Gene expression profiling of a cold-shocked earthworm Eisenia andrei.

To identify genes that are modulated under cold-stress conditions in the earthworm Eisenia andrei, we performed a genome-wide analysis of gene expression in cold-shocked earthworms by using Serial Analysis of Gene Expression (SAGE). We identified 5,977 and 5,407 unique SAGE tags under normal and cold-stressed conditions, respectively. The majority of the SAGE tags did not match to any known expressed sequences, due to a paucity of expression data in earthworms. We converted the statistically significant SAGE tags for the cold-stressed condition into expressed sequence tags (ESTs), and the results showed that particular genes associated with energy homeostasis, cellular defense mechanisms, and ion balance were up-regulated or down-regulated. We constructed a regulatory network of some of these genes and identified rps-6 as a core gene in the cold-response regulatory-gene network. Our data provide a baseline for gene expression studies of cold shock in the Lumbricidae.

Effects of gonadotropins on in vitro maturation and of electrical stimulation on parthenogenesis of canine oocytes.

The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 mus with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 mus without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.

Parthenogenetic induction of canine oocytes by electrical stimulation and Ca-EDTA.

In this study, we investigated parthenogenetic induction of canine oocytes by electrical stimulation following Ca-EDTA treatment. Oocyte maturation, parthenogenetic development, and cleavage rate in canine after various electrical stimulations (1.5, 1.8, 2.1 kV/cm) for 50 mus with single DC pulse following 1 mM Ca-EDTA treatment were investigated. In oocyte activated electrically at the voltage of 1.5 kV/cm after 1 mM Ca-EDTA treatment, the rate of pronucleus and two-cell was 4.1% and 2.7%, respectively. Although electrical stimulation could parthenogenetically induce immature oocyte to cleavage stage, degeneration rate in all experimental groups was more than 60%. This means that electrical stimulation after Ca-EDTA treatment could cause canine oocytes to be degenerated. However, two-cell in canine oocyte by parthenogenesis was for the first time induced. Therefore, we suggested that electrical stimulation for canine oocytes could induce parthenogenetically early embryonic cleavage. This result can be used as a basic data for parthenogenesis study in canine. Also, to perform more developed embryonic development, further study to parthenogenesis in canine need to be developed.

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes.

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.

Metallothionein suppresses collagen-induced arthritis via induction of TGF-beta and down-regulation of proinflammatory mediators.

Metallothionein is a low molecular weight, cysteine-rich, stress response protein that can act as an antioxidant and as an immunosuppressive agent in instances of antigen-dependent adaptive immunity. In this context, we assessed the therapeutic potential and mechanisms of action of metallothionein in a collagen-induced arthritis model. Repeated administration of metallothionein-I + II during the course of disease dramatically reduced the incidence and severity of the disease. Joint tissues isolated from boostered paws of metallothionein-I + II-treated mice expressed significantly reduced levels of proinflammatory mediators, such as tumour necrosis factor (TNF)-alpha and cyclooxygenase-2, when compared with those of control-treated mice. Lymph node cells obtained from metallothionein-I + II -injected mice exhibited a significant decrease in the proliferative response and a remarkable increase in tumour growth factor (TGF)-beta production in response to type II collagen. Taken together, these results suggest that metallothionein-I + II promote the development of type II collagen-specific, TGF-beta-producing cells to antagonize the expansion of arthritogenic cells. This could lead to local suppression of inflammatory responses by inhibiting the expression of proinflammatory molecules. Thus, this study demonstrates the suppressive effects of metallothionein on collagen-induced arthritis, and indicates that there may be a potential therapeutic application for manipulation of metallothionein during the treatment of autoimmune disorders.

Neoplastic transformation and tumorigenesis associated with overexpression of phospholipase D isozymes in cultured murine fibroblasts.

Phospholipase D (PLD) has been suggested to play an important role in a variety of cellular functions. PLD activity has been shown to be significantly elevated in many tumours and transformed cells, suggesting the possibility that PLD might be involved in tumorigenesis. In this study, we have established stable cell lines overexpressing PLD1 and PLD2 from fibroblast cells. These cells, but not control cells, showed altered growth properties and anchorage-independent growth in soft agar. Both PLD1 and PLD2 also induced an up-regulation of the activity of matrix metalloprotease-9 as detected by zymograms. Furthermore, both PLD1 and PLD2 transformants, but not vector-transfectants, induced undifferentiated sarcoma when transplanted into nude mice. Both PLD1- and PLD2-mediated cell cycle distributions in stable cell lines revealed an increased fraction of cells in the S phase compared with control cells. Interestingly, the level of cyclin D3 protein, known as an activator of G(1) to S phase transition in the cell cycle, was aberrantly high in cells overexpressing PLD1 and PLD2 compared with control cells. These results suggest that overexpression of PLD isozymes may play an important role in neoplastic transformation.

Expression of recombinant human granulocyte macrophage-colony stimulating factor (hGM-CSF) in mouse urine.

We have generated transgenic mice expressing human granulocyte macrophage-colony stimulating factor (hGM-CSF) in urine. In particular, the expression plasmid DNA containing mouse uroplakin II promoter was used to direct uroepithelium-specific transcription of transgene. In this study, hGM-CSF transcript was detected only in bladder uroepithelium as determined by northern blot analysis. Furthermore, hGM-CSF protein was detected in the suprabasal layer of the uroepithelium and ureter by immunohistochemistry. The hGM-CSF was secreted into urine at high level (up to 180 ng/ml), and enhanced proliferation of hGM-CSF-dependent human acute monocyte leukemic cells, suggesting that transgenic urine-derived hGM-CSF was bioactive. This is the first case of demonstrating biological activity of a cytokine produced in the urine of a transgenic animal. Our results demonstrate that bladder can be used as a bioreactor to produce biologically important substances. In addition, it suggests a potential application of bladder expression system to livestock for high-yield production of pharmaceuticals.

Identification of cDNA encoding a serine protease homologous to human complement C1r precursor from grafted mouse skin.

We isolated a cDNA clone from grafted mouse skin that encodes a serine protease homologous to human C1r. The C1r protease is involved in the activation of the first component of the classical pathway in the complement system. In order to identify novel transcripts whose expression is regulated in grafted mouse skin, we first performed differential display reverse transcription polymerase chain reaction analysis and obtained 18 partial cDNA clones whose protein products are likely to play an important role in allograft rejection. One of these showed significant sequence homology with human complement C1r precursor. The other clones displayed no homology to any known sequences, however. Northern blot analysis demonstrated that the level of this transcript was upregulated in day 8 postgrafted skin. The full-length cDNA 2121 nucleotides in length obtained from screening a mouse skin cDNA library contained a single open reading frame encoding 707 amino acid residues with a calculated molecular weight of 80,732 Da. Its deduced amino acid sequence revealed an 81% identity and 89% similarity to the human C1r counterpart. In particular, mouse C1r contained His501, Asp559, and Ser656, which were conserved among this group of serine proteases. This protein was thus designated as mouse C1r. We have expressed a truncated fragment of C1r protein without the N-terminal hydrophobic sequence in Escherichia coli and generated a polyclonal antibody against it. Subsequent immunohistochemical analysis confirmed that mouse C1r was significantly expressed 8 d after the skin graft in both allografted and autografted skins, compared with normal skins. These collective data suggest that a component of the complement system, C1r, might contribute to the graft versus host immune responses in mice.

Overexpression of membrane-type matrix metalloproteinase-1 gene induces mammary gland abnormalities and adenocarcinoma in transgenic mice.

To investigate the role of membrane-type matrix metalloproteinase-1 (MT1-MMP) in mammary gland development and tumorigenesis, transgenic mice overexpressing MT1-MMP in mammary gland under the control of the mouse mammary tumor virus long terminal repeat-promoter were generated. The mouse mammary tumor virus/MT1-MMP transgenic mice displayed abnormalities in 82% of female mammary glands. The abnormalities were verified as lymphocytic infiltration, fibrosis, hyperplasia, alveolar structure disruption, dysplasia, and adenocarcinoma. Northern and reverse transcription-PCR analyses demonstrated that MT1-MMP mRNA was overexpressed in mammary glands exhibiting abnormalities. Western blot analysis and immunohistochemical studies have revealed that the protein expression level was also increased in these glands. In addition, the beta-casein gene as a functional epithelial cell marker was poorly expressed in the mammary glands of transgenic mice exhibiting abnormalities. Gelatin zymography showed significantly increased MMP-2 activation in these mammary glands. These results showed that overexpression of MT1-MMP induced remodeling of the extracellular matrix and tumor formation in the mammary glands of transgenic mice. Therefore, we suggest that overexpression of MT1-MMP may play a key role in development and tumorigenesis in mammary glands.

Circling mouse: possible animal model for deafness.

Mutant mice with abnormalities are potentially useful as models for studying human defects. Here we report a group of mice with abnormal behavioral patterns. A new spontaneous mutant mouse exhibited hyperactive behavior at about seven days of age, followed by tight circling behavior. Breeding studies suggest that this mutation is caused by a single gene defect inherited in an autosomal recessive manner. Consequently, this mutation is referred to as a circling (cir) mouse mutation with the gene symbol cir. Auditory test results identified clearly the hearing loss of the cir, compared with wild-type mice. Pathologic studies confirmed developmental defects in cochlea and spiral ganglions that were correlated to the abnormal behavior observed in the cir mice. Thus, cir mice may be useful as a model for studying inner ear abnormalities and deafness in humans.

Promotion of development of bovine embryos produced in vitro by addition of cysteine and beta-mercaptoethanol to a chemically defined culture system.

The aim of this research was to determine the effects of L-cysteine and beta-mercaptoethanol on the in vitro development of bovine embryos that had been produced in vitro. A 2 x 3 factorial arrangement of treatments was used to evaluate the effect of 0.63 or 6.9 microM L-cysteine and 0, 10, or 100 microM beta-mercaptoethanol on the development of bovine embryos in a chemically defined medium. Embryos containing 6 to 8 cells were randomly allocated to one of the six treatment combinations and were cultured for 7 d. Both beta-mercaptoethanol and L-cysteine increased the number of embryos that reached the blastocyst stage of development, although no interaction was observed between the compounds. Embryos that were cultured in the presence of beta-mercaptoethanol had more cells at the blastocyst stage than did embryos cultured in medium without beta-mercaptoethanol. These findings provide evidence that beta-mercaptoethanol and L-cysteine promote increased embryonic development and that beta-mercaptoethanol increases the number of cells in bovine embryos produced in vitro and cultured in a cell-free, protein-free culture system.

beta-mercaptoethanol enhances blastocyst formation rate of bovine in vitro-matured/in vitro-fertilized embryos.

The objectives of this research were to determine the effects of beta-mercaptoethanol (beta-ME) and fetal bovine serum (FBS) on in vitro development of bovine embryos derived from oocytes matured and fertilized in vitro and to examine the mechanism through which beta-ME may influence embryo development. A 2 x 2 factorial treatment arrangement was used to evaluate the effect of 0 or 100 microM beta-ME and 0% or 10% FBS on embryos cultured in Medium 199 (M199) in the absence of somatic cells. Embryos were randomly allocated within stage of development (< 8 cells or 8-16 cells) to one of four treatment combinations and were cultured for 6 days. Both beta-ME and FBS promoted increased (p < 0.01) development of embryos to the blastocyst stage, and their effects were greater (p < 0.01) in 8- to 16-cell embryos than in embryos having fewer than 8 cells at the initiation of treatment. The cysteine and cystine content of M199, with and without beta-ME, were determined by HPLC. Medium supplemented with beta-ME contained neither cysteine nor cystine, and it is suggested that these compounds were converted into a mixed disulfide between cysteine and beta-ME. These results indicate that beta-ME is capable of enhancing bovine embryo development in a cell-free, serum-free culture system.