Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.     

Mechanism of self-sterility in a hermaphroditic chordate

Hermaphroditic organisms avoid inbreeding by a system of self-incompatibility (SI). A primitive chordate (ascidian) Ciona intestinalis is an example of such an organism, but the molecular mechanism underlying its SI system is not known. Here, we show that the SI system is governed by two gene loci that act cooperatively. Each locus contains a tightly linked pair of polycystin 1-related receptor (s-Themis) and fibrinogen-like ligand (v-Themis) genes, the latter of which is located in the first intron of s-Themis but transcribed in the opposite direction. These genes may encode male- and female-side self-recognition molecules. The SI system of C. intestinalis has a similar framework to that of flowering plants but utilizing different molecules.     

Gene Expression and Fungal Morphology in Submerged Culture Using Whole Barley of Aspergillus kawachii

The authors described a novel submerged batch culture system that produced high levels of amylase by Aspergillus kawachii using whole barley （WB）, the surface of which is covered by its husk. In this study, detailed analyses determining the amylase activities, residual sugars, fungal morphology and expression levels of genes were performed in a submerged culture using WB to address the mechanism underlying high amylase productivity in A. kawachii. High levels of glucoamylase and acid-stable u-amylase were produced in this culture, and expression levels of amylases, as well as glucose-repressive genes including high-affinity glucose transporter and peroxidase/catalase were also high. On the other hand, the morphology of mycelia was altered, with swollen, bulbous, multi-septum hyphae and conidiophores that normally form in a solid culture being partially generated. Furthermore, cell cycle and post-translational modification-related gene expression levels were altered, and were similar to those in the solid culture. These findings suggest that high amylase productivity in the submerged culture using WB is accompanied by both the up-regulation of amylase genes and activation of post-translational modifications due to fungal morphological changes being brought closer to those in the solid culture.     

Caffeic acid derivatives in the roots of yacon (Smallanthus sonchifolius)

Five caffeic acid derivatives were found in the roots of yacon, Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson, Asteraceae, as the major water-soluble phenolic compounds. The structures of these compounds were determined by analysis of spectroscopic data. Two of these were chlorogenic acid (3-caffeoylquinic acid) and 3,5-dicaffeoylquinic acid, common phenolic compounds in plants of the family Asteraceae. Three were esters of caffeic acid with the hydroxy groups of aldaric acid, derived from hexose. The structure of the aldaric moiety was determined by hydrolysis and comparison of NMR spectra with those of standard aldaric acids. The compounds were novel caffeic acid esters of altraric acid: 2,4- or 3,5-dicaffeoylaltraric acid, 2,5-dicaffeoylaltraric acid, and 2,3,5- or 2,4,5-tricaffeoylaltraric acid.     

Practical Application of Miyazaki Jitokko Chickens Selected for a Superior Allele at a Single Nucleotide Polymorphism Site in the Cholecystokinin Type A Receptor Gene

This study aimed to examine 1) whether selection for a superior allele at a single nucleotide polymorphism site (SNP; {"type":"entrez-nucleotide","attrs":{"text":"AB604331","term_id":"315258605","term_text":"AB604331"}}AB604331, g.420 C>A) of the chicken cholecystokinin type A receptor (CCKAR) gene in Miyazaki Jitokko chickens is detectable in commercial poultry farms, and 2) whether the reproductive traits of the Kyushu Rhode hens, as a maternal stock line of the Miyazaki Jitokko chickens, are affected by SNP selection. Conventional and A-allele fixed (improved) Miyazaki Jitokko chicks were hatched on the same day and raised in a battery cage until 7 days of age. The chicks were then deposited at two commercial poultry farms and reared until slaughter at 126 and 163 days for cockerels and pullets, respectively. Body weight on the day of hatching (day 0), at 5 days of age, and at slaughter were measured. The differences in the body weights of the farm and test groups at slaughter were analyzed using the generalized linear model. A-allele fixation increased the body weight at slaughter by approximately +123.5 g and +131.9 g in cockerels and pullets, respectively. No significant differences between the conventional and improved hens were detected in terms of egg-laying rate, fertilization rate, and hatchability in the Kyushu Rhode hens. The data suggest that fattening chicks can be supplied as usual, even if Kyushu Rhode hens are switched from the conventional to improved type. In conclusion, genetic improvements using the CCKAR SNP site as a marker were effectively established in terms of the growth of the Miyazaki Jitokko chickens in commercial farms and the reproductive traits of the Kyushu Rhode hens.     

Changes in single unit activity in the lateral hypothalamic area of goats during feeding

In order to clarify the location of feeding centers in the ruminant brain, this study used a single-unit activity (SUA) recording electrode to investigate the existence of appetite-regulating neurons in the lateral hypothalamic area (LHA) in goats. Seven male Japanese Saanen goats were used in the experiment. The animals were fed twice daily, once in the morning (1000 to 1200) with 1.5 kg of roughly crushed alfalfa hay cubes, and once in the afternoon (1600 to 1800) with 200 g of commercial ground concentrate feed. The animals were allowed free access to drinking water. In this study, the animals were surgically operated on to position the recording electrode in the LHA. Recordings of SUA were carried out continuously over a 2.25-h period beginning 15 min prior to the commencement of morning feeding. The eating rates of crushed alfalfa hay cubes were highest 10 min after feeding commencement, but decreased sharply by the time 40 min had elapsed. The cumulative feed intake after the completion of the 2-h feeding period was 1164 +/- 38 g. The cumulative water intake upon the conclusion of the 2-h feeding period was 2422 +/- 107 mL. This study recorded 31 units, of which five showed a response to feeding and altered their firing rates. In response to a sharp increase in eating rates, all five units increased their firing rates to a level higher than that of prefeeding (P < 0.05). As the animals reached a level of satiety (eating rates declined to very low levels), firing of units I and II stopped completely, while the firing rates of units III, IV, and V decreased. Examination of a serial histological section confirmed that the five units in which changes in firing rates with feeding were observed were all located in the dorsolateral hypothalamic area close to the fornix. The LHA neurons recorded in this experiment characteristically showed neuronal activity increases at high levels of feeding, but decreases at low levels. The results suggest that there are cells located in the LHA of goats that are active in the physiological regulation of hay (dry forage) intake.     

Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.     

Molecular phylogenetic analysis of bovine papular stomatitis viruses detected in Saga,Japan

In this study, we performed a molecular phylogenetic analysis of six bovine papular stomatitis virus (BPSV) field strains detected from Japanese beef calves kept on a farm in Saga prefecture, a southwest part of Japan, from 2017 to 2020. The phylogenetic analysis based on a partial B2L gene (554-nt) showed that these field strains were divided into two lineages, a lineage (A-lineage) constructed by a Saga strain and strains obtained from various regions of Japan and the world, and other lineage (B-lineage) constructed by five Saga strains and strains obtained from France, USA and Iwate prefecture (a north part of Japan). Furthermore, a Saga field strain named BPSV_SAGAbv2 and strains obtained from USA and Iwate prefecture belonged to a sub-lineage blanched from B-lineage. This is the first report elucidating molecular epidemiological characters of field BPSVs obtained from Saga prefecture. The existence of the multiple lineages was thought to be related to a history of calf introduction from various regions of Japan into Saga prefecture.     

A binding protein for fungal signal molecules in the cell wall of Pisum sativum

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules.     

Biodegradation of nonlignocellulosic substances II: physical and chemical properties of sawdust before and after use as artificial soil

The physical properties of sawdust including porosity, water retention, and water drainage were analyzed to prove its suitability for use as an artificial soil in the automatic decomposer-extinguisher (GADE) machine. The physical and chemical properties of residual sawdust from the GADE machine were also analyzed, the mechanical abrasion of sawdust in the GADE machine was tested, and the morphology of this residue was observed through a scanning electron microscope to investigate changes of these properties in the medium of decomposing garbage. Sawdust, which showed a lower specific gravity and larger porosity than soil, is considered capable of supplying air to bacteria. It was found that sawdust became worn from the operation of the machine. The spaces of residual sawdust were still observed, but water drainage decreased. The portion of hollocellulose in residual sawdust decreased, although the extractives in it increased. Results indicated that the capacity of sawdust to function as an artificial soil in the GADE machine was decreased owing not only to the destruction of sawdust grain but also to the adherence of products from decomposition, such that sawdust needed to be replaced every few months.Part of this report was presented at the 43rd annual meeting of the Japan Wood Research Society, Morioka, April 1993