A Trial to Cryopreserve Immature Medaka (Oryzias latipes) Oocytes after Enhancing Their Permeability by Exogenous Expression of Aquaporin 3

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.     

Effect of iron-quercetin complex on reduction of nitrite in in vitro and in vivo systems

This study investigated whether reducing agents such as quercetin and iron(II) facilitate formation of nitric oxide (NO) gas from orally ingested nitrite in an vivo study. When 3 mg/kg Na (15)NO2 was orally administered to rats with or without iron(II) or quercetin, Hb (15)NO, which is indicative of systemic (15)NO, was detected in the blood, with the maximum blood concentration of Hb (15)NO at 15 min after nitrite or nitrite plus quercetin treatment, whereas after administration of nitrite plus iron(II) or nitrite plus iron(II) and quercetin, the time was shortened to 10 min. Interestingly, iron(II), quercetin, or iron(II) plus quercetin did not affect the total amount of Hb (15)NO generated from orally administered Na (15)NO2. However, the systemic nitrite concentration was significantly decreased in the presence of iron(II) or iron(II) plus quercetin. These results may indicate that iron(II) is critical to the generation of NO gas from nitrite, whereas quercetin contributed little under the in vivo experimental conditions.     

Larval survival,development and growth in the horsehair crab,Erimacrus isenbeckii,cultured under different photoperiod conditions

Larval sinking causes larval mass mortality during seed production in the horsehair crab, Erimacrus isenbeckii. Under normal light conditions, horsehair crab larvae generally show negative phototactic behaviour and sink in their rearing seawater. It has been proposed that culturing larvae in the dark may prevent larval sinking. Herein, we examined the effect of photoperiods on horsehair crab larval survival and development to facilitate the development of larval rearing techniques that prevent sinking. Newly hatched larvae were reared with Artemia to the first crab stage in 2‐L beakers under five photoperiods: 0L:24D, 6L:18D, 12L:12D, 18L:6D and 24L:0D. Larvae survived and molted to the first crab stage under all tested photoperiod conditions. The survival rate improved with increasing light period, whereas the developmental period for each larval stage decreased with increasing light period. Longer light periods increased the carapace length at the first crab stage. Our results suggest that larvae could be cultured to the first crab stage in large‐scale tanks under constant darkness. However, significantly improved larval performance under longer photoperiodic conditions indicates a need for developing alternative culture techniques to control larval behaviour in the seed production tank.     

Anthracnose of bacopa caused by Colletotrichum destructivum

Severe spotting and blighting of leaves were found on bacopa (Sutera cordata), a scrophulariaceous ornamental, in greenhouses in Gunma Prefecture, Japan, from January through February 2007. After we isolated and identified the causal fungus as Colletotrichum destructivum and inoculated host plants with the isolate to confirm pathogenicity, we named this new disease anthracnose of bacopa.     

Seasonal Changes in Luteal Progesterone Concentration and mRNA Expressions of Progesterone Synthesis-related Proteins in the Corpus Luteum of Mares

Although circulating progesterone (P(4)) levels tend to change with the season, little is known about the seasonal changes of P(4) synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P(4) concentration, was used to evaluate the seasonal changes in P(4) synthesis. The luteal P(4) concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) were lowest during early winter and increased during late winter. These results suggest that P(4) synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P(4) synthesis-related proteins in mares.     

Grain yield responses of lowland rice varieties to increased amount of nitrogen fertilizer under tropical highland conditions in central Kenya

Tropical highland conditions in Mwea Kenya, ensure the high radiation and the large day–night temperature differences. Such conditions are generally believed to promote rice growth and yield, but the current grain yield is lower than the expectation. In the current standard N fertilizer practice in Mwea, 75 kg nitrogen (N) ha^?1 is applied in three splits at fixed timing. The effects of increases in N fertilizer amount (125, 175, and 225 kg N ha^?1) on rice growth and yield were evaluated to test the hypothesis that unachieved high rice grain yield in Mwea is due to insufficient amount of N fertilizer. Two popular lowland varieties in Mwea (Basmati 370 and BW196) and two varieties reported as high yielding in other countries (Takanari and IR72) were used. Shoot dry weight (DW) increased with increases in the amount of N fertilizer applied in three splits at fixed timing, irrespective of variety. It reached approximately 20 t ha^?1 under increased N conditions (>75 kg N ha^?1) in several cases, indicating that high biomass production could be achieved by increasing N application rate. However, the increased biomass did not increase grain yield, due to decreased grain filling under high N conditions in all varieties. Thus, N amounts above 75 kg ha^?1 were ineffective for increasing grain yields in Mwea, where N fertilizer was applied in three splits at fixed timing. Increasing influence of low temperature under high N conditions may be one of the reasons for the decreased grain filling in Mwea.     

Characterization of canine caspase-3

The canine caspase-3 gene was cloned and sequenced. The canine caspase-3 cDNA clone was 1473 base pairs in length and encoded 277 amino acids. The predicted amino acid sequence showed 88.4%, 88.0%, 85.9%, 65.9% and 60.6% homology with that of human, pig, mouse, chicken and zebra fish caspase-3, respectively. The caspase-3 mRNA was confirmed to express in skin, lymph node, bone marrow, small intestine and lung from a healthy dog by RT-PCR analysis.     

Expression analyses of sex steroid-regulated genes in neonatal rat hypothalamus

Estrogen plays an important role in sexual differentiation of the brain in rats during the perinatal period. To elucidate molecular mechanisms underlying sexual differentiation of the brain, in this study we investigated genes differentially expressed between sexes or induced to express by estrogen in neonatal rat hypothalamus using DNA microarray analysis in combination with real-time RT-PCR. It was found that the levels of expression of the genes encoding glutamic acid decarboxylase 65 and coronin 1b were higher in male than female hypothalamus on postnatal day (PN) 5 and those of collagen type 3 alpha1 and thioredoxin reductase 2 genes in female hypothalamus on PN5 were decreased and increased, respectively, by treatment with estradiol on PN2. Then the developmental changes in the expression of these 4 genes were examined from 1 day before the parturition to PN9, and they all showed sexual dimorphic patterns. In addition, dependence of the expression of these genes on either estradiol, testosterone or dihydrotestosterone during the neonatal period was confirmed. These results suggest that these four genes are involved in sexual differentiation of the rat brain, and that androgen per se as well as estrogen may take part in the processes.