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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).

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).

Comunicación presentada en The International Symposium on Genetics in Aquaculture XII, celebrado en Santiago de Compostela, España, del 21 al 27 de junio de 2015, Kisspeptin 1-10, encoded by the kiss1 gene has emerged as a major actor in the neuroendocrine regulation of the reproductive axis in mammals. Although the existence of two kiss genes (kiss1 and kiss2) and to kiss receptors (kissr1 and kissr2), issued from early whole genome duplication events, is well-documented in teleosts, the functions of kisspeptins are still very unclear in fishes. This is partly due to the existence of several models species, belonging to orders that have diverged evolutionary over a long period of time. This lecture intends to summarize the information that we have obtained in a wellstudied fish model, the European seabass (Dicentrarchus labrax) a fish of high commercial interest in Europe. In this species, the neuroanatomical distribution of kiss neurons has been studied in details showing the widespread distribution in the forebrain, notably in the habenula the preoptic area and the mediobasal hypopthalamus. This expression is subject to changes according to the reproductive activity, which is not surprising as populations of kiss neurons express estrogen receptors. Interestingly, kiss2 fibres exhibit a widespread distribution that overlaps quite well with the distribution of kissr2. Kiss1. The relationships between the kiss systems and the GnRH neurons have received much attention showing that while kissr2 are expressed in a variety of neurons, notably in somatostatinergic neurons, none of the GnRH1, GnRH2 or GnRH3 neurons was shown to express kissr2. This result is surprising as there is recent evidence that kiss2-12 given intracerebraly is able to stimulate gonadotrophin release and the expression of gnrh1. These results indicate that kissspeptins probably have a wide spectrum of actions in the brain of teleosts including some indirect actions on the reproductive axis that could involve intermediate neurons, potentially NO synthase expressing cells. On another hand, kiss1 mRNAs and kiss2 fibres are present in the pituitary where direct effects of kisspeptins on gonadotrophin release have been documented., Supported by the EU project LIFECYCLE (FP7-222719-1) to O.K. and S.Z., the EU INTEREG TC2N to O.K. and the GV PROMETEO10/2010/003 and II/2014/051to SZ.

Trabajo presentado en el 8th International Symposium on Fish Endocrinology (ISFE), celebrado en Gotemburgo (Suecia), del 26 de junio al 2 de julio de 2016, Kisspeptins are key players in the neuroendocrine control of puberty and other reproductive processes in mammals. Contrary to placental mammals, where only one KISS1 and one KISS1R gene are present, a second ancient paralog of Kiss1, referred to as Kiss2, has been described in different vertebrate lineages and species, including European sea bass (Dicentrarchus labrax). Both genes are expressed in the brain and pituitary of this teleost species, but their differential roles in the central control of fish reproduction are only beginning to be elucidated.In this study, we investigated whether the brain represents the only site of kisspeptin action on reproductive function, and also the potential actions of kisspeptin at the pituitary level could be considered. First, we examined the effects of intracerebroventricular injections of the highly active sea bass peptides Kiss1-15 and Kiss2-12 on spermiating male sea bass. Physiological saline, Kiss1-15, or Kiss2-12 were injected into the third ventricle. Blood samples were collected at different times after injection to analyze the effects of kisspeptins on the release of gonadotropins (Lh and Fsh) and androgens (testosterone and 11-ketotestosterone). Sperm samples were also collected to study the effects on the sperm quality. Our results showed that Kiss2-12 provoked a marked effect on plasma luteinizing hormone (Lh) levels, which in turn had a strong stimulatory effect on the release of androgens and on sperm quality parameters.On the other hand, both synthetic peptides, Kiss1-15 and Kiss2- 12, were used to stimulate dispersed sea bass pituitary cells obtained from mature males. Our results showed that Kiss2-12 induced Lh and folliclestimulating hormone (Fsh) release, whereas Kiss1-15 had no effect on gonadotropin secretion. Furthermore, the distribution and origin of Kiss2 and its potential interactions with the gonadotropins in the pituitary were analyzed using dual fluorescence immunohistochemistry. Kiss2 cells were found in the proximal pars distalis and colocalized with gonadotropin immunoreactive cells. In summary, our results provide, for the first time in a teleost species, functional and neuroanatomical evidence that Kiss2 may act through different routes to directly modulate the activity of gonadotrophs, either as a brain neuropeptide or as an autocrine/paracrine factor in the pituitary., Funded by grants: LIFECYCLE FP7-22719-1; AGL2011-28890; PROMETEOII-214/051.

Kisspeptins are key players in the neuroendocrine control of puberty and other reproductive processes in mammals. Several studies have demonstrated that the KISS/GPR54 system is expressed by gonadotrophs, but in vitro studies assessing the direct stimulatory effects of kisspeptin on gonadotropin secretion in the pituitary have provided conflicting results. In this study, we investigated whether kisspeptin directly influences the reproductive function of sea bass pituitary. First, the highly active peptides Kiss1-15 and Kiss2-12 were used to stimulate dispersed sea bass pituitary cells obtained from mature males. Our results show that, first, Kiss2-12 induced luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) release, whereas Kiss1-15 had no effect on gonadotropin secretion at full spermiation stage. Second, the distribution and nature of Kiss2 and its potential interactions with the gonadotropin-releasing hormone 1 (Gnrh1) system in the pituitary were analyzed using dual fluorescence immunohistochemistry. Kiss2 cells were found in the proximal pars distalis and colocalized with gonadotropin-immunoreactive cells. In summary, our results provide, for the first time in a teleost species, functional and neuroanatomical evidence that Kiss2 may act through different routes to directly modulate the activity of gonadotrophs, either as a hypophysiotropic neuropeptide or as an autocrine/paracrine factor.

Trabajo presentado en el 10º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Castellón (España), del 23 al 25 de septiembre de 2015, There are a wide variety of environmental factors that change seasonally and that may modulate the activity of the reproductive axis. Of all, photoperiod is the main environmental signal that most temperate fish species use to predict the changing seasons and therefore anticipate spawning time through activation of neuroendocrine pathways which, in turn, stimulate gonadotropic axis and gonadal growth. However, many molecular and endocrine mechanism involved in the onset of puberty and the influence of environmental conditions, such as photoperiod signalling, are not well understood in fish. In this study, 1 year-old male European sea bass (Dicentrarchus labrax) were exposed to three different photoperiod conditions, natural photoperiod (NP), advanced photoperiod (AP) and continuous light (LL) in combination with size-sorting and studied from August to February. The obtained results clearly indicated that AP and LL were able to train the rhythms of both kiss1 and gnrh2 mRNA levels in the forebrainmidbrain (FB-MB), suggesting that the photoperiodic signalling may modulate the activity of the habenular Kiss1 and the synencephalic Gnrh2 neurons to drive reproductive axis. Also AP and LL regimes affected both plasma Fsh and 11-KT profiles, which in turn would explain the phase shift and reduction of gonadal recrudescence under AP and LL, respectively. In conclusion, the present study reveals a strong relation between environmental control of puberty and the Kiss1 and Gnrh2 systems in agreement with previous ?ndings observed in other teleost species, including European sea bass. We also hypothesize that very likely, as a result of the modulatory role of the photoperiod on the Kiss1 and Gnrh2 activity, this is able to shift the hormone profiles and hence advance the onset and progression of gametogenesis under AP, whereas under LL, the unbalanced production of 11-KT, regulated by an altered Fsh profile, could be limiting the stimulation of germ cell proliferation at the testicular level and thus prevent the normal progression of the spermatogenesis.

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.

Some teleost species, including European sea bass, harbor two different kisspeptin coding genes: kiss1 and kiss2. Both genes are expressed in the brain, but their differential roles in the central control of fish reproduction are only beginning to be elucidated. In this study, we have examined the effects of intracerebroventricular injections of the highly active sea bass peptides Kiss1-15 and Kiss2-12 on spermiating male sea bass. Physiological saline, Kiss1-15, or Kiss2-12 was injected into the third ventricle. To establish the gene expression cascade involved in the action of kisspeptins, the expression of the two sea bass kisspeptin receptor genes (kiss1r and kiss2r) and the three sea bass Gnrh genes (gnrh1, gnrh2, and gnrh3) were analyzed in the forebrain-midbrain and the hypothalamus. In addition, the protein levels of hypothalamic and pituitary Gnrh1 were measured. Blood samples were collected at different times after injection to analyze the effects of kisspeptins on the release of gonadotropins (Lh and Fsh) and androgens (testosterone and 11-ketotestosterone). The present results provide the first evidence that the effects of Kiss2 on central regulation of reproductive function involve the neuroendocrine areas of the forebrain-midbrain in teleost fish. The marked effect of Kiss2 on kiss2r and gnrh1 expression in the forebrain-midbrain and on Gnrh1 release suggest that this neuronal system is involved in the neuroendocrine regulation of gonadotroph activity. This hypothesis was confirmed by a surge of plasma Lh in response to Kiss2, which presumably has a strong stimulatory effect on testosterone release, and thus on sperm quality parameters.

Comunicación presentada en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014, Puberty is the process by which an immature animal acquires the ability to reproduce for the first time and its onset occurs soon after sexual differentiation and is characterized by the beginning of gametogenesis in both sexes. In fish, the mechanisms underlying puberty and its triggering actors are still largely unknown. In the European sea bass, the recent isolation of genes coding for kisspeptins and their receptors allowed for expression studies that are clarifying something more the general endocrine picture of puberty of this species. We present here new insights on when and how the onset of male sea bass puberty occurs, its dependence of a critical size and how it can be controlled by the photoperiod. The suppression of plasma temporal patterns of key reproductive hormones in fish exposed to continuous light, revealed the existence of a photolabile period in September. Moreover, delayed puberty in male sea bass is an important economic issue because marketing time coincides with its puberty onset. Constant long photoperiod was highly effective in delaying puberty, which was linked to a phase difference in the rhythms of hormones that regulate gametogenesis. Indeed, the study of hormone daily rhythms revealed that its core values had equivalence with the seasonal rhythms so that the daily could be considered as the functional units of the seasonal rhythms. Regarding size, apparently only large fish attain the ability to carry out gametogenesis while the small ones do not succeed. Could this imply that to initiate and conclude puberty fish need to achieve an optimal threshold in hormone production? Studies performed with fish of different size demonstrate that small fish exhibit lower plasma hormonal levels than large fish confirming this assumption. Besides, size-photoperiod combined studies demonstrate a differential role of kisspeptins; apparently while Kiss1 is more linked to the photoperiodic signaling, Kiss2 is mostly involved in the reproductive processes. Is it necessary to accomplish a critical body size/fat stores/energy level in order to launch puberty attaining the hormonal signal able to initiate gametogenesis? Preliminary data show that a long-term restricted feeding regime did not prevent the onset of puberty. Besides, hormonal analysis revealed an upregulation of the kisspeptin system and Fsh, suggesting that the fish is able to maintain the reproductive function even at the expense of other functions., Supported by LIFECYCLE (FP7-222719-1) and PUBERCONTROL AGL2006-0472).

Comunicación presentada en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014, Kisspeptins are considered the key players in the neuroendocrine control of puberty and reproduction in mammals. Kiss genes, as well as kiss receptor genes, are present in most classes of vertebrates and derived from an ancestral gene that underwent gene duplication followed by gene losses. In most fish species two kiss genes (kiss1 and kiss2) and two kiss receptor genes (kiss1r and kiss2r) are identified but the organization and the functions of these duplicated systems are still poorly understood. Whit Withthe aim to investigate whether both kiss genes are involved in the neuroendocrine control of reproduction in fish we have characterized the neuroanatomical organization of both systems in zebrafish (Danio rerio, Cyprinodontiform), and the European sea bass (Dicentrarchus labrax), a highly evolved Perciform fish intensively farmed in Southern Europe. We also have investigated whether Kiss neurons have any special relationships with the GnRH neurons and express estrogen receptors. Finally, the nature of the very abundant cells expressing kiss2r was studied using combination of in situ hybridization and immunohistochemistry. The main kiss1 expression site is the habenula, whereas kiss2 mRNA is mostly observed at the lateral recess in both species. No evident sexual dimorphism was observed but an additional kiss1 expressing cells population was reported into the mediobasal hypothalamus in sea bass during the spawning period. In both teleosts, few kiss2 cells of the preoptic region could make synaptic contact with the hypophysiotrophic GnRH neurons. In both species, the kiss population sensible to estrogens is the one placed into the ventral hypothalamus corresponding to a kiss1 cell population in sea bass and to kiss2 expression site in zebrafish. Altogether our findings suggest that kiss2 in zebrafish and kiss1 gene in sea bass would be more likely to participate in the regulation of reproduction through the mediobasal hypothalamus kiss population sensitive to estrogens., Supported by EU Project LIFECYCLE (FP7-222719-1)

Comunicación presentada en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014, The follicle stimulating hormone (Fsh) and the luteinizing hormone (Lh) are central endocrine regulators of gonad function in vertebrates. These gonadotropins act by binding and activating their specific receptors that are located in certain cell types of the gonads. In fish, the differential roles of these hormones are being progressively elucidated with the development of different tools for their study. In the case of European sea bass Dicentrarchus labrax, the isolation of the genes coding for the gonadotropin subunits and receptors allowed in first instance for expression studies. Later, to overcome the limitation of native hormones, recombinant dimeric gonadotropins have been generated, which show different functional characteristics depending on the cell system and DNA construct. In addition, single gonadotropin beta-subunits have been produced and used as antigens for antibody production. This last approach has allowed the development of detection methods for native gonadotropins being the European sea bass one of the few species where both gonadotropins can be detected in their native form. The generation of antibodies has also allowed for their cellular localization and co-localization with other factors, helping to a better understanding of their function. By administration of the recombinant gonadotropins to gonad tissues cultured in vitro we have studied their effects on steroidogenesis, the activated intracellular pathways and their target genes. Their administration in vivo has also been tested both for basic studies and as a biotechnological approach. In this frame, it is known from different studies in mammals that exogenous administration of native hormones in cases of deficiency, including assisted reproduction, can be unsatisfactory due to their rapid clearance from circulation or limited availability. Other than the production of recombinant hormones gene-based therapies by using somatic gene transfer are offered as an alternative. Such an approach has been tested in sea bass for gonadotropin delivery in vivo. The hormones produced by the injected genes were functional and have allowed for different studies on gonadotropin action in spermatogenesis. All together, the use of gene therapy for hormone replacement in fish is a real alternative to the production of recombinant gonadotropins for in vivo use, due to the low cost of production and the high persistence of the injected DNA, and has a broad range of potential applications such as its use in out-of-season breeding programs or reproductive dysfunctions in fish species., Supported by MINECO (AGL2011-28890) and GV (ACOMP/2013/085).

Resumen del trabajo presentado en el 17th International Congress of Comparative Endocrinology (ICCE 2013), celebrado en Barcelona (España) del 15 al 19 de julio de 2013., In vertebrates the kisspeptin system plays a key role in the neuroendocrine control of puberty and reproduction by stimulating the GnRH neurons and the subsequent synthesis and release of gonadotropins. In the European sea bass a marine fish, two ligands and two receptors encoded by four different genes have been identified and molecularly characterized. This study was conducted in the brain and it aimed to map the kiss systems and identify their correlation with kisspeptin receptors (kiss-r1 and kiss-r2). The use of kiss2 specific antibodies, confirms that kiss2 neurons are mainly located in the hypothalamus and widely projected to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus and the neurohypophysis. These results were compared to the expression of kiss-r1 and kissr2 mRNAs by in situ hybridization showing a tight correlation between the wide distribution of kiss2-positive fibers and that of kiss-r2 expressing cells. The kiss-r2 messengers were highly expressed in ventral telencephalon and the preoptic area where kiss2 projections were also observed. Additionally, in the mediobasal hypothalamus, kiss-r2 expressing cells were detected in the ventral part of the nucleus of the lateral recess (NRLv), in line with the localization of kiss2 projections and the immunodetection of kiss2 cells. Instead, kiss-r1 was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Finally, as showed by our double staining experiments, GnRH1 neurons in the preoptic area did not appear to express kiss-r1 or kiss-r2 messengers. Our findings present detailed information regarding kiss2 fibers localization which suggest that kisspeptins are likely involved in neuronal systems related to several functions in the brain of sea bass., Funded by EU projects LIFECYCLE (FP7-222719-1) and INTEREG TC2N and Generalitat Valenciana (REPROBASS; PROMETEO/2010/003).

Resumen del trabajo presentado en el 17th International Congress of Comparative Endocrinology (ICCE 2013), celebrado en Barcelona (España), del 15 al 19 de julio de 2013, Kispeptins exert a dynamic control of gonadotropin secretion via stimulation of GnRH neurons which ultimately results in gonad maturation and release of gametes. In the brain of teleost, including European sea bass, two different kisspeptin coding genes kiss1 and kiss2 have been found but their differential roles in the central control of fish reproduction are only beginning to be elucidated. In this study, the acute effects of brain intracerebroventricular (icv) injections of the highly active sea bass peptides kiss1-15 and kiss2-12 in 3 years old spermiating males were examined. Physiological saline, kiss1-15 or kiss2-12 were injected into the third ventricle. Blood samples were collected at different times post-injection (from 30 min to 72h) to analyze the effect on the release of gonadotropins (FSH and LH) and sexual steroids (T and 11KT). Moreover, hypothalamus and pituitary were collected at 6, 9 and 12 hours post-injection to evaluate the role of the kisspeptins on GnRH-I synthesis and release. Finally, the effect of both kiss peptides on milt volume, sperm concentration and motility was analyzed at 3, 7 and 14 days after treatment. Injections of kiss1-15 and kiss2-12 evoked associated GnRH-I release into the pituitary. Both peptides provoked the release of LH, albeit to a lesser degree for kiss1-15. Neither of the two peptides caused release of FSH. Only kiss2- 12 increased the plasma levels of sexual steroids. Finally, kiss2-12 treatment stimulated a significant increase in milt volume peaking on day 7 and returning to control values at day 14 post treatment. In conclusion, these data show that the icv administration of kiss2-12 potently stimulates the hypothalamic-pituitary-gonadal (HPG) axis via the hypothalamic gonadotropinreleasing- hormone system and increases spermiation in male sea bass. Supported by the EU LIFECYCLE (FP7-222719-1) and the GV (REPROBASS; PROMETEO/2010/003)., Supported by the EU LIFECYCLE (FP7-222719-1) and the GV (REPROBASS; PROMETEO/2010/003).

Resumen del trabajo presentado en el 9º Congreso de la Asociación Ibérica de Endocrinología Comparada, celebrado en Barcelona (España), los días 13 y 14 de julio de 2013, Kisspeptins are key players in the neuroendocrine control of puberty and other reproductive processes in mammals. It has been suggested that the effect of kisspeptins occurs exclusively at the level of hypotalamic GnRH secretion, since the stimulatory effect of kisspeptin on gonadotropin release is blocked by a GnRH antagonist. On the other hand, several studies have demonstrated that the KiSS/GPR54 system is expressed by the gonadotrops, but in vitro studies assessing the direct stimulatory effects of kisspeptin on gonadotropin secretion in the pituitary have given conflicting results. In this study, we aimed at investigating the direct effect of sea bass kiss1 and kiss2 on the pituitary. The highly active peptides kiss1-15 and kiss2-12 were used to stimulate dispersed sea bass pituitary cells obtained from mature males. In the presence of kiss2-12, a peak of LH release was observed after 8h of incubation, while the same concentration of this peptide induced a significant release of FSH in 4h. On the other hand, kiss1-15 had no effect on LH or FSH release. These results were different depending on the hormonal (GnRH or sexual steroids) milieu of the cells. Thus, kiss2-12 had a synergic effect with GnRH on LH release. In the presence of sexual steroids (estradiol and testosterone) the stimulatory effect of kiss2-12 on LH and FSH release was inhibited. In conclusion, our results provide unequivocal evidence that kisspeptin exert direct pituitary effects in spermiating male sea bass and suggests a possible autocrine/paracrine mode of action., Supported by the EU LIFECYCLE (FP7-222719-1) and the GV (REPROBASS; PROMETEO/2010/003).

Poster presentado en el 7th International Symposium on Fish Endocrinology celebrado en Buenos Aires del 3 al 6 de septiembre de 2012, Kisspeptins are key players in the neuroendocrine control of puberty and reproduction. Kiss genes, as kiss receptor genes, are present in vertebrates and derived from an ancestral gene that underwent gene duplication followed by gene losses. In most fish two kiss genes (kiss1 and kiss2) and two kiss receptor genes (kiss1r and kiss2r) are identified but the organization and functions of these systems are still poorly understood. In order to know whether both kiss genes are involved in the neuroendocrine control of reproduction in fish we studied Kisspeptin/GPR54 expression in the European sea bass (Dicentrarchus labrax), a Perciform fish. We also investigated whether Kiss neurons have any special link with the GnRH neurons and express estrogen receptors. Finally, the nature of the cells expressing kiss2r was studied using combination of in situ hybridization and immunohistochemistry. The main kiss1 expression site is the habenula, whereas kiss2 mRNA is mostly seen at the lateral recess. No sexual dimorphism is observed but an additional kiss1 expressing cells population is reported into the mediobasal hypothalamus during the spawning period. A few kiss2 cells of the preoptic region could make synaptic contact with the hypophysiotrophic GnRH neurons. The kiss population sensible to estrogens is in the ventral hypothalamus and corresponds to a kiss1 cell population. Our findings suggest that both kiss1 and kiss2 genes in sea bass would be likely to participate in the regulation of reproduction., Supported by EU LIFECYCLE (FP7-222719-1)

Ponencia presentada en el XIII Congreso Nacional de Acuicultura celebrado en Barcelona del 21 al 24 de noviembre de 2011, Kisspeptin and its receptor Gpr54 play an important role in mammalian reproduction. To date, up to three distinct kiss and four gpr54 genes have been identified in vertebrates. In fish, the kiss1and/or kiss2 and their cognate kisspeptin receptors (gpr54-1b and/or gpr54-2b) have been characterized in a number of teleosts including, Beloniformes, Cypriniformes, Mugiliforms, Pleuronectiforms, Tetraodontiforms, Gasterosteiforms and other Perciforms such as sea bass. From them, only the medaka, zebrafish, goldfish and sea bass have two kiss and two gpr54 genes whereas other fish possess one form of ligand and receptor. Because these genes possess conserved structural organization, it is suggested that these genes originated from a common ancentral form. Phylogenetic and synteny analyses support this hypothesis. Evidence exits this neuroendrocrine system plays a relevant role in fish reproduction. Presently studies in sea bass include the characterization of this system in the brain and the ligand-receptor interactions as well as quantitative monitoring of kiss/gpr54 gene expression patterns under different environmental/physiological conditions., Este trabajo fue financiado por los proyectos GV06/268 y PROMETEO/2010/003 de la Generalitat Valenciana, LIFECYCLE (FP7222719) de la Comunidad Europea y AGL2009-11086 del MICINN.

Poster presentado en el 8º Congreso de la Asociación Ibérica de Endocrinología Comparada celebrado en Madrid del 5 al 7 septiembre 2011, The Kiss/Gpr54 system is considered to be an essential regulator of the reproductive axis in most vertebrate species. The existence of paralogous kisspeptin genes as well as kisspeptin receptor genes has quite recently been reported in several fish and amphibian species. Thus, the fishes may provide excellent animal models for the study of general principles underlying the kisspeptin and gpr54 receptor systems of vertebrates. The pharmacological profile of sea bass gpr54-1b and gpr54-2b was examined to characterize the ligand-receptor interactions and the signaling pathways of two gpr54 in sea bass (Dicentrarchus labrax). For this aim, the open reading frames of sea bass gpr54-1b and gpr54-2b were subcloned in the expression vector pcDNA3 and these plasmids were transiently transfected into Chinese hamster ovary cells in combination with pCRE-luc or pSRE-luc reporter vectors. In these reporter vectors the luciferase gene (luc) is transcriptionally regulated by a cyclic AMP response element (CRE) or by a serum response element (SRE), allowing to differentiate between protein kinase C and protein kinase A signal transduction pathways. We tested graded concentrations (10-5 to 10-10M) of sea bass kiss1 and kiss2 decapeptides as well as longer kisspeptins (kiss1-15 and kiss2-12) on the double transfected cells. The recorded luciferase activities showed that sea bass gpr54-1b exhibits high sensitivity toward kiss1-derived peptides, with a preferential binding to the kiss1-15. Activation of this receptor generated both SRE and CRE specific signalling, although the stimulation of the CRE pathway was more effective. On the other hand, a different ligand-receptor interaction is observed for the sea bass gpr54-2b that clearly appears to discriminate between long and short kisspeptin forms, showing higher potency and affinity for longer ones. It can be concluded that this receptor is activated by the two ligands, however it exhibits preferential binding for the kiss2-12. In this case, activation of SRE driven luciferase was slightly higher than CRE-luc. All together these data show distinct binding preferences of these receptors for sea bass kisspeptins and different activation levels of the studied signaling pathways., Supported by GV (PROMETEO/2010/003 to S.Z.), EU (LIFECYCLE, FP7222719 to S.Z.». F.E. was supported by a JAE Predoctoral fellowship (CSIC).

Poster presentado en el 9th International Symposium on Reproductive Physiology of Fish celebrado en Cochin (India) del 9 al 14 de agosto de 2011, Small fish displayed a remarkable delay in the progression of testicular development that prevented them from achieving spermiation in February. Large fish entered in spermatogonial mitosis towards meiosis in September, almost 1.5 month earlier than S group attaining full spermiogenesis in February. The highest FSH plasma levels were observed in L fish which almost doubled the FSH values of S fish at this time (p

Trabajo presentado en el 25th Conference of European Comparative Endocrinologists, celebrada en Pécs (Hungría), del 31 de agosto al 4 de septiembre de 2010, Kisspeptins are a family of peptides encoded by the KISS1 gene, in mammals, and kiss1 and kiss2 in fish. They were first identified by their capacity to inhibit tumor metastasis through the receptor GPR54, also termed KISSR. Nowadays, it is largely demonstrated that the kiss system plays an essential role in the neuroendocrine control of puberty and reproduction in several mammalian species, stimulating GnRH secretion at the hypothalamus. In the sea bass, an economically-important marine teleost, kiss1 and kiss2 coding sequences and those of their cognate receptors have been recently identified. Thus, this is the first study aimed to elucidate the neuroanatomical distribution of the cells expressing the two kiss genes and their receptors in adult sea bass brain by in situ hybridization. Our findings indicate the mediobasal hypothalamus, notably the nucleus of the lateral recess, as an important area of expression of both kiss1 and kiss2 genes. Furthermore, the kiss1expressing cells are also located at the level of the habenular region. Interestingly, the kisspeptin receptors-expressing cells are mostly placed in the same regions as the corresponding ligands. These results are in line with those obtained in the other fish species investigated so far. In conclusion, this work represents the first neuroanatomical analysis of the kisspeptin systems in the sea bass brain and suggests a putative involvement of the kisspeptins in the control of reproduction in this species., Supported by the EU Project LIFECYCLE (FP7-222719-1). Sebastián Escobar was sponsored by JAE-Predoc CSIC. Spain.

The onset of puberty is characterized by activation of the brain-pituitary-gonad axis. However, the molecular and endocrine mechanism involved in the process of puberty and the influence of environmental conditions, such as photoperiod signalling, are not well understood in fish. In this study, 1-year-old male European sea bass (Dicentrarchus labrax) were exposed to photoperiod manipulation in combination with size-sorting. Two treatment groups, a puberty accelerating photoperiod (AP) group and a continuous light (LL) group, were studied from August to February. Our results indicate that AP and LL are able to entrain the rhythms of both kiss1 and gnrh2 mRNA levels in the brain, while kiss2 and gnrh1 mRNA expression does not seem to be directly affected by the photoperiod, at least during testicular growth. It is likely that AP and LL photoperiod regimes affected both plasma Fsh and 11-KT profiles, which might explain, respectively, the phase shift and reduction of testes maturation seen under these conditions. We therefore hypothesize that the unbalanced production of this androgen regulated by circulating Fsh might be limiting the stimulation of germ cell proliferation in European sea bass males. In summary, our study establishes that photoperiod modulates the expression of kiss1 and gnrh2 in the forebrain-midbrain, which may be involved in the translation of the light stimulus to activate the reproductive axis., This work was supported by EU (LIFECYCLE, FP7-222719-1) and Generalitat Valenciana (PROMETEOII/2014/051) projects awarded to S.Z. F.E. was supported by a JAE Predoctoral fellowship (CSIC).