Search

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.

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: The European eel (Anguilla anguilla) is a highly valued species targeted for aquaculture production. However, it is considered a critically endangered species by CITES, and reproduction in captivity seems to be the only realistic alternative to solve both economic and ecological problems (Asturiano, 2020). It is possible to obtain gametes for artificial fertilization but it requires hormonal treatment, which is a time-consuming and expensive process (Gallego et al., 2012). Moreover, the variable response to these treatments results in high mortality rates during the early stages of development, which could be caused (at least in part) by low sperm quality and uncontrolled epigenetic factors (Herráez et al., 2017). Considering the environmental conditions experienced during eels oceanic migration, the effects of low temperature combined with hormonal treatment have been used to better understand the onset of eel maturation (Peñaranda et al., 2016). Thus, the present study considers the use of low temperature as a pre-treatment before starting a standard hormonal treatment and evaluates its impact on eel maturation and male reproductive performance., [Material and Methods]: Eighty-eight male European eels were maintained for 3-4 days in freshwater at 20 ºC. Then, 6 males were sacrificed, as initial control, and the rest were changed to seawater at 10 ºC, except the control group, which was changed to seawater but maintained at 20 ºC. Low water temperature pre-treatments were applied during 1, 2 or 4 weeks to groups of 20 males. Once finished each pre-treatment, 8 fish per group were sacrificed for sampling. The temperature of the water of the remaining males was increased to 20 ºC in 3-4 days and they started receiving standard hormonal treatment (weekly administration of recombinant chorionic human gonadotropin, rhCG; Ovitrelle, Spain, 1.5 IU/g fish). From sacrificed fish (controls and pre-treated) biometric parameters were measured. Blood and testis samples were used to perform steroid (T, 11-KT and E2) analyses and histology, respectively. Once eels were spermiating, sperm volume, motility and kinetic parameters were evaluated weekly using a CASA system., [Results]: The eye, fin and hepatosomatic indexes from 1-week, 2- and 4-weeks pre-treated fish were significantly higher than those of the control group. The gonadosomatic indexes registered in the control group were unusually high and significantly differed with 1-week pre-treatment group but not with the 2- and 4-weeks groups. Androgen levels (T, 11-KT) were significantly higher after 4-weeks of pre-treatment in comparison with the control. The E2 levels did not show the same increasing profile. The control and 1-week pre-treated males showed significantly higher percentages of undifferentiated and differentiated type A spermatogonia in comparison with early spermatogonia type B. The males after 2- and 4-weeks of pre-treatment revealed a significantly increased proportion of differentiated spermatogonia type A. After the rhCG treatment, males from the 4-weeks pre-treatment group started to produce sperm earlier than males from the other groups. The sperm volume in the 4-weeks pre-treatment group increased during the first weeks of spermiation and in the control group at the end of the experiment, but no significant differences were found. Sperm motility and kinetics parameters were low in pre-treated groups throughout the whole experiment while in the control group increased significantly at the end of the hormonal treatment., [Discussion]: The onset of maturation in the European eel is controlled by environmental conditions found during their migration. The application of pre-treatments can be important to induce maturation in aquaculture eels, which do not encounter these conditions (Palstra and van den Thillart, 2009). Several factors such as temperature, salinity, forced swimming and pressure have been used as part of pre-treatments applied to eels (reviewed by Asturiano et al., 2020). Rozenfeld et al. (2019) evaluated the effects of cold seawater in European eel maturation without hormonal treatment. In eels treated at 10 ºC, the T and 11-KT levels were higher and there was an increase in the proliferation of differentiated type A spermatogonia. Our results also corroborate that low-temperature pre-treatment (4-weeks at 10 °C) induces an early sexual development, thus leading to the release of androgens which promote the proliferation of type A spermatogonia. However, the low sperm quality found in the pre-treated groups revealed that thermal pre-treatment followed by rhCG treatment at high temperatures impairs spermatogenesis. Low-temperature pre-treatments induce testis cell proliferation and synchronization but inhibit further maturation. Peñaranda et al. (2016) suggested that low seawater temperature inhibits the gene expression of enzymes responsible for the synthesis of maturation-inducing steroids (MIS) during eels oceanic migration. Thus, the premature maturation of the testis is prevented before eels arrive to the spawning areas and a temperature threshold is reached allowing the change of the steroidogenic pathway to change from androgens to MIS. In our experiment, lowtemperature pre-treated males had a time-dependent and fast response to rhCG treatment (in comparison with control males maintained at 20 °C), but their spermiation period was shorter and their sperm motility parameters were significantly lower. If this is evidencing a lower rate of gene expression in the testis, and thus limiting the final sperm maturation process, is an open question that requires further research.

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.

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., The European eel (A. anguilla) is a valued species targeted for aquaculture production. However, it is considered a critically endangered species and its reproduction in captivity seems to be the only alternative to solve both economic and ecological problems. Although it is possible to obtain eel gametes for artificial fertilization, it requires longterm hormonal treatments. Recent studies found that cold seawater stimulates the brainpituitary-gonad axis in eel males, inducing the start of spermatogenesis. These results suggested that cold seawater could be used as a pre-treatment before the standard hormonal therapy applied to induce sexual maturation, to obtain higher sperm quantity and quality. Thus, our study evaluates the effects of cold seawater pre-treatments of different duration (1, 2 and 4-weeks) on reproductive performance of eel males. Biometric parameters from control and pre-treated fish were measured. Blood and testis samples were collected for steroid (T, 11-KT and E2) and histology analyses, respectively. Once eels were spermiating, sperm parameters were evaluated using a CASA system. Eel males pre-treated with cold temperature showed increased biometric indexes, androgens and E2 plasma levels. Males maintained at 10 ºC for 4-weeks showed a significantly higher percentage of differentiated type A spermatogonia in comparison to undifferentiated ones. However, the low sperm quality found in the pre-treated groups revealed that pretreatments followed by recombinant human chorionic gonadotropin (rechCG) treatment at high temperatures impairs spermatogenesis. Cold temperature pre-treated males had a time-dependent and fast response to rechCG treatment, but their spermiation period was shorter and sperm parameters were significantly lower. Further research is needed to study the effect of thermic pre-treatments on gene regulation controlling spermatogenesis., Funded by the Spanish MICIU (Project EELGONIA; RTI2018-096413-B-I00). LF and MM have contracts funded by Generalitat Valenciana (GRISOLIAP/2020/063 and APOSTD/2020/053). VG had a grant from the MICIU (IJCI2017-34200).

Topic Coordinators: María Begoña García ( IPE, CSIC ); Pedro Jordano ( EBD, CSIC )

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.

La idea inicial consiste en el diseño, fabricación y comercialización de un automóvil deportivo de tipo biplaza con chasis tubular, reutilizando y adaptando, en la medida de lo posible, los componentes empleados en los prototipos de las competiciones en las que participa la asociación de la universidad “Escudería UC3M”, muy especialmente en la denominada “Formula Student”, con el objetivo de reducir tiempo y dinero en el desarrollo. Este objetivo nace de la creación durante los últimos años de un nicho de mercado para deportivos ligeros, productos que buscan ofrecer sensaciones muy parecidas a un monoplaza a un precio contenido. Muy asequibles, tanto respecto tanto a los superdeportivos, como a los deportivos de producción en serie a gran volumen. Una de las ventajas competitivas del producto nace de entender que estos deportivos suelen apartar a mucha gente por el hecho de no ofrecer un aislamiento mayor de los elementos -requieren conducirlos con casco-, que sí buscan un vehículo que tenga las mismas prestaciones dinámicas. El objetivo complementario sobre el que discurre este proyecto es además aprovechar la infraestructura existente en la Universidad, utilizando como nexo esta plataforma "Escudería UC3M" para crear un modelo de desarrollo que integre estudiantes de diferentes departamentos de la Universidad dentro de una empresa. Ingeniería en Tecnologías Industriales

En los vertebrados, las gonadotrofinas (FSH y LH) son hormonas esenciales en el control de la gametogénesis y esteroidogénesis gonadal. Sus acciones están mediadas por la activación de sus respectivos receptores. A pesar de su importancia en la función reproductora, en teleósteos se dispone de muy poca información relativa a estos receptores. En este trabajo se investigaron las características moleculares, estructurales y funcionales de los receptores de gonadotrofinas (FSHR y LHR) de un teleósteo marino, la lubina (Dicentrarchus labrax). Además, se evaluó la especificidad de unión al ligando de estos receptores. Usando un método de RT-PCR cuantitativo, se midieron los perfiles de expresión de los receptores de gonadotrofinas durante la primera maduración gonadal de machos y hembras de lubina. Asimismo, se investigaron posibles relaciones entre estos perfiles, los de la proteína responsable de la respuesta aguda de la esteroidogénesis (StAR) y los perfiles plasmáticos de esteroides sexuales. Estudios recientes realizados en diversas especies de peces sugieren que el receptor de TSH (TSHR), que comparte características estructurales con los receptores de gonadotrofinas, podría estar directamente implicado en la fisiología gonadal. Por ese motivo, en este trabajo se investigó una posible participación del TSHR en la función reproductora de la lubina. Los resultados obtenidos contribuyen a entender en que momento del ciclo reproductor las células germinales en crecimiento son sensibles a las señales procedentes de la hipófisis (gonadotrofinas). Además, se ha demostrado que, a diferencia de otros peces, las interacciones entre los receptores de gonadotrofinas de lubina y sus respectivos ligandos son específicas. Asimismo, podemos decir que en la lubina, los receptores de gonadotrofinas se expresan en momentos diferentes del ciclo reproductor. Esto apoya la idea de que la FSH y la LH poseen diferentes acciones en el control de la función gonadal. Desde de un punto de vista aplicado, estos conocimientos podrían ser de gran importancia en la manipulación del ciclo reproductor de esta especie de gran valor económico. Por lo tanto, estos estudios son relevantes tanto por su componente de investigación básica como por las posibles aplicaciones que pueden derivarse., In vertebrates, the pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are the main regulators of gametogenesis and gonadal hormone production. To exert their biological actions, gonadotropins must bind to, and activate specific cell-surface receptors. The primary aim of the studies underlying this thesis was to investigate the role of gonadotropins in the sea bass (Dicentrarchus labrax) reproductive function by studying their cognate receptors. Two cDNAs encoding a FSH receptor (sbsFSHR) and a LH receptor (sbsLHR) were cloned and characterized. Their gene structure, expression, and functional activity were evaluated. Sea bass FSHR and LHR mature proteins display typical features of the glycoprotein hormone receptor family members, but the sbsFSHR also contains some remarkable differences when compared with other fish or mammalian FSHRs. Among them, a distinct extracellular N-terminal cysteine domain as regards to its length and cysteine number, and the presence of an extra leucine-rich repeat. sbsLHR is more conserved than sbsFSHR when comparing their genomic primary structures with other vertebrate gonadotropin receptors, particularly in the extracellular domain. Furthermore, two alternately spliced variants of the sbsFSHR were amplified. Both correspond to transcripts originated by removal of exons from the extracellular domain. Expression analysis revealed that the sbsFSHR is exclusively expressed in gonads, specifically in the wall of previtelogenic and early-vitelogenic follicles. On the contrary, sbsLHR mRNA was found to be widely distributed in somatic tissues. Recombinant sbsFSHR was specifically stimulated by bovine FSH, while sbsLHR was activated by both bovine LH and FSH. Nevertheless, specific stimulation of both sea bass FSHR and LHR was observed when recombinant sea bass gonadotropins were used advancing specific interactions. This behaviour differs from studies in other teleost where promiscuous activation was reported. The expression profiles of sea bass gonadotropin receptors were investigated in the gonads of both sexes during a complete reproductive cycle, in parallel with the expression of steroidogenic acute regulatory protein (StAR) gene and reproductive hormones. While in male sea bass, both gonadotropin receptors show different expression patterns, in females their expression patterns were somehow similar. Expression of the sbsFSHR was connected with early stages of gonadal development, but also with the spermiation period in males and the maturation-ovulation period in female. The expression profile of the sbsLHR, in both sexes, supports the already suggested involvement of LH in the regulation of the final stages of fish gamete maturation and ovulation/spermiation. sbsStAR expression was strongly correlated with sbsLHR expression. In addition, the expression profile obtained for sbsStAR suggests that this protein mediates the rapid delivery of sterol substrate for the synthesis of gonadal maturation inducing steroids. No strong relationship between gonadotropin receptor expression and plasma levels of the analysed sex steroids was seen. Further studies will be needed to understand how gonadotropins and sex steroids interact to regulate sea bass reproduction.Recent studies reported abundant expression of TSHR transcripts in both the ovary and testis of some fish species. To help elucidate the physiological role that TSHR may have in the gonadal function of sea bass, a cDNA encoding a TSHR was isolated from the gonads of this fish species. The mature protein displays typical features of the members of the glycoprotein hormone receptor family. By RT-PCR analysis we demonstrate the extra-thyroidal expression of sbsTSHR in numerous tissues of the sea bass. Seasonal changes in sbsTSHR mRNA levels in female and male sea bass during puberty suggest that in females the TSHR could participate in active vitellogenesis and in the regulation of gamete maturation and ovulation, whereas in males, the TSHR would be involved in the regulation of early stages of the gonadal development and also of gamete maturation and spermiation.

Este trabajo abarca distintos aspectos de la metodología de la transferenciagénica en lubina, tanto in vitro como in vivo. Por una parte, se ha centrado en el estudioy evaluación de la transferencia génica somática como posible sustitución a latransgénesis estable en distintas aplicaciones. Por otra parte, se ha evaluado el sistemade regulación por tetraciclina para la regulación exógena de genes en peces. Por último,se ha probado que una línea celular de lubina puede utilizarse como sistema deexpresión de genes exógenos.Por primera vez se demuestra que la inyección de DNA plasmídico es eficientepara transferir genes exógenos a músculo de lubina. Los factores que favorecen laexpresión son, dosis altas de plásmido, el uso de un promotor adecuado, y un estadometabolicamente activo del músculo. La expresión que se obtiene es transitoria,alcanzándose el máximo cinco días después de la inyección. Una limitación de estatécnica es la incapacidad de reproducir en los distintos individuos inyectados, lamanera exacta en que la aguja de inyección entra en el paquete muscular, lo queorigina una gran variabilidad en cuanto a la toma de DNA por parte de las células, ypor tanto en el nivel de expresión del transgen. Esto se puede mejorar aumentando elnúmero de animales inyectados para disminuir así el error debido a la técnica, o usandouna segunda construcción para normalizar la transfección.La técnica de inyección de DNA en músculo se ha ensayado como método devacunación en peces. Sin embargo, ésta es la primera vez, que se prueba esta técnica enpeces para secretar una hormona en sangre. Como gen a inyectar se ha diseñado yconstruido un gen híbrido que codifica una hormona gonadotropa de cadena única delubina. Tras su inyección en músculo de lubina se pudo detectar la proteína en plasma.Para sistemas de origen piscícola, no se dispone hasta el momento de un métodoque permita controlar la expresión de genes exógenos de manera fiable. Por primeravez, hemos probado la utilidad del sistema de regulación por tetraciclina para estamisión. Tras valorar diferentes versiones del sistema en células de pez in vitro y enmúsculo in vivo, podemos concluir que la opción más eficiente es el uso deltransactivador tTA bajo el control de un promotor estable.Al igual que en células de mamíferos, hemos comprobado que las células de peztambién presentan dificultades para mantener el sistema de regulación por tetraciclinade una manera funcional; como resultado, encontrar clones con un grado de regulaciónóptimo y un nivel bajo de expresión basal es difícil. Sin embargo, el uso de unaestrategia basada en el uso del plásmido nuevo pTet-luc+-pur/GFP facilita esta tarea, ypermite obtener un alto porcentaje de clones regulables.A lo largo de este trabajo se han comparado los promotores CMV, de origenviral, y el promotor de la actina ß de carpa, para dirigir la expresión de distintos genestanto en las líneas celulares EPC y SBL, como en músculo de lubina in vivo. Losresultados nos permite concluir que el promotor de la actina ß de carpa, es más débil encuanto al nivel de expresión génica que logra promover, y menos estable a la hora demantenerse en una línea celular que el promotor CMV.Por último, los resultados obtenidos con la línea celular SBL permiten señalar aesta línea como un sistema de lubina apto para la expresión de genes exógenos. Estascélulas son capaces de secretar los productos de los genes introducidos, con lo quepodrían ser utilizadas para la producción de proteínas recombinantes., This work focuses on different aspects of the gene transfer methodology in sea bass.On one hand, the use of the somatic gene transfer has been evaluated as an alternative to theuse of stable transgenesis in different applications. Somatic gene transfer has been used asmethod of delivery of a recombinant single-chain gonadotropin into the blood stream in seabass. On the other hand, we have evaluated the use of the tetracycline regulated system tocontrol exogenous gene expression in fish. The results showed that the best choice was the useof the rtTA transactivator under the control of a stable promoter. Finally, a sea bass cell linehas proven to be an efficient system for the expression of exogenous gene.

El proceso reproductivo en peces, al igual que en el resto de vertebrados, está regulado por una cascada hormonal en la que están implicados el cerebro, la hipófisis y las gónadas, conocido como eje CHG. Dos de las principales hormonas implicadas en el control de la reproducción son las gonadotrofinas FSH (hormona estimuladora del folículo) y LH (hormona luteinizante), ambas producidas en la hipófisis y liberadas tras el estímulo con las hormonas liberadoras de gonadotrofinas (GnRH) y con acción a nivel gonadal. Desde la perspectiva de la producción animal en general, y de la acuicultura en particular, es esencial conocer en la especie a domesticar los mecanismos básicos de control endocrino. Este conocimiento básico es de gran utilidad para poder manipular la reproducción a conveniencia y al mismo tiempo poder desarrollar estrategias terapéuticas para solventar problemas de fertilidad que en ocasiones presentan los stocks de reproductores. Sin embargo, como consecuencia de la limitación en la disponibilidad de herramientas para su estudio, para una gran mayoría de las especies cultivables de peces se desconocen los mecanismos particulares que controlan su reproducción. En esta Tesis se optimiza una alternativa nueva de administración de gonadotrofinas recombinantes, la tecnología conocida como transferencia génica somática. En los capítulos 3 y 4 se inyectaron adultos y juveniles de lubina con construcciones de DNA codificante de las gonadotrofinas de cadena única Lh y Fsh, respectivamente, y se evaluó el incremento de estas hormonas en el torrente sanguíneo. En ambos experimentos las inyecciones de DNA se compararon con la inyección directa de la gonadotrofina recombinante correspondiente. Estos capítulos muestran que las construcciones de DNA se incorporan a las células musculares en el lugar de inyección, y las hormonas codificadas son sintetizadas y liberadas al torrente sanguíneo. Los capítulos 3 y 4 confirman que la transferencia génica somática es una herramienta poderosa para realizar estudios funcionales in vivo con gonadotrofinas, siendo además útil como terapia hormonal en peces, presentándose como alternativa a los tratamientos tradicionales con hCG o GnRH. La funcionalidad de las hormonas recombinantes sintetizadas por las células musculares de lubina tras la incorporación del DNA, se determinó estudiando su efecto en el desarrollo de los testículos. En los capítulos 4 y 5 se evaluó como la acción de la Fsh es esencial para iniciar el proceso de espermatogénesis, promoviendo la proliferación de las espermatogonias, siendo necesaria la acción posterior de la Lh para culminar la espermiación. Los individuos inyectados sólo con el plásmido de Fsh presentaron una espermatogénesis parcial, sin indicios de espermiación, estado que sólo alcanzaron aquellos animales que se inyectaron de manera secuencial con los plásmidos de Fsh y Lh. Estos experimentos también demuestran que la Lh por si sola no es capaz de promover la maduración testicular. Además en los análisis de expresión génica se observó que los tratamientos con Fsh incrementaron la expresión de marcadores de células germinales como piwi, pcna y scp3, mientras que el efecto de la Lh se detectó en enzimas esteroidogénicas como cyp17a1 y cyp17a2. En esta Tesis también se describe cómo una de las acciones más directas de la Fsh es la estimulación de la producción de 11KT. Con el fin de analizar más en detalle la acción específica de la Fsh en la activación de la esteroidogénesis, y de definir mecanismos de regulación conservados entre diferentes especies de peces, se estudió in vitro la acción de la Fsh en cultivo primario de testículo y ovario de lubina y pez cebra. En el capítulo 6 se describe cómo la Fsh emplea principalmente la ruta del AMPc/PKA para transmitir su señal una vez activado su receptor, no siendo ésta la única vía, ya que también es importante la actividad de las MAPK. Haciendo uso de inhibidores y estimuladores de ambas rutas se ha perfilado un escenario en el que la Fsh utiliza de manera independiente las rutas del AMPc/PKA o las MAPK para estimular la transcripción de genes implicados en la esteroidogénesis, como star o cyp19a1a ABSTRACT The reproductive process in fish, as in all vertebrate species, is tightly regulated by a hormonal axis, called BPG, involving the brain, pituitary and gonads. Two major hormones that control reproduction are the gonadotropins FSH (follicle-stimulating hormone) and LH (luteinizing hormone) are both produced at the pituitary level, released to the bloodstream after the stimulus of the GnRH (gonadotropin release hormone), and with action at the gonad level. From a fish farming perspective, it is essential to fully understand the endocrine mechanism underlying reproductive performance. This knowledge is important to manipulate reproductive behaviour and for the development of therapeutic strategies aiming to solve fertility issues frequently present in the broodstocks. However, due to the limited tools available for the vast majority of the cultivable species, the mechanisms controlling their reproduction are not well characterized. This thesis describes an alternative administration of recombinant gonadotropins using the technique of somatic gene transfer. In Chapters 3 and 4, adult and juvenile sea bass were injected with a plasmid DNA encoding Lh and Fsh gonadotropin sequences in a single chain form, and the levels of either GtHs were analysed in the bloodstream. In both experiments the plasma levels of GtHs attained by plasmid DNA injected fish were compared with plasma levels of fish injected with the equivalent recombinant GtH. Both Chapters clearly show that plasmid DNA was efficiently incorporated by the cell muscle, and the encoded hormones were synthetized and released to the bloodstream. These results confirm that somatic gene transfer can be a powerful tool for in vivo functional studies of gonadotropin. In addition, this technique can be used for hormone therapy in fish, and can offer a useful alternative to classic treatments with hCG or GnRH. The functionality of the recombinant hormones synthetized by cell muscles after plasmid DNA injection was evaluated by studying the development of testes. Chapters 4 and 5 describe the crucial role of Fsh in starting the process of spermatogenesis, promoting the proliferation of spermatogonia cells, and its requirements for Lh to reach the spermiation process. Fish receiving only Fsh plasmid injections showed partial spermatogenesis, with no evidence for spermiation. Only the fish injected with Fsh and Lh plasmid sequentially reached this late step of the spermatogenesis process. Fish receiving Lh plasmid alone showed that GtH cannot trigger testis maturation alone. Furthermore, gene expression analysis highlighted the action of Fsh on promoting cell proliferation by increasing the expression of germ cell markers such as piwi, pcna piwi, pcna and scp3. By contrast, the effect of Lh was more obvious in transcript levels of steroidogenic enzymes such as cyp17a1 and cyp17a2. This thesis additionally describes a direct action of Fsh on the stimulation of 11KT production. In order to analyse the specific activity of Fsh on the steroidogenesis process in detail, and to define regulatory mechanisms conserved across fish species, the action of Fsh in vitro was studied using primary gonad tissue culture from sea bass and zebrafish. Chapter 6 describes the use of the signalling pathway cAMP/PKA by Fsh to transduce the signal after the activation of its receptor not clear. Importantly, cAMP/PKA is not the only pathway used by Fsh, the MAPk route is also involved in Fsh signal transduction. The use of cAMP/PKA and MAPk inhibitors and stimulators suggest the possibility that Fsh can use both routes independently to activate the transcription of some steroidogenic genes such as star and cyp19a1a

La kisspeptina es el producto peptídico del gen KISS1. Los primeros estudios que se llevaron a cabo en mamíferos, concretamente en humanos y en ratones, revelaron que ciertas mutaciones que inactivaban al receptor de la kisspeptina (GPR54) daban lugar al desarrollo de hipogonadismo hipogonadotrópico. Este hecho sugirió que el sistema KISS1/GPR54 juega un papel importante en la función reproductiva de los mamíferos. En los mamíferos placentarios hay un solo gen KISS1 y otro que codifica GPR54. Sin embargo en diferentes especies de teleósteos se ha descrito un segundo parálogo ancestral tanto de kiss1 como de gpr54-1b, denominados kiss2 y gpr54-2b, respectivamente. Los mecanismos funcionales, tanto moleculares como endocrinos, que desencadenan las kisspeptinas en el cerebro de los peces teleósteos en relación con la pubertad y la fertilidad, permanecen inexplorados en gran medida. Por ello, el objetivo general de esta Tesis Doctoral ha sido ampliar el conocimiento acerca del sistema Kiss/Gpr54 y su función en la reproducción de peces teleósteos, tomando como modelo de estudio la lubina europea (Dicentrarchus labrax). Nuestros resultados muestran que los receptores de kisspeptinas de lubina poseen selectividad diferencial por el ligando. Así, el receptor Gpr54-1b se activa preferentemente por los péptidos derivados de Kiss1, mientras que el receptor Gpr54-2b presenta una unión preferencial por los péptidos derivados de Kiss2, aunque también se activa en presencia de los péptidos derivados de Kiss1. Por otro lado, en el estudio se muestran evidencias funcionales de que las áreas neuroendocrinas del cerebro anterior y medio, el telencéfalo y el área preóptica, están implicadas en el control del eje reproductivo vía Kiss2/Gnrh1, modulando al eje reproductivo y en última instancia influyendo en la calidad del esperma. Además se proporcionan evidencias funcionales y neuroanatómicas sobre el papel de Kiss2 en la modulación del eje reproductivo de esta especie de teleósteo marino, mediante su acción como hormona autocrina/paracrina, a nivel de la hipófisis. Estos resultados sugieren que el sistema Kiss/Gpr54 juega un papel destacado en el control del eje reproductivo de los peces teleósteos, no solo como neuropéptido hipofisiotrópico sino también como hormona autocrina/paracrina. Por otro lado, esta memoria de Tesis Doctoral también muestra que el sistema kisspeptina está implicado en la regulación de la secreción de las gonadotrofinas y los andrógenos durante distintos periodos de desarrollo testicular de la primera reproducción sexual o pubertad de la lubina. Así mismo, existe una fuerte correlación entre el control ambiental de la pubertad y el sistema Kiss/Gpr54 en el cerebro anterior y medio de la lubina.