Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Present state of imposex in neptune whelk Neptunea arthritica inhabiting shallow waters around Hokkaido, Japan

ABSTRACT:   The state of imposex in Neptunea arthritica from seven sites along the coast of Hokkaido, Japan was examined in 2002 based on the criteria: (i) relative penis size index (RPSI); (ii) imposex frequency; (iii) stage frequency distribution of imposex in adult and immature whelks; and (iv) sex ratio. RPSI differed from site to site, although values from all sites were low (0.186–5.294). In particular, the RPSI values for four sites were very low (<1.0). In sites where immature whelks were also collected, the frequency of imposex was considerably lower in immature (7.7%–55.6%) than in adult whelks (50%–95.2%) except at one site. The imposex stage frequency distribution also differed among sites, and the trend in the adult whelks corresponded with their RPSI value. Female whelks showing severe imposex (Stage 3 and 3+) were restricted to large individuals.     

Color vision, spectral sensitivity, accommodation, and visual acuity in juvenile masu salmon Oncorhynchus masou masou

ABSTRACT:   Color vision, spectral sensitivity, accommodation, and visual acuity were examined in juvenile masu salmon Oncorhynchus masou masou to obtain fundamental information about the visual system. Two types of S-potentials were recorded from 415 horizontal cells in isolated retinas from 34 cultured freshwater masu salmon (114–219 mm standard length, SL). Although horizontal cells recording S-potentials were not identified, the horizontal cells were confirmed because their responses were maintained while the stimulus remained. The variety of chromaticity (C)-type S-potentials indicated well-developed color vision. The analysis of the luminosity (L)-type S-potentials indicated that the peak spectral sensitivity was at 522 nm. S-potentials were also recorded in response to ultraviolet light. The direction and extent of lens movement induced by electrical stimulation was measured in 12 cultured masu salmon (99.0–142.5 mm SL). The results indicated that the visual axis was upward and forward, and that the range of accommodation was from 0.79 × SL in front of the eye to infinity. In histological analysis of the retinas of five wild smolts (100–118 mm SL), the maximum cone densities (276–345 cones/0.01 mm²) were detected in the ventral to temporal regions. The visual acuities assessed by histological methods were 0.069–0.075.     

Effects of entrance design on catch efficiency of arabesque greenling traps: a field experiment in Matsumae, Hokkaido

ABSTRACT:   A field experiment was conducted in the Matsumae area of Hokkaido, Japan, during June and July 2002, to investigate the effects of different entrance designs on the catch efficiency of fish traps by fishing with commercial traps (entrance inclination angle [α] = 37°; funnel length of entrance [ L _f ] = 22 cm) and experimental traps. The experimental traps were of the same size and similar design as commercial traps, with different entrance inclination angles (trap E1: α = 46°; E2: α = 27°; E3: α = 0°; all L _f  = 22 cm) or funnel lengths (E4: α = 37°, L _f  = 8 cm). In total, 2200 fish during 200 trap hauls were captured. The catch was significantly higher using both traps E2 and the commercial trap than with trap E3 ( P  < 0.05), and the catch of trap E2 was higher than that of the commercial trap. There were no significant differences in mean fish body length or the frequency distributions of body length among trap types (E1, E2, E3 and commercial). The funnel length of the entrance also affected the catch of traps. Trap E4 had significantly higher catches than the commercial trap ( P  = 0.04) when traps were deployed for a 1-day soak time. Fish body length frequency distributions did not differ between trap E4 and the commercial trap. The results showed that catch can be greatly affected by trap entrance designs.     

Liquid culture and transformation of hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan’s (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containingβ-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicidebar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene. The generous supply of AVF-B from Dr. B.J. Nairn, Tasman Forestry, NZ is also appreciated.     

Comparative Analysis of mRNA Expression of Surface Antigens between Histiocytic and Nonhistiocytic Sarcoma in Dogs

Background

Definitive diagnosis of histiocytic sarcoma (HS) in dogs is relatively difficult by conventional histopathological examination because objective features of HS are not well defined.

Hypothesis

Quantitative analysis of mRNA expression of selected cellular surface antigens (SAs) specific to HS in dogs can facilitate objective and rapid diagnosis.

Animals

Dogs with HS (n = 30) and dogs without HS (n = 36), including those with other forms of lymphoma (n = 4), inflammatory diseases (n = 6), and other malignant neoplasias (n = 26).

Methods

Retrospective clinical observational study. Specimens were collected by excisional biopsy, needle core biopsy, or fine needle aspiration. To determine HS detection efficacy, mRNA expression levels of selected SAs specific to HS in dogs, including MHC class IIα, CD11b, CD11c, and CD86, were quantitatively analyzed using real‐time quantitative polymerase chain reaction.

Results

Each SA mRNA expression level was significantly higher in HS dogs than in non‐HS dogs (P = .0082). Cutoff values for discriminating between HS and non‐HS dogs based on these expression levels were calculated on the basis of receiver‐operating characteristic analysis. Accuracy of the cutoff values, including MHC class IIα, CD11b, CD11c, and CD86, was 87.9, 86.4, 86.4, and 84.8%, respectively.

Conclusions and Clinical Importance

Our results suggest that quantitative analysis of mRNA expression of the selected SAs could be an adjunctive diagnostic technique with high diagnostic accuracy for HS in dogs. Substantial investigation is required for exclusion of diseases with similar cell types of origin to lymphoma.     

Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas

KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.     

In vitro study on the increase of trypsin resistance in the rat testicular sperm head.

The alteration of trypsin resistance in rat sperm head was investigated after the sperm were incubated in the culture media (199-Earele, 1% BSA) containing the inhibitors of protein synthesis. The head of non-incubated testicular sperm was easily digested by trypsin (1 mg/ml) while the head of the incubated sperm showed resistance to trypsin digestion after 3 hr-incubation. Trypsin resistance in epididymal sperm head did not change after the sperm was incubated. Neither cycloheximide (100 micrograms/ml) nor chloramphenicol (100 micrograms/ml) affected the trypsin resistance in the testicular sperm head.