Effects of Trichostatin A on In Vitro Development and DNA Methylation Level of the Satellite I Region of Swamp Buffalo (Bubalus bubalis) Cloned Embryos

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82–85%), cleavage (79–84%) and development to the 8-cell stage (59–65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.     

An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E₂ (PGE₂) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE₂ at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.     

Effects of sampling errors on abundance estimates from virtual population analysis for walleye pollock in northern waters of Sea of Japan

ABSTRACT:   Effects of sampling errors on abundance estimates from virtual population analysis (VPA) were quantified with the bootstrap method for stock of walleye pollock in the the Sea of Japan. In the bootstrap method, individual fish measurements were resampled. A total of 1000 bootstrap samples were produced for each year from 1991 to 2001. The coefficients of variation (CV) of catch at age in 2001 ranged 6.1–33.1%. The CV of an abundance estimate in 2001 ranged 9.0–35.7%. Abundance estimates of the oldest age and the latest year, which had larger CVs than the other estimates, were sensitive to sampling errors. Effects of sampling errors became smaller when the catch at age had been accumulated over a few years. Although VPA includes various types of errors, only the sampling errors have room for improvement in reality. Quantifying the effect of sampling error on VPA estimates is essential for sound and efficient stock management, and is emphasized in this study.     

Genes expressed in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> spleen: analysis of genes involved in immune function

ABSTRACT:   Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and for profiling gene expression. A cDNA library developed from messenger RNA of the Japanese flounder, Paralichthys olivaceus spleen was constructed by directional cloning, in order to isolate functional genes involved in immunity in fish. A total of 1010 ESTs from the library were sequenced and compared with sequences in the GenBank database. Of the 1010 ESTs, 618 ESTs (61%) were identified as being homologous with known genes from many organisms by BLAST searches, whereas 392 (39%) appeared to be unknown and are likely to represent newly described genes. Of the identified genes, 105 (17%) encoded proteins associated with cell/organism defence and homeostasis. Of these 105 genes, 21 were identified for the first time in Japanese flounder. These included macrophage inflammatory protein (MIP)-3 α, granulin-A, novel immune-type receptor (NITR), interferon regulatory factor (IRF)-10, presenilin-like protein-2, and antizyme inhibitor. A comparison of ESTs derived from spleen, kidney, liver and leukocytes suggested that expression of immune-related genes in different tissues, organs, or cells are different.     

Comparison of behavioral characteristics of hatchery-reared and wild red tilefish Branchiostegus japonicus released in Maizuru Bay by using acoustic biotelemetry

ABSTRACT:   In order to establish techniques for the stock enhancement of red tilefish Branchiostegus japonicus , it is important to understand the behavioral characteristic of both hatchery-reared and wild fish. Four hatchery-reared and six wild fish were released and tracked using acoustic biotelemetry in Maizuru Bay, Kyoto, Japan, from August 2003 to February 2004. Data for the four wild fish released and tracked from January 2003 to May 2003 were also referred to for analysis. The released fish moved around in relatively large areas within about 10 days after the release, and then three hatchery-reared and two wild fish settled and stayed within limited areas. Fish of both origins showed strong site fidelity and diel activity patterns, i.e. they were detectable in the day but not at night, probably due to hiding behavior in their burrows at night. However, some discrepancies between fish of both origins were detected by comparing their track terms and activity patterns in detail. These discrepancies are probably attributable to the differences in environmental conditions that the fish had experienced before release.     

Directed movements and diel burrow fidelity patterns of red tilefish <Emphasis Type="Italic">Branchiostegus japonicus</Emphasis> determined using ultrasonic telemetry

ABSTRACT:   Wild red tilefish Branchiostegus japonicus were tracked and/or monitored for up to nearly 3 months in Western Wakasa Bay and Maizuru Bay, Japan, using ultrasonic telemetry to examine their movements and burrow fidelity patterns. In Western Wakasa Bay, it is guessed that fish released in relatively shallow waters, about 30 m deep, actively moved into more suitable deeper habitats after their release. In Maizuru Bay, the fish did not undertake large-scale movements and showed burrow fidelity about 1 month after their release. That is, the fish primarily remained in the burrow, which was constructed in the sea-bottom, during the night and moved outside the burrow during the day. The marked circadian rhythm from Fourier analysis indicated a clear diel movement pattern of the red tilefish.     

A genetic linkage map of the Japanese flounder, Paralichthys olivaceus

We report the first genetic linkage map of the Japanese flounder (Paralichthys olivaceus) constructed with 111 microsatellite markers and 352 AFLP fragments. The parental male linkage map consisted of 25 linkage groups while the female map consisted of 27 groups, with an average resolution of 8 and 6.6 cM, respectively. We have identified linkage among 96% of the markers and the total map length was estimated to be around 1000–1200 cM. This study reports the first low-density linkage map for the Japanese flounder and describes differences in sex recombination. Recombination rates were higher in male flounder compared to the female (7.4:1), a rare condition among vertebrates. This map is a starting point for the mapping of single loci and quantitative traits in flatfish species.