Application of laser-assisted zona drilling to in vitro fertilization of cryopreserved mouse oocytes with spermatozoa from a subfertile transgenic mouse

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.     

Effect of gestational exposure to nonylphenol on the development and fertility of mouse offspring

Nonylphenol (NP), a kind of environmental chemical, is thought to imitate endogenous hormones, inhibit the actions of hormones, and induce reproductive abnormalities. A number of experimental animals, usually rats, have been used to evaluate the potential reproductive toxicity of NP. However, the findings of previous studies were contradictory in some cases. Therefore, we used ICR mice as a biomodel for in utero study of NP. After mating, 8- to 12-week-old females were assigned to four groups (n=8) for subcutaneous injections from day 5 to 20 of gestation. Group I animals received corn oil alone as a control, while the mice of groups II, III and IV received NP at concentrations of 1/1000, 1/100 and 1/10 of the LD(50), respectively. A dose-dependent decrease was observed in terminal body weights of males of the F1 generation; however, a very small negative effect was only found in the females of the NP1/10 group. No significant effect was found on the liver weights of both sexes. The weights of the testis and epididymis were slightly decreased in the NP1/10 group. The NP1/100 treatment increased ovary weight considerably. The uterus weight tended to be increased in the NP treatment groups; however, there were large variations. The gestational exposure of the groups had no significant effect on the rate of pregnancy (94.4-100%) and the number of fetuses per litter (13.6-14.3 males, 12.3-13.7 females) compared with the control group. However, the overall mortality of fetuses/embryos was increased considerably in the NP1/100 (male: 13.9%) and NP1/10 (female: 9.8%) groups. These results suggest that exposure to NP in utero possibly affects the body weight and some reproductive organ weights, but does not influence the potential fertility of the F1 generation.     

Practicable group testing method to evaluate weight/weight GMO content in maize grains

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.     

Asynchronous diversification in a specialized plant-pollinator mutualism

Most flowering plants establish mutualistic associations with insect pollinators to facilitate sexual reproduction. However, the evolutionary processes that gave rise to these associations remain poorly understood. We reconstructed the times of divergence, diversification patterns, and interaction networks of a diverse group of specialized orchids and their bee pollinators. In contrast to a scenario of coevolution by race formation, we show that fragrance-producing orchids originated at least three times independently after their fragrance-collecting bee mutualists. Whereas orchid diversification has apparently tracked the diversification of orchids' bee pollinators, bees appear to have depended on the diverse chemical environment of neotropical forests. We corroborated this apparent asymmetrical dependency by simulating co-extinction cascades in real interaction networks that lacked reciprocal specialization. These results suggest that the diversification of insect-pollinated angiosperms may have been facilitated by the exploitation of preexisting sensory biases of insect pollinators.     

Sequence differences in the internal transcribed spacer 1 and 5.8S ribosomal RNA among three Moniezia species isolated from ruminants in Japan

This study was designed to clarify the differences in the internal transcribed spacer (ITS) 1 and 5.8S nucleotide sequences of Moniezia expansa, M. benedeni and M. monardi isolated from ruminants in Japan and to determine their phylogenetic relationships. A 98% similarity in the 5.8S sequences was observed among the 3 Moniezia species, whereas many nucleotide indels and substitutions were observed in the ITS1 sequences among the three Moniezia species. These results suggest that the ITS1 region could serve as a potential marker for discriminating the 3 Moniezia species. In the phylogenetic tree based on the ITS1 sequences, M. monardi and M. benedeni showed genetically closer relationship to each other than to M. expansa.     

Isolation of endophytic Frankia from root nodules of Casuarina equisetifolia and infectivity of the isolate to host plants

Abstract

A method which uses the pressure head to predict unsaturated hydraulic conductivity is presented to calculate the soil water flux in a field. Hydraulic conductivities in the primary drying and wetting processes were measured with core samples in the laboratory and the hysteresis between the hydraulic conductivity and pressure head was taken into account. Hydraulic gradients were measured every hour with tensiometers installed in the field. This method was applied to analyze the water movement at 94-cm depth in Hydric Hapludands. Downward or upward flow of water by summing soil water fluxes was examined using the water balance method. Amounts of downward flow determined by our method after heavy rain in a wet soil were slightly larger in the soybean plot but smaller in the bare plot than those obtained by the water balance method due to non-uniform infiltration. Water balance equation which used values of upward flow across a 94-cm depth estimated reasonably well the evapotranspiration from the soybean plot and the evaporation from the bare plot during a dry period. Effect of initial soil water conditions on downward flux by rainfall was well monitored by our method.