Molecular mechanism of fertilization in the pig

At fertilization, the sperm triggers resumption from the arrest, extrusion of the second polar body and pronuclear formation, the events of which are collectively acknowledged as ‘oocyte activation’. In all species up to date, oocyte activation requires a fertilization‐associated increase in the intracellular concentration of calcium. Especially in mammals, the signal of intracellular calcium rise at fertilization consists of periodical rises, which are also referred to as calcium oscillations. Our recent results suggest that these calcium oscillations have an important role in not only oocyte activation but also development of mammals. Pigs are animals of great agricultural value and ones in which assisted reproductive techniques, including somatic cell nuclear transfer, to produce gene‐modified pigs. Although reconstructed embryos require artificial activation stimuli which mimic fertilization‐associated increase of intracellular calcium in the oocytes, it has been known that the developmental ability of the oocytes after artificial activation is low and the regimen seems to be required for improvement. Recently we focused on two molecules, phospholipase C zeta and inositol 1,4,5‐triphosphate receptor which have important roles in regulation of calcium oscillations during fertilization in mammals, including pigs. In this review, we will discuss the present status and future perspective of molecular mechanisms during fertilization in pigs.     

Genetic diversity of wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. et Zucc.) and Japanese cultivated soybeans [<Emphasis Type="Italic">G. max</Emphasis> (L.) Merr.] based on microsatellite (SSR) analysis and the selection of a core collection

Wild soybeans, Glycine soja, are a source of genetic variation to improve soybeans. To improve the efficiency evaluation of conserved germplasm a core or mini-core collection approach that maximizes allelic diversity in a proportion of the whole collection has frequently been advocated. The genetic diversity of a wild soybean collection (1,305 accessions) plus Japanese cultivated soybeans (53 accessions) were analyzed at 20 SSR marker loci. Higher levels of allelic diversity were found in wild soybeans (28 alleles per locus) than Japanese cultivated soybean (five alleles per locus). The genetic distance between wild soybeans from different regions reflected their proximity. Accessions from Russia consisted of a diverse array of alleles resulting in accessions being spread further apart in a PCA plot than accessions from other regions. Accessions of wild soybean from Korea included many rare alleles and thus had a high representation in the core collection. The two core collections developed here, traditional and mini, consisted of 192 accessions with 97% of the allelic diversity (14% of the whole collection) and 53 accessions with 62.4% of the allelic diversity (5% of the whole collection), respectively.     

A new taxonomic treatment for some wild relatives of mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilcz.) based on their molecular phylogenetic relationships and morphological variations

Mungbean (Vigna radiata (L.) Wilcz.) is an important leguminous crop cultivated mainly in Asia. Its wild relatives are considered useful genetic resources for mungbean breeding. However, the taxonomic history of mungbean and its wild relatives is complicated and some confusion is still present in recent publications. In this study, we examined the rDNA-ITS sequences and morphological characteristics of 83 gene bank accessions closely related to mungbean. As a result, we classified the 83 accessions into five species and one unclassified accession. The proper taxon name for each species was determined (Vigna grandiflora (Prain) Tateishi et Maxted, Vigna mungo (L.) Hepper, V. radiata, Vigna subramaniana (Babu ex Raizada) Raizada, and Vigna trinervia (Heyne ex Wight et Arn.) Tateishi et Maxted) based on a review of their taxonomic history and morphological comparisons between gene bank accessions and type specimens. A new taxonomic treatment is proposed and a morphological key has been prepared. In this treatment, Vigna radiata (L.) Wilcz. var. setulosa (Dalz.) Ohwi et Ohashi is treated as a synonym of Vigna radiata (L.) Wilcz. var. sublobata (Roxb.) Verdc., and Vigna hainiana Babu, Gopinathan et Sharma is a synonym of V. subramaniana. Accession ‘NI1135’ was revealed to be most closely related to, but is considerably differentiated from, mungbean (V. radiata) based on its rDNA-ITS sequences. It also has distinguishing morphological characters. Plants with similar morphologies and DNA profiles might be distributed in the Indian Himalaya. However, since only one accession is available at present, the taxonomic classification of ‘NI1135’ needs to be reconsidered in the future.     

Morphological Studies on Zoospores of Aphanomyces cochlioides and Changes during Interaction with Host Materials

The behavioral and morphological diversity of intact zoospores of Aphanomyces cochlioides and their dynamic morphological changes when interacting with the host or a host-specific zoospore attractant were studied by light and electron microscopy. The reniform-ovate zoospore has two heterokont flagella, both inserted in a ventral groove. The anterior flagellum possesses two rows of tripartite tubular hairs (TTHs) distributed throughout its length, whereas the posterior flagellum was ornamented with two rows of highly dense fine tubular hairs, except on its tapered terminal part. The tip of the posterior flagellum has a bunch of similar fine hairs like those on the flagellum shaft. The biflagellated zoospores quickly aggregated on the host (spinach) root, adhered to the root surface, encysted by shedding or retracting the flagella, germinated at a fixed point to form germ tubes, and finally invaded the root tissues via the appressoria within 50–60 min. This precise homing response of A. cochlioides zoospores reflects guidance by a host-specific signal(s) for locating potential infection sites, differentiation of zoospores to cystospores, formation of infection structures and/or germ-tube tropism for completing pre-infection events in haste. Prior to the encystment, the posterior flagellum may be involved in successful docking on the root surface by tip contact followed by shedding or retraction. Received 22 January 2001/ Accepted in revised form 28 May 2001     

Studies on herbicidal carbamoyltetrazolinone derivatives: Selection of fentrazamide

1-Phenyl-5(4 H )-4-dialkylcarbamoyltetrazolinones represent a new class of herbicides that are active against barnyardgrass ( Echinochloa spp.) in paddy fields. To select the most favorable compound as a paddy herbicide, herbicidal activity and selectivity of the carbamoyltetrazolinone compounds to transplanted rice were determined in greenhouse tests. Higher herbicidal activities were associated with the compounds having a 2-Cl- or 2-Cl-6-Me-substituted or an unsubstituted phenyl ring, and a combination of C_2–3 linear alkyl group and a C₄ branched-chain alkyl or a C_5–6 cycloalkyl group on the nitrogen atom in the carbamoyl structure. In addition, the compounds carrying a total of seven to nine carbon atoms in the substituents on the carbamoyl nitrogen exhibited a good selectivity between barnyardgrass and transplanted rice plants. From the results of soil thick-layer chromatography, the good crop compatibility of carbamoyltetrazolinones was correlated to the mobility of the compounds in paddy soil. The investigation resulted in the selection of an excellent compound, 4-(2-chlorophenyl)- N -cyclohexyl- N -ethyl-4,5-dihydro-5-oxo-1 H -tetrazole-1-carboxamide (common name: fentrazamide), as a rice paddy herbicide for controlling barnyardgrass.     

Genetic analysis of shiga-toxigenic Escherichia coli isolates from cattle in a limited region

The ecology of shiga‐toxigenic Escherichia coli (STEC) is important in the animal production environment. We investigated fecal shedding of STEC in one town in Miyagi, Japan by multiplex polymerase chain reaction (PCR) targeting shiga toxin gene 1 (stx₁), gene 2 (stx₂) and malB promoter gene, and analyzed the PCR products of stx₁ or stx₂ (54 samples) by direct sequencing. Three of 46 (6.5%) beef cattle in the University Farm of Tohoku University (Kawatabi Farm) and 11 of 70 (15.7%) calves in neighboring dairy farms carried STEC. Rate of detecting genes of stx₁, stx₂ and stx₁₊₂ was 3.4% (4/116), 8.6% (10/116) and 0.9% (1/116), respectively. Serotyping indicated that STEC contaminated farms at different times or through different routes. Isolates harbored no mutation among stx₁, but six (Kawatabi Farm) and 38 (neighboring farms) base substitutions among stx₂, respectively. The diversity of substitutions of stx₂ was observed among farms or even in a farm. Phylogenic analysis revealed that STEC detected in the area were classified into three clusters by the variety of stx₂. Sequence analysis of stx₂ will be one of the tools for clarifying the source of outbreaks and the route of contamination of STEC.     

Novel polymorphism of the canine dopamine receptor D4 gene intron II region

Various dog breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of these characteristics are under genetic control. However, little is known about genes related to behavioral traits in canine species. In humans, it has been reported that the dopamine receptor D4 gene (DRD4) includes polymorphism at several regions that relate to personality or psychiatric disorders. In an earlier study by the authors of the present study, the polymorphisms in canine DRD4 exon III and exon I regions were reported. In the present study, a novel polymorphism in canine DRD4 intron II was found based on a 17 base pair insertion/deletion, and the two alleles detected were named P (shorter allele) and Q (longer allele). The allelic distribution in 28 breeds of dog, including a total of 1114 unrelated individuals, were then investigated. Both P and Q alleles were detected in most of the breeds investigated; however, the frequencies of P and Q differed greatly between breeds. With respect to classification based on breed origin, P and Q alleles were frequent in Occidental and Oriental breeds, respectively. Furthermore, two subspecies of wolves, the ancestors of dogs, were analyzed for the comparison of allele frequencies with dogs, and the P allele was predominant in both European and Chinese wolves.     

The effects of cataract stage, lens-induced uveitis and cataract removal on ERG in dogs with cataract

OBJECTIVE: The purpose of this study was to determine the effects of cataract stage, lens-induced uveitis and cataract removal on the electroretinogram (ERG) of dogs with cataract. ANIMALS STUDIED: Fifty-seven dogs diagnosed with unilateral or bilateral cataract whose ERG was recorded at Rakuno Gakuen University Teaching Animal Hospital from 2001 to 2004. PROCEDURES: Four responses were recorded during the ERG: rod ERG, standard combined ERG, single-flash cone ERG and 30-Hz flicker ERG. Cataracts were divided into four stages: incipient, immature, mature and hypermature, and with or without lens induced uveitis (LIU). Noncataractous eyes of dogs with unilateral cataract were used as the control. We compared ERG amplitude, implicit time, and the b- to a-wave amplitude ratio of cataractous vs. noncataractous eyes, preoperative vs. postoperative cataractous eyes, and cataractous eyes with and without LIU. RESULTS: No significant difference was found in ERG amplitude between incipient, immature and hypermature cataractous eyes, while in mature cataractous eyes decreased amplitude was confirmed in all responses compared with control eyes. However, no significant difference in b/a ratio was found at any stage of cataract. In postoperative eyes, increased amplitude was recorded in all responses compared to preoperative values. In eyes with LIU a decreased amplitude in the rod ERG and b-wave of standard combined ERG was recorded and, furthermore, a significant decline was confirmed in b/a ratio. CONCLUSION: ERG values were influenced by cataract stage and LIU. LIU was associated with a reduction in the b/a ratio.