It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates) and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase (EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar H -ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellinecdysteroid-phosphates complex and the dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.

We have conducted the first phylogenetic study to our knowledge of Zoanthus in the northern hemisphere by sequencing and analysing the mitochondrial cytochrome oxidase subunit 1 (COI) gene. Various unidentified Zoanthus specimens and samples of what have been assumed to be four discrete species (Z. pacificus, Z. sansibaricus, Z. gnophodes, Z. erythrochloros) were collected from four field sites in Kagoshima Prefecture, Japan. Based on our obtained COI gene sequences, all but one of our collected Zoanthus samples appear to be conspecific, with nearly 100.00% base pair matching. Genetic results are further backed up by collected polyp diameter, tentacle count, and mesentary count data. These results indicate a need to reconsider and re-analyze current Zoanthus classification and identification. Possible reasons for the large morphological variation in the same genotype in Zoanthus are also discussed.

Chemical modification of glycerinated stalks of Vorticella with TNM is used to investigate the role of tyrosine residues in the Ca² -induced contraction of the spasmoneme. Tetranitromethane (TNM) is often employed as a specific reagent for the nitration of tyrosine residues in a protein at neutral and slightly alkaline pHs although TNM can also oxidize cysteine residues in the acidic and neutral pH range. Prior incubation with Ca² of stalks to be treated with TNM can protect the spasmoneme from irreversible denaturation. On the other hand, TNM treatment in the absence of free Ca² causes an irreversible denaturation of the spasmoneme. It was revealed by us that an isolated Ca² -binding protein called spasmin could not bind with Ca² after TNM treatment, even if the treatment was performed in the presence of Ca² . In an additional experiment, we confirmed that the chemical modification of cysteine residues in the spasmoneme with N-7-dimethyl-amino-4methyl-coumarinyl-maleimide (DACM) has no effect on the contractibility. These results suggest that tyrosine residues in spasmin are essential for spasmoneme contraction and are protected from TNM in the presence of Ca² when spasmin binds with its receptor protein in the spasmoneme.

We describe development from fertilization to metamorphosis of the enteropneust hemichor-date Balanoglossus misakiensis. This is the first report to describe the complete development of an indirect-developing hemichordate under laboratory conditions. Mature adults were induced to spawn by shifting the temperature of seawater from 23 to 28°C. Eggs (200 μm diameter) were enclosed within a nonmucilaginous membrane, and dispersed readily in seawater. After artificial insemination, a fertilization envelope was elevated from the egg surface beneath the egg membrane; this was followed by the formation of the first and second polar bodies within the envelope. Zygotes cleaved at 20-min intervals to form blastulae, and gastrulation started 9 h after fertilization. Embryos hatched 1 day after fertilization to become typical feeding tornaria larvae. The larvae metamorphosed 7–10 days after fertilization without undergoing the first (Müller) or forth (Krohn) stage of indirect-developing hemichordate development. Larvae that were not fed failed to metamorphose. Juveniles completed adult body formation within a week of settling in sand at the bottom of the culture tube. We discuss heterochronical modifications of B. misakiensis development, and make the case for this species as a potential model organism for the investigation of indirect-developing hemichordates.

Retinoic acid (RA), the active derivative of vitamin A, is essential for normal embryonic development of vertebrates because both the lack and excess of RA result in developmental malformations. We previously reported that aryl hydrocarbon receptor (AHR) is also required for vascular and bone formation by regulating cytochrome P450 expression. However, little is known about the roles of retinoic acid receptors (RAR) and retinoid X receptors (RXR) in the embryonic development of blood vessels and molecular cross-talk between RAR/RXR and AHR. We report for the first time that RA and RAR/RXR are required for expression of AHR mRNA and the embryonic development of blood vessel and bone. The embryonic organogenesis of medaka fish was specifically inhibited by an inhibitor of RA synthesis (diethylaminobenzaldehyde), antagonists of RAR (Ro41-5253) and RXR (Ro71-4595), agonist (β-naphthoflavone) and antagonist (α-naphthoflavone) of AHR, and excess RA. These reagents are useful for future studies to elucidate molecular mechanisms for vascular and bone formation in the medaka embryogenesis. Our results also show that medaka embryos may be useful for screening inhibitors of vascular formation for anti-cancer drugs.

The phylogenetic position of ascidians near the base of the chordate tree makes them ideal organisms for evolutionary developmental studies of programmed cell death (PCD). In the present study, the following key features of an apoptotic form of PCD are described in Boltenia villosa: fragmentation of DNA, increases in plasma membrane permeability, decreases in mitochondrial activity, production of reactive oxygen species (ROS), and caspase activation. First, evidence is presented for apoptosis of cells within the ovary. Later in development, during the early phase of larval tail resorption at the beginning of metamorphosis, some notochord nuclei showed DNA fragmentation and their cell corpses were rapidly eliminated from the larval body. In striking contrast to the rapid demise of notochord cells, larval muscle cells persisted for more than a week within developing juveniles. Rhodamine 123 and MTT experiments suggest that mitochondria within some of the resorbed larval tail muscle cells were metabolically active for more than a week. Furthermore, resorbed tail muscle cells contained a muscle-specific intermediate filament, termed p58, despite relatively high levels of ROS activity and the ubiquitination of their plasma membranes at day two. Corpses of larval tail muscle cells containing aggregated pigment granules survived within juveniles for more than a month, in contrast to the rapid elimination of notochord cells. Evidence consistent with the formation of larval muscle cell apoptotic bodies is presented. The most surprising result of the present study was that caspase-8, usually associated with apoptotic signaling, was activated in larval endoderm cells that develop into adult structures. When the present results were compared to features of PCD previously reported in other ascidians, significant species differences in PCD were revealed.

Changes in serum 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-DP) levels around the gestation time of normal pregnant and experimentally non-pregnant females were investigated in the black rockfish, Sebastes schlegeli, a viviparous teleost. The serum 17,20β-DP in both females showed similar changes and levels, increasing from the early to late gestation periods and declining just before parturition in pregnant females and egg-release in non-pregnant females, respectively. These results suggest that the maintenance of high serum levels of 17,20β-DP after oocyte maturation is not correlated with gestation or parturition, but occurs spontaneously in this species. The decline of 17,20β-DP levels prior to egg-release in non-pregnant females tended to occur one week earlier than those prior to parturition in pregnant females, suggesting that both a decline in 17,20β-DP levels in mothers and some response from embryos are needed for a smooth parturition. The post-ovulatory follicles were maintained throughout the gestation period and produced a considerable amount of 17,20β-DP in vitro (3.44–6.96 pg/ml/mg tissue), but little estradiol-17β (0.92–1.66 pg/ml/mg tissue). The production of 17,20β-DP tended to be enhanced by the addition of a precursor steroid, pregnenolone, in the pre-, early and mid-gestation periods. These results strongly suggest that the follicle cells in black rockfish have the ability to synthesize 17,20β-DP during the post-ovulatory period, and high serum 17,20β-DP during gestation is supplied by the post-ovulatory follicles, which in Sebastes are considered to be functionally homologous to the mammalian corpus luteum.

We investigated the photoperiodic response of serotonin- and galanin (GA)-immunoreactive (ir) cells in the paraventricular organ (PVO) and infundibular nucleus (IF) of the Japanese quail and the interaction of these cells with gonadotropin-releasing hormone (GnRH)-ir neurons in the hypothalamus. Serotonin-ir cells were located in series from the PVO to the IF, and were connected with each other. The number of serotonin-ir cells differed significantly between light and dark phases on the short days (SD), but did not differ between light and dark phases on long days (LD). GA-ir cells were also found in the PVO and IF. The number of GA-ir cells under SD conditions was significantly greater than under LD conditions but did not change diurnally. Both serotonin-ir and GA-ir fibers ran along the GnRH-ir cells in the nucleus commissurae pallii. Serotonin-ir and GA-ir fibers were connected with the GnRH-ir fibers in the external layer of the median eminence (ME). We confirmed that GA-ir fibers were closely associated with serotoninir neurons in the PVO and IF. GA-ir neurons have at least 2 routes of regulating GnRH neurons directly, and indirectly via the serotonin-ir cells in the PVO and IF.

All termite species possess a distinct sterile-soldier caste in their colonies, although reproductive soldiers, with soldier characteristics and reproductive ability, have been reported from several species of the family Termopsidae. Such intercastes have been considered the primitive-soldier caste, and based on this many researchers have discussed the evolutionary origin of termite soldiers. We investigated whether such soldier-reproductive intercastes also exist in the Japanese rotten-wood termite Hodotermopsis sjostedti. Abnormal individuals with soldier-like characteristics were found and designated as soldier-like intercastes, which appeared to have both soldier and reproductive characteristics. Based on our morphometric analyses and histological examinations, we suggest that the developmental origin of this inter-caste is a pseudergate, nymph or sixth-instar larva. In addition, the intercaste was found to have relatively well-developed gonads, although mature oocytes and spermatozoa were not found in female ovaries and male seminal vesicles, respectively. We conclude that the soldier-like intercaste of H. sjostedti does not have reproductive ability, which is different from all other known soldier-like intercastes in Termopsidae.

Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were 1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b genes on the phylogenetic tree of the mammalian MyHC genes. These results suggest that an ancestral MyHC-2b gene had lost its function and changed to a pseudogene during the course of horse history. The MyHC genes in some ungulates were analyzed through the PCR amplifications using the MyHC isoform-specific primers to confirm the presence of the MyHC-2b and -2x genes. The exon coding the 3′ untranslated region of the MyHC-2x was successfully amplified from the all ungulates examined; however, that of the MyHC-2b gene was amplified only from horses, pigs and lesser mouse deer. The PCR analyses from rhinoceros, sika deer, moose, giraffes, water buffalo, bovine, Japanese serow and sheep genes implied the absence of the MyHC-2b-specific sequence in their genomes. These results suggest that the MyHC-2b gene independently lost its function in some ungulate species.