly until 11 weeks and their gonads were examined by histology. The percentages of females at 17 degrees C, 24 degrees C, and 29 degrees C were 100%, 73%, and 0%, respectively. Sex differentiation occurred earlier and at a smaller body size at higher temperatures in both sexes. The first signs of ovarian differentiation were observed at 4 and 7 weeks at 24 degrees C and 17 degrees C, respectively, and those of testicular differentiation at 4 and 7 weeks at 29 degrees C and 24 degrees C, respectively. Body or gonadal growth rates before sex differentiation were not proportional to temperature and showed no sexual dimorphism at 24 degrees C, where both sexes were present. Thus, differential growth rate is probably not a factor in TSD or histological sex differentiation in pejerrey. Blood vessels were formed before sex differentiation in both sexes and at all temperatures, and may be important for sex differentiation. No signs of intersexuality were found in any of the groups, and this characterizes pejerrey as the differentiated type of gonochorist even at feminizing and masculinizing temperatures. Ovaries were formed by the same histological processes at feminizing (17 degrees C) and neutral (24 degrees C) temperatures and without any pathological features such as germ cell degeneration. The process of testicular formation was generally similar at 24 degrees C and 29 degrees C, but some fish at 29 degrees C had widespread germ cell degeneration before sex differentiation. This suggests that pathological processes leading to germ cell death, such as heat-induced dysfunction of the supporting somatic cells, could be involved in masculinization of the genetic females at high temperatures.

Age Factors↗

PubMed2005

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells.

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.

Animals↗

PubMed2004

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress.

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.

Animals↗

PubMed2004

Characterization of multiple members of the HSP70 family in platyfish culture cells: molecular evolution of stress protein HSP70 in vertebrates.

A shift from 28 to 37 degrees C in the incubation temperature of a culture of the platyfish fibroblast cell line, EHS cells (platyfish fibroblast cell line), induced a set of stress proteins. A two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed that the cells expressed three genetically distinct forms of heat-shock protein 70 (HSP70) family proteins: heat-inducible forms of HSP70, the constitutively expressed heat-shock cognate protein 70 (HSC70) and its phosphorylated isoform, and the glucose-regulated protein 78 (GRP78). Three different clones encoding two major isoforms of heat-inducible HSP70, platyfish HSP70-1 and HSP70-2, and of the HSC70 were isolated from a platyfish cDNA library. We compared the deduced amino acid sequences of the platyfish HSP70 and HSC70 proteins with those of other vertebrates. Phylogenetic analysis showed that vertebrate HSP70 could be classified into four cluster groups: (a) fish HSP70, with two isoforms of heat-inducible HSP70 in fish, fish HSP70-1 and HSP70-2; (b) the mammalian testis-specific HSP70-related protein HST70; (c) the mammalian heat-inducible HSP70B'; and (d) the mammalian major histocompatibility complex (MHC)-linked HSP70, including the MHC-linked heat-inducible HSP70 and the testis-specific HSP70-related protein. These findings suggest that vertebrate HSP70 was derived from a single ancestral HSP70 gene during vertebrate evolution and that multiple copies of heat-inducible HSP70 were probably evolved during genetic divergence in fish and higher vertebrates.

Amino Acid Sequence↗

PubMed2004

An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal.

Dietary administration of a cytochrome P450 aromatase (P450arom) inhibitor (fadrozole) in genetic female juveniles of zebrafish (Danio rerio) was performed at 15-40 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, treated with 0, 10, 100 and 1000 microg fadrozole g(-1) diet(-1) were 0, 62.5, 100 and 100%, respectively. Rearing at high water temperature in genetic all-females was performed at 15-25 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, at water temperatures of 28.5, 35 and 37 degrees C were 0, 68.8 and 100%, respectively. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive oocytes of early diplotene and perinucleolar stages in fadrozole-treated genetic females (1000 microg g(-1) diet(-1)) were observed at 15-40 days post-hatching during sex-reversal. In contrast, apoptotic oocytes of early diplotene stage in high temperature-treated genetic females (at 35 and 37 degrees C) during sex-reversal and presumptive males of wild-type fish during sex differentiation were found at 15-27 days post-hatching. Our findings indicate that oocyte apoptosis, depletion of P450arom activity and differentiation of spermatogonia during gonadal sex-reversal are caused by treatments of aromatase inhibitor or high water temperature.

Animals↗

PubMed2004

Cloning and characterization of two distinct isoforms of rainbow trout heat shock factor 1. Evidence for heterotrimer formation.

To elucidate the molecular mechanism underlying the heat shock response in cold-water fish species, genes encoding heat shock transcription factors (HSFs) were cloned from RTG-2 cells of the rainbow trout Oncorhynchus mykiss. Consequently, two distinct HSF1 genes, named HSF1a and HSF1b, were identified. The predicted amino acid sequence of HSF1a shows 86.4% identity to that of HSF1b. The two proteins contained the general structural motifs of HSF1, i.e. a DNA-binding domain, hydrophobic heptad repeats and nuclear localization signals. Southern blot analysis showed that each HSF1 is encoded by a distinct gene. The two HSF1 mRNAs were coexpressed in unstressed rainbow trout RTG-2 cells and in various tissues. In an electrophoretic mobility shift assay, each in vitro translated HSF1 bound to the heat shock element. Chemical cross-linking and immunoprecipitation analysis showed that HSF1a and HSF1b form heterotrimers as well as homotrimers. Taken together, these results demonstrate that in rainbow trout cells there are two distinct HSF1 isoforms that can form heterotrimers, suggesting that a unique molecular mechanism underlies the stress response in tetraploid and/or cold-water fish species.

Amino Acid Sequence↗

PubMed2003

Cold-inducible expression of the cell division cycle gene CDC48 and its promotion of cell proliferation during cold acclimation in zebrafish cells.

A member of the ATPases associated with diverse cellular activities (AAA) family, the cell division cycle gene CDC48/VCP (valosin-containing protein)/p97, was cloned from zebrafish and found to be a major cold-inducible protein in fish cells. CDC48 mRNA levels increased significantly after reducing the temperature from 30 to 15 degrees C for 25 days. CDC48 protein levels also increased 2.5-fold after 30 days at cold temperatures. When fish cells overexpressing CDC48 were exposed to a temperature of 15 degrees C, cell proliferation was markedly enhanced in comparison with control cells. By contrast, expression of a mutant molecule with a tyrosine-805 to alanine substitution at the C-terminal phosphorylation site inhibited cell proliferation and induced apoptosis at low temperatures. Therefore, CDC48 may promote cell cycling and cell proliferation via C-terminal tyrosine phosphorylation during cold acclimation in fish cells.

Acclimatization↗

PubMed2003

Histological process and dynamics of germ cell degeneration in pejerrey Odontesthes bonariensis larvae and juveniles during exposure to warm water.

Elevated temperature causes degeneration and disappearance of the germ cells in the males of scrotal mammals. It was recently shown that heat-induced germ cell degeneration occurs also in fish but, unlike in mammals, it occurs not only in males but also in females. The purpose of this study was to clarify the histological process and dynamics of heat-induced germ cell disappearance in pejerrey Odontesthes bonariensis larvae and juveniles. Monosex and mixed-sex fish produced by thermal manipulation of sex (temperature-dependent sex determination) were subjected to 29 degrees C for periods between 1 and 12 weeks, and used to analyze, by histological methods, the changes in gonadal size and the number of normal and degenerating germ cells. Groups exposed to 29 degrees C for 8-12 weeks were subsequently transferred to 24 degrees C to verify if any gonadal damage would be permanent. Germ cell degeneration, histologically characterized by nuclear pyknosis or eosinophilia and cytoplasmic eosinophilia, was observed with increasing frequency at higher temperatures (29>24> 17 degrees C) and more in males than in females. Clear degenerative changes in the germinal epithelium usually began within one week of exposure to 29 degrees C and appeared clearer in females than in males. Complete loss of germ cells was observed only in individuals exposed for periods of 8-12 weeks to 29 degrees C but no treatment produced 100% sterile fish. Germ cells that remained in the gonads after exposure to 29 degrees C retained the capacity to rapidly recolonize germ cell-depleted areas, suggesting that the associated somatic cells in the gonads are little or not affected by this temperature.

Animals↗

PubMed2003

Apoptosis in zebrafish development.

Apoptosis (programmed cell death) is important in normal biological processes and in pathogenesis in vertebrates. This review focuses on some of the prominent features of apoptosis during fish development. Caspases and other apoptosis-regulating genes have been cloned from zebrafish (Danio rerio) and other fish species. Elucidation of in vivo functions of apoptosis is focused on development, morphogenesis and sex differentiation. In an attempt to elucidate cause and effect relationships between caspase and development, transgenic zebrafish overexpressing procaspase-3 were generated. Stress-induced apoptosis in zebrafish embryos can be monitored by whole mount TUNEL staining and caspase assay. Thus, zebrafish is a useful experimental model animal for investigation of apoptosis in vivo.

Animals↗

PubMed2002

Purification of mummichog (Fundulus heteroclitus) gonadotropins and their subunits, using an immunochemical assay with antisera raised against synthetic peptides.

To detect mummichog gonadotropins (GtHs) and their subunits immunochemically, fragment peptides with amino acid sequences corresponding to cDNA data were synthesized, and antisera were raised against them. In the case of GtH-IIbeta, large loops such as the second loop and the "seat belt" structure (deduced from the hCG 3D structural data) were considered to be favorable regions for antigen, although further examination is needed to determine if this is the case of GtH-Ibeta and GtH-alpha. In the purification process, glycoprotein was extracted from acetone-dried mummichog pituitary and separated by various liquid chromatography procedures. Each fraction was assayed by immunoblotting with the appropriate antisera against synthetic peptides. Subunits (GtH-alpha, GtH-Ibeta, and GtH-IIbeta) were obtained through gel filtration, anion-exchange chromatography, and reverse-phase HPLC. Intact bioactive GtH-I and GtH-II were obtained through gel filtration, anion-exchange chromatography, and hydrophobic chromatography. Both GtH-I and GtH-II dissociated into subunits under acidic conditions. Nominal MW of each subunit was estimated from SDS-PAGE as 23,000 for GtH-alpha from GtH-I, 22,000 for GtH-alpha from GtH-II, 18,000 for GtH-Ibeta, and 21,000 for GtH-IIbeta.

Amino Acid Sequence↗

PubMed2002

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin.

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.

Animals↗

PubMed2002

Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish.

Large numbers of apoptotic early diplotene oocytes were observed during the transition from ovary-like undifferentiated gonadal tissue to testes during sex differentiation in presumptive males of the zebrafish (Danio rerio). The percentage of terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL)-positive apoptotic oocytes in the gonads of presumptive males was approximately eight- to 12-fold higher than in genetic all-females. By 29 days post-hatching, all oocytes had disappeared from the gonads of presumptive males. In these males, we also observed apoptotic somatic cells in the ovarian cavity between 23 and 35 days post-hatching. Therefore, the disappearance of oocytes and the decomposition of the ovarian cavity caused by apoptosis during sex differentiation were male-specific events. In genetic all-females, apoptosis in a proportion of early diplotene oocytes was found in the undifferentiated gonads at 15-19 days post-hatching, probably as a result of programmed oocyte loss during ovarian development. These findings suggest that oocyte apoptosis is the mechanism of testicular and ovarian differentiation in zebrafish.

Animals↗