Genetic linkage groups in the Japanese brown frog (Rana japonica)

To elucidate the genetic linkage groups of the Japanese brown frog (Rana japonica) and compare them with those of other amphibians, we analyzed the inheritance of alleles at 15 enzyme and blood protein loci and one pigment locus (Blk) in 3,298 offspring derived from 37 crosses using 28 males heterozygous at these loci. Of 63 pairs of loci tested for linkage, 55 pairs showed independent assortment in all crosses examined. In another six pairs, that is, between FUM/ME-1, alpha-GDH/MDH-2, MDH-1/ME-1, MDH-2/ME-2, MDH-2/PEP-C, and ME-2/PEP-C, all the offspring analyzed were parental, and none were recombinant. In the other two pairs, that is, between GPI/PEP-D and AAT-1/Blk, most of the offspring were parental, and some were recombinants with recombination rates ranging from 4.8% to 8.0%. Thus the following four linkage groups comprising 11 loci were established in R. japonica. The first group included the loci for alpha-GDH, MDH-2, ME-2, and PEP-C; the second group included the loci for MDH-1, ME-1, and FUM; the third group included the loci for GPI and PEP-D; and the fourth group included the loci for AAT-1 and Blk. No linkage between the other five loci--ADA, MPI, PEP-A, PGM, and Alb--was observed in the present study.     

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

Possible involvement of phosphatidylinositol 3-kinase, but not protein kinase B or glycogen synthase kinase 3beta, in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica

It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3beta (GSK3beta) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3beta are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3beta, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3beta is a component of the PI3K signal transduction pathway.     

Isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica)

We report the isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica). These markers were tested in 24 individuals each collected from three distinct populations in Ichikai-machi, Tochigi Prefecture. The number of observed alleles per locus ranged from 3 to 24 across all populations, and the values of observed and expected heterozygosities ranged from 0.130 to 1 and from 0.125 to 0.941, respectively. These microsatellite loci will be useful for investigating the intraspecific genetic variation and population structure of this species.     

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.     

A novel regulatory element determines the timing of Mos mRNA translation during Xenopus oocyte maturation

Progression through vertebrate oocyte maturation requires that pre-existing, maternally derived mRNAs be translated in a strict temporal order. The mechanism that controls the timing of oocyte mRNA translation is unknown. In this study we show that the early translational induction of the mRNA encoding the Mos proto-oncogene is mediated through a novel regulatory element within the 3' untranslated region of the Mos mRNA. This novel element is responsive to the MAP kinase signaling pathway and is distinct from the late acting, cdc2-responsive, cytoplasmic polyadenylation element. Our findings suggest that the timing of maternal mRNA translation is controlled through signal transduction pathways targeting distinct 3' UTR mRNA elements.     

Conservation of RNA polymerase during maturation of the Rana pipiens oocyte.

DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5% of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.     

Fluctuation in abundance and age structure of a breeding population of the Japanese brown frog,Rana japonica gunther (Amphibia,anura)

A breeding population of Rana japonica was studied at a marsh on the campus of Hiroshima University in Higashi-Hiroshima during the five years 1995-1999. The mark-recapture study showed that the size of the breeding population varied from year to year, and increased more than twofold in 1999 in comparison with the preceding years. The sex ratio of the breeding population (male/female) was from nearly 1.0 to 1.6. Frogs of both sexes were estimated to breed for the first time at the age of one or two years, and their maximum age was four years according to skeletochronology using phalanges and mark-recapture. Modes of the estimated ages were one year for males during the study years except 1997, but one or two years for females. Two thirds of breeding frogs, irrespective of their sex, were estimated to breed only once throughout their lives.     

Molecular cloning of a cDNA encoding atypical antimicrobial and cytotoxic Brevinin-1Ja from the skin of the Japanese brown frog, Rana japonica

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).     

Amino acid sequence of a lectin from Japanese frog (Rana japonica) eggs

The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented. The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide. The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus. Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111. The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin. They are also homologous to human angiogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases.     

Vascular endothelial growth factor-induced expression of its receptors and activation of the MAPK signaling pathway during ovine oocyte maturation in vitro

The vascular endothelial growth factor (VEGF) has beneficial effects on ovine oocytes during in vitro maturation and their subsequent early embryonic development, but the biochemical pathway underlying this effect has not been elucidated. Therefore, the focus of the present study was to investigate the activation of the mitogen-activated protein kinase (MAPK) pathway in response to the addition of VEGF to the maturation medium, and to study the subcellular localization of VEGF and its receptors during ovine oocyte maturation. We concluded that: (1) VEGF mainly localized in the cytoplasm, whereas its receptors, fms-tyrosine kinase-1 and kinase domain region (KDR), were localized on the plasma membrane of oocytes; (2) the addition of 5 ng/mL VEGF increased the percentage of oocytes with extruded first polar bodies (50.9 ± 2.2% vs. 34.6 ± 2.9%; treatment vs. control, respectively; P < 0.01) and the rate of oocytes competent to undergo nuclear maturation (70.6 ± 0.9% vs. 62.9 ± 1.9%, P < 0.01); and (3) as the expression of VEGF, fms-tyrosine kinase-1, and KDR increased after supplementation with 5 ng/mL, expression of VEGF, mitogen-activated protein kinase kinase (MEK), and MAPK mRNA, as well as MAPK phosphorylation, were stimulated in a time-dependent manner. We inferred that, in a paracrine manner, exogenous VEGF bound to KDR, its main receptor, and then activated the MAPK signaling pathway, which promoted maturation of ovine oocytes. However, the VEGF system also had an autocrine regulatory loop that contributed to creating an environment optimal for oocyte maturation.     

MyoD binds to Mos and inhibits the Mos/MAP kinase pathway

When ectopically expressed, the serine/threonine kinase Mos can induce oncogenic transformation of somatic cells by direct phosphorylation of MAP kinase/ERK kinase (MEK1), activating the mitogen-activated protein kinases ERK1 and ERK2. On the other hand, overexpression of Mos in C2C12 myoblasts is not transforming. Mos activates myogenic differentiation by promoting heterodimerization of the MyoD/E12 proteins, increasing the expression of myogenic markers and the positive autoregulatory loop of MyoD. In this study, we show that in myogenic cells, the mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway is suppressed by MyoD through the formation of a heterotrimeric complex.     

The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important post-translational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through over-expression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Over-expression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.     

Calcium-dependent chloride transient currents in the immature oocyte of the frog, Rana esculenta

Transient currents of chloride were studied in the plasma membrane of immature frog oocyte in voltage clamp conditions. The transients appeared to be activated by an influx of Ca2+ from the external medium. The mechanism leading to a surge of intracellular Ca2+ concentration needed at least 30 sec before full recovery. It was inhibited by substituting Ba2+ for Ca2+ in the external medium, or in the presence of La3+, Co2+ and Cd2+, or when external Na+ was replaced by Li+. Verapamil proved ineffective. The data suggest that an intracellular system of Ca-activated Ca-release is present in the frog oocyte, which can be primarily activated by membrane hyperpolarization via an influx of Ca2+ through non-selective channels.     

Migration of epidermal melanophores to the dermis through the basement membrane during metamorphosis in the frog, Rana japonica

The number of epidermal melanophores of the skin decreases dramatically during metamorphosis in the frog, Rana japonica. This decrease may represent an adaptation for rapid color change, a property which the animal acquires after metamorphosis. We concluded that the decrease was due to the migration of epidermal melanophores to the dermis. Epidermal melanophores and epidermal cells are tightly associated with each other in the young tadpole. The association becomes looser at the metamorphic stage and, occasionally, small breaks in the basement membrane are seen. These breaks may facilitate the migration. The migration was observed exclusively at the metamorphic stage, in spite of careful observation of other stages under the electron microscope. The migration of epidermal melanophores was induced by treatment with thyroxine of cultured skin from tadpoles at stage 15, and this hormone may act directly on epidermal melanophores. Until now, the increase in the number of dermal melanophores during metamorphosis has been explained by the differentiation of dermal melanophores from melanoblasts and by their mitotic division. Our results show that the migration of epidermal melanophores to the dermis may be a factor which accounts for the increase in the number of dermal melanophores.     

Intramitochondrial transformations during oocyte maturation in the mouse

An electron microscope study of oocyte maturation in the mouse revealed that some mitochondria undergo gradual transformation in their ultrastructural appearance. In very young oocytes these mitochondria were already found to contain vacuoles, one in each such organelle. In somewhat older oocytes more mitochondria displayed vacuoles which generally appeared to be getting larger. These intramitochondrial vacuoles were found to be essentially optically empty structures surrounded by a single membrane. In favorable sections someof the developing vacuoles had a bottle-shaped appearance, the constricted end of which was attached to the inner limiting membrane of the mitochondrion. With further maturation of the oocytes vacuoles having a pear-shaped appearance became evident. An hypothesis was presented outlining the mode of formation of these vacuoles by expansion of the individual cristae. Intramitochondrial transformations occuring during both oogenesis and spermatogenesis in mammals were reviewed.