Characterization and expression of DNA sequences encoding the growth hormone gene in African Pygmy Mouse (Mus minutoides)

We determined the nucleotide sequence of the growth hormone (Gh) gene in Mus minutoides, one of the smallest mammals, where was predicted to be distinct in the functional regions between M. minutoides and Mus musculus. To investigate the evolutionary characteristics of Gh in M. minutoides, we constructed a phylogenetic tree based on the putative amino acid sequences of Gh, suggesting that the Gh of M. minutoides diverged earlier than M. musculus. Furthermore, the Gh gene expressed higher in M. minutoides than in M. musculus. Our results suggest that the specific feature of the Gh in M. minutoides is in rather the regulatory mechanism than the sequence.     

Differential responses to high temperature during maturation in heat-stress-tolerant cultivars of Japonica rice

High-temperature stress during the grain-filling stage reduces grain quality of rice, and this is a serious problem in Japan, especially in the Kyushu region. To solve this problem, various heat-tolerant cultivars have been bred, such as ‘Nikomaru’, ‘Kumasannochikara’, ‘Genkitsukushi’, ‘Sagabiyori’, and ‘Otentosodachi’. When cultivated under high temperature after flowering, these heat-tolerant cultivars had lower percentages of chalky grains than in the heat-sensitive cultivar ‘Hinohikari’. All the heat-tolerant cultivars markedly decreased the nonstructural carbohydrate content in the stem under the high temperature compared to control condition during early grain-filling stage, which is considered to be a common trait of heat tolerance. Notably, ‘Sagabiyori’, ‘Genkitsukushi’, and ‘Nikomaru’ maintained a nucellar epidermis at 17 days after flowering (DAF) under high temperature, whereas the nucellar epidermis disappeared in ‘Hinohikari’. In addition, the expression of AGPS2b, thought to be a rate-limiting enzyme in starch synthesis, in ‘Kumasannochikara’, ‘Otentosodachi’, and ‘Nikomaru’ did not decrease under high temperature, whereas ‘Hinohikari’, ‘Sagabiyori’, and ‘Genkitsukushi’ could not maintain expression of the gene at 17 DAF. Moreover, the expression of Amy3E, a starch-degradation-related gene considered to induce grain chalkiness, in ‘Kumasannochikara’ at 17 DAF was not increased by high temperature. These results suggest that the heat-stress-tolerant cultivars have various mechanisms for dealing with high-temperature stress.     

Effects of cryopreservation on sperm structure in Japanese pearl osyter <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis>

To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol+18% fetal bovine serum+72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately −20 °C/min to −50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9±4.2%, and that of cryopreserved spermatozoa was 24.0±1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6±3.9%, while in fresh spermatozoa this was 8.7±2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6±5.2%, while in fresh spermatozoa this was only 0.9±0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4±3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.     

Studies on the bacteria isolated anaerobically from paddy field soil IV. Model experiments on the production of branched-chain fatty acids

Based on the fact that the clostridia isolated from paddy field soil were mostlyproteolytic and that they produced some branched-chain volatile fatty acids on peptone media, the occurrence and the substrates for the production of such acids in paddy soil were investigated. The results obtained are as follows:

• 1) It was revealed that two branched-chain volatile fatty acids, iso-valeric and iso-butyric acids, were detected In fresh paddy field soil.

• 2) The paddy soil supplemented with peptone solution produced significant amounts of branched-chain volatile fatty acids-iso-butyric and iso-valeric acidsin addition to straight-chain volatile fatty acids-acetic, propionic, and butyric acids. When glucose was added instead of peptone, not branched-chain fatty and propionic acids were detected but only acetic and butyric acids. These facts indicate that the branched-chain fatty acids may originate from amino acids.

• 3) When a pair of amino acids, such as proline and leucine or proline-and valine, were supplemented in addition to peptone, the production of iso-valeric or iso-butyric acids was increased, respectively. Even In the case of glucose, the supplementation of a pair of amino acids supported the production of branched-chain fatty acids. These facts indicate that the Stickland reaction may take place in paddy field soil.

• 4) All the strains of strict anaerobes isolated from the soil and supplemented with peptone formed iso-valeric and iso-butyric acids in addition to two or three kinds of volatile fatty acids when they were cultured in medium containing peptone. All of them were classified as belonging to genus Clostridium. No strains of facultative anaerobes isolated from the same soil produced any branched-chain fatty acids, but produced only acetic acid.

• 5) In conclusion, these results reveal that the branched-chain fatty acid in paddy field soil is produced only by clostridia through the Stickland reaction.

     

Motility of spermatozoa obtained from testes of Japanese pearl oyster <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis>

Effects of NH₃ concentration in sea water and pH of sea water on the motility of spermatozoa obtained from testes were examined in the Japanese pearl oyster Percent motility at 30 s after dilution increased with increasing NH₃ concentration in sea water from 0.75–2.0 mM. When spermatozoa were diluted with sea water containing 0.75 mM NH₃, which is widely used as the insemination fluid in the hatchery of this species, the percent motility increased with time elapsed after dilution, and peaked at 5 min. For spermatozoa diluted with sea water containing 2.0 mM NH₃, the percent motility increased rapidly and peaked at 30 s. The pH of sea water increased with increasing NH₃ concentration from 8.2 (0 mM NH₃) to 9.9 (5.0 mM NH₃). When spermatozoa were diluted with artificial sea water at various pH (buffered without NH₃ at 6.0–10.0), only spermatozoa diluted with artificial sea water of pH 10.0 were motile, and the percent was considerably lower than those in ammonical sea water. These results indicate that sea water containing 2.0 mM NH₃ is a suitable solution for evaluating sperm motility, and that NH₃ and/or ammonium ions may activate sperm motility in this species.     

Cryoprotective effects of shrimp head protein hydrolysate on gel forming ability and protein denaturation of lizardfish surimi during frozen storage

ABSTRACT:   The effects of shrimp head protein hydrolysate (SHPH) from three species of shrimp (northern pink shrimp [ Pandalus eous ], endeavour shrimp [ Metapenaeus endeavouri ], black tiger shrimp [ Penaeus monodon ]) on gel forming ability and protein denaturation of lizardfish surimi during frozen storage at −25°C were evaluated. The quality of lizardfish surimi with 5% (dried matter) of any of the three SHPH or sodium glutamate (Na-Glu) was examined in terms of gel strength, whiteness, Ca-ATPase activity and the amount of unfrozen water, comparing with those of surimi without additive as the control. The residual Ca-ATPase activity and gel strength of surimi with SHPH were higher than those of the control throughout 180 days of frozen storage, regardless of shrimp species. The highest effect was found in surimi with Na-Glu. The gel strength and Ca-ATPase activity found a high positive correlation. The addition of SHPH to surimi also increased the amount of unfrozen water by approximately 1.29–1.36 fold higher than the control, however kamaboko gels of the control was significantly whiter. From these results, freeze-induced denaturation of lizardfish muscle protein could be lessened by the addition of SHPH, resulting in a high gel strength and Ca-ATPase activity.     

Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytes

Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic‐polyadenylation‐element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT‐PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant‐negative mutant pCPEB1 (AA‐CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA‐CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A.     

Calf production from vitrified bovine sexed embryos following in‐straw dilution

The objective of this study was to develop an in‐straw dilution method suitable for direct transfer of vitrified bovine sexed embryos. Embryo sexing was performed by molecular diagnosis. Several sexed and vitrified‐warmed embryos were transferred after evaluation of morphologically embryonic survival at warming and in‐straw dilution (Evaluation group). The other embryos were immediately directly transferred to recipients without first being expelled from the straws after in‐straw dilution (Non‐evaluation group). The pregnancy rates of vitrified sexed embryos were 38.7% and 34.8% in the Evaluation group and Non‐evaluation group, respectively, which were not significantly different. The viability of lower quality embryos before vitrification tended to be lower (P = 0.087) than that of the higher quality embryos regardless of evaluating embryos after warming and in‐straw dilution. The abortion rates were similar, and there was no difference between the two groups (13.9% and 12.5%, respectively). These results demonstrate that vitrified bovine sexed embryos can be vitrified and diluted by the in‐straw method and that the vitrified and warmed sexed embryos can develop to term.     

Competition as a predisposing factor of crown dieback in a secondary forest of <Emphasis Type="Italic">Betula maximowicziana</Emphasis> in Hokkaido,northern Japan

We analyzed the probability that Betula maximowicziana Regel (monarch birch) would suffer crown dieback (crown-dieback probability) and the basal area growth rate (GB), which was found to be a predisposing stress factor making birch trees susceptible to crown dieback. First, we analyzed the relationship between the probability that birch trees would suffer from crown dieback in 1999 and GB from a period prior to the occurrence of crown dieback (1985–1987), using a data set of repeated measurements on 217 trees. Logistic regression analysis revealed that monarch birch had a larger crown-dieback probability when GB was low in the preceding period. Hence, there were predisposing stress factors that reduced GB and continued to affect trees for at least a decade. Next, we analyzed GB in the same period in relation to symmetrical and asymmetrical competition between trees and found that GB was reduced by symmetrical competition, suggesting that this was one of the predisposing factors for crown dieback. Based on these results, we used selected models for crown-dieback probability and GB to calculate crown-dieback probabilities for individuals with different initial basal areas and experiencing different intensities of symmetrical competition. The predicted crown-dieback probability decreased with decreasing symmetrical competition between trees. We discuss a possible process of crown dieback to death for monarch birch and the use of thinning as a method to reduce the risk of crown dieback.