Implication of extracellular matrix related molecules in the immune response and spermatogenesis in the teleost fish gilthead seabream / Regulación de la espermatogénesis y respuesta inmunitaria por moléculas de la matriz extracelular en peces teleósteos (original) (raw)
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Identification of a β1 integrin isoform with restricted tissue expression in a teleost fish
Reproduction, fertility, and development, 2011
The composition and organisation of extracellular matrix (ECM)-related molecules change during 8 development. These components interact with different cell surface receptors to modulate the transduction of 9 signals for cell growth, differentiation, migration, proliferation and apoptosis. Previous findings in the teleost 10 fish gilthead seabream (Sparus aurata L., Teleostei), a marine protandrous hermaphrodite fish, showed that 11 endocrine and immune stimuli are able to modulate the expression of ECM-related molecules, as well as 12 specific correlations between them. In the present study, quantitative reverse transcription-polymerase chain 13 reaction was used to examine the gene expression profile of β 1 integrin isoform b (ITGB1b) and its possible 14 role in reproductive physiology, especially in relation to spermatogenesis. Expression profiles were analysed 15 in the context of the reproductive cycle (RC) and in relation with other ECM-related molecules, including 16 matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, Tissue-specific inhibitor of metalloproteinase 17 (TIMP)-2a, TIMP-2b, collagen (COL1A1) and ITGB1a. Expression of ITGB1b was found in the testis and 18 brain and, to some extent, in endothelial cells. In contrast, ITGB1a was expressed ubiquitously. In the testis, 19 the ITGB1b expression peaked during spermatogenesis, whereas the expression of the other ECM-related 20 molecules is induced mainly during the post-spawning stage, when marked tissue remodelling occurs during 21 the first and second RC in males. In addition, in fish exposed to the endocrine disruptor 17α-22 ethynyloestradiol, ITGB1b expression in the testis was inhibited in a dose-and time-dependent manner and 23 was related to reduced serum levels of testosterone. Together, these results suggest a different functionality 24 for the two ITGB1 isoforms in the gilthead seabream, where ITGB1b is more specifically involved in 25
Journal of Leukocyte Biology, 2005
It has been shown previously that professional phagocytic granulocytes are present in the testis of the gilthead seabream, a seasonal breeding teleost that offers an excellent model for studying the testicular regression process that occurs in seasonal testicular involution and sex change. It is unexpected that testicular granulocytes produce interleukin-1, a regulator for spermatogonia proliferation in mammals, but are not involved in the elimination of degenerative germ cells. Here, we show that phagocytosis and reactive oxygen intermediate (ROI) production were suppressed dramatically in testicular phagocytic granulocytes, compared with their level of activity in the head-kidney, the main hematopoietic organ in fish. Furthermore, testicular-conditioned media modulated migration, phagocytosis, and ROI production of head-kidney phagocytic granulocytes, and the addition of testicular cells impaired their ROI production capacity. Until now, monocytes/ macrophages were believed to be the only innate immune cells able to develop into functional subsets, whereas neutrophils only infiltrate the tissues upon infection or inflammation. Our findings demonstrate, however, that fish professional phagocytic granulocytes also display functional adaptation to different microenvironments and strongly suggest a role for these cells in the reorganization of the testis during post-spawning. J. Leukoc. Biol. 78: 000 -000; 2005.
Molecular Immunology, 2008
Immune responses in the testis are regulated in a way that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. In addition, germ cells are potent targets for the growth factors and cytokines which regulate the reproductive process. Our study analyzes for the first time the pattern of expression of several immune-relevant genes in the gonad of a seasonal breeding teleost fish. The immune molecules analyzed include (i) inflammatory molecules, such as interleukin-1b (il1b), il6, tumor necrosis factor-a (tnfa), cyclooxygenase-2 (cox2) and the NADPH oxidase subunit p40 phox (ncf4 gene); (ii) the anti-inflammatory cytokine transforming growth factor-b1 (tgfb1) and its type 2 receptor tgfbr2; (iii) innate immune receptors, including toll-like receptor 9 (tlr9), tlr5, tlr22 and macrophage-colony stimulating factor receptor (mcsfr); (iv) lymphocyte receptors, such as the  subunit of T-cell receptor (Tcrb) and the heavy chain of immunoglobulin M (ighm); (v) the anti-bacterial molecules lysozyme (lyz), hepcidin (hamp) and complement component 3 (c3); (vi) the anti-viral molecule myxovirus (influenza) resistance protein (mx); and (vii) molecules related to leukocyte infiltration, including the CC chemokine ccl4, the CXC chemokine il8 and the leukocyte adhesion molecule E-selectin (Sele). Notably, all of them show a pattern of expression that depends on the reproductive stage of the first two reproductive cycles when the fish develop and function as males. Furthermore, we demonstrate that some of these immune-relevant molecules, such as Il1b and Mcsfr, are produced by germ cells (Il1b) and ovarian and testicular somatic cells (Mcsfr). These data suggest that, as occurs in mammals, there is a critical balance between immune molecules and that these may play an essential role in the orchestration of gametogenesis and the maintenance of gonad tissue homeostasis in fish. (A. García-Ayala).
Distribution of laminin β2, collagen type IV, fibronectin and MMP-9 in ovaries of the teleost fish
Journal of Molecular Histology, 2010
Extracellular matrix in the ovarian follicle has been characterised for several mammalian species but there are no reports that describe the immunolocalisation of the extracellular matrix elements, matrix metalloproteinases, and its relation to plasma 17b estradiol levels and follicular apoptosis during the teleost's reproductive cycle. The present study used immunohistochemistry to characterise the distribution of laminin b2, collagen type IV, fibronectin and matrix metalloproteinases-9 (MMP-9). The TUNEL in situ technique was used to quantify apoptosis and indirect immunofluorimetric to determine plasma 17b estradiol levels. The TUNEL-positive reaction associated with morphological features exhibited follicular apoptosis. During postovulatory follicle involution, the drop in plasma 17b estradiol levels after spawning contributed to the intense apoptosis observed. By immunohistochemical analysis, laminin b2 and collagen type IV were identified as the major constituents of the basement membrane. The loss of integrity of the basement membrane occurred due to lyses of the major constituents, and coincides with increased follicular apoptosis. The integrity of the basement membrane is important for the survival of follicular cells. Furthermore, the MMP-9 results suggest that this enzyme is involved in final oocyte maturation and regression of postovulatory follicles. Fibronectin was observed on the surface of follicular cells of the postovulatory follicle in P. argenteus, this being important for maintaining normal cell adhesion to extracellular matrix. In conclusion, our results suggest that the structure and composition of the extracellular matrix, and plasma 17b estradiol levels related to apoptosis, play an important role during the follicular development and post-spawning involution in teleost fishes.
Developmental and Comparative Immunology, 2010
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 1a) 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.
Reproductive Biology and Endocrinology, 2007
Background: Leukocytes are found within the testis of most, if not all, mammals and are involved in immunological surveillance, physiological regulation and tissue remodelling. The testis of seasonal breeding fish undergoes a regression process. In the present study, the second reproductive cycle (RC) of the protandrous seasonal teleost fish, gilthead seabream, was investigated and the presence of leukocytes analysed. Special attention has been paid to the testicular degenerative process which is particularly active in the last stage of the second RC probably due to the immediacy of the sex change process.
Journal of Endocrinology, 2003
In mammals, a complex interaction between the immune and the reproductive systems has been described, in which testicular immune cells produce cytokines and growth factors which modulate gonad functions, while specific gonad cells influence the immune response in this organ. In this study we describe the presence of acidophilic granulocytes in the testis of the hermaphrodite teleost fish gilthead seabream (Sparus aurata L.) by using a specific monoclonal antibody. During the post-spawning stage of the testis, this cell type appears in the germinal compartment, accumulates interleukin (IL)-1 and does not seem to be involved in the phagocytosis of degenerating cells. Moreover, in vitro, 11-ketotestosterone and 17 -oestradiol, the principal fish sexual steroids, regulate the respiratory burst activity of acidophilic granulocytes obtained from the head-kidney (the bone marrow equivalent in fish) and the intracellular accumulation of IL-1 by these cells. It is likely, therefore, that IL-1 produced by testicular acidophilic granulocytes regulates important functions of the testis in fish.
Collagen regulates the activation of professional phagocytes of the teleost fish gilthead seabream
Molecular Immunology, 2009
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-1 , 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.
Journal of Reproduction and Development, 2009
A great deal is known regarding the process of sex differentiation in fish. However, little is known about the presence of immune cells and cytokines in this process. In the gilthead seabream, both immune cells and cytokines play an important role in the tissue reorganization of the gonads during the adult reproductive cycle. We have studied, using light microscopy and immunocytochemistry, the ontogenetic development of the gilthead seabream gonads, focusing on the presence of immune cells and cytokines. We show that the testicular area is quickly differentiated and becomes functional in specimens less than a year old, while the ovarian area differentiates later and continues to develop during the first two years of life. Throughout the morphogenesis process, acidophilic granulocytes were present in the gonad. Interleukin-1b (Il1b) is produced in the testicular area in juveniles and male fish, but not in the ovarian area. Macrophage-colony stimulating factor receptor (Mcsfr) is not produced in the undifferentiated gonad and is only found once the testicular area is well developed.
Early ovarian regression was analyzed in the neotropical freshwater teleost, curimatã-pacu (Prochilodus argenteus), in order to evaluate follicular apoptosis, basement membrane morphology, and integrin β 1 and collagen type IV immunostainning in postovulatory follicles. Mature females were induced to spawn by using carp pituitary extract for study of ovarian regression up to 5 days post-spawning. Morphometric analyses showed that the postovulatory follicle area decreased progressively after spawning and was coupled to the gonadosomatic index (r=0.92). During ovarian regression, follicular cells detached from the neighboring cells and basement membrane and then died by apoptosis. The follicular basement membrane became thicker and diffuse and was breached during regression of the postovulatory follicles. Follicular apoptosis was detected by TUNEL, histology, and electron microscopy. The ladder pattern of apoptotic DNA was revealed by agarose gel electrophoresis. The apoptotic index for the follicular cells increased until 3 days postspawning and decreased thereafter. Immunohistochemistry reactions detected caspase 3, integrin β 1 , and collagen type IV in the follicular layer of the postovulatory follicles. Labeling for integrin β 1 and collagen type IV decreased significantly, whereas a peak in cell death occurred 3 days post-spawning. At 4-5 days post-spawning, the connective theca was more thickened and vascularized. Simultaneously, granulocytes migrated toward the follicular lumen. Thus, follicular apoptosis contributes to early ovarian regression in P. argenteus. Additionally, our findings suggest integrin β 1 and collagen type IV as possible survival factors for follicular cells in teleost ovary.