Mechanical properties of the compass depressors of the sea-urchin Paracentrotus lividus (Echinodermata, Echinoidea) and the effects of enzymes, neurotransmitters and synthetic tensilin-like protein

The compass depressors (CDs) of the sea-urchin lantern are ligaments consisting mainly of discontinuous collagen fibrils associated with a small population of myocytes. They are mutable collagenous structures, which can change their mechanical properties rapidly and reversibly under nervous control. The aims of this investigation were to characterise the baseline (i.e. unmanipulated) static mechanical properties of the CDs of Paracentrotus lividus by means of creep tests and incremental force-extension tests, and to determine the effects on their mechanical behaviour of a range of agents. Under constant load the CDs exhibited a three-phase creep curve, the mean coefficient of viscosity being 561±365 MPa.s. The stress-strain curve showed toe, linear and yield regions; the mean strain at the toe-linear inflection was 0.86±0.61; the mean Young's modulus was 18.62±10.30 MPa; and the mean tensile strength was 8.14±5.73 MPa. Hyaluronidase from Streptomyces hyalurolyticus had no effect on creep behaviour, whilst chondroitinase ABC prolonged primary creep but had no effect on secondary creep or on any force-extension parameters; it thus appears that neither hyaluronic acid nor sulphated glycosaminoglycans have an interfibrillar load transfer function in the CD. Acetylcholine, the muscarinic agonists arecoline and methacholine, and the nicotinic agonists nicotine and 1-[1-(3,4-dimethyl-phenyl)-ethyl]-piperazine produced an abrupt increase in CD viscosity; the CDs were not differentially sensitive to muscarinic or nicotinic agonists. CDs showed either no, or no consistent, response to adrenaline, L-glutamic acid, 5-hydroxytryptamine and γ-aminobutyric acid. Synthetic echinoid tensilin-like protein had a weak and inconsistent stiffening effect, indicating that, in contrast to holothurian tensilins, the echinoid molecule may not be involved in the regulation of collagenous tissue tensility. We compare in detail the mechanical behaviour of the CD with that of mammalian tendon and highlight its potential as a model system for investigating poorly understood aspects of the ontogeny and phylogeny of vertebrate collagenous tissues.(undefined)info:eu-repo/semantics/publishedVersio

New Insights into Mutable Collagenous Tissue: Correlations between the Microstructure and Mechanical State of a Sea-Urchin Ligament

The mutable collagenous tissue (MCT) of echinoderms has the ability to undergo rapid and reversible changes in passive mechanical properties that are initiated and modulated by the nervous system. Since the mechanism of MCT mutability is poorly understood, the aim of this work was to provide a detailed morphological analysis of a typical mutable collagenous structure in its different mechanical states. The model studied was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was characterized in different functional states mimicking MCT mutability. Transmission electron microscopy, histochemistry, cryo-scanning electron microscopy, focused ion beam/scanning electron microscopy, and field emission gun-environmental scanning electron microscopy were used to visualize CDLs at the micro- and nano-scales. This investigation has revealed previously unreported differences in both extracellular and cellular constituents, expanding the current knowledge of the relationship between the organization of the CDL and its mechanical state. Scanning electron microscopies in particular provided a three-dimensional overview of CDL architecture at the micro- and nano-scales, and clarified the micro-organization of the ECM components that are involved in mutability. Further evidence that the juxtaligamental cells are the effectors of these changes in mechanical properties was provided by a correlation between their cytology and the tensile state of the CDLs

Matrix metalloproteinases in a sea urchin ligament with adaptable mechanical properties

Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states ("standard", "compliant" and "stiff") was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability

The elusive role of l-glutamate as an echinoderm neurotransmitter: evidence for its involvement in the control of crinoid arm muscles

Although l-glutamate is the most widespread excitatory neurotransmitter in vertebrate and invertebrate nervous systems, there is only sparse evidence that it has this role in echinoderms. Following our previous finding that l-glutamate is widely distributed in the arms of the featherstar (crinoid echinoderm) Antedon mediterranea and initiates arm autotomy (defensive detachment), we now provide evidence of glutamatergic involvement in the control of the arm muscles of the same species using immunocytochemical and physiological methods. Immunofluorescence and immunoenzymatic techniques, which employed the same polyclonal antibody against l-glutamate conjugated to glutaraldehyde, revealed a high level of glutamate-like reactivity in the brachial muscles. By recording the mechanical responses of isolated arm pieces, we found that l-glutamate, l-aspartate and elevated [K(+)](o) induced rhythmic muscle contractions, while glycine, \u3b3-aminobutyric acid, adrenaline and acetylcholine had either no, or no consistent, effect. The frequency and duration of the dominant component of the rhythmic contractions indicated that these may be responsible for the rhythmic activity of the arms that occurs during swimming and after autotomy. We conclude that it is highly likely that l-glutamate has at least a neuromodulatory role in the neural pathways controlling the brachial muscles of A. mediterranea

Effects of exposure to ED contaminants (TPT-Cl and Fenarimol)on crinoid echinoderms: comparative analysis of regenerative development and correlated steroid levels

13 pages, 7 figures.Regenerative phenomena reproduce developmental processes in adult organisms and are regulated by neuro-endocrine mechanisms. They can therefore provide sensitive tests for monitoring the effects of exposure to endocrine disrupter contaminants (EDs) which can be bioaccumulated by the organisms causing dysfunctions in steroid hormone metabolism and activities and affecting reproduction and development. Echinoderms are prime candidates for this new ecotoxicological approach, since (1) they offer unique models to study physiological regenerative processes and (2) in echinoderms vertebrate-type steroids can be synthesized and used as terminal hormones along the neuro-endocrine cascades regulating reproductive, growth and developmental processes. We are currently exploring the effects on the regenerative potential of echinoderms of different classes of compounds that are well known to have ED activity. The present paper focuses on the possible effects of well-known compounds with suspected androgenic activity such as TPT-Cl (Triphenyltin-chloride) and Fenarimol [(± )-2,4-dichloro-α-(pyrimidin-5-yl) benzhydryl alcohol]. The selected test-species is the crinoid Antedon mediterranean a tractable and sensitive benthic filter-feeding species which represents a valuable experimental model for investigation on the regenerative process from the macroscopic to the molecular level. The present investigation employs an integrated approach which combines exposure experiments and biological analysis utilizing microscopy, immunocytochemistry and biochemistry. The experiments were carried out on experimentally induced arm regenerations in semistatic controlled conditions with exposure concentrations comparable to those of moderately polluted coastal zones. The bulk of results obtained so far provide indications of significant sublethal effects from exposure to TPT-C1 and Fenarimol and mechanisms of toxicity related to developmental physiology, which are associated with variations in steroid levels in the animal tissues. The results indicate that these two substances (1) affect growth and development by interfering with the same basic cellular mechanisms of regeneration, such as cell proliferation, migration and differentiation/dedifferentiation, which are possibly controlled by steroid hormones; and (2) can induce a number of significant modifications in the timing, modalities and pattern of arm regeneration, which may involve the activation of cell mechanisms related to steroid synthesis/metabolism.The present work has received financial support from the EU (COMPRENDO Project n EVK1-CT-2002-00129). The authors are particularly grateful to Dr Ulrike Shulte-Oehlmann for her valuable coordinating activity and to all the partners of the COMPRENDO project for their direct or indirect support and advice. Special thanks are addressed to Drs Simona Ceriani and Angelita Doria for their valuable help and technical assistance. All the experiments carried out for the research work are in accord with the current laws of our country. The authors are grateful to the anonymous reviewers for their invaluable suggestions and careful revision of the manuscript.Peer reviewe

Gametogenesis correlated with steroid levels during the gonadal cycle of the sea urchin Paracentrotus lividus (Echinodermata: Echinoidea)

9 pages, 6 figures.The specific mechanism regulating reproduction in invertebrates is a field of topical interest which needs to be explored in detail considering also the intriguing possible comparison with vertebrates. In this paper levels of Testosterone (T) and Estradiol (E2) and their reciprocal ratios were determined in ovaries and testis of the echinoid model species Paracentrotus lividus during the year 2004 by taking into account a putative relationship between steroid levels and reproductive cycle. T levels appeared to significantly vary during male reproductive cycle, thus suggesting a possible role of this hormone in regulation of spermatogenesis as demonstrated for other echinoderms. E2 levels were lower in males with respect to females; consequently E2 involvement in oogenesis is hypothesized. In parallel with steroid levels evaluation, variations in P450- aromatase activity and its possible role on regulation of gametogenesis were also considered. Clear correlations between steroid levels and gonad index (GI), as well as between GI and reproductive cycle were not detected, suggesting that GI alone is not a reliable parameter in describing the reproductive status of the gonads. Altogether the results obtained so far confirm the presence of a relationship between steroid levels and reproductive cycle as suggested by previous results on different echinoderm species.The present work has received financial support from the EU (COMPRENDO Project no. EVK1-CT-2002-00129). The authors are particularly grateful to Dr Ulrike Shulte-Oehlmann for her valuable coordinating activity and to all the partners of the COMPRENDO project for their direct or indirect support and advice. All the experiments carried out for the research work are in accord with the current laws of our country. The authors are grateful to the anonymous reviewers for their profitable suggestions and careful revision of the manuscript.Peer reviewe

Larval development in the feather star Antedon mediterranea

Echinoderms have been used often as experimental models in developmental biology and evolutionary biology studies. Numerous data on echinoid and asteroid development are available, whereas little is known about crinoid larval biology. This contribution focussed on the life cycle of the Mediterranean feather star Antedon mediterranea. Light and electron microscopy were used to characterize, in detail, the morphology and behaviour of the main larval stages. Similarities and differences with respect to what is already known for other crinoids, and echinoderm species, were explored. In view of the importance of serotonin during settlement and morphogenesis, analyses of the distribution of this molecule were carried out on swimming larvae. Immunolabelling results suggested a role for serotonin in A. mediterranea development, underlining the ancestral importance of this conserved neurotransmitter in deuterostome evolution