Establishment of a Conditional Transgenic System Using the 2A Peptide in the Female Mouse Germline

Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken β-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.     

Thinning alters crown dynamics and biomass increment within aboveground tissues in young stands of Chamaecyparis obtusa

The stand density of a forest affects the vertical distribution of foliage. Understanding the dynamics of this response is important for the study of crown structure and function, carbon-budget estimation, and forest management. We investigated the effect of tree density on the vertical distribution of foliage, branch, and stem growth, and ratio of biomass increment in aboveground tissues; by monitoring all first-order branches of five trees each from thinned and unthinned control stands of 10-year-old Chamaecyparis obtusa for four consecutive years. In the control stand, the foliage crown shifted upward with height growth but the foliage quantity of the whole crown did not increase. In addition, the vertical distribution of leaf mass shifted from lower-crown skewed to upper-crown skewed. In the thinned stand in contrast, the foliage quantity of individual crowns increased two-fold within 4 years, while the vertical distribution of leaf mass remained lower-crown skewed. The two stands had similar production rates, numbers of first-order branches per unit of tree height, and total lengths of first-order branches. However, the mortality rate of first-order branches and self-pruning within a first-order branch were significantly higher in the control stand than in the thinned stand, which resulted in a higher ratio of biomass increment in branch. Thinning induced a higher ratio of biomass increment in foliage and lower in branch. The increased foliage quantity and variation in ratio of biomass increment after thinning stimulated stem growth of residual trees. These results provide information that will be useful when considering thinning regimes and stand management.     

Effect of improvement cutting on the community structure of aphyllophoraceous fungi on Okinawa Island

Improvement cutting, which decreases tree density by cutting and thinning while leaving logging residues, has been practiced on Okinawa Island since the 1970s and changes not only the vegetation composition but also the amount and composition of woody debris on the forest floor. To quantitatively compare the effect of improvement cutting with the effects of woody debris and vegetation on the community structure of aphyllophoraceous fungi, we conducted a field survey of vegetation composition, the amount and composition of woody debris, and the aphyllophoraceous fungi flora on Okinawa Island in December 2006. We established 3 plots in forest stands without logging and 13 plots in a forest that had undergone improvement cutting from 1 to 22 years before our field survey. The number and volume of woody debris tended to be highest soon after improvement cutting. We recorded 1,172 fungal samples belonging to 81 species in four orders. Fungal species richness was correlated with the composition of the woody debris. Fungal species composition in forest plots 1 year after improvement cutting differed from most of the other logged forests. Variation partitioning revealed that the effects of improvement cutting explained 14.1 % of the total variation in fungal species composition, and that differences in woody debris and vegetation not related to improvement cutting explained 40.4 %. Changes in the composition of woody debris caused by improvement cutting explained at most 2.7 % of the variation. This suggests that improvement cutting does not have a strong effect on fungal community structure, but woody debris does.     

The sex determining gene of medaka: a Y-specific DM domain gene (DMY) is required for male development

Although the sex-determining gene Sry has been identified in mammals, no comparable genes have been found in non-mammalian vertebrates. To clone positionally the sex-determining region of the medaka, Oryzias latipes, we generated a Y congenic strain to highlight the genetic differences between the X and Y chromosomes from inbred strains of medaka. We used recombinant breakpoint analysis and deletion analysis of the Y chromosome of a congenic XY female to restrict the sex-determining region to 250-kb stretch of the Y chromosome. Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed during sexual differentiation. However, only one gene was Y specific. The full-length cDNA sequence of this gene encodes a putative protein of 267 amino acids, including the highly conserved DM domain. We thus named it DMY. To establish a role for DMY during sexual differentiation, we screened wild medaka populations for naturally occurring DMY mutants. Two XY females with distinct mutations in DMY were found in separate populations. The first heritable mutant – a single insertion in exon 3 and the subsequent truncation of DMY – resulted in all XY female offspring. Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY female offspring. Furthermore, during normal development, DMY is expressed only in somatic cells of XY gonads. These findings strongly suggest that the sex-specific DMY is required for normal testicular development and is a prime candidate for the medaka sex-determining gene.     

Is experience on a farm an effective approach to understanding animal products and the management of dairy farming?

The understanding of animal products and dairy farming is important for the promotion of dairy farming. Thus, to examine the effects of farm experience on the understanding of animal products and the management of dairy farming, the interaction between students and dairy cows was investigated in groups of first‐year veterinary nursing students in 2011 and 2012 (n = 201). These students included 181 women and 20 men. Nine items about dairy cows were presented in a questionnaire. The survey was performed before and after praxis on the educational farm attached to the authors' university. After praxis on the farm, increases occurred in the number of positive responses to the items involving the price of milk, dairy farming and the taste of milk. For these items, a significant difference (P < 0.05) was found between the scores obtained before and after training. The results of the study suggested that farm experience is useful for improving the understanding of animal products and dairy farming.     

Determination of bacteria constituting ruminal fibrolytic consortia developed on orchard grass hay stem

To determine the relationship between Fibrobacter succinogenes and other rumen bacteria, the bacterial community structure on fiber was analyzed by using two different materials. These were ruminally incubated orchard grass hay stems without and with preincubation with F. succinogenes (natural and artificial consortia, respectively). The natural consortium mainly consisted of Firmicutes (56.6%) and Bacteroidetes (33.1%), while the artificial consortium showed a significantly higher proportion of Firmicutes (85.5%) and a lower proportion of Bacteroidetes (4.6%). At species or genus level, Butyrivibrio fibrisolvens, the U2 group, Ruminococcus albus and Lachnospiraceae incertae sedis made up a higher proportion in the artificial consortium. The most dominant bacterial group was the Butyrivibrio‐Pseudobutyrivibrio‐Lachnospiraceae incertae sedis group, which accounted for 19.7% in the natural and 29.5% in the artificial consortium. Within the genus Butyrivibrio, the phylogenetic groups SA and VA2 and phylogeny‐undefined Butyribivrio, but not VA1, were detected at high frequency in the artificial consortium. These results suggest that ecological and possibly functional relationships exist in the rumen among F. succinogenes, a subset of B. fibrisolvens, the U2 group, R. albus and Lachnospiraceae incertae sedis.