Junji Ueda

Population Research of Genetic Polymorphism at Amino Acid Position 631 in Chicken Mx Protein with Differential Antiviral Activity

A single amino acid substitution between Asn and Ser at position 631 in the chicken Mx protein has been reported to determine resistant and sensitive antiviral activity. In this study, we investigate whether various kinds of chicken breeds and jungle fowls carry the resistant or sensitive Mx allelic gene by using the mismatched PCR-restriction fragment length polymorphism (RFLP) technique. In total, 271 samples from 36 strains of 17 chicken breeds and from 3 kinds of jungle fowls were examined. The rates of the resistant Mx gene and sensitive gene were 59.2% and 40.8%, respectively. Only a Red jungle fowl captured in Laos carried the resistant Mx gene, and the other three Red jungle fowls from Indonesia and Gray and Green jungle fowls all had the sensitive Mx gene. These results were confirmed by the determination of amino acid sequences in the GTPase effector domain of jungle fowls.

Effects of culture time, ovarian activity, cumulus cells and sera on the nuclear and cytoplasmic maturation of pig oocytes in vitro

Pig cumulus-oocyte complexes (COC) were used in this study to assess the effect of length of culture, ovarian activity, cumulus cells and sera on maturation and fertilization in vitro. In Experiment 1, the incidence of nuclear maturation increased (P<0.01) from 24 to 48 h of culture and the ability for male pronucleus (MPN) formation peak at 42 h (86.6%). The penetrability of immature COC and those at the metaphase 2 (M2) stage appeared to be not influenced by the stage of meiosis (75.3–89.3%). In Experiment 2, no significant difference in the nuclear maturation (84.8–88.0%) and penetration (82.1–92.0%) rates of COC collected from ovaries with or without corpora lutea (CL) or corpora albicans (CA) was observed, though MPN formation differed (P<0.05) between O1 (82.6%) and O3 (61.5%) oocytes. In Experiment 3, significant differences (P<0.01) on the nuclear maturation and MPN formation of oocytes cultured with cumulus cells versus those without or lacking cumulus cells was observed. It shows that the presence of cumulus cells is important in supporting maturation in vitro. In Experiment 4, serum supplementation of the medium improved the rate at which COC reached the M2 stage (P<0.05) and formed MPN except at the 5% level when using fetal bovine serum (FBS) and calf serum (CS). The use of porcine serum (PS) consistently supported the maturation of COC. Serum supplementation is thus necessary for maturation in vitro and the concentration and type of serum to be used is important.

Timing of sequential changes in chromosome configurations during the second meiotic division and cytoplasmic events of pig oocytes matured and fertilized in vitro

Abstract The present study determined the specifics of early events of fertilization of pig oocytes matured in vitro (IVM) and subsequently fertilized in vitro (IVF). The IVF system used for sperm capacitation, time of fertilization and fixation of eggs at 1–3 h intervals during the 30 h period after insemination precisely measured the timing of each critical stage of fertilization with precision. The first evidence of sperm penetration was observed 3 h after insemination. Attachment of the spermatozoa in the ooplasm caused resumption of meiosis (A2/T2 stage of second meiotic division), and was commonly observed from 4 to 5 h after insemination. The sperm heads of penetrating spermatozoa started to decondense, tails became detached and completion of the second meiosis (second polar body extrusion) occurred from 6 to 9 h. Further decondensation of the sperm head and female chromosomes took place from 9 to 12 h. Opposing and apposing pronuclei were frequently observed from 12 to 18 h and 18 to 24 h after insemination, respectively. Syngamy and prophase stage of first mitotic division, including first cleavage were observed from 24 to 30 h.

A Functional Truncated Form of c-kit Tyrosine Kinase Is Produced Specifically in the Testis of the Mouse But Not the Rat, Pig, or Human

The complete nucleotide sequence of mouse-truncated mRNA of c-kit, tr-kit, has been determined using the CD1 strain. In this study, the nucleotide sequences of tr-kit from AKR/N, C57BL/6, and ICR strains of mice were determined and found to be identical, although many silent variations were found compared with the sequence in a database for CD1. Tr-kit protein consists of 12 amino acids encoded by the 16th intron and the following 190 amino acids of c-kit. In the sequences of tr-kit encoding 12 specific amino acids, no substitution was detected among the three strains and CD1. Furthermore, RT-PCR analysis clearly showed that tr-kit mRNA expression was present only in testis. No nucleotide mutation in two important regions of the presumptive promoter for tr-kit mRNA was detected within the 16th intron of the mouse strains examined. However, no functional form of tr-kit was found in the rat, pig, or human by sequence analysis and homology testing.

Specific Intracellular Localization and Antiviral Property of Genetic and Splicing Variants in Bovine Mx1

In bovine Mx1, only an amino acid substitution between Ile and Met at position 120 was detected by the nucleotide sequence and mismatched PCR-RFLP technique. The Ile variant was assumed to distribute mainly in the bovine population since the gene frequency was 79.3%. Furthermore, we cloned water buffalo Mx1 cDNA, which showed 51 nucleotide and 20 amino acid substitutions in comparison with that of the cow. Another kind of Mx1 cDNA, bovine Mx1B cDNA, was found and it was deduced to cause 27 amino acid substitutions at the N-terminus compared to the original Mx1 by alternative splicing. However, no variation was detected in 27 amino acids specific for Mx1B among 29 cows and a water buffalo. We established four kinds of mRNA-expressing 3T3 cells and Vero cells. When infection experiments were performed using recombinant vesicular stomatitis virus (VSVDeltaG*-G), bovine Ile and Met types and water buffalo Mx1 mRNA-expressing cell lines showed equally positive antiviral activities (p < 0.05). On the other hand, bovine Mx1B mRNA-expressing cell lines did not have activity against VSVDeltaG*-G. Intracellular localization of bovine Mx1 and Mx1B proteins was examined by a transiently GFP-fused expression system in 3T3 cells. Bovine Mx1 was localized in the cytoplasm, while bovine Mx1B was mainly localized in the nucleus. An arginine-rich nuclear localization signal was found in 27 amino acids specific for Mx1B. N-terminus-deleted Mx1B was only localized in the cytoplasm, and the deleted Mx1B-expressing cell lines showed significantly positive antiviral activities (p < 0.05) against VSVDeltaG*-G.

Structural and functional analysis of the bovine Mx1 promoter.

The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFκB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction. Several artificial deletion mutants were prepared to determine the important regulatory elements responsible for the promoter activity, and it was found that ISRE has a key function in IFN response. The proximal ISRE1 showed potential induction by IFN. Furthermore, the proximal GC boxes were found to be essential for IFN response in the bovine Mx1 promoter with the deletion mutants. In this case, the 2 GC boxes exhibited a synergistic activation in the IFN response. Mithramycin A, an agent that inhibits gene expression selectively by coating GC boxes, was used, and Mx mRNA expression in MDBK cells was suppressed by this chemical. Therefore, GC boxes were also shown to be essential for IFN response in the bovine Mx1 gene.

Blastocyst formation of pig embryos derived from in vitro fertilization of in vitro matured pig oocytes in the amniotic fluid of a developing chick embryo

In this study, the acquisition for developmental competence of early-stage pig embryos derived from in vitro fertilization of in vitro matured pig oocytes was determined by culturing in a modified Whittens medium (mWM) or amniotic fluid of a developing chick embryo (CEAm fluid). Of 1080 oocytes inseminated, 420 were fixed after 15 h revealing a nuclear maturation rate of 95.2% (400420) with a fertilization rate of 84.3% (354420). The monospermic fertilization rate was 36.4% (129354). Of 660 oocytes that were allowed to continue development, 74.2% (490660) cleaved. Of these, 220 and 157 embryos were cultured in mWM and CEAm fluid with 19.5% and 25.7% progressing to blastocyst stage, respectively. The mean (±standard error of the mean) cell number of blastocyst stage embryos derived from CEAm fluid (52.0 ± 3.0) was significantly higher (P < 0.001) than those obtained from mWM (39.0 ± 1.7). The results suggest that CEAm fluid is a better in vitro culture system for pig embryo development than mWM.

Analysis of specific factors generating 2-cell block in AKR mouse embryos.

The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR x C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.

Polymorphic Study of Equine Antiviral MXA Gene

The interferon-induced MX protein is known to generate innate immunity of hosts against viral infection (Lee and Vidal, 2002). The mouse Mx1 protein, the first protein of the Mx family to be reported, confers resistance to influenza virus (Staeheli et al., 1986). The mouse Mx1 protein localizes in the nucleus, but the Mx2 protein localizes in the cytoplasm and inhibits the replication of vesicular stomatitis virus (VSV) and hantavirus but not influenza virus (Jin et al., 1999, 2001). On the other hand, the human MXA protein has been reported to have a broader antiviral spectrum, because it could inhibit the replication of many kinds of viruses, such as several negative single-stranded RNA viruses, a few positive RNA viruses, and even a DNA virus (Frese et al., 1995; Landis et al., 1998; Gordien et al., 2001). No antiviral activity of human MXB protein, however, has been found for any viruses, and both human MXA and MXB proteins localize in the cytoplasm (Pavlovic et al., 1990). In the horse, both MXA and MXB proteins have also been reported to localize in the cytoplasm (Horisberger and Gunst, 1991), but only the nucleotide sequence of the MXA cDNA has been reported (Chesters et al., 1997). In the present study, we tried to detect polymorphisms of the equine MXA gene by determining the nucleotide sequences of MXA cDNA.

An Analysis of Age Patterns of Dams and their Relationships with Age of Home-bred and Foreigen-bred Sires in Hokkaido Dairy Herds

北海道乳牛群の交配構造を把握し,今後の育種計画を策定するために必要な情報を得る目的で,前報で父牛の年齢構成を調査したが,本研究では,母牛の年齢構成について調査分析した.1970～1983年に北海道で生まれ,しかも1960～1981年に生まれた北海道産母牛からの658,810頭の乳牛の最統登録情報を用いた.乳牛は父牛の生産地(北海道,アメリカ,カナダ)によって3群に区分し,各群内で分析し比較した.北海道産父牛と交配した母牛は3歳で最も多くの娘牛を生産し,その後年齢とともに急激にその数を減少した.これに対して,アメリカ産の父牛に交配した母牛は4歳で最も多いが年齢に伴う減少は緩やかであり,カナダ産の父牛に交配した母牛は2群の中間の年齢に伴う推移を示した.これら3群の母牛から娘牛への平均世代間隔はそれぞれ4.6,6.1,5.5年であった.しかし,2～3歳の母牛からの娘牛の比率は年次的に減少し,6～7歳からの比率が増加する傾向が3群で同様に認められた.父牛と母牛の年齢の相関は低いが有意な正の相関が認められ,また北海道産父牛への交配は外国産父牛よりも4～6月若い母牛に交配される傾向が認められた.