Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria‐rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo

In ascidian eggs, cytoplasmic and cortical reorganization, previously called ooplasmic segregation, occurs in two phases during the first cell cycle. In the second phase of reorganization, the mitochondria‐rich cytoplasm (myoplasm) moves to the future posterior side, concurrent with sperm aster migration along the egg cortex. Although this reorganization is the critical step for establishing the anteroposterior axis, its molecular mechanism is not fully understood. In this study, we showed that low concentrations of the mitochondrial inhibitor sodium azide (NaN₃), which showed the low toxicity in sperm, inhibited the second phase of reorganization without the microtubule depolymerization. In the NaN₃‐treated embryo, the sperm aster was not attracted to the cortex and altered its migration pathway; therefore, the myoplasm remained at the vegetal pole. Consequently, the anteroposterior axis was not established. Another mitochondrial inhibitor, oligomycin, did not affect these processes. These results suggest that NaN₃ inhibits unknown molecules that are important for the second phase of reorganization. Identifying the target molecule of NaN₃ will lead to a molecular understanding of cytoplasmic and cortical reorganization.     

Biological cycle of silicon in moso bamboo (Phyllostachys pubescens) forests in central Japan

Silicon (Si) has various biogeochemical functions, such as regulating soil formation and species composition, not only in terrestrial ecosystems but also in aquatic ones. Bamboo stands accumulate large quantities of amorphous Si. Evaluating Si dynamics in moso bamboo (Phyllostachys pubescens) forests, which are currently spreading through eastern Asia, is important in understanding their biogeochemical function as a supply source of phytoliths. We conducted a study on the organic accumulation and biological cycle of Si in three P. pubescens stands in central Japan with different site characteristics. The amounts of Si accumulation aboveground and underground were 200–360 and 180–460 kg/ha, respectively. These values indicate that Si accumulation underground was comparable to that aboveground. Silicon supply to the forest floor through litterfall was 77–330 kg/ha/year corresponding to 165–706 kg/ha/year as phytoliths (SiO₂), and 72–88 % was supplied as leaf litter. These results showed that a huge biogenic Si pool derived from bamboo plants exists in the floor of bamboo forests. Furthermore, we estimated the Si turnover time in P. pubescens forests as being 1.3–12.2 years, although this variation may depend on forestry conditions such as soil water content or stem density.     

Inhibition of JNK in HaCaT cells induced tight junction formation with decreased expression of cytokeratin 5, cytokeratin 17 and desmoglein 3

Epidermal keratinocytes proliferate in the basal layer, differentiate, migrate through the spinous layer, granular layer and cornified layer, and finally are peeled off from the surface of skin with layer-specific expression of differentiation markers, including cytokeratins and cell–cell junction proteins such as desmogleins. Basal cells express CK5, CK14 and Ki67. In contrast, the suprabasal cells in the spinous and granular layers express CK1 and CK10 without Ki67. Inhibition of c-Jun NH₂-terminal protein kinase (JNK) in HaCaT cells, a human epidermal keratinocyte cell line, induced the formation of tight junctions, which occurs in the granular layer in vivo. These cells lost their expression of CK5 and CK17, exhibited decreased expression of desmoglein 3 and had no Ki67 labeling in the nucleus. These results suggest that inhibition of JNK causes HaCaT cells to differentiate from basal- and spinous-like cells to granular-like cells. The inhibition of JNK in HaCaT cells provides a useful in vitro model system to study the differentiation of epidermal keratinocytes.     

Spatiotemporal variation in N2O flux within a slope in a Japanese cedar (Cryptomeria japonica) forest

Topographic factors affect nitrogen cycling in forest soils, including nitrous oxide (N₂O) emissions, which contribute to the greenhouse effect. We measured the N₂O flux at 14 chambers placed along a 65-m transect on a slope for 1 year at 2- to 3-week intervals. We applied a hierarchical Bayesian model with a conditional autoregressive (CAR) model to assess the spatiotemporal N₂O flux along a slope and quantify the effects of environmental factors on N₂O emissions. N₂O fluxes at chambers located at lower positions along the slope were relatively greater than those at higher positions. During the non-soil-freezing period, N₂O fluxes fluctuated seasonally depending on soil temperature. The soil temperature dependency of N₂O fluxes at each chamber increased with descending slope position (the median of the Q₁₀ equivalent simulated from posterior distribution ranged from 1.18 to 3.64). According to the Bayesian hierarchical model, this trend could be partially explained by the C/N ratio at each chamber position. During the soil-freezing period, relatively high N₂O fluxes were observed at lower positions along the slope.     

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head-thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG(2,3)-AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG(2,3)-AG(1-7) complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG(2,3)-AG(1-7) complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.     

Effect of exposure to high isoflavone-containing diets on prenatal and postnatal offspring mice

Isoflavone (IF), a type of phytoestrogen, has multiple beneficial effects, but too much phytoestrogen can have adverse effects on offspring. To examine whether chronic exposure to high IF has adverse effects on reproductive development, mice offspring were exposed to IF through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning until sacrifice. In male offspring, there was no difference between the IF group and controls; however, in female offspring in the IF group, remarkably earlier puberty and induction of multioocyte follicles on postnatal day (PND) 21 were observed. Gene expression levels of estrogen receptor beta decreased in the ovary and vagina on PND 21. These results suggest that chronic exposure to higher than normal levels of IF induces alterations in the reproductive development of female mice through an estrogenic effect.     

Tektin 4 is located on outer dense fibers, not associated with axonemal tubulins of flagella in rodent spermatozoa

Tektins, which are thought to be the constitutive proteins of microtubules in cilia, flagella, basal bodies, and centrioles, have been reported to be involved in the stability and structural complexity of axonemal microtubules. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin 2 and Tektin 4, have been verified to be present in sperm flagella. To elucidate the molecular localization of Tektin 4 in flagella of rodent spermatozoa, we performed immunocytochemistry, fractionation study followed by immunoblot analysis, and immunogold electron microscopy. Confocal laser scanning microscopy and immunogold electron microscopy indicated that Tektin 4 was associated with outer dense fibers (ODFs) in both the middle and principal piece of flagella in rat and mouse spermatozoa. Tektin 4 in rat spermatozoa is completely released by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Pre-embedding immunoelectron microscopy demonstrated that Tektin 4 located on the abaxial (convex) surface of ODFs in flagella, not associate with axonemal microtubules. Our data strongly suggested that Tektin 4 is not associated with axonemal tubulins but an ODFs-affiliated molecule in rodent spermatozoa.     

Presence of a cerebral factor showing summer-morph-producing hormone activity in the brain of the seasonal non-polyphenic butterflies Vanessa cardui, V. indica and Nymphalis xanthomelas japonica (Lepidoptera: Nymphalidae)

Three species of nymphalid butterflies, Vanessa cardui, V. indica and Nymphalis xanthomelas japonica , do not exhibit seasonal polyphenism in wing coloration. To determine whether seasonal non-polyphenic butterflies possess a cerebral factor affecting wing coloration, we used a Polygonia c-aureum female short-day pupal assay for detection of summer-morph-producing hormone (SMPH) activity in P. c-aureum. When 2% NaCl extracts of 25 brain-equivalents prepared from the pupal brains of V. cardui, V. indica or N. xanthomelas japonica were injected into Polygonia female short-day pupae, all recipients developed into summer-morph adults with dark-yellow wings, and the average grade score (AGS) of summer morphs showing SMPH activity was 3.8, 3.7 and 4.0, respectively. In contrast, when acetone or 80% ethanol extracts prepared from pupal brains were injected into Polygonia pupae, all recipients developed into autumn-morph adults with a dark-brown coloration and each exhibited an AGS of less than 0.5. Our results indicate that a cerebral factor showing SMPH activity is present in the pupal brain of seasonal non-polyphenic nymphalid butterflies, suggesting that a SMPH and cerebral factor showing SMPH activity occur widely among butterfly species. This finding will improve our understanding of the presence of cerebral factors showing interspecific actions of SHPH.     

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.