We investigated gamete longevity in two sea urchin species, Echinometra mathaei (Em) and Tripneustes gratilla (Tg), via laboratory experiments. The longevity of dry sperm at different ages (6, 12, 18, 24, and 30 h after collection) was tested at 20°, 25°, and 30°C. Sperm viability was determined by the percent cleavage of eggs after in-vitro fertilization. Dry sperm of Em remained viable longer than dry sperm of Tg at all temperatures. The viability of dilute sperm and eggs was also determined at 20°, 25°, and 30°C over varying durations (for sperm, 5, 45, and 85 min; for eggs, 1, 3, 6, 12, and 24 h). The viability of dilute Em sperm remained relatively constant over time (nearly 100%) at 20° and 25°C, but decreased rapidly at 30°C. The viability of Tg sperm declined gradually over time at 20°C and decreased sharply at 25° and 30°C. Eggs of Em and Tg remained fertilizable for 12 and 6 h, respectively. Most eggs of Em and Tg underwent normal development when exposed for less than 6 h and 3 h, respectively. These results may illuminate the different fertilization strategies of these two sea urchin species.

As with zebrafish, attention has focused on the teleost medaka Oryzias latipes as an experimental animal representative of non-mammalian vertebrates in various fields of biological science. To enable real-time analyses of the dynamics of nuclei and chromosomes in living medaka cells, we produced a transgenic medaka expressing a fusion protein between histone H2B and green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter. Since the nuclei and chromosomes of transgenic medaka cells are labeled with GFP, their morphological changes can be instantly monitored throughout the mitotic cell cycle progression under a fluorescent microscope without any fixation and staining of samples. However, GFP-labeling of nuclei and chromosomes is not successful during early embryonic development until zygotic expression begins and during the meiotic cell cycle progression, because the CMV promoter does not work in these stages. In addition, histone H2B-GFP fusion proteins are expressed in an organ-specific manner; strong and ubiquitous expression occurs in cells comprising the gut and fin, whereas the expression is restricted to certain types of cells in the liver and brain. These findings suggest that the CMV-driven expression of the histone H2B-GFP transgene is modified depending on the integration site of the transgene in the genome. Nevertheless, easy and precise monitoring of cytological changes in nuclei and chromosomes in the majority of mitotic cells by using the transgenic medaka will greatly contribute to a better understanding of control mechanisms of nuclear and chromosomal behaviors in vertebrate cells.

We studied testes size in free-living and laboratory-born commensal and non-commensal populations of various Mus species (M. musculus musculus, M. m. domesticus, M. spicilegus, M. spretus, M. macedonicus, and laboratory mice). We found no apparent differences between wild-caught and laboratory-born individuals, or among commensal, non-commensal, and laboratory populations of M. musculus. There were, however, considerable differences among the species studied. The highest values of relative testes size were found in the aboriginal species M. spicilegus (4.4% and 2.9% for wild and laboratory populations, respectively), followed by those of M. macedonicus (from 1.7% to 0.9% for various samples) and M. spretus (1.5%). All thirteen samples representing various populations of Mus musculus exhibited smaller testes (0.7–1.0%), and finally the three lowest mean values came from laboratory mice (0.5–0.7%). It is very surprising that aboriginal species, in particular M. spicilegus, which is widely considered to be monogamous, have relatively larger testes than the polygynous/promiscuous M. musculus. This result is in apparent contradiction to the current views on evolutionary forces affecting testes size, and suggests that there could be another uncontrolled factor obscuring the relationship between testes size and multiple paternity. This raises a question concerning the proper interpretation of social organisation in the genus Mus.

The Mississippi River Basin supports the richest fish fauna in eastern North America and has played a key role in the maintenance of fish biodiversity, especially as a refuge for freshwater fishes during glaciations. In this study, we investigated the phylogeography of the bluegill sunfish, Lepomis macrochirus, in eastern North America, using complete sequence of the mitochondrial ND1 gene from 369 samples collected at 15 sites. Phylogenetic analysis revealed two major lineages (northern and southern clades) in a parsimony network. A sympatric distribution of the lineages was widely observed in the Mississippi Basin. Sequence diversity in the two lineages was significantly lower in glaciated regions around the Great Lakes than in unglaciated regions. The two lineages were estimated to have diverged in the Kansan glaciation, and refugia for both existed around the Ouachita Highlands. The southern clade dispersed during the Yasmouth Interglacial, prior to the dispersal of the northern clade during the Sangamon Interglacial. In the northern clade, low genetic diversity and population fragmentation inferred by nested clade analysis (NCA) were considered due to bottleneck events in the Wisconsin glaciation, while the southern clade showed isolation by distance in a Mantel test. A difference in demographic fluctuation suggests that sympatry of the two lineages has resulted from recent secondary admixture through the range expansion of the northern clade in the post-Pleistocene. Large-scale admixture of multiple mtDNA lineages in L. macrochirus, which has not been recorded in other fishes in the Mississippi River Basin, may result from their high vagility.

The salamander Hynobius yatsui from southwestern Japan was formerly regarded as a small-sized group of H. naevius, but has recently been resurrected as a distinct species. We investigated the phylogeography of H. yatsui from Kyushu Island using partial sequences of the mitochondrial cytochrome b gene. We detected 49 haplotypes in 181 individuals from 24 localities covering the entire geographic distribution of this species on Kyushu. These haplotypes were grouped into two lineages, a northern lineage from northern and central regions, and a southern lineage from the southern region; no haplotypes were shared between the lineages. We surmise that the divergence of the two lineages was induced by volcanic activity that started in the Beppu-Shimabara Graben, between the areas occupied by these two lineages. From the results of a nested clade phylogeographical analysis, we surmise that the high intrapopulation genetic variation observed in the non-volcanic Kyushu Mountains was generated by alternation of contiguous range expansion or long-distance dispersal, and isolation. The current distribution and the observed complicated genetic structures of H. yatsui in Kyushu seem to have been affected first by volcanic activities since the late Pliocene, but subsequent climatic oscillations during the Pleistocene may also have some effects, although it is impossible at this time to differentiate the effects of these two factors.

The genetic structure of populations is not necessarily reflected in the geographical proximity of individuals, because environmental gradients such as those of vegetation or climate can function as cryptic barriers to gene flow. We examined polymorphisms at nine microsatellite loci to determine and discuss whether a distinctive genetic structure was detectable in a spatially continuous population of the sika deer (Cervus nippon) on the Boso Peninsula of central Japan. Spatially explicit Bayesian analysis revealed that two genetically distinctive clusters exist in the Boso population. The spatial boundary of the two clusters approximately conformed to the border defined previously from a mitochondrial DNA dataset. By combining information on the geomorphic features surrounding the boundary and that on the lineage of 1970s population, we propose a schematic scenario for characterizing the population genetic structure to the present. The current population consists of genetically different lineages, and spatially discontinuous clusters have come into contact in the vicinity of a local road running along a steep-walled ravine that could act as principal barrier to gene flow. Biological factors such as distribution of vegetation and philopatric behavior might also have helped strengthen the cryptic genetic structure of the Boso population.

Gravitactic bioconvective patterns created by Tetrahymena pyriformis in a Hele-Shaw apparatus were realized and compared with theoretical results. There were found to be two thresholds for bio-convection development: the first indicates the transition from the diffusion to the steady convection state; the second corresponds to the transition from the steady to the unsteady convection state. The results showed that the Hele-Shaw apparatus may be used as a physical analogy of porous media to study 2D bioconvection, with possible extensions to larger scale biological systems where population growth and distribution are driven by similar bio-physical interactions.

SnoN, which belongs to the ski family of nuclear proteins, is a novel oncoprotein; it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. SnoN is an important regulator of signal transduction of the transforming growth factor beta super-family. The present study determined the ovarian localization and regulation of SnoN protein levels in neonatal mice, and in gonadotropin-induced immature mice during follicular development, atresia, and luteinization. In the postnatal mice, positive staining for SnoN was detected for the first time in the interstitial compartment adjacent to the follicles at 7 days and the pattern of immunostaining remained constant. As theca cells differentiated from the stroma, the theca externa layers stained positively for SnoN, and this immunostaining in the theca externa layers persisted in preantral, antral, and preovulatory follicles, even in atretic follicles. Interestingly, the theca interna layers did not contain detected levels of SnoN until the large antral stage of follicular development. In follicular development, SnoN was not expressed in granulosa cells of the healthy follicles but in those that became atretic. After the initiation of luteinization with hCG, SnoN was detected within the luteinizing granulosa cells, and the levels of SnoN were higher during the luteinization process of granulosa cells. Together, our data indicate that SnoN is expressed in a cell-specific manner during ovarian follicular development, atresia, and luteinization and that SnoN might play essential roles in these physiological processes. The present study is the first to investigate SnoN localization and regulation in mouse ovary.

In the genus Oryzias, the morphologies of the dorsal and anal fins are typical secondary sex characters. In the Japanese medaka (Oryzias latipes) and Thai medaka (Oryzias minutillus), androgen receptor (AR) expression levels in the dorsal, anal, and pectoral fins were higher in males than in females. Conversely, in both species estrogen receptor (ER) β expression levels in the dorsal and anal fins were higher in females than in males. AR and ERβ expression levels in the dorsal and anal fins of sex-undeterminable individuals of Thai medaka were intermediate between those in normal male and female Thai medaka. There was no difference in the bone morphogenic protein (Bmp) 2b expression level between male and female Japanese medaka. In contrast, the Bmp2b expression level in the dorsal fin of sex-undeterminable individuals was lower than in normal male and female Thai medaka. It is thus clear that androgen and estrogen regulate the sex-dependent characters of fin morphology in both Oryzias species. In sex-undeterminable individuals of Thai medaka, the low levels of Bmp2b expression in the dorsal fin are evidence that androgen and estrogen are necessary for adequate expression of Bmp2b in the normal development of at least the dorsal fin.

The Chinese brown frog (Rana chensinensis) is a special amphibian in northern China, as it has been used widely in traditional Chinese medicine. The skin of the Chinese brown frog is also a promising resource for producing diverse antimicrobial peptides. To obtain a more comprehensive view of the metabolism and effective pharmacological components of Chinese brown frog skin, we constructed a non-normalized cDNA library from the skin. By sequencing cDNA clones at the 5′ end, we obtained 5,976 high-quality EST sequences, which clustered into 512 contigs and 1379 singletons (in all 1,891 clusters). After BLAST searches of the protein and nucleic acid databases in GenBank, we found 46.7% of clusters to have significant similarity to known sequences; 28% matched Xenopus tropicalis ESTs and 29.1% matched Xenopus laevis ESTs. Gene annotation results indicated that genes related to secretion and defensive function, such as ubiquitin, lectin, and proteinase inhibitors, are highly expressed in the skin. Whey acidic-domain proteins are also highly abundant in the skin. Furthermore, both a β-defensin and a lysozyme are transcribed in the frog skin, providing antibacterial protection. Analyses of gene ontology and KEGG metabolic pathways indicated the physiological roles of Chinese brown frog skin.

We studied variation in morphometric and meristic characters and color pattern in the salamander Hynobius kimurae, examining 282 males from 24 localities encompassing the whole distributional range of the species in Honshu, the mainland of Japan. Multivariate analyses of 24 morphometric characters resulted in the separation of two groups, (1) eastern populations from the Kanto District to Shizuoka Prefecture of the Chubu District, and (2) central-western populations from Aichi Prefecture of the Chubu District westwards. Similar groups were recognized in meristic characters and color pattern. These geographic patterns of morphological variation coincided with the pattern of genetic differentiation inferred from allozymes in this species, except for the position of one population from the intermediate region. Some of the morphometric and meristic characters significantly correlated with environmental parameters of sampling sites, and suggested effects of differential habitat conditions among populations on the geographic morphological variation in this species.