Intracellular Localization and Sedimentation Coefficient of DNA Ligase in Oocytes of the Starfish, Asterina pectinifera

When immature oocytes of Asterina pectinifera were separated into karyoplasts (nuclear fragments) and cytoplasts (anuclear fragments) by cytochalasin B treatment and centrifugation in a sucrose gradient, almost all their DNA ligase activity was recovered in the karyoplasts. Thus, DNA ligase seems to be localized in the germinal vesicles or their vicinity in starfish oocytes. No ontogenic change in the activity of DNA ligase per oocyte, egg or embryo, or in its sedimentation coefficient (4.1 S) was observed during oocyte maturation, fertilization, or early development.     

Origin of Germ Cells and Early Differentiation of Gonads in the Starfish, Asterina pectinifera

The origin of the germ cells and the development of the genital system in the annually spawning starfish, Asterina pectinifera , were studied by light and electron microscopy. Characteristic germ cells were first characterized in gonads after spawning: the gonia are larger than somatic cells, have large nuclei (with electron-lucent nucleoplasm), and show mitochondrial aggregation associated with nuage (electron-dense bodies). In young starfish without gonads similar cells were detected in the haemal sinus, where they were termed primordial germ cells (PGCs). Brachiolariae and metamorphosed juveniles had a cellular cluster in the coelomic epithelium, near the hydroporic canal. The cluster was comprised of cells endowed with the above-mentioned characteristics of the germ cells. The germ cell counts indicated that PGCs migrate from the aboral haemal sinus near the hydroporic canal, through the haemal sinus to the gonads, where they settle, proliferate, and differentiate into gonia.     

Spawning Inhibitor in Gonads of the Starfish,Asterina pectinifera

A biologically active substance which inhibits spawning of the starfish, Asterina pectinifera, has been isolated from gonads of the same organism and identified as l-glutamic acid.     

Nature of the 1-Methyladenine-Requiring Phase in Maturation of Starfish Oocytes

Reinitiation of meiosis in starfish oocytes requires the continuous presence of 1-methyladenine (1-MeAde) in the surrounding medium for a definite period. The length of the 'hormone-dependent phase' (HDP) in Asterina pectinifera , which was defined as the time necessary for induction of 50% germinal vesicle breakdown (GVBD), was found to be about 11 min at 17°C, and 8 min at 20°C. Repeated treatments for shorter periods with 1-MeAde revealed that the action of this agent was cumulative, and that stable intermediate states between the unstimulated and fully stimulated levels existed during the HDP. Measurement of the stiffness of oocytes also demonstrated this stable intermediate state. Thus, there may be a factor(s) in the cytoplasm that accumulates continuously during the HDP and triggers GVBD when it reaches a critical level(s). When dithiothreitol (DTT) was used as an artificial maturation-inducing agent, the intermediate state was far less stable, suggesting a difference in the modes of action of 1-MeAde and DTT. Isotonic CaCl₂, the Ca²⁺ ionophore (A 23187) and methylxanthines, which are known to cause increase in intracellular Ca²⁺, had additive effects with 1-MeAde. These results suggest that part of the action of 1-MeAde is to release Ca²⁺ in the oocyte cytoplasm.     

Intracellular Localization of DNA Polymerases in the Oocyte of Starfish, Asterina pectinifera

Oocytes of the starfish, Asterina pectinifera , were separated into karyoplasts and cytoplasts by centrifugation in a sucrose density gradient in the presence of cytochalasin B. More than 90% of the DNA polymerase activity of whole oocytes was recovered in the karyoplasts, and less than 10% in the cytoplasts. DNA polymerase α was specifically localized in karyoplasts. Of the DNA polymerase β activity of whole oocytes, 70–80% was found in the karyoplasts, and about 15% in the cytoplasts. The problem of whether DNA polymerases are localized within germinal vesicles in starfish oocytes is discussed.     

Germ Cell Differentiation in Starfish: The Posterior Enterocoel as the Origin of Germ Cells in Asterina pectinifera

The origin of germ cells of Asterina pectinifera was traced back to the posterior enterocoel (PE) of 2-day bipinnaria by two steps. First, the cellular cluster, composed of presumptive germ cells in the coelomic epithelium at brachiolaria stage, was confirmed to be the origin of the aboral haemal sinus located near the hydroporic canal (HC-AHS) by continuous observation of the formation process of HC-AHS. Second, the origin of the cluster was traced back to the PE of 2-day bipinnaria by comparison of the number of the presumptive germ cells in microsurgically PE-removed bipinnariae with that of non-operated control larvae. A summary of the differentiation of germ cells in Asterina pectinifera is given/presented.     

Cloning,Heterologous Expression,and Enzymatic Characterization of Cathepsin L from Starfish (Asterina pectinifera)

Methyl esters of 3-epi-GA₃ and 3-epi-GA₁ were efficiently prepared from methyl esters of GA₃ and GA₁ respectively, by highly selective epimerization of the 3-hydroxyl function with a base in a low-polar aprotic medium.     

Seasonal variations in polyhydroxysteroids and related glycosides from digestive tissues of the starfish Patiria (=Asterina) pectinifera

Seasonal variations in the concentrations of individual polyhydroxysteroids and related low molecular weight glycosides in pyloric caeca and stomach of the starfish Patiria (=Asterina) pectinifera collected at one location near Vladivostok have been studied. HPLC analysis on the fractions containing these substances showed a fairly constant composition of steroids in digestive tissues of P. pectinifera in spite of small seasonal variations in the relative concentrations of individual compounds.     

Seasonal variations in the levels of polyhydroxysteroids and related glycosides in the digestive tissues of the starfish Patiria (Asterina) pectinifera

Seasonal variations in the levels of polar steroids including polyhydroxylated steroids and related glycosides in digestive organs of the starfish Patiria (=Asterina) pectinifera have been studied. The concentration of polar steroids is related to the annual reproductive cycle of the starfish and periods of active feeding. Two peaks in concentrations of polar steroids in pyloric caeca and stomach were found, the first in winter during reorganization and the second in summer during intensive gametogenesis before spawning. Probable biological functions of polyhydroxysteroids and related glycosides are discussed. The data support the hypothesis these compounds are involved in digestion in the starfish.     

Oocyte Competence to Maturation-inducing Hormone. I. Breakdown of Germinal Vesicles of Small Oocytes in Starfish,Asterina pectinifera*

1-Methyladenine (1-MeAde) is the endogenous maturation-inducing substance (MIS) in starfish. However, small oocytes have no competence to 1-MeAde even at the concentration of 10^?5M. Furthermore, when they were injected with cytoplasm of fully-grown (large) and maturing (1-MeAde-treated) oocytes, known to contain maturation-promoting factor (MPF), they did not undergo germinal vesicle breakdown (GVBD). On the other hand, germinal vesicles (GV) of the small oocytes underwent nuclear breakdown when the small oocytes were fused with the large maturing oocytes. Therefore it is concluded that the GV of the small oocytes are capable of undergoing nuclear breakdown in the presence of the sufficient MPF, but that the small oocytes can not amplify the injected MPF. Fused cells displayed particular shape changes during the course of nuclear breakdown of both the large and the small oocytes.     

Archenteron-Forming Capacity in Blastomeres Isolated from Eight-Cell Stage Embryos of the Starfish, Asterina pectinifera

Starfish blastomeres are reported to be totipotent up to the 8-cell stage. We reinvestigated the development of blastomeres of 8-cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8-cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8-cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4-cell stage embryos along the animal-vegetal axis so that all the blastomeres in the 8-cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single-layered embryos at the 8-cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.     

The Role of Cell Adhesion in the Differentiation of Mesendodermal Tissues in the Starfish, Asterina pectinifera

The role of cell-to-cell adhesion in the early embryogenesis of the starfish Asterina pectinifera was studied by using concanvalin A (ConA), an agent known to weaken cellular contact by binding to glycosides at the cell surface. The major change in morphology was a diminution in the volume of the endodermal tissues (the digestive tract) of the treated larvae. It was found by pulse treatment that this effect of ConA was stage-specific, and that the effective period corresponded to the stage when blastomeres become more cohesive. The number of cells in the mesodermal tissues, however, was relatively constant while the volume of the endodermal tissue varied considerably. It was suggested that cell-to-cell adhesion during this stage is involved in the allocation of endodermal tissues. In contrast, mechanisms other than cell adhesion were considered to be important for the differentiation of the mesodermal tissues.     

The Reaction of the Starfish Asterias amurensisand Patiria pectinifera (Asteroidea) from Vostok Bay (Sea of Japan) to a Salinity Decrease

The reactions of the starfish Asterias amurensis and Patiria pectinifera that live in Vostok Bay at the salinity of 32–33 to a salinity decrease were studied under laboratory conditions. The lower limits of the desalination tolerance range of A. amurensis and P. pectinifera were, respectively, 24 and 20. A. amurensis proved to be less resistant to desalination. Under experimental conditions, all specimens of this species survived the salinity of 22, while those of P. pectinifera tolerated 18. At the same time, A. amurensis responded more actively than P. pectinifera to unfavorable changes in the environment. Turned to their dorsal side and exposed to a salinity of 16 to 32, the former reverted to the normal position within a shorter time than the latter. Being a more euryhaline species, P. pectinifera endured a salinity decrease to 6 or 8 over, respectively, 21 or 28 h. However, only 30–40% of all specimens could recover locomotory activity 12 or 8.5 h after being placed into water of normal salinity.     

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in various body components of the starfish Patiria (=Asterina) pectinifera

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in different body components of the Far-eastern starfish Patiria (=Asterina) pectinifera has been studied. It was shown that free sterol fractions from aboral and oral body walls, gonads, stomach and pyloric ceca contained Delta(7) sterols with a preponderance of 5alpha-cholest-7-en-3beta-ol. All these body components had also toxic steroid oligoglycosides. However, polyhydroxysteroids and related low molecular weight steroid glycosides were found in stomach and pyloric ceca only. In pyloric ceca, the sulfated monoside 'asterosaponin' P(1) was identified as a main polar steroid, whereas 6-sodium sulfate of cholestane-3beta,4beta,6alpha,7alpha,8,15beta,16beta,26-octaol predominated in the stomach. Probable biological functions of polar steroids and free sterols in this starfish were discussed. It was suggested that some polyhydroxysteroids and related monoglycosides play the same biological role as bile alcohols and bile acids do in vertebrates.     

Changes in the Composition of Celomic Fluid Metabolites of the Black Sea Urchin Mesocentrotus nudus (Echinoidea) and the Starfish Asterina pectinifera (Asteroidea) under Conditions of Hypoxia Stress

Biology Bulletin - The composition of metabolites in the coelomic fluid of the starfish Asterina pectinifera and sea urchin Mesocentrotus nudus was studied under normal and hypoxic conditions using...     

Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina) pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated sterols and monoglycoside asterosaponin P1 in feeding experiments

For the first time, it is experimentally established that the dietary cholesterol and cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low molecular weight glycosides in starfishes. These deuterium labeled precursors were converted into partly deuterated 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol, and steroid monoside asterosaponin P₁ in result of feeding experiments on the Far Eastern starfish Patiria (=Asterina) pectinifera. The incorporations of deuterium were established by MS and NMR spectroscopy. Scheme of the first stages of biosynthesis of polar steroids in these animals was suggested on the basis of inclusion of three from six deuterium atoms and determination of their positions in biosynthetic products, when [2,2,3,4,4,6-²H₆]cholesterol 3-sulfate was used as precursor. It was also shown that labeled cholesterol is transformed into Δ⁷-cholesterol (lathosterol) in digestive organs and gonads of the starfish.     

Characterization of O-GlcNAcylation in starfish (Asterina pectinifera) development from fertilization to bipinnaria larva

Though O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins has been found in many multicellular organisms, its presence or absence in Echinodermata is unknown. Here we report the occurrence of O-GlcNAcylation in starfish (Asterina pectinifera) oocytes and the apparent O-GlcNAcylation pattern in starfish early development. O-GlcNAcylation might participate in the regulation of starfish development at the mid-blastula stage and thereafter.     

Effect of Estradiol Dipropionate on Protein Synthesis in Oocytes and Ovary of the Sea Urchin Strongylocentrotus intermedius at Different Stages of the Reproductive Cycle

We studied protein synthesis in the oocytes and ovary of the sea urchin Strongylocentrotus intermedius at different stages of the reproductive cycle after treatment with estradiol dipropionate. During the early development of oocytes and active gametogenesis, this estrogen induced the incorporation of ³H-leucine in the oocytes. The changes in synthetic activity of cells were accompanied by an elevated efficient incorporation of free amino acid in proteins due to its increased pool, increased tissue permeability for precursors, and increased rate of protein synthesis. Before spawning, estradiol dipropionate did not cause any changes in protein synthesis. After estradiol dipropionate treatment, the inhibitors of protein synthesis, puromycin and actinomycin D, decreased the intensity of ³H-leucine incorporation in the oocytes and protein synthesis in the ovary. The involvement of estradiol dipropionate in the regulation of protein synthesis in the sea urchin gonad is discussed.     

PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.