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Trabajo presentado en el 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.

Trabajo presentado en el 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.

Trabajo presentado en 6th Genomics in Aquaculture Symposium, celebrado en Granada (España) del 04 al 06 de mayo de 2022.

Resumen del trabajo presentado en 6th Genomics in Aquaculture Symposium, celebrado en Granada (España) del 04 al 06 de mayo de 2022., Growth is one of the most desirable traits in an aquaculture species, which is in turn closely related to early maturity, a complex biological process that reduces the final yield of fish farming operations. Thus, while the selection of rapid-growth fish is highly attractive for the aquaculture sector, it is also common to select against early-maturing fish. The correlation between body size and age at maturation is still not entirely understood in fish, and several studies have reported different associations between the two traits. In this study, biometric and physiological parameters are being evaluated in order to determine the relationship of small and large sized female sea bass and its ovarian growth. Fish were individually tagged and growth and gonadal development were evaluated during the second and third year of age. Fish have been sacrificed at the end of each sexual cycle and performance indicators are being restored to specific point-in time based on the oocyte development. Temporal hormonal profiles, including Fsh, sex steroids, vitellogenin and Igf-1 plasma levels are being determined. Furthermore, genes with a putative role in the relationship between both growth and reproductive systems will be analysed. Results show the existence of early differential growth within the female sea bass. Thus, slow- (small-sized) and fast- (large-sized) growing female sea bass have been identified and their relationship to gonadal maturity has been considered. We have identified four female phenotypes as follows: a) small-sized and immature gonads, b) small-sized and maturing gonads, c) large-sized and immature gonads and d) large-sized and maturing gonads, over the second and third sexual cycles of this species. Accordingly, physiological aspects and also molecular factors are being considered to elucidate key events contributing to the relationship between these traits. A comprehensive understanding of the relationship between growth and maturation in sea bass will contribute to gain new insights for improving breeding conditions and production of this teleost species., Project funded from the Ministry of Science and Innovation (AGL2016-75400, PID2019-109548RB-I00). L.S. supported by a FPI fellowship from MICINN (Spain).

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.

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.

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., Several studies suggest that body weight in teleost during critical periods affects the proportion of fish that reach maturation in a given reproductive season. Although there is a large variation in the age of maturity, this trait depends on genetic, physiological and environmental factors that contribute to gonadal growth. While interactions between growth and reproductive competence in male fish have been reported, much is still to be resolved in females. In the sea bass a substantial proportion of males mature precociously during the first year of life suggesting that it is only allowed in those fish which have reached a certain threshold of development or accumulated enough lipid reserves. Precocious males, which are usually larger in size, exhibit a significant increase in 11-KT presumably induced by Fsh that is able to block amh expression, a meiosis inhibiting factor, thus evoking spermatogenesis progression. Little is known about the oocyte growth in prepubertal female sea bass and its relationships with body growth; however it has been reported that advanced puberty can also affect captivereared females. Recently, we have monitored the growth history of female sea bass and the degree of advancement in oocyte development during the first two years of life. Some body indexes and circulating vitellogenin served as markers of advanced ovarian growth. Over the second year of age, females could be divided into four distinct groups: females that remained at perinucleolar stage, females with oocytes in varying degrees of cortical alveoli incorporation and no lipid droplets, females with oocytes in secondary growth including oocytes in varying degrees of lipid droplet incorporation and no yolked droplets and females with oocytes with both lipid and yolked droplets inside. Consequently, our objective is to determine changes in ovarian gene influencing the rate of oocyte development in this aquaculture finfish species., This research was financially supported by the Spanish Ministry of Science and Innovation (AGL2016-75400, PUBERTRAIT).

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., European sea bass (Dicentrarchus labrax L.) is a teleost fish species that presents sexual dimorphism affecting growth. Under culture condition, a substantial proportion of fish develop as males and those exhibiting larger size usually mature earlier during its first year of age (i.e., precocious males). This situation presents economic problems for producers and thus the production of highly female-biased stocks is considered as an opportunity to benefit its aquaculture. Females grow faster than males and mature later, around 3 years old, reaching a marketable size (∼400 g) after 2-year production cycle. Nevertheless, there is limited information about relationships among body weight in females and how this may affect the rate of oocyte development. The objective of this work is to determine those factors that are important links in the mechanisms by which growth influences ovarian development. To this aim, the evolution of growth and reproductive performance of prepubertal females has been determined in a total of seven batches of fertilized eggs that were reared over the first two years of age. Firstly, results show that there are significant differences of growth in females. Secondly, prepubertal females with slower growth remain immature while those fish with faster growth display advanced oocyte development. It evidences that sea bass females are able to undergo advanced vitellogenesis at 2 years of age although the rate of fish entering into advanced puberty varies among sea bass batches. Significant changes among body weight and plasma Fsh, E2, and Igf-1 levels are also observed between immature and advanced females. These findings suggest that these factors are important links that signals growth and nutritional status to the reproductive axis. The present results show that growth performance in female sea bass has indeed some interesting features that merit to be investigated to face its profitability in aquaculture., This research was financially supported by the Spanish Ministry of Science and Innovation (AGL2016-75400, PUBERTRAIT).

Trabajo presentado en el XVII Congreso Nacional de Acuicultura, celebrado en Cartagena (España) del 7 al 10 de mayo de 2019, [ES] La precocidad en machos de lubina (Dicentrarchus labrax L.) está bien documentada en la literatura y se conocen los efectos negativos que esta situación trae consigo para el sector acuícola. Sin embargo, la incidencia en hembras es menos conocida y el impacto que esta situación puede acarrear a nivel productivo no se ha evaluado. Por ello, en el presente trabajo se analizó el porcentaje de hembras prepúberes que, bajo condiciones de cultivo, son capaces de alcanzar un estadio de vitelogénesis avanzada (VTGa) a los dos años de edad. Un hecho que puede considerarse como un proceso de maduración gonadal adelantada en hembras de esta especie. Para ello, se establecieron tres lotes de lubinas en los que periódicamente se evaluó el crecimiento, los valores de ciertos índices biométricos y los niveles plasmáticos de hormonas clave en reproducción. La evaluación del estado gonadal se llevó a cabo mediante técnicas histológicas. Los resultados obtenidos indican que existen hembras prepúberes de dos años de edad que poseen ovocitos en estadio de VTGa y que este porcentaje puede llegar a alcanzar valores hasta del 25-28%. Estas hembras son un 17% más grandes en peso que aquéllas que poseen ovocitos inmaduros (crecimiento primario y/o vitelogénesis incipiente). Asimismo, estas hembras en VTGa poseen un índice gonadosomático mayor que el de las hembras inmaduras (i.e., 3,05% vs 0,49%) que se correlaciona con valores bajos de sus índices viscerosomáticos. A nivel hormonal, los valores plasmáticos de vitelogenina muestran diferencias significativas asociadas con el estado de desarrollo gonadal de las hembras., [EN] Precocious puberty in sea bass males (Dicentrarchus labrax L.) has been widely reported in the literature and the negative effects that it has in aquaculture are known. However, the incidence in females is little known and its impact in the sector has not been evaluated. In the present work, we analysed the percentage of prepuber females that, under rearing conditions, are able to develop oocytes in advanced vitellogenic stage (VTGa) at two years of age. This could be considered as females with an early gonadal development in this species. Three different trials were established and fish were periodically measured for somatic growth and biometric indexes, while plasmatic levels of reproductive hormones were also evaluated. The gonadal development was performed by histological analysis. Results demonstrated that prepuber females are able to exhbit VTGa up to 25-28% at 2 years old. These females are 17% heavier in weight than those with immature oocytes (i.e., primary oocytes and early vitelogenesis). Females in advanced vitellogenic stage have a gondosomatic index higher than that of immature females (i.e., 3.05% vs 0.49%) in correlation with the low values of their viscersomatic indexes. Plasmatic levels of vitellogenin showed significant differences according to ovarian stage of female fish.

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)

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, The follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of gametogenesis in vertebrates. Gonadotropin-releasing hormones (Gnrh) have been postulated as the main regulators of the synthesis and secretion of Fsh and Lh since long. In addition, sex steroids produced by the gonads 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 (August), showed that Gnrh injections stimulated Lh synthesis and release, but had no effect on the expression of the Fsh beta-subunit gene (fshb). At the same time, different steroid implants almost suppressed basal fshb expression, while activated the expression of lhb in the pituitary. 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 estrogen, androgen and progesterone receptors, in relation with the different stages of spermatogenesis, the pituitary gonadotropin content and the circulating levels of sex steroids. In addition, immunohistochemistry studies have been performed to identify the pituitary cells containing steroid receptors. Next, we have used an in vitro pituitary primary cell culture stimulated with Estradiol, Testosterone Dihydroxyprogesterone and a Luteinizing Hormone-Releasing Hormone analogue (LHRHa) to analyse their impact on gonadotropin synthesis and release. It has been confirmed the strong effect of LHRHa on sea bass Lh release, while this effect is more weak on Fsh release and decreases with the advance of spermatogenesis. On the other hand, the main evidence of repression for both gonadotropins is due to Estradiol, with major intensity at the beginning of spermatogenesis., Founded by MINECO (AGL 2015-67477-C2-1-R), CSIC (201640E073) and GV (PROMETEO II-2014/051)

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)