Metalloproteinase inhibitor GM6001 delays regeneration in holothurians.

The effect of the GM6001 metalloproteinase inhibitor on the regeneration of ambulacral structures in Eupentacta fraudatrix has been investigated. Inhibition of proteinase activity exerts a marked effect on regeneration, being dependent on the time when GM6001 is injected. When administration of the inhibitor begins on day 3 post-injury, regeneration is completely abolished, and the animals die. This means that early activation of proteinases is crucial for triggering the regenerative process in holothurians. When GM6001 in first injected on day 7 post-injury, the regeneration rate decreases. However, this effect has proven to be reversible: when inhibition ceases, the regeneration resumes. The effect of the inhibitor is manifested as a retarded degradation of the extracellular matrix, the lack of cell dedifferentiation, and, probably, a slower cell migration. The gelatinase activity is detected in all the regenerating organs of E. fraudatrix. In the holothurian Cucumaria japonica, which is not capable of healing skin wounds and ambulacrum reparation, no gelatinase activity was observed at the site of damage. A suggestion is made that proteinases play an important role in regeneration in holothurians. The most probable morphogenesis regulators are matrix metalloproteinases with gelatinase activity.

Muscle regeneration in holothurians.

The muscle system of holothurians includes visceral (coelomic epithelium) and somatic (longitudinal muscle bands, retractors of aquapharyngeal complex) musculature. Visceral musculature regeneration is achieved by the transformation of myoepithelial cells via their dedifferentiation, migration, proliferation, and redifferentiation. During somatic muscle regeneration the new muscle bundles are formed due to dedifferentiation, migration, and immersion of the coelomic epithelial cells into the connective tissue. While submerging, the epithelial cells transform into myocytes and begin to produce myofibrils in their cytoplasm. Concomitantly, a basal lamina is formed around the group of myogenic cells, separating them from the surrounding extracellular matrix. The myohistogenesis is accompanied by a conspicuous DNA-synthetic activity. Proliferation is insignificant and seems to be of no essential importance for muscle regeneration. The synthesis of DNA followed by no cytokinesis results in an increase in the amount of DNA of myocyte nuclei.

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.