Xenopus embryonic epidermis as a mucociliary cellular ecosystem to assess the effect of sex hormones in a non-reproductive context.

BACKGROUND: How important are sexual hormones beyond their function in reproductive biology has yet to be understood. In this study, we analyzed the effects of sex steroids on the biology of the embryonic amphibian epidermis, which represents an easily amenable model of non-reproductive mucociliary epithelia (MCE). MCE are integrated systems formed by multiciliated (MC), mucus-secreting (MS) and mitochondrion-rich (MR) cell populations that are shaped by their microenvironment. Therefore, MCE could be considered as ecosystems at the cellular scale, found in a wide array of contexts from mussel gills to mammalian oviduct. RESULTS: We showed that the natural estrogen (estradiol, E2) and androgen (testosterone, T) as well as the synthetic estrogen (ethinyl-estradiol, EE2), all induced a significant enhancement of MC cell numbers. The effect of E2, T and EE2 extended to the MS and MR cell populations, to varying degrees. They also modified the expression profile of RNA MCE markers, and induced a range of "non-typical" cellular phenotypes, with mixed identities and aberrant morphologies, as revealed by imaging analysis through biomarker confocal detection and scanning electron microscopy. Finally, these hormones also affected tadpole pigmentation, revealing an effect on the entire cellular ecosystem of the Xenopus embryonic skin. CONCLUSIONS: This study reveals the impact in vivo, at the molecular, cellular, tissue and organism levels, of sex steroids on non-reproductive mucociliary epithelium biogenesis, and validates the use of Xenopus as a relevant model system in this field.

Identification of a ß1 integrin isoform with restricted tissue expression in a teleost fish.

The composition and organisation of extracellular matrix (ECM)-related molecules change during development. These components interact with different cell surface receptors to modulate the transduction of signals for cell growth, differentiation, migration, proliferation and apoptosis. Previous findings in the teleost fish gilthead seabream (Sparus aurata L., Teleostei), a marine protandrous hermaphrodite fish, showed that endocrine and immune stimuli are able to modulate the expression of ECM-related molecules, as well as specific correlations between them. In the present study, quantitative reverse transcription-polymerase chain reaction was used to examine the gene expression profile of ß(1) integrin isoform b (ITGB1b) and its possible role in reproductive physiology, especially in relation to spermatogenesis. Expression profiles were analysed in the context of the reproductive cycle (RC) and in relation with other ECM-related molecules, including matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, tissue-specific inhibitor of metalloproteinase (TIMP)-2a, TIMP-2b, collagen (COL1A1) and ITGB1a. Expression of ITGB1b was found in the testis and brain and, to some extent, in endothelial cells. In contrast, ITGB1a was expressed ubiquitously. In the testis, the ITGB1b expression peaked during spermatogenesis, whereas the expression of the other ECM-related molecules is induced mainly during the post-spawning stage, both stages of marked tissue remodelling during the first and second RC in males. In addition, in fish exposed to the endocrine disruptor 17α-ethynyloestradiol (at 5 and 50 µg g(-1) food during 7, 14 and 21 days), ITGB1b expression in the testis was inhibited in a dose- and time-dependent manner and was related to reduced serum levels of testosterone. Together, these results suggest a different functionality for the two ITGB1 isoforms in the gilthead seabream, where ITGB1b is more specifically involved in reproduction. This is the first report of an ITGB1 gene isoform whose expression is restricted to endocrine-related tissues in vertebrates.

A role for specific collagen motifs during wound healing and inflammatory response of fibroblasts in the teleost fish gilthead seabream.

Specific sites and sequences in collagen to which cells can attach, either directly or through protein intermediaries, were identified using Toolkits of 63-amino acid triple-helical peptides and specific shorter GXX'GEX″ motifs, which have different intrinsic affinity for integrins that mediate cell adhesion and migration. We have previously reported that collagen type I (COL-I) was able to prime in vitro the respiratory burst and induce a specific set of immune- and extracellular matrix-related molecules in phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). It was also suggested that COL-I would provide an intermediate signal during the early inflammatory response in gilthead seabream. Since fibroblasts are highly involved in the initiation of wound repair and regeneration processes, in the present study SAF-1 cells (gilthead seabream fibroblasts) were used to identify the binding motifs in collagen by end-point and real-time cell adhesion assays using the collagen peptides and Toolkits. We identified the collagen motifs involved in the early magnesium-dependent adhesion of these cells. Furthermore, we found that peptides containing the GFOGER and GLOGEN motifs (where O is hydroxyproline) present high affinity for SAF-1 adhesion, expressed as both cell number and surface covering, while in cell suspensions, these motifs were also able to induce the expression of the genes encoding the proinflammatory molecules interleukin-1ß and cyclooxygenase-2. These data suggest that specific collagen motifs are involved in the regulation of the inflammatory and healing responses of teleost fish.

Correlated expression profile of extracellular matrix-related molecules during the inflammatory response of the teleost fish gilthead seabream.

Extracellular matrix (ECM) components, in addition to their structural functions, interact with cell surface receptors and intracellular components to modulate the transduction of signals for cell growth, differentiation, migration, proliferation, polarization, apoptosis and inflammation. Our previous findings in the gilthead seabream (Sparus aurata L.), a marine seasonal hermaphrodite teleost fish, have shown that both endocrine and immune stimuli modulate the expression of matrix metalloproteases (MMPs) and tissue inhibitors of MMP (TIMPs). In addition, collagen type I (COL1) induces the expression of some pro-inflammatory cytokines and MMPs in professional phagocytes. Consequently, in this study we use real-time RT-PCR to analyze the gene expression profile of several ECM-related molecules (MMP-2, -9 and -13, TIMP-2a, and -2b, COL1A1, and integrin beta1a) in different organs of adult specimens as well as in response to innate immune challenges. Our results showed that liver had the lowest basal levels of them, although they were clearly modulated during injury and infection. In the same way, ECM-related molecules seem to participate in pro-inflammatory processes, being of particular interest COL1 which is synthesized by immune cells and is able to act as autocrine/paracrine stimulus for them. Lastly, we propose that the observed correlations between ECM-related molecules during the inflammatory response should be considered to obtain a more accurate picture of their roles in this process.

Collagen regulates the activation of professional phagocytes of the teleost fish gilthead seabream.

The innate immune system mediates the initial inflammatory response that follows infection or injury. Although the innate immune response of fish to infection has been relatively well characterized during recent years at both cellular and molecular levels, no studies have examined the role of extracellular matrix (ECM) in the regulation of innate immunity and inflammation. We report here that collagen and gelatin in vitro were able to prime the respiratory burst of phagocytes from the bony fish gilthead seabream. In addition, collagen and gelatin induced a specific set of immune-related and ECM remodelling enzymes that substantially differed from that induced by pathogen-associated molecular patterns. Notably, both collagen and gelatin induced the expression of interleukin-1beta, chemokine (C-C motif) ligand 4 and matrix metalopeptidases (MMP) 9 and 13 in acidophilic granulocytes and macrophages but were unable to significantly increase the expression of other pro-inflammatory genes. Furthermore, it was found that the MMP2/MMP9 inhibitor V had a dose-dependent inhibitory effect on seabream phagocyte activation by either collagen or gelatin. In contrast, pre-treatment of collagen and gelatin by collagenase resulted in a higher stimulatory capacity compared to non-digested proteins. Collectively, these results indicate that collagen fragments produced by the action of different host proteases, and probably released by infectious agents, are sensed by fish phagocytes. Therefore, we propose that, besides to the well-established response to infection, the innate immune system of fish is able to respond to tissue injury.