Sergio Liarte

17Beta-Estradiol Triggers Postspawning in Spermatogenically Active Gilthead Seabream (Sparus aurata L.) Males

Abstract The testis is a tightly controlled dynamic tissue. In mammals, there is growing evidence that estrogen plays a role in the regulation of testicular functions. In teleosts, high levels of 17beta-estradiol (E2) in serum correlate with the end of spermatogenesis, spawning, and the initiation of postspawning stages when spermatogonia are the main cell types in the testis. Moreover, E2 modulates leukocyte functions in several teleost species. We hypothesized, therefore, that E2 would induce the infiltration of acidophilic granulocytes and cause a resumption of testicular cell proliferation in spermatogenically active gilthead seabream males. Several studies of this species have reported that supraphysiological doses of E2 are needed to induce histological and developmental changes in males. In fact, as gilthead seabream is a protandrous hermaphrodite teleost, long exposures (6–14 wk) to high doses of E2 result in feminization of the males. Taking all this into account, we sharply increased E2 levels during short times by i.p. injecting E2 diluted in coconut oil as the vehicle and sampled the fish after 7, 13, and 18 days to assess the effects that E2 had on spermatogenesis. It was observed that E2 levels in plasma increased, while 11-ketotestosterone (11-KT) and testosterone (T) levels remained unaltered. However, 11-KT and T levels strongly increased in control fish 18 days postinjection. The most relevant result of our study was that E2 accelerates the final events of spermatogenesis, inhibits the proliferation of spermatogonia in early stages, and induces some of the processes that usually occur during postspawning, such as the infiltration of acidophilic granulocytes and the apoptosis of primary spermatogonia. Strikingly, neither the shedding of spermatozoa nor an increase in the proliferative rate of spermatogonia stem cells was observed, probably because of the lack of other necessary stimuli, such as the increase in T levels that takes place during normal postspawning.

Pattern of expression of immune-relevant genes in the gonad of a teleost, the gilthead seabream (Sparus aurata L.).

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 beta 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.

17α-Ethynylestradiol alters the immune response of the teleost gilthead seabream (Sparus aurata L.) both in vivo and in vitro.

There is increasing public attention concerning the effect of endocrine disruptor chemicals (EDCs) on the immune system. One important group belonging to EDCs are the environmental estrogens. Commonly found in the effluents in wastewater treatment plants, 17α-ethynylestradiol (EE(2)) which is used in contraceptive pills, is an endocrine disruptor with strong estrogenic effects. This study aims to investigate the capacity of EE(2) to modulate in vivo and in vitro the innate immune response of the gilthead seabream (Sparus aurata L.), a teleost species of great commercial value. For this purpose, adult specimens were bath-exposed to EE(2) (0, 5 and 50 ng/L) and then immunized with hemocyanin in the presence of the adjuvant aluminum. The results indicate that, after 15 days of EE(2)-exposure, the disruptor was able to inhibit in a dose-dependent manner the induction of interleukin-1β (IL-1β) gene expression, but did not significantly alter the specific antibody titer. To shed light on the role played by EE(2) into seabream immune response, leukocytes were exposed in vitro to several concentrations of EE(2) (0, 0.5, 5, 50 and 500 ng/ml) for 3, 16 and 48 h and the production of reactive oxygen intermediates, the phagocytic activity and the gene expression profile of these cells were analyzed. EE(2) was seen to inhibit both cellular activities and to alter the immune gene expression profile in primary macrophages. Thus, low concentrations of EE(2) increase the mRNA levels of IL-1 β, IL-6, tumour necrosis factor α and tumour growth factor β in non-activated macrophages. In contrast, EE(2) treatment of activated macrophages resulted in the decreased expression of pro-inflammatory genes and the increased expression of genes encoding anti-inflammatory and tissue remodeling/repair enzymes. Taken together, our results suggest that EE(2) might alter the capacity of fish to appropriately respond to infection although it does not behave as an immunosuppressor.

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration

BackgroundLeukocytes 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.MethodsSexually mature specimens (n = 10–18 fish/month) were sampled during the second RC. Some specimens were intraperitoneally injected with bromodeoxyuridin (BrdU) before sampling. Light and electron microscopy was used to determine the different stages of gonadal development and the presence of leukocytes and PCR was used to analyse the gene expression of a testis-differentiating gene and of specific markers for macrophages and B and T lymphocytes. Immunocytochemistry and flow cytometry were performed using a specific antibody against acidophilic granulocytes from the gilthead seabream. Cell proliferation was detected by immunocytochemistry using an anti-BrdU antibody and apoptotic cells by in situ detection of DNA fragmentation.ResultsThe fish in the western Mediterranean area developed as males during the first two RCs. The testis of all the specimens during the second RC underwent a degenerative process, which started at post-spawning and was enhanced during the testicular involution stage, when vitellogenic oocytes appeared in the ovary accompanied by a progressive increase in the ovarian index. However, only 40% of specimens were females in the third RC. Leukocytes (acidophilic granulocytes, macrophages and lymphocytes) were present in the gonad and acidophilic granulocyte infiltration occurred during the last two stages. At the same time DMRT1 gene expression decreased.ConclusionsThe results demonstrate that innate and adaptive immune cells are present in the gonads of gilthead seabream. Moreover, the whole fish population underwent a testicular degenerative process prior to sex change, characterized by high rates of apoptosis and necrosis and accompanied by an infiltration of acidophilic granulocytes and a decrease in DMRT1 levels.

17β-Estradiol regulates gilthead seabream professional phagocyte responses through macrophage activation

In mammals, estrogens regulate the immune system, either directly or indirectly via several leukocyte types through autocrine/paracrine mechanisms. In the gilthead seabream (Sparus aurata L.) gonad, an intensive remodeling process accompanied by the massive infiltration of acidophilic granulocytes (AG) is partially triggered by 17β-estradiol (E(2)). Once AG infiltrated the gonad, show impaired activities. In this study we first demonstrate that neither testicular nor head-kidney AG express any of the three estrogen receptor (ER) genes (ERa, ERb1 and ERb2) described in the gilthead seabream, while head-kidney macrophages (Mc) and lymphocytes (Ly) constitutively express ERa gene. Moreover, Mc are important in the immune-modulatory role of E(2), as suggested by its ability to induce ERb2 gene expression and up-regulate the expression of genes coding for ERa, ERb1, pro-inflammatory cytokines, chemokines and tissue remodeling molecules. Furthermore, the soluble factors produced by E(2)-treated Mc decreased in head-kidney phagocytes, their phagocytic ability and capacity, while no effects were observed on their reactive oxygen intermediate (ROI) production or their migratory capabilities. However, the role of Ly in the regulation of AG migration and the modulation of phagocytic and ROI production activities triggered by E(2) can not be ruled out, so that further studies are necessary to clarify these issues.

Natural and synthetic estrogens modulate the inflammatory response in the gilthead seabream (Sparus aurata L.) through the activation of endothelial cells.

Sex steroids are known to deeply alter processes other than fish reproduction, including fish growth, intermediary metabolism, osmoregulation and immunity. We have previously reported that 17β-estradiol (E(2)), the main fish estrogen, promotes the mobilization of acidophilic granulocytes from the head kidney, the bone marrow equivalent in fish, to the gonad in the bony fish gilthead seabream (Sparus aurata L.). The aim of this study was to investigate the effects of E(2) and 17α-ethinylestradiol (EE(2)), an endocrine disruptor with strong estrogenic effects commonly found in the aquatic environment, on the ability of gilthead seabream endothelial cells (ECs) to promote leukocyte infiltration. E(2) and EE(2) were seen to affect ECs in different ways. Thus, E(2) was able to increase the production of nitric oxide (NO) and up-regulate the expression of the key activation markers, interleukin-1β, CC chemokine ligand 4, interleukin-8, E-selectin and matrix metalloproteinase 9, when used alone or combined with bacterial DNA. In contrast, EE(2) failed to affect NO release and reduced the up-regulation of the above genes promoted by bacterial DNA. Moreover, we found that leukocyte adhesion to ECs was enhanced by E(2) treatment. Collectively, these results suggest that estrogens modulate fish leukocyte trafficking during an inflammatory process by activating ECs.

Estrogen-responsive genes in macrophages of the bony fish gilthead seabream: A transcriptomic approach

The role of sex steroids in the modulation of fish immune responses has received little attention. Previous studies have demonstrated that 17β-estradiol (E(2)) is able to alter the response of gilthead seabream leukocytes to infectious agents. We have used suppression subtractive hybridization to identify genes upregulated by E(2) (50 ng/ml) in macrophage cultures from gilthead seabream. We isolated 393 up-regulated cDNA fragments that led to the identification of 162 candidate estrogen-responsive genes. Functional analyses revealed the presence of several enriched immune processes and molecular pathways. The E(2) up-regulation of some immune-relevant genes was further confirmed by real time RT-PCR. Bioinformatics analysis revealed the ability of E(2) to orchestrate profound alterations in the macrophage expression profile, especially immune-related processes and pathways. This is the first report on E(2)-dependent modifications of fish macrophage transcriptome and lends weight to a suggested role for estrogen in the immune system, the possible significance of which is discussed.

TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity

As an organism is exposed to pathogens during very early development, specific defense mechanisms must take effect. In this study, we used a germ-free zebrafish embryo model to show that osmotic stress regulates the activation of immunity and host protection in newly hatched embryos. Mechanistically, skin keratinocytes were responsible for both sensing the hyposmolarity of the aquatic environment and mediating immune effector mechanisms. This occurred through a transient potential receptor vanilloid 4/Ca2+/TGF-β–activated kinase 1/NF-κB signaling pathway. Surprisingly, the genes encoding antimicrobial effectors, which do not have the potential to cause tissue damage, are constitutively expressed during development, independently of both commensal microbes and osmotic stress. Our results reveal that osmotic stress is associated with the induction of developmental immunity in the absence of tissue damage and point out to the embryo skin as the first organ with full capacities to mount an innate immune response.

Estrogen receptor 2b deficiency impairs the antiviral response of zebrafish.

Although several studies have demonstrated the ability of some endocrine disruptive chemicals (EDCs) to alter the physiology of zebrafish, the immune-reproductive interaction has received little attention in this species. In this study, we used a homozygous line carrying an insertion of 8 amino acids in the ligand-binding domain of the estrogen receptor 2b gene (esr2b) to further understand the role of estrogen signaling on innate immunity. Adult mutant fish showed distorted sexual ratios related with alterations in testicular morphology and supraphysiological testosterone and 17β-estradiol (E2) levels. Immunity-wise, although esr2b mutant fish showed unaltered antibacterial responses, they were unable to mount an effective antiviral response upon viral challenge. RT-qPCR analysis demonstrated that mutant fish were able to induce the genes encoding major antiviral molecules, including Ifnphi1, Ifnphi2, Infphi3, Mxb and Mxc, and the negative feedback regulator of cytokine signaling Socs1. Notably, although esr2b mutant larvae showed a similar resistance to SVCV infection to their wild type siblings, waterborne E2 increased their viral susceptibility. Similarly, the exposure of adult wild type zebrafish to E2 also resulted in increased susceptibility to SVCV infection. Finally, the administration of recombinant Ifnphi1 hardly reversed the higher viral susceptibility of esr2b mutant zebrafish, suggesting that elevated socs1 levels impair Ifn signaling. All together, these results uncover an important role for E2 and Esr signaling in the fine-tuning of sexual hormone balance and the antiviral response of vertebrates.

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.