Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus

The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes withinphysiological timescales, the only major exception being the reversible destiffening of the mammalianuterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (seaurchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions intimescales of around a second to minutes. Elucidation of the molecular mechanism underlying suchmutability has implications for the zoological, ecological and evolutionary field. Important informationcould also arise for veterinary and biomedical sciences, particularly regarding the pathological plasti-cization or stiffening of connective tissue structures. In the present investigation we analyzed aspectsof the ultrastructure and biochemistry in two representative models, the compass depressor ligamentand the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three differentmechanical states. The results provide further evidence that the mechanical adaptability of echinodermconnective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher gly-cosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compassdepressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues.The possible involvement of GAG in the mutability phenomenon will need further clarification. Duringthe shift from a compliant to a standard condition, significant changes in GAG content were detected onlyin the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling)and biochemistry (two alpha chains) were found between the two models and mammalian collagen.Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLASTalignment highlighted the uniqueness of sea urchin collagen with respect to mammalian collagen

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

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

Exploring endocrine regulation of sea urchin reproductive biology : effects of 17 f-oestradiol

Although several authors have suggested a plausible involvement of steroids in the reproductive biology of echinoderms, their definitive role is still poorly understood. In this paper we focused on oestradiol (E2), whose presence and variations were previously revealed in different echinoderm tissues. The aim of this investigation was to provide further information on the scarcely known role of this hormone in the reproductive biology of sea urchins. We injected two different concentrations (5 ng ml 121 and 50 ng ml 121) of 17 f-oestradiol into specimens of the common Paracentrotus lividus for 10 weeks. The E2 treatment did not influence the maturation stage of the gonads and the development of the gametes; it caused a slight decrease in the gonad index and an increase in lipid content. Our present results suggest that E2 could have a function different from that reported for vertebrates and suggested for other echinoderms such as asteroids

The spherule cells of Holothuria polii Delle Chiaie, 1823, (Aspidochirata, Holothuroidea) during brown body formation: an ultrastructural study

Spherule cells are specific types of coelomocytes found in both the coelomic fluids and the connective tissues of many echinoderm groups and are characterised by large membrane-bound inclusions which completely fill their cytoplasm. In holothurians they are present in massive number in the coelomic fluids and are employed in brown body formation. Brown bodies are products of encapsulation and mainly consist of phagocytic amoebocytes and spherule cells: they surround foreign particles too large to be ingested by circulating phagocytes. During brown body formation, phagocytic amoebocytes flatten out over the surface of foreign particles to form unpigmented nodules which eventually aggregate into a single brown body in which many spherule cells are entrapped. Morphological modifications of spherule cells were studied in Holothuria polii following the induction of brown body formation by intracoelomic injection of sheep erythrocytes. Our ultrastructural observations provide evidence that the granules undergo typical exocytosis after previous disorganisation of their content and suggest a specific secretory activity for the spherule cells. The possible functional role of the secreted vacuolar material in brown body formation is discussed

Endocrine disrupting compounds and echinoderms : new ecotoxicological sentinels for the marine ecosystem

Echinoderms are valuable test species in marine ecotoxicology and offer a wide range of biological processes appropriate for this approach. In spite of this potential, available data in literature are still rather limited, particularly with regard to the possible effects of endocrine disrupter compounds (EDCs). This review presents echinoderms as useful models for ecotoxicological tests and gives a brief overview of the most significant results obtained in recent years, particularly in the context of the COMPRENDO EU project. In this research project two different aspects of echinoderm physiology, plausibly regulated by humoral mechanisms, were investigated: reproductive biology and regenerative development. Selected EDCs suspected for their androgenic or antiandrogenic action were tested at low concentrations. The results obtained so far showed that different parameters such as regenerative growth, histological pattern, egg diameter and gonad maturation were affected by the exposure to the selected compounds. These results substantiate that reproductive and regenerative phenomena of echinoderms can be considered valuable alternative models for studies on EDCs and confirm that these compounds interfere with fundamental physiological processes, including growth, development and reproductive competence

Cellular and molecular mechanisms of arm regeneration in Crinoid Echinoderms.

Crinoid echinoderms can provide a valuable experimental model for studying all aspects of regenerative processes from molecular to macroscopic level. Recently we carried out a detailed study into the overall process of arm regeneration in the crinoid Antedon mediterranea and provided an interpretation of its basic mechanisms. However, the problem of the subsequent fate of the amputated arm segment (explant) once isolated from the animal body and of its possible regenerative potential have never been investigated before. The arm explant in fact represents a simplified and controlled regenerating system which may be very useful in regeneration experiments by providing a valuable test of our hypotheses in terms of mechanisms and processes. In the present study we carried out a comprehensive analysis of double-amputated arm explants (i.e. explants reamputated at their distal end immediately after the first proximal amputation) subjected to the same experimental conditions as the regenerating donor animals. Our results showed that the explants undergo similar regenerative processes but with some significant differences to those mechanisms described for normal regenerating arms. For example, whilst the proximal-distal axis of arm growth is maintained, there are differences in terms of the recruitment of cells which contribute to the regenerating tissue. As with normal regenerating arms, the present work focuses on (1) timing and modality of regeneration in the explant; (2) proliferation, migration and contribution of undifferentiated and/or dedifferentiated/transdifferentiated cells; (3) putative role of neural growth factors. These problems were addressed by employing a combination of conventional microscopy and immunocytochemistry. Comparison between arm explants and regenerating arms of normal donor adults indicates an extraordinary potential and regenerative autonomy of crinoid tissues and the cellular plasticity of the phenomenon.

Biologia riproduttiva ed ormoni steroidei nel comune riccio di mare Paracentrotus lividus (Lamarck 1816)

Il riccio di mare Paracentrotus lividus \ue8 uno dei modelli pi\uf9 noti, e storicamente pi\uf9 utilizzati, nel campo biologia riproduttiva e dello sviluppo. Ciononostante i meccanismi ormonali che regolano lo sviluppo della gonade e dei gameti sono poco conosciuti. Questo lavoro si focalizza sul ruolo degli steroidi e in particolare dell\u2019estradiolo (E2), uno dei principali regolatori della biologia riproduttiva dei vertebrati. Negli echinodermi, questo ormone \ue8 stato rilevato in diversi tessuti, dove presenta variazioni stagionali e differenze sesso-specifiche. Il nostro protocollo sperimentale prevede l\u2019iniezione di 17 f-estradiolo in esemplari adulti di Paracentrotus lividus, a tre diverse concentrazioni (2 ng/mL, 20 ng/mL and 200 ng/mL) e per un periodo di 2 e 12 settimane. La concentrazione minore risulta prossima a quella fisiologica, precedentemente misurata in esemplari di campo. Sono stati misurati i seguenti parametri: 1) livelli ormonali nei fluidi e nella gonade, 2) attivit\ue0 di enzimi coinvolti in sintesi/metabolismo di E2, 3) sviluppo dimensionale (GI) della gonade, 4) stadio di maturazione delle gonade, 5) contenuto lipidico della gonade. Nonostante si sia rilevato un effettivo incremento di ormone circolante (pi\uf9 ridotto nel caso del tessuto gonadico), entrambi i trattamenti, a breve e lungo termine, non hanno indotto evidenti variazioni n\ue9 nel GI (apparentemente pi\uf9 influenzato dall\u2019alimentazione) n\ue9 nello stadio maturativo della gonade sia femminile sia maschile. Al contrario, il contenuto lipidico \ue8 risultato negativamente correlato ai livelli di ormone misurato nella gonade, suggerendo un\u2019inibizione di questo parametro da parte dell\u2019E2. Infine l\u2019attivit\ue0 degli enzimi coinvolti nella sintesi/metabolismo di E2 suggerisce la presenza di meccanismi omeostatici che mantengono i livelli endogeni di ormone entro certi valori critici, proteggendo i tessuti da eccessivi sbalzi ormonali. Nel complesso i risultati ottenuti forniscono un contributo alla conoscenza dell\u2019endocrinologia degli echinoidei. In particolare, si evidenzia come, diversamente dai vertebrati e dagli echinodermi asteroidei, l\u2019E2 non influenzi direttamente la biologia riproduttiva di questa specie regolandone lo sviluppo della gonade e la maturazione dei gameti. Piuttosto, l\u2019ormone sembra essere coinvolto in altre funzioni fisiologiche specifiche correlate alla riproduzione, quali ad esempio la regolazione della composizione biochimica e dell\u2019accumulo di sostanze di riserva all\u2019interno della gonade