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Trabajo presentado en 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology, celebrado en Faro (Portugal) del 04 al 08 de septiembre de 2022., Puberty comprises the developmental period that leads to the first successful reproduction. In female European sea bass (Dicentrarchus labrax) puberty occurs at the end of the third year of life, although intensive rearing leads to the appearance of precocious maturation one year earlier. This study is aimed at the characterization of the onset of precocious puberty at the gonad level in sea bass females through a hemigonadectomy approach. Hemigonadectomy allows the study of early events occurring in the ovaries prior to the appearance of any histological changes induced by the onset of puberty. With this purpose, by the time puberty was about to start the left ovary of a group of fish was removed by precise surgery. These ovaries were frozen and kept for further gene expression and ovarian steroid analysis. After the established gonadal maturation period of sea bass, histological observation of the developmental stage achieved by the remaining ovary was used to classify the fish into precocious or non-precocious. Blood samples were taken monthly to analyze plasma levels of sex steroids, follicle-stimulating hormone, luteinizing hormone and vitellogenin using specific ELISAs. Gene expression was analyzed by RT-qPCR.In February, the ovaries of non-precocious fish remained in previtellogenesis, while those of precocious animals attained different stages of secondary growth or oocyte maturation. Plasma hormonal profiles during the experiment showed differences between the two groups, and vitellogenin showed the most divergent pattern. Ovarian steroids and gene expression analyses revealed also differences between non-precocious and precocious fish. RNA samples were subsequently hybridized on a sea bass specific microarray for the identification of differentially expressed genes involved in early events of precocious female puberty. One of these genes is ncoa7 that encodes a nuclear receptor co-activator that, in mammals, increases the transcriptional activity of the estrogen receptor. We cloned the complete cDNA of sea bass ncoa7 and studied its tissue expression and its annual expression pattern in gonads of both sexes. Functional analyses of Ncoa7 in HEK293 cells cotransfected with estrogen, androgen or progesterone receptors, and exposed to 17b-estradiol, testosterone, 11-ketotestosterone or 17,20ß-P, showed that Ncoa7 is able to increase the transcriptional activity of all the different receptors. Moreover, immunoprecipitation and targeted mutation analysis showed which Ncoa7 domains are involved in direct binding to the nuclear receptors. All together our data indicate that in sea bass ovaries entering secondary growth for the first time the progesterone receptor is the most probable partner of Ncoa7, and therefore progestogens may have a decisive role in gonadal precocious puberty in females sea bass.

Trabajo presentado en la Joint 30th Conference of the European Society for Comparative Endocrinology and of the 9th International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022., Fluoxetine (FLX) is a highly prescribed selective inhibitor of serotonin-reuptake and an emerging pollutant affecting fish behaviour, stress and reproduction, but little is known about possible actions and mechanisms in barrier tissues. We combined in vivo and in vitro approaches to demonstrate multi-level impacts of FLX on the sea bass (Dicentrarchus labrax) skin-scale barrier and on the estrogenic system. Juvenile sea bass intraperitoneally injected with FLX had significantly increased levels of FLX and its metabolite nor-FLX. In contrast to the natural estrogen E2, FLX did not increase plasma calcium, phosphorus (P) or vitellogenin, although a slight decrease in scale P content was detected. Quantitative SWATH-MS proteomics of the scales identified 134 proteins that were affected by FLX. Modified proteins were mainly related to extracellular matrix and protein turnover and energy production, 31 of which were also affected by E2. Multiple estrogen receptors and genes related to serotonin activity, transport and degradation were expressed in sea bass scales and transcript abundance of some of them was modulated by E2 and/or FLX. Using a minimally invasive in vitro bioassay with cultured sea bass scales and adhering epithelia we showed direct effects of FLX exposure on enzymatic activity associated with mineral mobilization, while the expression of estrogen receptors was not significantly affected. In in vitro receptor-reporter assays, FLX alone did not activate any of the three sea bass nuclear estrogen receptors but had antiestrogenic effects on Esr1/2b when in co-treatment with E2, and directly activated both plasma membrane Gprotein-coupled estrogen receptors. The combination of in vitro and in vivo assays substantiated the notion that FLX disrupted scale physiology through several different processes, with probable consequences for fish health, and revealed that some of the mechanisms of disruption can result from direct interaction with multiple estrogen ., Projects UIDB/04326/2020, PTDC/AAG-GLO/4003/2012 and DL57/2016/CP1361/CT0015 from FCT (Pt); EU Interreg FR-UK project RedPol; grant AGL2015-67477-C2-1- R (Sp).

Trabajo presentado en 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the 9 International Society for Fish Endocrinology, celebrado en Faro (Portugal) del 04 al 08 de septiembre de 2022., Puberty is the developmental period during which an individual becomes sexually mature for the first time and its regulation is not completely known in teleosts. The gonadal soma-derived factor (Gsdf) gene was found to be downregulated in precocious testis of male European sea bass and proposed as an early gonadal marker of puberty. Gsdf is a member of the TGFbeta superfamily, it is found in tetrapods, excluding mammals, and it is apparently involved in the proliferation of type A spermatogonia. The genome of sea bass contains two gsdf duplicates, gsdf1 and gsdf2, whose encoded proteins share 87% identity and have gonad-specific expression.In the present study we aimed at characterizing the Gsdf genes of European sea bass. First, we have confirmed that expression of gsdf1 and gsdf2 decreases in testis of sea bass males that enter precocious puberty compared with their siblings that remain immature. To achieve this, prepuberal one-year -old male European sea bass of two different sizes (Small or Large) were sampled during the months corresponding to onset of gametogenesis in adults (August to November). The males from the Small groupdid not arrive to full spermiation and showed higher gsdf1 expression than the ones of the Large group during all the experiment, with significant differences in August and October. Most of the animals of the Large group spermiated the next winter. In adult males, gsdf1 showed maximum expression levels in premeiotic (immature) testis, that decreased as spermatogenesis progressed. The expression of gsdf2 followed the same trend but was 5-fold lower. This expression profile matched with the presence of Gsdf protein in testis extracts, and with its location in Sertoli cells surrounding type A spermatogonia, as revealed by IHC with a species-specific antibody.In adult female ovaries, mRNAs from gsdf1 and gsdf2 are present to a much lower level than in testis, and the highest levels correspond to gsdf2 in post-ovulatory ovaries and also in isolated follicular cells. In fact, Gsdf1/2 was located in follicular cells surrounding previtellogenic oocytes. The sea bass gsdf1 and gsdf2 genes are located in the same chromosome, and their coding sequences are placed in different strands and transcription directions. The functional data obtained so far point to common regulatory elements for both genes, but also to a sex-specific regulation for gsdf1 and gsdf2 connected to males and females respectively., Funded by Spanish MCIN/AEI/10.13039/501100011033 and EU-FEDER through grant RTI2018-094667-B-C22. A.M. is supported by a PhD contract from GV (GRISOLIAP/2020/129).

Resumen del trabajo presentado en la Joint 30th Conference of the European Society for Comparative Endocrinology and of the 9th International Society for Fish Endocrinology, celebradas en Faro (Portugal) del 04 al 08 de septiembre de 2022., Funded by Spanish MCIN/AEI/10.13039/501100011033 and EU-FEDER through grants AGL2015-67477-C2-1-R and RTI2018-094667-B-C22. A.M. has a PhD contract from GV (GRISOLIAP/2020/129).

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., In higher vertebrates, Anti-Müllerian Hormone (AMH) is required for involution of the Müllerian ducts during male sexual differentiation and for negatively regulating gonadal development in both sexes. AMH signals through a transmembrane AMH type II receptor (AMHR2) with serine-threonine kinase activity. Despite the absence of Müllerian ducts in teleosts, orthologues of mammalian AMH have been described in fish, and a role of this hormone in sex determination and gonad differentiation has been demonstrated. In adult teleost gonads, Amh exerts a role at early stages of germ cell development in both males and females, however, the exact mechanism of Amh signaling and its implication in gonad development are poorly investigated. Recently, in sea bass, we have demonstrated that Amh signals through Amhr2, and that Amh is localized in Sertoli cells surrounding early germ-cell generations. In addition, information available of amh and amhr2 expression during sea bass ovarian development suggest a role of Amh during vitellogenesis. However, still knowledge is lacking about the function and the specific mechanisms of Amh signaling during gametogenesis. As tool for studying the mechanisms of Amh action, we have produced specific recombinant mature forms of this hormone, and have used them in in vitro transactivation experiments to study the intracellular signaling pathways of sea bass Amhr2. In addition, we have performed in vitro tissue cultures of ovaries and testis treated with recombinant Amh, and in vivo experiments through Amh injection in fish, to study the impact of this hormone in steroidogenesis, and its interaction with Fsh, both by analyzing steroid production and gene expression in gonads. We finally concluded that Amh promotes steroid production in immature testis and early vitellogenic ovaries, showing a clear involvement of sea bass Amh in gametogenesis., Funded by MICINN (AGL2015-67477-C2-1-R, RTI2018-094667-B-C22) and EU (LIFECYCLE FP7-22719-1).

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., There are increasing concerns regarding the accumulation of pharmaceuticals in the environment as even low dose exposures can interfere with the endocrine system of fish and other non-target aquatic organisms. In this study, we carried out different in vitro approaches using European sea bass tissues or estrogen receptors to investigate possible estrogenic-disrupting effects of fluoxetine (FLX), a widely used antidepressant and emerging environmental contaminant. Using an in vitro scale bioassay, a slight but significant increase in the enzymatic activity of osteoclast marker TRAP was detected upon exposure to FLX or to the natural estrogen 17b-estradiol (E2) for 24h, while the activity of osteoblast marker ALP was unaffected. The transcript expression of different estrogen receptor subtypes was detected in sea bass scales (nuclear receptors esr2a and esr2b and membrane receptor gperb) but this was not affected by the in vitro exposure to both compounds. Using an in vitro estrogen-response element reporter gene assay, FLX alone did not activate any of the three sea bass nuclear estrogen receptors (Esr1, Esr2a, Esr2b) but when combined with E2 showed antiestrogenic effects on the transactivation through Esr1 and Esr2b. Using an in vitro cAMP response element-luciferase reporter gene assay, significant induction of both membrane estrogen receptors (Gpera and Gperb) was achieved with FLX or E2 alone, but no synergistic effects were detected using the mixture of the two compounds. This combination of in vitro assays suggests different estrogenic and antiestrogenic mechanisms through which FLX may interfere with the scales and other fish tissues and provides promising tools for risk assessment of other potential endocrine disrupting compounds., Work supported by the Portuguese Foundation for Science and Technology (FCT) through projects UIDB/04326/2020 and PTDC/AAG-GLO/4003/2012 and by EU project Interreg France England RedPol. FCT funded the Researcher contract to PP under “Norma Transitória”-DL57/2016/CP1361/ CT0015 and the Spanish Ministry of Science funded grant AGL2015-67477-C2-1-R to AG.

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-β superfamily that classically has been correlated to the regression of Müllerian ducts in higher vertebrates. Despite the absence of Müllerian ducts in teleosts, orthologues of mammalian AMH have been described in some fish species, and a role of this hormone in sex determination and gonad differentiation has been demonstrated. In adult teleost gonads, Amh acts at early stages of germ cell development in both males and females, however, the exact mechanism of Amh signaling and its implication in gonad development are poorly investigated. We have recently demonstrated that Amh signals through a transmembrane AMH type II receptor (Amhr2) with serine-threonine kinase activity, and that Amh is localized in Sertoli cells surrounding early germ-cell generations, but still lacking knowledge about the function of this growth factor. As tool for studying the mechanisms of Amh action, we have produced specific recombinant mature forms of this hormone, and have used them in in vitro transactivation experiments to study the intracellular signaling pathways of sea bass Amhr2. In addition, we have performed in vitro tissue cultures of ovaries and testis treated with recombinant Amh to study the impact of this hormone in steroidogenesis, and its interaction with Fsh, both by analyzing steroid production and gene expression. We finally concluded that Amh promotes steroid production in immature testis and early vitellogenic ovaries, showing a clear involvement of sea bass Amh in gametogenesis., Supported by funds from MICINN (AGL2015-67477-C2-1-R, RTI2018-094667-B-C22) and EU (LIFECYCLE FP7-22719-1). C. Z. was recipient of a postdoctoral fellowship from the Ministerio de Ciencia e Innovación (Programa Juan de la Cierva).

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., Puberty is the developmental period during which an individual becomes sexually mature for the first time and its regulation is not completely known in teleosts. The gonadal soma-derived factor (Gsdf) was found to be downregulated in precocious testis of male European sea bass (Dicentrarchus labrax) and proposed as an early gonadal marker of puberty. The genome of sea bass contains two gsdf duplicates, gsdf1 and gsdf2, whose encoded proteins share 87% identity and have gonad-specific expression. In the present study, we have confirmed that expression of gsdf1 and gsdf2 decreases in testis of 1-year old sea bass males that enter precocious puberty compared with their siblings that remained immature. In adult males, gsdf genes showed high expression levels only in premeiotic testis, and gsdf2 expression was 5-fold lower than that of gsdf1. This expression profile matched with the presence of Gsdf protein in testis extracts, and with its location in Sertoli cells surrounding type A spermatogonia. In adult females, gsdf1 and gsdf2 are expressed to a much lower level than in testis, and the highest expression found corresponds to gsdf2 in post-ovulatory ovaries and also in isolated follicular cells. Indeed, the protein was located in follicular cells surrounding previtellogenic oocytes. To functionally characterize this gonadal factor, a sea bass recombinant Gsdf has been produced in CHO cells, exhibiting the same size as the native protein. Sea bass gsdf1 and gsdf2 are located in the same chromosome, and their coding sequences are placed in different strands and transcription directions. The functional data obtained so far point to common regulatory elements for both genes, but also a sex-specific regulation for gsdf1 and gsdf2 connected to males and females respectively, Funded by MICINN grant RTI2018-094667-B-C22. AM is supported by a PhD contract from GV (GRISOLIAP/2020/129)

The follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of gametogenesis in vertebrates, and gonadotropin-releasing hormones (Gnrh) have been postulated as the main regulators of their synthesis and secretion. Gonadal sex steroids have a feedback effect modulating the availability of gonadotropins. All these effects at the level of pituitary have a direct impact in gametogenesis progression. Previous in vivo studies in sea bass, during the sexual resting period, showed that Gnrh injections stimulated Lh synthesis and release, but had no effect on the expression of the Fsh beta-subunit gene. At the same time, different steroid implants repressed fsh-b expression, but activated the expression of lh-b in the pituitary. To elucidate how this system is organized in the pituitary of male sea bass, we analysed the annual expression profile of the three nuclear estrogen receptors (Esrs) in male pituitary and the circulating levels of estradiol in relation with the different stages of spermatogenesis. Also, immunohistochemistry studies have been performed to identify the pituitary cells containing steroid receptors and their relationship with gonadotrophs and GnRh1 fibers. Moreover, we have used an in vitro pituitary primary cell cultures stimulated with estradiol-17β (E2) to study its direct action on gonadotrophs, and E2 implants to study its effect in vivo on the pituitary and the gonad. We have concluded that E2 has an inhibitory effect on the gonadotropins, although with different specific actions, which is also reflected in the localization of the Esrs in the gonadotrophs, while Gnrh differentially regulates Lh and Fsh cells in male seabass., Funded by Spanish MICINN (AGL2015-67477-C2-1-R, RTI2018-094667-B-C22) and CSIC (201640E073). PP was supported by the Portuguese FCT through project UID/Multi/04326/2019 and a researcher contract under “Norma Transitória”-DL57/2016/CP1361/CT0015.

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: In nature, European sea bass males reach puberty by their second year of life; however, under intensive culture conditions, the sex ratio male:female increases (3:1), males generally are smaller than females and the precocious puberty occurs as a phenotypic response to enhanced growth conditions and feed availability (Espigares et al., 2015). Around 20-30% of farmed male European sea bass enter puberty at one year of age (Carrillo et al., 1995). Puberty is associated with a progressive reduction of the growth rate with age (Taranger et al. 2010), that is even more marked in precocious males compared to their non-precocious counterparts during the second year (Felip et al., 2006), resulting in considerable economic losses in fish farms. The gonadal soma-derived factor (Gsdf), which belongs to the transforming growth factor beta (TGFβ) superfamily, is exclusively found in teleosts and it is apparently involved in the proliferation of type A spermatogonia, with expression levels decreasing as spermatogenesis progresses (Schulz et al., 2010; Skaar et al., 2011). Therefore, it could be considered a decisive player in the onset of puberty and a possible marker for the early detection of precocious males., [Materials and methods]: All the animals used for experiments were kept under natural photoperiod and temperature conditions at the Instituto de Acuicultura Torre de la Sal (IATS) facilities. Juvenile (9-months-old) European sea bass males were subjected to hemigonadectomy in September, the period when spermatogonial proliferation towards differentiation may occur in precocious males (Molés et al., 2011). The left testicle was extracted from each animal, frozen in liquid nitrogen and stored at -80 °C until molecular analysis. Hemigonadectomized animals were kept until the spermiation period (February), when they were sacrificed and the right gonad collected for histological analysis. One-year-old male European sea bass kept in natural conditions were sampled every two weeks from August to November. In each sampling only the smallest 15% (Small group) and the largest 25% (Large group) fish were selected for analysis. Gonads were sampled for histological analysis or frozen in liquid nitrogen and stored at -80 °C to perform gene expression assays. Adult specimens of male sea bass (5-years-old) kept in natural conditions were sampled monthly during an entire annual reproductive cycle. Testes from all fish (9-months-old hemigonadectomized, 1-year-old and adult animals) were staged, according to Begtashi et al. (2004). Total RNA was extracted from whole testes and reverse-transcribed, and cDNAs were used as templates for quantitative real-time RT-PCR (qPCR) assays., [Results]: The hemigonadectomized 9-months-old animals were classified as “non-precocious” or “precocious” when right testes remained in stage I, or reached stage IV, respectively. The gsdf1 gene was found to be significantly downregulated in the left testes of precocious animals in September (p < 0.05) (Crespo et al., 2013). One-year-old males from the Small group did not arrive to full spermiation and showed higher gsdf1 expression than the ones of the Large group during all the experiment time, with significant differences in August and October. Most of the animals of the Large group spermiated the next winter. During the annual cycle of adult sea bass, gsdf1 showed maximum expression levels in premeiotic (immature) testis, that decreased as spermatogenesis progressed, with a slight increase in stage VI (post-spawning) in preparation for the next reproductive cycle., [Discussion]: In juvenile 9-months-old sea bass males, precocious puberty is negatively correlated with gsdf1 expression. According to Begtashi et al. (2004), during their first year of life, precocious males are significantly larger than non-precocious one. Thus, in the group of 1-year-old large fish there are significantly more precocious animals than in the group of small specimens. In accordance with these results, small males (supposed not precocious) showed higher gsdf1 expression than the large ones. In adult males, high expression levels of the gene are only detected in premeiotic stages, suggesting a role for gsdf1 in spermatogonial stem cells, or in proliferation of undifferentiated type A spermatogonia. Once spermatogenesis starts, a number of undifferentiated type A spermatogonia halt the self-renewal process and enter the differentiation pathway toward meiosis, which would explain the lower expression levels of gsdf1 in adult males after premeiotic stages and in juvenile precocious animals (Crespo et al., 2013). In summary, this study confirms gsdf1 as marker of precocious puberty and as a potential target for puberty manipulation., Funded by MICINN grants CSD2007-00002 and RTI2018-094667-B-C22, and by EU project LIFECYCLE (FP7-222719-1). A.M. is supported by a PhD contract from GV (GRISOLIAP/2020/129).

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: Estrogens are involved in the regulation of a wide range of processes in teleost reproduction (Lubzens et al, 2010; Schulz et al, 2010), and exert their functions mainly through ligand-activation of their specific cognate receptors. Nuclear estrogen receptors are transcription factors that bind to estrogen response elements (EREs) on gene promoters to regulate their expression (Tsai et al, 1994), and three subtypes of these receptors have been identified in teleosts. Differences in binding affinity and seasonal expression patterns in reproductive tissues among estrogen receptor subtypes suggest different roles during oogenesis, vitellogenesis and testicular development along the brain-pituitary-gonad axis. It is known that endocrine-disrupting compounds (EDCs) may act as agonists or antagonists of regulatory actions of steroid receptors or of the production of the receptors themselves. Thus many different points in the endocrine control of fish reproduction can be potential targets for the actions of EDCs. In aquaculture, the presence of estrogenic compounds in water is as worrying as its presence in new formulation of commercial fish diets (Arpin-Pont et al, 2016; Nacher-Mestre et al, 2013; Quesada-García et al, 2012). Nevertheless, the lack of information available on the role of each nuclear estrogen receptor in teleosts, including European sea bass, makes it difficult to have an accurate knowledge on the impacts of EDCs on reproduction. This study focused on investigating the role of the three nuclear estrogen receptor subtypes in European sea bass, on evaluating the impacts of endocrine-disrupting compounds on nuclear estrogen receptors functions and on their use as tools for risk assessment., [Material and methods]: To investigate the role of the three nuclear estrogen receptors subtypes in European sea bass, analysis of the expression of their coding genes during a whole reproductive cycle was investigated by qPCR in testis, ovary and pituitary. Localization of the nuclear estrogen receptors along the brain-pituitary-gonad axis was performed by immunohistochemistry using specific antibodies for each subtype. Using the human embryonic kidney cell line HEK293, we have performed transient transfections (1) to characterize nuclear estrogen receptors (Esr1, Esr2a and Esr2b) by using transactivation of an ERE- luciferase reporter gene assay; (2) to study the effect of two possible EDCs, genistein – a phytoestrogen also present in fish meals – and fluoxetine – an antidepressant, mainly constituent of Prozac - on all nuclear estrogen receptors from sea bass., [Results]: The coding genes of the three nuclear estrogen receptor subtypes of sea bass are highly expressed in reproductive-related tissues such as pituitary and gonad. Quantification of esr1, esr2a and esr2b expression in the gonad and pituitary during a whole reproductive cycle showed different expression patterns depending on stage and subtype. Localization of the three nuclear estrogen receptors along the pituitary-gonad axis showed differences among subtypes depending on the gonadal stage. The functional characterization of the nuclear estrogen receptors showed that there are different ligand affinities among the nuclear estrogen receptor subtypes, which also translate into differential receptor responses when we evaluate the effect of potential EDCs, such as genistein and fluoxetine., [Conclusion]: The results suggest that the three nuclear estrogen receptors of European sea bass are not redundant and have differential roles in the regulation of gametogenesis, as proposed also in other teleosts, which means that the effects produced by EDCs can induce potential adverse effects at reproductive level. The results also show that in vitro bioassays using nuclear estrogen receptors are a good tool for risk assessment of potential endocrine-disrupting compounds.

Trabajo presentado en el 8th Young environmental scientists meeting, celebrado en Ghent (Bélgica) del 5 al 10 de febrero de 2019, Oestrogens are important regulators of multiple physiological processes. Across vertebrates, the oestrogenic action is mediated by the binding to several oestrogen receptors. Specific transcription factors designated nuclear oestrogen receptors (Esrs) have been mainly associated with the classical activation of gene expression by oestrogens or structurally similar compounds. More recently,membrane receptors, such as the G protein-coupled oestrogen receptor (Gper), have been related to rapid, non-genomic responses. Both signaling pathways are present in fish, which express additional gene duplicates for both receptor types. Using the human embryonic kidney cell line HEK293, transient transfections were performed (1) to study the effects of two chemicals, genistein - a phytoestrogen - and fluoxetine (Prozac®) – an antidepressant - on the activation of the sea bass Esrs, using an ERE-luciferase reporter gene assay; and (2) to characterize the sea bass Gper duplicates, using a cAMP response element-luciferase reporter gene assay. Results indicate that genistein and fluoxetine affect each nuclear receptor in a different manner: Fluoxetine rather triggers an anti-oestrogenic response, while genistein behaves as an oestrogen mimic in the transactivation of the three Esrs. Oestradiol was able to induce luciferase activity in cells transfected with both membrane receptors, confirming, for the first time, that both teleost gene duplicates are functional. This study also demonstrates the suitability of the luciferase reporter assay as a tool to assess the oestrogenic potency and mechanisms of action of a wide variety of chemicals. Potential risks of the exposure of fish to various compounds in the environment or in aquaculture can be identified efficiently.

Póster presentado en el XII Congress of Iberian Association for Comparative Endocrinology, celebrado en Faro (Portugal) del 26 al 28 de septiembre de 2019., The follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of gametogenesis in vertebrates, and gonadotropin-releasing hormones (Gnrh) have been postulated as the main regulators of their synthesis and secretion. In addition, gonadal sex steroids have a feedback effect modulating the availability of gonadotropins. All these effects at the level of pituitary have a direct impact in gametogenesis progression. Previous in vivo studies in sea bass, during the sexual resting period, showed that Gnrh injections stimulated Lh synthesis and release, but had no effect on the expression of the Fsh beta-subunit gene. At the same time, different steroid implants repressed fsh beta expression, but activated the expression of lh-beta in the pituitary. In this study we have focused on the action of estradiol in males in different moments of gametogenesis. To elucidate how this system is organized in the pituitary of sea bass, we analysed in a first step the annual profile of male pituitary expression of the three nuclear estrogen receptors, in relation with the different stages of spermatogenesis, the pituitary gonadotropin content and the circulating levels of estradiol. In addition, immunohistochemistry studies have been performed to identify the pituitary cells containing steroid receptors and their relation with gonadotrophs and GnRh1 fibers. Next, we have used an in vitro pituitary primary cell cultures stimulated with estradiol to study its direct action on gonadotrophs, and estradiol implants to study its in vivo effect on the pituitary and the gonad. We have concluded that estradiol has a general an inhibitory effect on the gonadotropins, although with different specific actions, which is also reflected in the distribution of the estradiol receptors in the gonadotrophs, while Gnrh differentially regulates Lh and Fsh cells in male seabass., Funded by MICINN (AGL2015-67477-C2-1-R, RTI2018-094667-B-C22) and CSIC (201640E073)

Accumulation of chemicals in aquatic ecosystems remains a concern, despite extensive legislation to control discharges. In this study, the impacts of in vivo exposures of juvenile sea bass (Dicentrarchus labrax) to the antidepressant fluoxetine (FLX), and to the natural estrogen 17βestradiol (E2) were investigated. A significant increase in E2 and FLX plasma levels confirmed the effectiveness of exposure by injection. Plasma estrogen-responsive parameters including calcium, phosphorus and vitellogenin were significantly altered in response to E2 but not FLX, while enzyme activities related to mineral turnover in scales were not affected. The scale proteome of fish exposed to E2 and FLX for 5 days revealed that 213 proteins had significantly altered levels compared to the control group, 31 of which were altered by both E2 and FLX. In vitro transactivation assays revealed antiestrogenic activities of FLX via nuclear estrogen receptors when combined with E2 and indicated one of the potential mechanisms through which FLX may interfere with scales., Supported by FCT through projects PTDC/AAGGLO/4003/2012, UID/Multi/04326/2019 and UID/NEU/04539/2019, researcher contracts under “Norma Transitória”-DL57/2016/CP1361/CT0015 to PP and DL57/2016/CP1361/CT0011 to LA and grant SFRH/BD/88419/2012 to CS, and by the Spanish Ministry of Science through grant AGL2015-67477-C2-1-R to AG.

Comunicación presentada en el Society of Environmental Toxicology and Chemistry Europe - SETAC Europe 29th Annual Meeting, celebrado en Helsinki (Finlandia) del 26 al 30 de mayo de 2019., Oestrogenic compounds are well known endocrine disruptors. In aquatic ecosystems, these compounds may derive from natural (e.g. phytoestrogens) or anthropogenic sources (e.g. pharmaceutical products such as contraceptives or antidepressants) and a continuous exposition to these pollutants may threaten animals’ health. Oestrogens are important regulators of multiple physiological processes and oestrogenic actions are mediated across vertebrates by binding to several oestrogen receptor types. Specific transcription factors, designated nuclear oestrogen receptors (Esrs), have been mainly associated with the classical activation of gene expression by oestrogens or structurally similar compounds. More recently, membrane receptors such as the G protein-coupled oestrogen receptor (Gper) have been associated with rapid, non-genomic responses to estrogens. Both signaling pathways are present in fish, which express additional gene duplicates for both receptor types, although relative oestrogenic activities to the different receptors may differ between species. Using transient transfections in the human embryonic kidney cell line HEK293 and an ERE-luciferase reporter gene assay, the activation of sea bass Esrs was studied in response to of two compounds, the phytoestrogen genistein and the antidepressant and emerging pollutant fluoxetine (Prozac®). In addition, the activation of the two recently identified sea bass Gper duplicates was evaluated using a cAMP response element-luciferase reporter gene assay. Results indicate that genistein and fluoxetine affect each nuclear receptor in a different manner: Fluoxetine rather triggers an anti-oestrogenic response, while genistein behaves as an oestrogen mimic in the transactivation of the three Esrs. Oestradiol was able to induce luciferase activity in cells transfected with both membrane receptors, confirming, for the first time, that both teleost gene duplicates are functional. Furthermore, several xenoestrogens also stimulated luciferase activity and G1 and G15 were confirmed as agonist and antagonist of sea bass Gper, respectively. This study also demonstrates the suitability of the luciferase reporter assay as a tool to assess the oestrogenic potency and mechanisms of action of a wide variety of chemicals. Potential risks of the exposure of fish to various compounds in the environment or in aquaculture can be efficiently identified.

Trabajo presentado en el 11th International Symposium on Reproductive Physiology of Fish, celebrado en Manaus (Brasil), del 3 al 8 de junio de 2018

Trabajo presentado en el 6th International Workshop on the Biology of Fish Gametes, celebrado en Vodňany (República Checa), del 4 al 7 de septiembre de 2017, In higher vertebrates, anti-Müllerian hormone (AMH) is required for involution of the Müllerian ducts during male sexual differentiation and for negatively regulating gonadal development in both sexes. AMH signals through a transmembrane AMH type II receptor (AMHR2) with serine-threonine kinase activity. Despite the absence of Müllerian ducts in teleosts, orthologues of mammalian AMH and AMHR2 have been described in some fish species, and a role of this hormone in sex determination and gonad differentiation has been demonstrated. In addition, it seems that Amh inhibits germ cell proliferation and differentiation, as well as steroidogenesis, in adult gonads of both sexes. However, the mechanism of Amh signaling and its implication in gonad development are poorly investigated. In European sea bass, a recent study showed higher expression levels of amh and amhr2 during early and final stages of spermatogenesis, and immunolocalization of Amh in Sertoli cells surrounding early germ-cell generations, in line with its role in germ cell proliferation and differentiation. Available information of amh and amhr2 expression during sea bass ovarian development shows an opposite profile for these two genes. Although this information may suggest a role of Amh in early stages of oogenesis, the specific mechanisms of Amh signaling during ovarian development still have to be investigated. As tool for this kind research recombinant sea bass Amh would be needed. We know from previous studies that this hormone is processed in a similar way to mammalian AMH, becoming a biologically active protein able to bind and activate the sea bass Amhr2. Here, we report the production of an His-tagged recombinant sea bass Amh in a Pichia pastoris expression system, where Amh is endogenously cleaved and secreted as a mature peptide into the culture media. Bioactivity of this recombinant sea bass Amh has been demonstrated using COS7 cells co-transfected with the sea bass amhr2 cDNA and the BRE-luc reporter plasmid. Localization of Amh in granulosa cells of vitellogenic ovarian follicles was shown using a specific antibody. To investigate how Amh regulates sea bass ovarian development, we performed an in-vitro tissue culture of vitellogenic ovaries treated with recombinant Amh. First, we analyze steroid release in the medium by using a specific EIA. Finally, several candidate genes regulated by Amh were analyzed by real time qPCR., Funded by MINECO (AGL2015-67477-C2-1-R, AGL2011-28890), EU (LIFECYCLE FP7- 22719-1) and GV (PROMETEOII-2014/051)