Specific binding of [3H]17α,20β-dihydroxy-4-pregnen-3-one to oocyte cortices of rainbow trout (Oncorhynchus mykiss)

Specific binding of [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) to plasma membranes prepared from defolliculated oocytes of rainbow trout (Onchorhynchus mykiss) was identified and characterized. Binding was rapid and reached equilibrium in 30 min. 17α,20β-DP strongly inhibited [³H] 17α,20β-DP binding in a competitive manner. Scatchard analysis revealed two different binding sites: a high affinity binding site with a K_d of 18 nM and a B_max of 0.2 pmoles/mg protein; and a low affinity binding site with a K_d of 0.5 μM and a B_max of 1 pmoles/mg protein. This binding activity was successfully solubilized with n-heptyl-β-D-thioglucoside. [³H]17α,20β-DP binding to solubilized preparations reached equilibrium in 1h, and was competitively inhibited with 17α,20β-DP and 17α,20β,21-trihydroxy-4-pregnen-3-one. However, Scatchard analysis showed a single binding site with a K_d of 0.3 μM. The reason for the disappearance of the high affinity binding site in solubilized preparations remains unclear. These results demonstrate that a specific binding site for 17α,20β-DP exists in the plasma membrane of rainbow trout oocytes.

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Résumé Une liaison spécifique de le [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), avec des membranes plasmiques d'ovocytes défollicularisés de truite arc-en-ciel (Onchorhynchus mykiss), a été identifiée et caractérisée. Sa cinétique est rapide et atteint son équilibre en 30 minutes. Le 17α,20β-DP inhibe fortement, et de manière compétitive, la liaison de la [³H] 17α,20β-DP. Une étude de Scatchard a mis en évidence deux sites diffŕents de liaison: un site de forte affinité, de K_d 18 nM et de B_max 0,2 pmoles/mg de protéine; et un site de faible affinité, de K_d 0,5 μM et de B_max 1 pmoles/mg de protéine. L'activité de liaison a été solubilisée, avec succés, par le n-heptyl-β-D-thioglucoside. Dans la fraction soluble, la liaison de le [³H]17α,20β-DP atteint un équilibre en 1h.; et elle est complétement inhibiée par la 17α,20β-DP et le 17α,20β,21-trihydroxy-4-pregnen-3-one. Cependant, une étude de Scatchard ne permet de déceler qu'un seul site de liaison, de K_d 0,3 μM. La disparition du site de liaison de forte affinité dans la fraction soluble reste inexpliquée. Ces résultats démontrent l'existence d'un site spécifique de liaison du 17α,20β-DP dans les membranes plasmiques des ovocytes de truite arc-en-ciel.

     

Variation in seed production among years and among individuals in 11 broadleaf tree species in northern Japan

Seed production is the most problematic part of tree regeneration and is the least amenable to control by silvicultural management. Understanding variability in seed production among years and among trees will allow better planning of seed collection for seedling production and natural regeneration. We estimated the extent of variability in seed production among years and within years among individual trees. Specifically, we measured individual annual seed production in 11 woody species in Hokkaido, northern Japan. We analyzed the coefficient of variation (CV) of seed production among years and among individual trees. We used population- and individual-based CVs (CV_pt and ) to estimate the variability in seed production among individual trees. Alnus hirsuta, in which these CVs among trees were maximal, will require further analyses of the spatial patterns of seed production among individual trees. Additionally, we used population- and individual-based CVs (CV_py and ) to estimate the variability in seed production among years. The was statistically higher than in 2 of the 11 species: Betula maximowicziana and Sorbus alnifolia. Activities for the regeneration of these species should regard the annual variation in seed production as more important than individual variation. For the nine species in which was similar to even if seed production by specific trees was not sufficient for regeneration in a particular year, other trees or stands often had high seed production in that year. We discuss the relative importance of annual and individual variability in determining efficient methods for artificial and natural regeneration of these woody plant species.     

Molecular endocrinology of oocyte growth and maturation in fish

Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17β to 17α,20β-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA. Estradiol-17β stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2⁺ gene product of fission yeast (p34^cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17α,20β-DP-induced oocyte maturation in fish.     

The effects of early and intense pruning on light penetration,tree growth,and epicormic shoot dynamics in a young hybrid larch stand

We examined the effect of early and intense pruning on light intensity under the canopy, individual growth, diameter–height relationships, and epicormic shoot dynamics in young hybrid larch (Larix gmelinii var. japonica × L. kaempferi) to establish a new effective management method for hybrid larch plantations. The objective is to produce high-quality wood while reducing silviculture costs using a combination of low-density planting and early and intense pruning. In a young hybrid larch plantation, we pruned branches to two different heights (2 and 4 m above ground level) using a no-pruning treatment as a control. Although the growth rates were lower in the heavy pruning treatment (4 m above the ground level) than in other treatments in the year following pruning, when measured 4 years later, growth did not differ between treatments. The number of epicormic shoots increased in the year following pruning, as did the relative photosynthetic photon flux density (rPPFD). The number of epicormic shoots was also dependent on the size of individual trees. However, survival of epicormic shoots was not sufficiently high to be problematic for high-quality timber production. If branches are pruned carefully such that the rPPFD does not rise above 20%, the emergence of epicormic shoots can also be controlled. Our results indicate that early and intense pruning is an effective component of a new management system for hybrid larch plantations.     

Identification and characterization of pathogenic bacteria isolated from dead masu salmon Oncorhynchus masou masou and antibacterial activity of probiotic Lactococcus lactis subsp. lactis strain K-C2 against isolated pathogens

We focused on developing an epidemic prevention method for circulating masu salmon aquaculture using the probiotic Lactococcus lactis strain K-C2. This study aimed to evaluate the antibacterial activities of strain K-C2 against pathogens isolated from dead Yamame and masu salmon. First, we identified pathogenic bacteria that were isolated from dead masu salmon based on phylogenetic analysis of the 16S rRNA gene sequence and biological and biochemical characterization. The antibacterial activity of strain K-C2 against seven pathogenic strains isolated from dead masu salmon in this study and two strains of Aeromonas salmonicida previously isolated from dead Yamame was tested using a double agar plate method. The results of a BLAST search using 16S rRNA partial sequence data (1200–1300 bp) revealed that six of the former strains and one of the latter strains showed high similarity to Vibrio anguillarum and Tenacibaculum maritimum, respectively. Strain K-C2 showed antibacterial activity against all pathogenic bacteria. In this study, pathogenic bacteria were newly isolated from dead seawater-acclimated masu salmon, and strain K-C2 was found to have antibacterial effects against these pathogens.

     

A weeding-duration model for <Emphasis Type="Italic">Larix kaempferi</Emphasis> plantations in Hokkaido,northern Japan

In this study, a weeding-duration model for Larix kaempferi plantations was developed that employs a generalized linear model. The number of years that weeding is necessary is the response variable, and elevation, slope, maximum snow depth, annual precipitation, geological type, soil type, site index, slope aspect, and vegetation type are explanatory variables. Among the explanatory variables, geological type, soil type, slope aspect, and vegetation type are categorical data. We assumed a Poisson distribution for the response variable. The link function was log. Among the models that could be developed from these variables, we chose the model with the smallest Akaike’s information criterion (AIC). The weeding-duration model can be written as follows: years that weeding is necessary = Exp (−0.0172833 × site index + 0.0014053 × maximum snow depth (cm) + 1.7417731). The results of this study imply that weeding of Larix kaempferi plantations is needed for more years as the maximum snow depth increases and fewer years as the site index increases. This model is useful for cost–benefit analyses of afforestation or reforestation with Larix kaempferi.     

Proteome analysis: a new approach to identify key proteins involved in acquisition of maturational competence and oocyte maturation of medaka oocytes

We have applied analytical methods of proteomics to identify key proteins that are involved in physiologically important processes on fish reproduction. We have improved methods of proteome analysis for fish oocytes and revealed changes of expression patterns of oocyte proteins and protein phosphorylation during acquisition of maturational competence and signal transduction of oocyte maturation of medaka oocytes.     

Laminin binds to myostatin and attenuates its signaling

Myostatin is a growth and differentiation factor and acts as a negative regulator of skeletal muscle mass. Although the mechanism whereby myostatin controls muscle cell growth is mostly clarified, the regulation of myostatin activity after its secretion into the extracellular matrix (ECM) is still unclear. In the present study, we investigated the interaction between laminin and myostatin and the effect of laminin on myostatin signaling in vitro. The surface plasmon resonance assay showed that laminin bound to mature myostatin and activin receptor type IIB (ActRIIB), but did not bind to latency‐associated protein, which remains non‐covalently linked to mature myostatin. Furthermore, kinetic analysis demonstrated that the affinity of mature myostatin for laminin was similar to that for ActRIIB. Next, we examined the action of laminin on the myostatin signaling pathway using a conventional reporter assay. The luciferase activity of myostatin‐treated cells was repressed significantly (P < 0.05) by coincubation of laminin. These results suggest that laminin has a potential to regulate myostatin activity through binding to mature myostatin and/or its receptor ActRIIB.     

Spawning induced by cubifrin in the Japanese common sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

The neuropeptide cubifrin-I and its derivative cubifrin-L have recently been shown to be potent substances that induce in vitro oocyte maturation and spawning in the Japanese common sea cucumber Apostichopus japonicus. We have examined the reproductive behavior of A. japonicus in vivo following the injection of cubifrin-L into the body cavity with the aim of determining the practical value of using this peptide to induce spawning in the hatchery setting. The responsiveness of ovarian fragments with oocytes >155 μm in diameter to cubifrin-L in vitro was well correlated with the spawning success induced in vivo by a cubifrin-L injection. Mature sea cucumbers injected with cubifrin-L displayed sequential reproductive behaviors, which comprised climbing up the side wall of the tank toward the water surface, waving of the head, and shedding of gametes. Gamete shedding started about 60 and 80 min after the injection of cubifrin-L in males and females, respectively, and was completed almost simultaneously in the two sexes about 2 h post-injection. Repeated injections of cubifrin-L at intervals of about 10 days successfully induced multiple spawnings in males and females. These results demonstrate that cubifrin-L can be used as an effective inducer of spawning in Japanese sea cucumber cultivation.