Igor Yu. Dolmatov

Muscle regeneration in the holothurian Stichopus japonicus

The regeneration of longitudinal muscle bands (LMBs) in the sea cucumber Stichopus japonicus was studied using light and electron microscopic and immunocytochemical methods. Previous investigations of holothurian organs showed the presence of some cytoskeletal proteins which were specific for LMBs only. One of them, the 98 KDa protein, was isolated by means of SDS-electrophoresis and used as an antigen to obtain polyclonal antibodies. When tested on paraffin sections of sea cucumber organs, the antibodies were shown to interact only with coelomic epithelial cells covering the LMBs. The antibodies were used to study LMB regeneration after transverse cutting. During regeneration no signs of myocyte dedifferentiation or mitotic division were observed. In the wound region, damaged myocytes degenerated and muscle bundles desintegrated. However, the coelomic epithelial cells dedifferentiated and began to invade the LMB. Just beneath the surface these cells formed clusters (muscle bundle rudiments). The number and size of the clusters gradually increased, the cells lengthened and developed contractile filaments. These observations suggest that new muscle bundles arise from coelomic epithelial cells covering the LMBs. The migration of coelomic epithelial cells into the damaged LMBs and their myogenic transformation are the basic mechanism of holothurian muscle regeneration.

Structures and biological activities of cladolosides C3, E1, E2, F1, F2, G, H1 and H2, eight triterpene glycosides from the sea cucumber Cladolabes schmeltzii with one known and four new carbohydrate chains

Eight new nonsulfated triterpene glycosides, cladolosides C3(1), E1(2), E2(3), F1(4), F2(5), G(6), H1(7) and H2(8) have been isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida) collected in the Vietnamese shallow waters. The structures of the glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. Glycosides 2, 3, 4, and 5 have pentasaccharide branched carbohydrate moieties and differ from each other by monosaccharide compositions and aglycone structures. At that, glycosides 2 and 3 contain three xylose, one 3-O-methyl-glucose and one quinovose residues, while glycosides 4 and 5 have two quinovose, two xylose and one 3-O-methyl-glucose residues. Compounds 1 and 6-8 are hexaosides differing from each other by aglycone structures and by the fifth monosaccharide residue, which proved to be glucose in cladoloside C3(1), xylose in cladoloside G(6) and quinovose in cladolosides H1(7) and H2(8). The presence of quinovose residue in the fifth position, as in 4, 5, 7 and 8 has never been earlier found in carbohydrate chains of triterpene glycosides from sea cucumbers. The carbohydrate chains with xylose in the fifth position of pentaosides and hexaosides are also very unusual for holothurious glycosides. All the substances demonstrate strong or moderate cytotoxic and hemolytic effects with hexaosides being more active than the corresponding pentaosides. Peculiarities of the biosynthesis and biochemical evolution of glycosides of this type are discussed.

Proteases from the Regenerating Gut of the Holothurian Eupentacta fraudatrix

Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2–4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed.

Metamorphosis and definitive organogenesis in the holothurian Apostichopus japonicus

Abstract The structure of the late doliolaria, pentactula and 1-month-old juvenile of the holothurian Apostichopus japonicus was studied using light microscopy and 3D reconstruction methods. It was shown that metamorphosis in this species consists in the reorganization of the shape of the body and the destruction of provisional organs. The late doliolaria has a spindle-like form, ciliary rings and hyaline spheres shifted relative to the anterior-posterior axis of the body. Some provisional organs (ciliary rings, hyaline spheres) are destroyed during settlement, and others (hydropore and hydroporic canal) remain after metamorphosis. Definitive organogenesis in A. japonicus begins long before metamorphosis. The late doliolaria already has well-developed water-vascular and digestive systems, and the ectoneural part of the nervous system. Muscle and hemal systems begin to form in the pentactula. Moreover, the calcareous ring and connective tissue part of the body wall develop at this stage. The pentactula has anlages of the hyponeural part of the nervous system, which form in the mid-ventral and dorsal nerve cords. The hemal ring of the pentactula is located on the inner wall of the water-vascular ring. It remains unclosed in the left ventral radius. One-month-old juveniles have all the major organ systems except respiratory and reproductive systems. The hemal vessels of the intestine are well developed and begin to form the rete mirabile. Differentiation of the intestine into regions due to differential specialization of the enterocytes begins in 1-month-old juveniles. Obviously, emergence of new types of enterocytes enables the animal to consume a wider range of food items and indicates its increased feeding activity.

Posterior regeneration following fission in the holothurian Cladolabes schmeltzii (Dendrochirotida: Holothuroidea)

The regeneration of the posterior portion of the body after fission was studied in the holothurian Cladolabes schmeltzii using electron microscopy methods. Following fission, the aquapharyngeal complex, gonad and anterior portion of the first descending part of the intestine remain in the anterior fragment of the body. The entire regeneration process is divided into five stages. In the first three stages, the digestive system and damaged ends of the longitudinal muscle bands regenerate. The intestine is formed through the rearrangement and growth of the remaining portion of the first descending part of the intestine. The gut anlage grows down the mesentery and joins the regenerating cloaca. The cloaca is formed from two sources: its posterior portion appears as a result of immersion of the epidermis, while the anterior portion develops from the terminal segment of the growing intestine. Regeneration of muscles progresses in the typical manner for echinoderms: through immersion and myogenic transformation of the coelomic epithelium. Respiratory trees appear in animals when the growth of the external part of the body has begun (fourth stage). They are formed as an outgrowth of the dorsal wall of the anterior portion of the cloaca. It was concluded that regeneration of the posterior portion of the body in the holothurian C. schmeltzii following fission is realized through morphallactic rearrangements of the remaining parts of organs. The main mechanism through which the digestive, respiratory, and contractile systems are formed is epithelial morphogenesis. Microsc. Res. Tech. 78:540–552, 2015.

Anterior regeneration after fission in the holothurian Cladolabes schmeltzii (Dendrochirotida: Holothuroidea).

The regeneration of the anterior portion of the body after fission was studied in the holothurian Cladolabes schmeltzii using electron microscopy methods. Following fission, the posterior portion of the digestive tube, cloaca, and respiratory trees remain in the posterior fragment of the body. The regeneration comprises five stages. In the first stage, connective‐tissue thickening (an anlage of the aquapharyngeal bulb) occurs on the anterior end between the torn‐off ends of the ambulacra. Most of the lost anterior organs developed in the second and third stages. The structures of water‐vascular system and nerve ring form through dedifferentiation, proliferation, and migration of cells of the radial water‐vascular canals and the radial nerve cords, correspondently. The lost digestive system portion is restored through the formation and merging of two anlagen. The digestive epithelium of the esophagus and pharynx develops from lining cells of microcavities near the central portion of the connective‐tissue thickening, which probably migrate from the epidermis. The second gut anlage develops through transformation of the anterior gut remnant portion. The enterocytes partly dedifferentiate, but the epithelium retains integrity. The gut anlage grows down the mesentery and joins the regenerating aquapharyngeal bulb. In the fourth and fifth stages, all lost organs are formed and have nearly normal structure. The regeneration was concluded to occur through morphallactic rearrangements of the remaining parts of organs. Epithelial morphogenesis is the key development mechanism of the digestive, water‐vascular, and nervous systems.

Digestive system formation during the metamorphosis and definitive organogenesis in Apostichopus japonicus

We studied the ultrastructure of the digestive system in late doliolaria, pentactula, and 1-month-old juvenile of the holothurian Apostichopus japonicus. In late doliolaria and pentactula, the digestive system is divided into three segments: pharynx, gut, and cloaca. Pharynx and the posterior part of cloaca are lined by cuticular epithelium and apparently of ectodermal origin. The anterior part of cloaca and the entire gut is not differentiated histologically and is lined by a single type of digestive cells, vesicular enterocytes I. These cells are characterized by large secretory granules, containing an acidic substance, found in cytoplasm. As the anterior part of the cloaca is lined by cells typical of the endodermal segment of digestive tube (vesicular enterocytes I), we suggest this part to be of endodermal origin and probably formed from the larval stomach. In 1-month-old juveniles, the structure of the digestive system grows more complicated. In addition to vesicular enterocytes I, three more types of enterocytes appear in luminal epithelium. The specific distribution of the four types of digestive cells divides the intestine into three parts, each probably performing its own function. All enterocytes develop long microvilli, which indicate the intensification of the extracellular digestion processes and an increased absorption of dissolved nutrients.

Wnt and frizzled expression during regeneration of internal organs in the holothurian Eupentacta fraudatrix

Several genes of the Wnt and Frizzled families in the holothurian Eupentacta fraudatrix are characterized, and the complete coding sequences of wntA, wnt4, wnt6, wnt16, frizzled1/2/7, frizzled4, and frizzled5/8 are obtained. The dynamics of expression of these genes during regeneration of internal organs after evisceration are studied. Evisceration and the associated damages supposedly induce the expression of wnt16 on third day after evisceration. Genes wntA, wnt4, wnt6, and frizzled1/2/7 up‐regulate during the period of active morphogenesis (5–7 days after evisceration) and might participate in regulation of tissue and organ formation. The signaling induced via Frizzled5/8 is could be necessary for formation of the anterior (ectodermal) part of the digestive system and development of the calcareous ring on 10th day after evisceration. Our data suggest that the Wnt signaling pathway plays a significant role in the regulation of regeneration of internal organs in holothurians.

Cladolosides I1, I2, J1, K1, K2 and L1, monosulfated triterpene glycosides with new carbohydrate chains from the sea cucumber Cladolabes schmeltzii

Six new monosulfated triterpene glycosides, cladolosides I1 (1), I2 (2), J1 (3), K1 (4), K2 (5) and L1 (6) were isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida). Structures of these glycosides were elucidated by 2D NMR spectroscopy and mass spectrometry. Four new types of carbohydrate chains have been found in 1-6. Cladolosides of the groups I and J are characterized by pentasaccharide carbohydrate chains sulfated at a terminal 3-O-methylglucose residue and branched by the position 4 of the first xylose residue, but differing from each other in the lengths of the main and side carbohydrate chains. Cladolosides of the groups K and L contain hexasaccharide chains with different positions of a sulfated terminal 3-O-methylglucose residue (as the fourth or the sixth monosaccharide residue). Sulfated hexasaccharide carbohydrate chains were found in the sea cucumbers glycosides for the first time. A pentasaccharide carbohydrate chain of cladoloside J1 (3) having a disaccharide moiety of glucose and a sulfated 3-O-methylglucose linked to the first xylose residue in a linear trisaccharide fragment is also unusual. All substances studied demonstrated strong or moderate hemolytic and cytotoxic effects.

Formation of the ectodermal organs during the metamorphosis and definitive organogenesis in the holothurian Apostichopus japonicus

The fine structure of the ectodermal organs (epidermis and nervous system) in late doliolaria, pentactula, and 1-month-old juveniles of the holothurian Apostichopus japonicus was studied. The definitive structure of the epidermis is shown to start forming prior to metamorphosis. The cell types of late doliolaria epidermis are the same as those in adult holothurian epidermis, consisting of support, secretory, and granular cells. The rivet-like structures are also present already at this stage of development. The ciliary rings of late doliolaria represent ribbon-like aggregations of tall ciliary cells. The destruction of ciliary rings is due to the programmed death of ciliary cells, which are then utilized by mesenchymal cells. Changes to the epidermis during metamorphosis are linked to the sinking of the cells into the underlying extracellular matrix, resulting in the formation of the typical sunken epidermis of the body and appendages. The nervous system in late doliolaria consists of a nerve ring, five radial nerve cords, and tentacle and tube foot nerves. The nerve ring and the radial nerve cords represent epithelial tubes. First hyponeural cells appear in the mid-ventral nerve cord during the late doliolaria stage. In the rest of the nerve cords, the hyponeural part begins developing only after the settlement. Functioning of the adult nervous system in holothurians begins only after metamorphosis, following the complete loss of provisory organs and the switch to benthic life.