Flounder metamorphosis: its regulation by various hormones

Metamorphosis in the flounder has often been compared with the transition of tadpoles into frogs. The dorsal fin rays of the Japanese flounder (Paralichthys olivaceus) elongate during prometamorphosis when thyroid hormone levels are low, and are resorbed during metamorphic climax when thyroid hormone levels are high. Using an in vitro system for the culture of the flounder fin rays, we have examined how various hormones affect the resorption process. Both thyroxine (T₄) and triiodothyronine (T₃) directly stimulated fin ray shortening, T₃ being more potent than T₄. Other hormones, such as prolactin, cortisol and sex steroids, did not directly affect the resorption process but modified the tissue's response to thyroid hormones. Similar observations were obtained from in vivo studies. We also monitored the changes in the whole body concentrations of various hormones during early development and metamorphosis, and related these with the thyroid hormone profiles in order to get a better picture of their interactions. The gaps in the present status of research on the role of thyroid hormones during metamorphosis in the Japanese flounder are also discussed.     

Changes in plasma prolactin and growth hormone concentrations during freshwater adaptation of juvenile chum salmon (Oncorhynchus keta) reared in seawater for a prolonged period

Freshwater adaptability of chum salmon was examined in juvenile fish reared in seawater for 4 months. The fish, weighing about 40g, were transferred directly to fresh water in October, when their cohorts are migrating in the North Pacific Ocean. Plasma sodium concentration decreased from 167 mM in seawater to about 130 mM during the first 24h, and increased gradually during 2–7 days after the transfer. No immunoreactive prolactin (PRL) was detected in the plasma of the seawater-adapted fish nor during the first 24h in fresh water. Significant levels of PRL were detected after 2–3 days. The maximal level (2.6 ng/ml) was observed after 5 days and became undetectable again after 7 days; no significant correlation was seen between the changes in plasma sodium and PRL levels during the transfer. Plasma growth hormone levels were relatively constant, except for a significant decrease 12h after the transfer. Although plasma thyroxine levels were highly variable during the experiment, a significant decrease and an increase were observed 12h and 5 days after the transfer, respectively. The present study indicates that juvenile chum salmon retain hyperosmoregulatory ability even after prolonged rearing in seawater. Examination of turnover rates, rather than changes in plasma levels, seems to be essential to clarify the osmoregulatory roles of the hormones.     

Effect of collar insertion on soil respiration in a larch forest measured with a LI-6400 soil CO2 flux system

Little information is available on the effect of root cutting by the collar pre-insertion technique on soil respiration. In this study, we found that soil respiration rates decreased with increasing depth of collar insertion in both the with live roots intact and with live roots severed treatments, but the rate of decrease was substantially higher in the former. The cutting of roots, especially fine roots, may be responsible for this result.     

Improvement of fiberboard made from acetylated fibers by ozonation I: effect of ozonation on mechanical properties

Acetylated fibers with a 24.8% weight gain by acetylation were ozonated with 0%–2.0% ozone on fibers. Fiberboards were then made from these treated fibers. The internal bond and bending strength of the acetylated fiberboards increased drastically with increasing ozone charge up to 0.5%–1.0%, whereas the thickness swelling of the fiberboards decreased. Ozone selectively cleaves the aromatic rings of hydrophobic lignin and introduces a hydrophilic carboxyl group into lignin. Thus, the wettability and thermoplasticity of acetylated fibers increased, and this structural modification improved the interfiber contact. Internal bond and bending strength increased as a result. In addition, steep density profiles were formed by the ozonation, resulting in high bending strength. The high compaction ratio accelerated the effect of ozonation. The optimum ozone charges for improving mechanical properties were 0.5%–1.0%.Part of this report was presented at the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Changes in intracellular and cell surface localization of Le(x) epitope during germ cell differentiation in fetal mice.

Changes in the intracellular and cell surface localization of Lewis x (Le(x)) determinants in germ cells during fetal development in mice were examined by light and electron microscopy. Light microscopically, in undifferentiated gonads on day 12 post coitum (p.c.), the anti-Le(x) monoclonal antibody (MAb) was specifically bound to the plasma membranes and to the cytoplasmic granule-like structures of germ cells. In the testes on day 13 p.c., most of the germ cells were enclosed within the testicular cords and showed an MAb-positive reaction which was restricted mainly to the cytoplasmic granule-like structures. The reaction on the plasma membranes almost disappeared. On the other hand, the ectopic germ cells still showed a positive reaction on their plasma membranes. In the ovaries on day 13 p.c., the germ cells also exhibited positive reactions both on the plasma membranes and on the granule-like structures. Immunoelectron microscopic observations agreed well with these light microscopic observations in such a way that both the plasma membranes and the "small dense bodies" (SDB) were positive in undifferentiated gonads on day 12 p.c. In the germ cells organized into the testicular cords, the reaction to anti-Le(x) MAb became restricted to the SDB. These results may indicate that such intracellular changes in Le(x) determinants during germ cell differentiation are associated with the enclosure of germ cells within the testicular cords.     

Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.     

Relationship among ovarian follicular status,developmental competence of oocytes,and anti‐Müllerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts

The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti‐Müllerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross‐sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.