Midori Matsumoto

Switching from asexual to sexual reproduction in the planarian Dugesia ryukyuensis: Bioassay system and basic description of sexualizing process

Abstract An assay system has been established for the sexual induction in the OH strain, an exclusively fissiparous (asexual) strain, of Dugesia ryukyuensis by feeding them with sexually matured worms of Bdellocephala brunnea, an exclusively oviparous (sexual) species. In this assay system, asexual worms gradually differentiated sexual organs, namely the ovary, testis, genital pore and yolk gland in this order, and eventually mated and laid cocoons filled with fertilized eggs. Although the OH strain worms were believed not to have any sexual organs, a pair of undeveloped ovaries with a few oogonia were detected by an intensive histological search. Along with the progression of sexualization, five distinct stages were histologically recognized: In the first stage, the ovaries became larger enough to be externally apparent; oocytes appeared first at stage 2; the primordial testes emerged at stage 3; a genital pore opened, yolk gland primordia developed and spermatocytes appeared at stage 4; and finally at stage 5 matured spermatozoa and yolk glands were formed. Worms in stages 1 and 2 but not in later stages returned asexual if feeding on B. brunnea was interrupted. Furthermore, when the worms at stage 3 onwards were cut posterior to the ovaries, all the tail regenerants developed eventually into fully sexualized worms. Taking these results in account, we have concluded that the process of sexualization has a point-of-no-return between stages 2 and 3. It is likely also that the testes, even the primordia, play an important role in the maintenance and development of sexuality.

Transcriptional control of the heme oxygenase gene in mouse M1 cells during their TPA-induced differentiation into macrophages

It has long been known that heme oxygenase (HO) is a key enzyme in heme catabolism and recently it was also found to acts as an oxidative stress protein to produce carbon monoxide (CO), which has similar actions to those of nitrogen monoxide (NO). Therefore, we examined transcriptional control of the HO gene in mouse M1 (myeloleukemia) cells during their differentiation into macrophages. Since the promoter region of this gene is known to have a TPA‐responsive element (TRE), its expression might be regulated by a C‐kinase signal transduction pathway. Then we investigated the activation of the HO gene after treatment of M1 cells with TPA and inhibitors of C‐kinase. When M1 cells were treated with TPA, they differentiated into macrophage‐like cells. Upon treatment with TPA, H2O2 was produced first, the nuclear proto‐oncogenes fos and jun were activated, and then the HO gene was activated. The extent of transcriptional activation of the fos, jun, and HO genes in M1 cells treated with TPA was reduced by a specific inhibitor of C‐kinase and a scavenger of oxygen radicals. When M1 cells were treated with H2O2 essentially the same level of transcription of the HO gene was observed, but the extent of transcriptional activation of the fos and jun genes was about half of the treatment with TPA. Super‐shift assays using the TRE of the HO gene revealed that the Fos and Jun proteins from nuclei of M1 cells treated with TPA bound to the TRE, and same assays using DNA with the NF‐kB motif also revealed that the active NF‐kB protein from M1 cells treated with H2O2 or TPA also bound to the corresponding motif. These results strongly suggest that the HO gene in M1 cells is activated by TPA through a production of H2O2, an oxidative activation pathway of NF‐kB, and a signal‐transduction pathway that involves C‐kinase during the differentiation of macrophages that occurs upon treatment with TPA.

Haploid specific activations of protamine 1 and hsc70t genes in mouse spermatogenesis

Protamine 1 and heat shock cognate 70 kDa protein (hsc70t) are known to be synthesized in haploid cells during spermatogenesis, and the mRNAs of these proteins have also been shown to accumulate in the haploid cells. However, it is unknown at which stage of spermatogenesis the genes for these proteins are actually activated. To examine this problem, we fractionated mouse adult testes cells at four different developmental stages, extracted their nuclei and carried out run-off assays with hsc70t and protamine 1 DNA probes. Results showed that both genes are mainly activated at the round spermatid stage. As the protein products of these genes accumulate at the later stage, it is interesting that these genes are regulated at the transcriptional and translational levels during spermatogenesis.

Transcriptional activation of heme oxygenase-1 gene in mouse spleen, liver and kidney cells after treatment with lipopolysaccharide or hemoglobin.

Heme oxygenase (HO)−1 catalyzes the conversion of heme to biliverdin, iron and carbon monoxide. HO−1 is induced by many reagents including heme, Hb and lipopolysaccharide (LPS). LPS is known to activate the HO−1 gene in cultured mouse liver and macrophage cells through oxidative activation of NF‐κB. But little is known about the effect of LPS and Hb on the HO−1 gene in living organisms. To study this issue, we examined the HO−1 and its mRNA levels in mouse liver, spleen and kidney after intravenous administration of LPS and Hb. On LPS treatment, the amount of HO−1 and its mRNA increased markedly mainly in mouse spleen, but on Hb treatment the amounts of HO‐1 and its mRNA increased slightly only in liver. Run‐off transcription assay supported the above results and band shift assays also revealed that LPS significantly activates an NF‐κB‐like factor in spleen cells, while Hb slightly activates it in liver cells. According to our previous study, a small amount of Hb injected to mouse is selectively taken up by liver as Hb—haptoglobin complex.

Notch homologue from Halocynthia roretzi is preferentially expressed in the central nervous system during ascidian embryogenesis

Abstractu2002We describe here the primary structure of HrNotch, an ascidian homologue of the Drosophila neurogenic gene Notch. HrNotch transcripts encode a protein of 2352 amino acids and share the principal features of the Notch gene family: extracellular epidermal growth factor (EGF)-like repeats, three Notch/Lin-12 repeats and six intracellular ankyrin repeats. Yet ascidian Notch contains only 33 EGF repeats in the putative extramembrane domain and specifically lacks the three EGF-like repeats. In situ hybridization shows that maternal HrNotch mRNA is distributed uniformly in the cytoplasm of the unfertilized egg. During cleavage, maternal HrNotch transcripts are ubiquitous in the ectoderm cells of the animal hemisphere, which contain less yolk granules. During gastrulation, maternal transcripts persist in most ectoderm lineage cells. Zygotic expression of HrNotch seems to start at the neural plate stage in both a-line cells (descendants of anterior-animal blastomeres) of the dorsal neuroectoderm and b-line cells (descendants of the posterior-animal blastomeres) that comprise the neural fold. Following this stage, transcripts are most evident in the descendants of these cells, that is, the brain lineage cells, precursors of a larval adhesive organ, and dorsal part of the nerve cord (roof plate). Brain lineage cells include the precursors of sensory pigment cells that are known to comprise an equivalence group in ascidian embryos. During tail elongation, transcripts disappear. Predominant expression of HrNotch in epidermal and neural cells is a common feature of chordate Notch genes. Furthermore, the timing of HrNotch expression in sensory pigment cell precursors suggests involvement in the determinative events in the sensory pigment cell equivalence group.

Sequence analysis of cDNAs encoding precursors of starfish asterosaps

To understand how starfish sperm activating peptides (asterosaps) are synthesized in the ovary, we cloned cDNAs encoding asterosaps and elucidated their nucleotide sequences. The mRNA encoding asterosaps was synthesized only in the oocytes, but not in the follicle cells, and the length was 3.7 kb. The cDNA clones contained multiple isoforms of asterosaps. We assume that asterosap precursors are large prepolypeptide chains with an unusual rosary-type structure made of 10 successive similar stretches of 51-55 residues. Each stretch finishes with a spacer of 17-21 residues immediately followed by the sequence of one asterosap isoform. The N-terminal of this precursor has 19-21 successive glutamine-rich repeating units. Maturation of the precursor may require endopeptidases that cleave both C- and N-sites of lysine-arginine.

Pertussis toxin-sensitive G protein participating in starfish oocyte maturation induced by 1-methyladenine

Summary 1-Methyladenine (1-MA) secreted from the follicle cells is the biological signal for meiosis reinitiation of starfish oocytes. The signal of-1-MA is transduced into cytoplasmic formation of maturation-promoting factor (MPF) that eventually induces a germinal vesicle breakdown (GVBD). Microinjection of pertussis toxin (PTX) inhibited 1-MA-induced GVBD in Asterina pectinifera and Asterina (Patina) miniata. PTX-inhibition of GVBD was rescued by the injection of MPF into PTX-preinjected oocytes. Most of the PTX- and MPF-double injected eggs were fertilized and underwent cleavage, suggesting the presence of a GTP-binding protein (G protein) specific for 1-MA signal transduction. Indeed, plasma membrane preparations of A. pectinifera oocytes contained a G protein consisting of 39-kDa α, 37-kDa β, and 8-kDa γ subunits. The α subunit contained a site for ADP-ribosylation catalyzed by PTX. It was also recognized by antibodies specific for a common GTP-binding site of mammalian α subunits or a carboxy-termi...

Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy

Heme is known to activate the HO (heme oxygenase) gene in cultured cells, but little is known about the effect of heme on the HO gene in intact organisms. The expressions of HO and its RNA in mouse liver were measured using mouse HO cDNA and HO antibody after injection of heme or splenectomy. The antibody was prepared against a beta-galactosidase-HO hybrid protein made in Escherichia coli. The HO mRNA level increased to a maximum 15 h after heme injection. In contrast, expression of HO was maximal about 45 h after heme injection. Essentially the same results were obtained in mice after splenectomy. These results suggest that the HO gene in mouse liver was activated by the injection of heme and splenectomy.