Primary Cell Cultures of Regenerating Holothurian Tissues

Bello, Samir A.; Abreu-Irizarry, Ricardo J.; García‐Arrarás, José E.

doi:10.1007/978-1-4939-1164-6_19

Cited by 14 publications

(10 citation statements)

References 13 publications

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“…Except for the morphology of living cells, which compared well with previous studies (e.g., Endean, 1958;Hetzel, 1963), comparison of our data with other studies is difficult because of differences in methods. Although other studies of coelomocytes in Holothuroidea have also analyzed morphometrics (Prompoon et al, 2015;Ramírez-G omez et al, 2010) and cell morphology of stained (D'Ancona & Canicattì, 1990;Prompoon et al, 2015;Ramírez-G omez et al, 2010) and SEM (Xing et al, 2008;Bello et al, 2015) preparations, these studies commonly use smeared cells instead cytocentrifugation. Taking into account that coelomocyte morphology, and consequently morphometry, may be affected by biotic or abiotic factors in smeared preparations (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…In some studies, two or more methods were used together (Canicattì et al, 1989b;Xing et al, 2008), which certainly improves the analysis. By contrast, data from other methods such as cytochemistry and scanning electron microscopy (SEM) have hardly ever been used to study coelomocyte morphology (Bello et al, 2015;D'Ancona & Canicattì, 1990). The use of morphometry in studies of coelomocytes in Holothuroidea is even scarcer, though it has been proven informative in studies of hemocytes in other invertebrates, including Mollusca and Arthropoda (Salimi et al, 2009;Silva et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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The use of an integrative approach to identify coelomocytes in three species of the genus Holothuria (Echinodermata)

Queiroz

Mauro

Arizza

et al. 2021

Invertebrate Biology

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Coelomocytes in the Holothuroidea are traditionally identified according to their morphology through light, fluorescence, or electron microscopy. Former studies have typically used only one method, with few works combining two or more approaches.Studies using cytocentrifugation to study these cells are scarcer. Thus, for the first time, an integrative approach was used to compare coelomocytes in Holothuroidea.This approach consisted of living and stained cells, scanning electron microscopy (for spherule cells), and accurate morphometric analyses. Specifically, we used specimens of Holothuria grisea, Holothuria arenicola, and Holothuria tubulosa to test whether cytocentrifugation could be valuable in comparative studies with coelomocytes, whether an integrative approach could help to understand spherule cell diversity, and whether closely related species, even those having distinct geographic distributions and ecological requirements, would have a similar population of coelomocytes. Our results showed seven distinct cell types in these species, including phagocytes, fusiform cells, morula cells, acidophilic spherulocytes, spherulocytes, progenitor cells, and crystal cells. Total and differential cell counts, along with morphometric parameters, were similar among species. Morphometric analyses of spherule cells revealed consistent differences among the diameter of their cytoplasmic spherules, as well as a set of different morphotypes in acidophilic spherulocytes and spherulocytes. Cytospin preparations proved to be quite useful because they provided constant morphological and morphometric data, allowing accurate identification of the cell types and comparisons among species. Moreover, this study highlighted (1) that the spherule diameter is a good parameter to separate spherule cells and (2) a putative maturation process to acidophilic spherulocytes and spherulocytes. Lastly, we showed that the cells of these species are very similar, regardless of their geographic distribution and ecology. Thus, our work contributes to a better understanding of the coelomocytes in Holothuria, a genus with a wide geographic distribution. The present study may be useful to establish these species as important model organisms, as well as bring insights into the functions of coelomocytes.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The use of an integrative approach to identify coelomocytes in three species of the genus Holothuria (Echinodermata)

Queiroz

Mauro

Arizza

et al. 2021

Invertebrate Biology

View full text Add to dashboard Cite

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“…Muscle tissues were obtained following the dissection protocol described by Bello, SA, et al 2015 [ 76 ], with some modifications. Briefly, animals were anesthetized by placing them on ice for 1 h, then washed with 10% sodium hypochlorite, ethanol 70% respectively for one minute, and left in purified/autoclaved sea water.…”

Section: Methodsmentioning

confidence: 99%

Antibiotics Modulate Intestinal Regeneration

Díaz-Díaz

Rosario-Meléndez

Rodríguez-Villafañe

et al. 2021

Biology

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The increased antibiotics usage in biomedical and agricultural settings has been well documented. Antibiotics have now been shown to exert effects outside their purposive use, including effects on physiological and developmental processes. We explored the effect of various antibiotics on intestinal regeneration in the sea cucumber Holothuria glaberrima. For this, holothurians were eviscerated and left to regenerate for 10 days in seawater with different penicillin/streptomycin-based cocktails (100 µg/mL PS) including: 100 µg/mL kanamycin (KPS), 5 µg/mL vancomycin (VPS), and 4 µg/mL (E4PS) or 20 µg/mL (E20PS) erythromycin. Immunohistological and histochemical analyses were performed to analyze regenerative processes, including rudiment size, extracellular matrix (ECM) remodeling, cell proliferation, and muscle dedifferentiation. A reduction in muscle dedifferentiation was observed in all antibiotic-treated animals. ECM remodeling was decreased by VPS, E4PS, and E20PS treatments. In addition, organisms subjected to E20PS displayed a significant reduction in the size of their regenerating rudiments while VPS exposure altered cell proliferation. MTT assays were used to discard the possibility that the antibiotics directly affect holothurian metabolic activity while bacterial cultures were used to test antibiotic effects on holothurian enteric microbiota. Our results demonstrate a negative effect on intestinal regeneration and strongly suggest that these effects are due to alterations in the microbial community.

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“…The eluted cell suspension was washed three times for 10 min each time under centrifugation (900 × g , 10 min, 4°C) in Ca 2+ and Mg 2+ ‐free artificial seawater solution with neomycin (10 mg/ml, N8090; Solarbio, Beijing, China) and amphotericin B (2.5 mg/ml, A8250; Solarbio, Beijing, China). The cell pellets were resuspended in a modified Leibovitz 15 medium (GNAM1300; Genom, Hangzhou, China) supplemented with human IGF‐1 (100 μg/ml), human FGF‐basic (0.1 μg/μL), vitamin E (1.75 mg/L), insulin (5 mg/L), and gentamycin (40 mg/L) at 1–10 × 10 6 cells in 1.5‐ml closed polyethylene tubes (Bello, Abreu‐Irizarry, & García‐Arrarás, ; with cells primary culture of regenerated intestinal tissue in vitro). For stress induction, cells after stabilization at 15°C for half an hour were immediately heated at 25°C for 4 hr in a metal bath reactor as an acute heat stress stimulus, whereas other cells were kept at 15°C to serve as the control group.…”

Section: Methodsmentioning

confidence: 99%

A potential antiapoptotic regulation: The interaction of heat shock protein 70 and apoptosis‐inducing factor mitochondrial 1 during heat stress and aestivation in sea cucumber

Wang

Chen

et al. 2018

J Exp Zool Pt A

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The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally induced aestivation in marine invertebrates. A characteristic feature of aestivation in this species is the degeneration of the intestine. In the current study, we hypothesized that energy conservation and cytoprotective strategies need to be coordinated in the intestine to ensure long-term survival during aestivation, and there was potential relationship between heat shock protein 70 (HSP70) and apoptosis-inducing factor mitochondrial 1 (AIFM1) during extreme environmental stress. AIFM1 is a bifunctional flavoprotein that is involved in the caspase-independent activation of apoptosis. The gene and protein expression profiles of AjAIFM1 and AjHSP70 in intestinal tissue during aestivation were analyzed and results showed an inverse correlation between them, AjAIFM1 being suppressed during aestivation whereas AjHSP70 was strongly upregulated. Comparable responses were also seen when intestinal cells were isolated and analyzed in vitro for responses to heat stress at 25°C (a water temperature typical during aestivation), compared with 15°C control cells. Combined with co-immunoprecipitation studies in vivo and in vitro, our results suggested that AjHSP70 protein may have potential interaction with AjAIFM1. To determine the influence of heat stress on apoptotic rate of intestinal cells, we also assessed the DNA fragmentation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and results also supported a potential antiapoptotic response in sea cucumber during heat stress. This type of cytoprotective mechanism could be used to preserve the existing cellular components during long-term aestivation in sea cucumber.

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Primary Cell Cultures of Regenerating Holothurian Tissues

Cited by 14 publications

References 13 publications

The use of an integrative approach to identify coelomocytes in three species of the genus Holothuria (Echinodermata)

The use of an integrative approach to identify coelomocytes in three species of the genus Holothuria (Echinodermata)

Antibiotics Modulate Intestinal Regeneration

A potential antiapoptotic regulation: The interaction of heat shock protein 70 and apoptosis‐inducing factor mitochondrial 1 during heat stress and aestivation in sea cucumber

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