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11th International Symposium on Reproductive Physiology of Fish (ISRPF-2018), New frontiers in reproductive diversity in a changing environment, 3-8 June 2018, Manaus, Brazil.-- 1 page, Introduction. Atlantic halibut are group-synchronous, periodic spawners and in captivity wild-caught females release 6-10 batches of eggs in the spawning season, which lasts from late February to late April in southwestern Norway. While wild-caught females generally adapt well in captivity, displaying high fecundity with egg batches spawned at regular intervals, hatchery-produced F1/F2 females appear to suffer from a reproductive dysfunction, releasing small batches of eggs at irregular intervals. Materials and methods. Reproductive performance of wild-caught halibut and farmed (F1) females was compared by strip-spawning and fertilization of eggs. Eggs were photographed for size measurements. For calculation of hatching rate, fertilized eggs were incubated in darkness at 6°C. Newly hatched larvae were photographed. Blood samples were taken from the same wild-caught and farmed females at 3-6 week intervals. After centrifugation of blood, plasma was frozen immediately on dry ice and stored at -80°C. Estradiol-17ß, testosterone, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) were analysed by ELISA. Results and Discussion. Realised fecundity was identical in wild-caught and farmed females, but ovulatory intervals were more irregular, and fertilization and hatching rates were lower in the farmed broodstock. Plasma E2 was elevated during autumn in both groups and reached peak levels in February. E2 remained high during the spawning period, then decreased and remained low for the rest of the sampling period. Plasma T concentrations were low until spawning, when a peak was observed. Farmed females had lower plasma Fsh concentrations during vitellogenesis than wild-caught fish. The individual variation was high, but Fsh concentrations decreased during spawning. This decrease was more pronounced in wild-caught females. Plasma Fsh increased after spawning. Plasma Lh concentrations were relatively high from September to December. Just before spawning, Lh concentrations appeared to decrease. Highest plasma Lh concentrations were seen during the spawning period. Conclusion. Farmed females of the F1 generation had a somewhat lower reproductive success and lower Fsh concentrations than wild-caught females. Individual variations were high, however, and further studies, as well as careful selection of breeders, are needed to improve reproductive development and spawning performance

11th International Symposium on Reproductive Physiology of Fish (ISRPF-2018), New frontiers in reproductive diversity in a changing environment, 3-8 June 2018, Manaus, Brazil.-- 1 page, Introduction. Sperm production of captive Senegalese sole, Solea senegalensis, remains a major hindrance to commercial production of this highly valued species. New biotechnological approaches involving efficient hormonal therapies that increase sperm production are thus needed to establish manageable in vitro fertilization protocols. Homologous recombinant gonadotropins, follicle stimulating and luteinizing hormones (rFsh and rLh, respectively), produced in mammalian host cells, have recently arisen as promising candidates. Previous trials on juvenile fish using these hormones indicated that treatment with rFsh stimulates spermatogenesis, whereas rLh potentiates spermatozoa differentiation. The aim of the present work was to set up a protocol using rFsh and rLh to enhance sperm production in adult sole males. Material and methods. Senegalese sole males (~1 kg) were injected with rFsh (9 or 18 μg/kg) each week for 5 weeks, and on the 6th week treated with a single injection of rLh (9 or 18 μg/kg). Twenty-four hours after rLh injection, gonadotropin and androgen levels were determined by enzyme-linked immunosorbent assay (ELISA), and semen quantity and quality were evaluated by computer-assisted sperm analysis (CASA). Trials were carried out in early spring (temperatures from 13ºC to 17ºC), and in autumn under a constant temperature of 12°C. Results and Discussion. Treatment of males with rFsh and rLh during spring was not effective at inducing sperm production, possibly because of an advanced stage of sexual maturation of the males, as reflected by the high basal plasma levels of Lh (17 ng/ml). However, under constant low temperature in autumn, when circulating Lh is much lower (3 ng/ml), the dose of 9 ug/kg rFsh and rLh generated a four-fold increase in sperm production, which was not observed with the 18 ug/kg dose. It seems likely that, as previously found in juvenile fish, sustained treatment with high doses of rFsh diminished Leydig cell survival. Nevertheless, the motility, progressivity and velocity of spermatozoa were enhanced after rLh treatment during both spring and autumn. Conclusion. The data indicate that combined treatments with rFsh and rLh increase sperm production and quality in Senegalese sole adult males. However, the effectiveness of these hormonal therapies appears to be highly dependent on the reproductive stage of the males, and the temperature during which rFsh-induced spermatogenesis takes place

11th International Symposium on Reproductive Physiology of Fish (ISRPF-2018), New frontiers in reproductive diversity in a changing environment, 3-8 June 2018, Manaus, Brazil.-- 1 page, Introduction. The flathead grey mullet (Mugil cephalus) is a catadromous fish that does not spawn spontaneously in captivity. In Mediterranean aquaculture, few males produce flowing milt and females have dysfunctions at the early stages of vitellogenesis and oocyte maturation and ovulation. Research has focused on and hormonal therapies to address final maturation and spawning, but has been less successful in gametogenesis. Recently, promising results with the use of recombinant gonadotropins in finfish species have induced gametogenesis. Therefore, this study aimed to induce vitellogenesis and spermatogenesis in grey mullet using homologous recombinant follicle-stimulating hormone (mugil-rFSH), produced in Chinese Hamster Ovary (CHO) cells. Methods: Two groups of 12 immature grey mullet (9♀and 3♂, mean weight 990 ± 212g) received weekly intramuscular injections either of CHO cells medium (control) or mugil-rFSH at a dose of 15 μg.kg-1. Approximately every two weeks blood and gonadal samples (with cannulas or abdominal pressure) were taken for analysis. Ovarian development was evaluated in situ examining the diameter of the most advanced vitellogenic oocytes (n=20) and in samples fixed for histology. The presence of milt was assessed and motility measured. Plasma levels of 11-ketoteststerone and 17β-estradiol (E2) were measured in plasma throughout the experiment. Females with oocytes greater than 350 μm and rFSH-treated males received exogenous luteinizing hormone (LH) or human chorionic gonadotropin (hCG) (Solea senegalensis-rLH, hCG, hCG+Progesterone) in different consecutive weeks depending on the ovarian response. Statistical comparisons between groups were made with the Student's t-test. Results and discussion: Initial significant increases (P, The project received funding from the European Union 7FP (GA603121, DIVERSIFY)

11 pages, In marine teleosts, such as the gilthead seabream, several aquaporin paralogs are known to be expressed during the hyperosmotic induction of spermatozoon motility in seawater. Here, we used immunological inhibition of channel function to investigate the physiological roles of Aqp1aa, Aqp1ab, and Aqp7 during seabream sperm activation. Double immunofluorescence microscopy of SW-activated sperm showed that Aqp1aa and Aqp7 were respectively distributed along the flagellum and the head, whereas Aqp1ab accumulated in the head and in discrete areas toward the anterior tail. Inhibition of Aqp1aa reduced the rise of intracellular Ca2+, which is independent of external Ca2+ and normally occurs upon activation, and strongly inhibited sperm motility. Impaired Aqp1aa function also prevented the intracellular trafficking of Aqp8b to the mitochondrion, where it acts as a peroxiporin allowing H2O2 efflux and ATP production during activation. However, restoring the Ca2+ levels with a Ca2+ ionophore in spermatozoa with immunosuppressed Aqp1aa function fully rescued mitochondrial Aqp8b accumulation and sperm motility. In contrast, exposure of sperm to Aqp1ab and Aqp7 antibodies did not affect motility during the initial phase of activation, but latently compromised the trajectory and the pattern of movement. These data reveal the coordinated action of spatially segregated aquaporins during sperm motility activation in a marine teleost, where flagellar-localized Aqp1aa plays a dual Ca2+-dependent role controlling the initiation of sperm motility and the activation of mitochondrial detoxification mechanisms, while Aqp1ab and Aqp7 in the head and anterior tail direct the motion pattern © 2015 by the Society for the Study of Reproduction, Inc., Supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) AGL2010-15597 and AGL2013-41196-R to J.C. M.B. was supported by a predoctoral (FPI) fellowship from Spanish MINECO

Jornada Anual de la Secció d’Aqüicultura de la Societat Catalana de Biologia, Avenços en Recerca en Aqüicultura, 12 de Juny de 2015, Barcelona.-- 2 pages, The aquaculture of Senegalese sole is hampered by the absence of methods to control reproduction, and in particular by the low amount and quality of the sperm produced by cultured males. In Senegalese sole, spermatogenesis is semi-cystic and its endocrine control is largely unknown. In recent years, we have made significant progress in the understanding of sole spermatogenesis by focusing on the mechanism of action of the major reproductive hormones, the gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). The isolation of the transcripts encoding the sole Fsh and Lh subunits, as well as the Fsh and Lh receptors (Fshra and Lhcgrba), allowed us to produce recombinant hormones and specific antibodies to investigate the role of Fsh and Lh during spermatogenesis. Cell-based functional assays and immunolocalization studies showed that the sole Fshra is promiscuos (i.e. activated by both Fsh and Lh), and expressed in Sertoli cells enclosing the germ cells as well as in the androgen-producing Leydig cells. By contrast, the sole Lhcgrba is Lh-specific and only present in Leydig cells and spermatids free in the tubular lumen. Both Fsh and Lh control similar steroidogenic pathways in the testis, although Fsh also regulates genes encoding Sertoli cell growth factors, and Lh specifically activates spermatid-specific genes encoding spermatozoon proteins through direct interaction with the Lhcgrba in these cells. Finally, we have developed specific enzyme-linked immunosorbent assays (ELISAs) for Fsh and Lh that are being employed to determine the plasma levels of both gonadotropins during the reproductive cycle. All these molecular tools will allow the design of novel methods based on recombinant hormones for the control of sperm production in Senegalese sole, and the establishment of biomarkers to monitor reproductive success in cultured stocks

Jornada Anual de la Secció d’Aqüicultura de la Societat Catalana de Biologia, Avenços en Recerca en Aqüicultura, 12 de Juny de 2015, Barcelona.-- 1 page, The saltwater salmon louse (Lepeophtheirus salmonis) is an ectoparasitic crustacean copepod that causes severe infections of salmonids across the Atlantic and Pacific regions. Increasing levels of resistance to established drugs requires alternative approaches to solving the salmon louse epidemic. In contrast to its salmonid host, which is an osmoregulator, the louse´s cell volume regulation breaks down in fresh and brackish waters. These features suggest that aquaporins, which facilitate the transmembrane transport of water and other small uncharged solutes could represent potential therapeutic targets. However almost nothing is known concerning the functional diversity of aquaporins in crustaceans. To redress this lack of knowledge, we screened the genomes of the salmon louse and the Atlantic salmon and phylogenetically characterised their aquaporin superfamily repertoires in relation to those present in 131 arthropod and 121 vertebrate genomes. Subsequently we isolated and cloned seven full-length aquaporin transcripts from the louse and determined the functional transport properties of the encoded channels using a heterologous Xenopus laevis oocyte expression system. Our data reveal that the aquaporin repertoires in crustaceans and other arthropods can be classified into 3 major grades of aquaporins (1) classical type aquaporins including Big brain and Prip-like channels (2) aquaglyceroporins and (3) unorthodox aquaporins, while vertebrates have an additional Aqp8-type grade of channels. Functional characterization of the louse channels, reveal that the permeation properties of the arthropod grades of aquaporin are largely conserved to the vertebrate counterparts. Transcript abundance analyses and subcellular localisation studies indicate that selective aquaporins may be suitable as therapeutic targets

Jornada Anual de la Secció d’Aqüicultura de la Societat Catalana de Biologia, Avenços en Recerca en Aqüicultura, 12 de Juny de 2015, Barcelona.-- 2 pages, IIn the marine teleost gilthead seabream (Sparus aurata) different aquaporin water channels, such as Aqp1aa, -1ab, -7 and -8b, are expressed during the hyperosmotic induction of spermatozoon motility in seawater (SW). However, the physiological roles of these channels are unknown. Single and double immunofluorescence microscopy studies indicate that Aqp1aa and -7 are respectively localized in the entire flagellum or the head, while Aqp1ab and -8b are both in the head and the anterior tail of ejaculated spermatozoa. Upon SW activation, Aqp1ab and -8b are rapidly phosphorylated and translocated to the head plasma membrane and the mitochondrion, respectively, whereas Aqp1aa and -7 remain unchanged. Immunological inhibition of Aqp1aa function reduced the rise of intracellular Ca2+ that normally occurs upon activation, and strongly inhibited sperm motility. Impaired Aqp1aa function also prevented the intracellular trafficking of Aqp8b to the mitochondrion, where it acts as a peroxiporin allowing H2O2 efflux and ATP production during flagellar motility. However, restoring the Ca2+ levels in spermatozoa with immunosuppressed Aqp1aa fully rescued mitochondrial Aqp8b accumulation and sperm motility. In contrast, exposure of sperm to Aqp1ab and -7 antibodies did not affect motility during the initial phase of activation, but latently compromised the trajectory and the pattern of movement. These data reveal the coordinated action of spatially segregated aquaporins during sperm motility activation in seabream, where flagellar-localized Aqp1aa plays a dual Ca2+ -dependent role controlling the initiation of motility and the activation of mitochondrial Aqp8b-mediated detoxification mechanisms, while Aqp1ab and -7 in the head and anterior tail direct the motion pattern. These findings provide new insights into the molecular mechanisms regulating sperm motility in marine teleosts and the causes of male infertility under culture conditions

38 pages, 6 pages, 3 tables, supporting information https://doi.org/10.1371/journal.pone.0113686, A major physiological barrier for aquatic organisms adapting to terrestrial life is dessication in the aerial environment. This barrier was nevertheless overcome by the Devonian ancestors of extant Tetrapoda, but the origin of specific molecular mechanisms that solved this water problem remains largely unknown. Here we show that an ancient aquaporin gene cluster evolved specifically in the sarcopterygian lineage, and subsequently diverged into paralogous forms of AQP2,-5, or-6 to mediate water conservation in extant Tetrapoda. To determine the origin of these apomorphic genomic traits, we combined aquaporin sequencing from jawless and jawed vertebrates with broad taxon assembly of >2,000 transcripts amongst 131 deuterostome genomes and developed a model based upon Bayesian inference that traces their convergent roots to stem subfamilies in basal Metazoa and Prokaryota. This approach uncovered an unexpected diversity of aquaporins in every lineage investigated, and revealed that the vertebrate superfamily consists of 17 classes of aquaporins (Aqp0-Aqp16). The oldest orthologs associated with water conservation in modern Tetrapoda are traced to a cluster of three aqp2-like genes in Actinistia that likely arose >500 Ma through duplication of an aqp0-like gene present in a jawless ancestor. In sea lamprey, we show that aqp0 first arose in a protocluster comprised of a novel aqp14 paralog and a fused aqp01 gene. To corroborate these findings, we conducted phylogenetic analyses of five syntenic nuclear receptor subfamilies, which, together with observations of extensive genome rearrangements, support the coincident loss of ancestral aqp2-like orthologs in Actinopterygii. We thus conclude that the divergence of sarcopterygian-specific aquaporin gene clusters was permissive for the evolution of water conservation mechanisms that facilitated tetrapod terrestrial adaptation, This work was supported by the Research Council of Norway (RCN) project #178837/40 and 224816/E40 to (RNF), the Spanish Ministry of Science and Innovation (MICINN) AGL2010-15597 to (JC) and by the National Science Foundation, USA (NSF) IOS 0844818, 1041885 and 1241312 to (CPC). FC was supported by a postdoctoral (Juan de la Cierva Programme) fellowship from MICINN and the RCN (224816/E40)

10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 page, Introduction Recent studies in mammals have inferred that multiple classes of aquaporins may play different roles for water and solute transport during spermatogenesis and spermiogenesis. In teleosts, immunolocalization experiments in gilthead seabream have demonstrated that seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the mature (spermiating) testis. In this work, we used recombinant piscine gonadotropins and steroid hormones to investigate the in vivo and in vitro endocrine regulation of aquaporin expression during gilthead seabream spermatogenesis. Methods Males were sampled during the resting phase, at the onset of spermatogenesis and during the spermiating period. Plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) were measured by EIA. Testicular subsamples were used for immunofluorescence microscopy using paralog-specific antibodies, or incubated in vitro in the presence of European seabass recombinant follicle-stimulating hormone (rFsh) or luteinizing hormone (rLh), T, estradiol (E2), 11-KT or 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Changes in aquaporin gene expression were determined by real-time qPCR. Results and Discussion During the resting phase, when plasma levels of T and 11-KT were low and the testis exclusively contained spermatogonia, only Aqp1ab was detected in the cytoplasm of spermatogonia, whereas Aqp9b and -10b were expressed in Leydig and Sertoli cells, respectively. At the onset of spermatogenesis and in the spermiating testis, androgen plasma levels progressively increased which correlated with the appearance of spermatocytes and spermatids, and few spermatozoa, in the seminiferous tubules. At both stages, Aqp0a and -9b were localized in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and spermatozoa. In vitro incubations of testis explants with rFsh and rLh indicated that both gonadotropins induced elevated expression levels of aqp0a, - 9b and - 10b at the onset of spermatogenesis, whereas aqp1aa and - 7 only increased in the presence of rLh. At the spermiating stage, gonadotropins only promoted the expression of aqp7 and - 8b. The response to the steroids was also different depending on the spermatogenic stage. Thus, at the onset of spermatogenesis aqp0a, - 1aa, - 7, - 9b and - 10b mRNA levels increased in the presence of 11-KT, and in the case of aqp9b also with E2 and 17,20β-P. In contrast, during spermiogenesis the mRNA levels of aqp0a, -8b, - 9b and - 10b were only stimulated with T, and 17,20β-P for aqp9b, whereas those of aqp7 were still responsive to 11-KT. Interestingly, aqp1ab expression was not regulated by gonadotropins or steroids at the onset of spermatogenesis or during spermiation, suggesting an earlier control of this channel. Conclusion These observations suggest a differential and complex gonadotropic and steroid regulation of aquaporin expression during gilthead seabream germ cell development

27th Conference of European Comparative Endocrinologists (CECE 2014), 25-29 August 2014, Rennes, France.-- 1 page, In both mammals and teleosts, spermiogenesis, i.e. the differentiation of postmeiotic spermatids to spermatozoa, is thought to be controlled by androgens produced in response to the activation of the LH/choriogonadotropin receptor (LHCGR) by the luteinizing hormone (LH) in the interstitial Leydig cells. Accordingly, LH controls the expression of genes involved in the steroidogenic cascade in Leydig cells. Interestingly, in some teleosts, such as the flatfish Senegalese sole (Solea senegalensis) LH can also regulate different genes implied in spermiogenesis and sperm maturation. However, a non-steroidal role of LH mediating the spermiogenic pathway in vertebrates remains unclear. In Senegalese sole, spermatogenesis occurs in spermatocysts which develop within semi-cystic seminiferous lobules (SLs), where round spermatids are released from the supporting Sertoli cells into the SL lumen (SLL) where they elongate and transform into spermatozoa. Using this model, here we show that sole spermatids released into the SLL express the homolog of the tetrapod LHCGR (Lhcgrba) in the plasma membrane. Immunodetection of both native Lh and intramuscularly injected recombinant Lh (rLh) bound to free spermatids in the SLL indicated that circulating gonadotropin can reach the intratubular compartment. In vitro incubation of isolated spermatids with rLh specifically promoted their differentiation into spermatozoa in a dose-dependent manner, while homologous recombinant follicle-stimulating hormone or steroid hormones had no effect. Using a repertoire of molecular markers and inhibitors, we find that the Lh-Lhcgrba induction of spermiogenesis is mediated through a cAMP/PKA signaling pathway that initiates the transcription of genes potentially involved in the function of spermatozoa. The Lhcgrba expression in late spermatocytes and/or spermatids was also found in distantly related fishes with cystic spermatogenesis, such as zebrafish (Danio rerio) and gilthead seabream (Sparus aurata), suggesting that this feature is likely conserved in teleosts regardless of the type of germ cell development . These data reveal a novel role of Lh in vertebrate germ cells, whereby a Lhcgrbaactivated signaling cascade in haploid spermatids directs gene expression and the progression of spermiogenesis, Funded by the Spanish Fundación Ramón Areces

10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 page, Introduction In marine teleosts, such as the gilthead seabream, aquaporin water channels are implicated in sperm activation through mediation of water efflux from ejaculated spermatozoa exposed to hyperosmotic seawater. We have recently shown that seabream spermatozoa express up to five different aquaporin paralogs (Aqp1aa, -1ab, -7, -8b and -10b) with a segregated spatial distribution. However, the specific contribution of each paralog to the initiation and maintenance of sperm motility remains unknown. Here, we used paralog-specific antibodies as functional inhibitors to investigate the different roles of Aqp1aa and -8b during the hyperosmotic activation of sperm motility in the gilthead seabream. Methods Sperm was collected during the natural spawning season and used for immunofluorescence microscopy or activation assays. Aqp1aa and -8b mediated transport of water, urea or ammonia was determined from ectopic expression in Xenopus laevis oocytes, in the presence or absence of Aqp1aa and -8b affinity-purified antibodies. Motility parameters on seawater-diluted sperm were recorded using the Integrated Semen Analysis System software (ISAS version 1) in the presence of IgG (controls) or Aqp1aa or -8b antisera. Results and Discussion Expression of Aqp1aa and -8b in X. laevis oocytes revealed that Aqp1aa is water-specific, whereas Aqp8b is permeable to water, urea and ammonia. Double immunolocalization of Aqp1aa and -8b in ejaculated immotile spermatozoa confirmed that Aqp1aa is exclusively localized along the flagellum, whereas Aqp8b is dispersed in the head and the anterior flagellum. Upon activation, Aqp1aa localization is maintained along the tail while Aqp8b almost completely accumulates in the mitochondrion of the spermatozoa. Exposure of oocytes expressing Aqp1aa and -8b to their corresponding antibodies inhibited the osmotic water permeability of oocytes in a dose-dependent manner, and no cross-reaction of the antibodies was detected. Using these antibodies on sperm activation assays in seawater, we found that inhibition of Aqp1aa decreased most of the motion parameters (% of motile sperm, velocity, progressive motility), whereas Aqp8b inhibition only reduced the % of motile and progressive sperm. Conclusion These data suggest for the first time that Aqp1aa and -8b play distinct roles in the activation of teleost sperm. Aqp1aa possibly facilitates flagellar beating while Aqp8b may be required for the maintenance of spermatozoa progressivity. The function of Aqp8b-mediated mitochondrial transport of solutes (i.e. ammonia, urea) in this mechanism is under investigation

12 pages, 5 figures, 4 tables, One of the major obstacles for the vitrification of mature porcine oocytes with ethylene glycol is their low permeability to this cryoprotectant, which results in osmotic stress-induced cell damage and low survival. Pig blastocysts, on the other hand, show enhanced water and cryoprotectant permeability, which has been related to the transcriptional activation of aquaporin-3 (AQP3) channels at this stage of development. In this study, we asked if expression of cRNAs encoding two aquaglyceroporins, human AQP3 (hAQP3) or the zebrafish Aqp3b-T85A mutant, in porcine oocytes can increase their permeability. Microinjection of germinal-vesicle-stage oocytes with enhanced green fluorescent protein (EGFP) or AQP3 cRNAs resulted in the expression of the corresponding proteins in ∼26% of the metaphase-II stage oocytes at 40-44hr of in vitro culture; co-injection of EGFP cRNA appeared to be a suitable marker for oocyte selection since all EGFP-positive oocytes also expressed the corresponding aquaporin. Using this method, we found that mature oocytes co-expressing EGFP and hAQP3 or EGFP and Aqp3b-T85A showed approximately a twofold increase of the hydraulic conductivity (Lp) with respect non-injected or EGFP alone-injected oocytes in a 0.43M sucrose or 1.3M ethylene glycol solution, whereas the ethylene glycol permeability (PEG) of EGFP+hAQP3 and EGFP+Aqp3b-T85A oocytes was 6.7- and 12-fold higher, respectively, than control oocytes. These data demonstrate that the artificial expression of aquaglyceroporins in porcine metaphase-II oocytes improves their permeability, and that the zebrafish Aqp3b-T85A mutant is more efficient than the human channel at increasing the oocyte permeability to ethylene glycol. © 2014 Wiley Periodicals, Inc.

Póster presentado en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014, [Introduction] The Senegalese sole (Solea senegalensis) is a highly valued flatfish in Southern European aquaculture, but the industrial production of this species is still impaired due to poor knowledge of its reproductive endocrinology. The main endocrine regulators of fish gametogenesis are the pituitary gonadotropins (GTHs), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), which are composed by a specific beta chain (Fshb, Lhb) and a common alpha chain (Cga). Although cDNAs encoding both GTHs have been isolated in some flatfishes, no specific assay has been developed in these species to measure their physiological levels. Since immunoassays for GTH dimer proteins may be more sensitive than those using isolated subunits, in this study we developed a specific enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh using a chimeric protein containing homologous Fshb coupled with chicken (Gallus gallus) Cga., [Methods] The methylotrophic yeast Pichia pastoris was used to produce a chimeric recombinant single-chain Fsh containing the coding sequence of the sole mature Fshb (SsFshb), followed by six His residues, the carboxyl-terminal peptide sequence of human chorionic gonadotropin b subunit as a linker, and the mature sequence of the chicken Cga (GgCga). Homologous recombinant single-chains of Fsh (SsFsh) and Lh (SsLh) were produced in CHO cells. Polyclonal antibodies against the chimeric construct were generated in rabbits, and specific SsFshb antibodies were further purified by affinity chromatography on GgCga-bound columns. Preadsorbed and GgCga adsorbed antibodies were tested in ELISA using SsFshb-GgCga for coating and standard curve generation., [Results and Discussion] ELISA using the preadsorbed antiserum showed a crossreactivity with GgCGa and SsLh of 46.0% and 10.1%, respectively, which was respectively reduced to 0.5% and 0.05% after purification. Using the purified antiserum, the ELISA standard curves for SsFshb-GgCga paralleled those of serially diluted plasma and pituitary extracts, as well as that of SsFsh. The sensitivity of the assay for Fsh was

17 pages, 9 figures, 1 table, In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility, Supported by the Spanish Ministry of Science and Innovation (MICINN; AGL2010-15597 to J.C.), with additional financial support from the Research Council of Norway (204813/F20 and 224816/E40 to R.N.F.), and from the Ministry of Agriculture and Aquaculture of the Puglia Region (Italy) under the program between the Region of Puglia and the University of Salento (to S.V.). F.C. and M.B. were supported by a postdoctoral (Juan de la Cierva Programme) and predoctoral (FPI) fellowship, respectively, from MICINN

Miniposter Session VI - Endocrine disruptors; International audience

Ovarian growth (vitellogenesis) in most lower vertebrates is mediated by estradiol-17beta (E2) secreted by the follicles in response to follicle-stimulating hormone (Fsh), whereas oocyte maturation and ovulation are mediated by progestins, such 17alpha,20beta-dihydroxypregn-4-en-3-one (17,20beta-P), produced in response to luteinizing hormone (Lh). In teleosts, follicular synthesis of 17,20beta-P at the time of maturation is due primarily to up-regulation of the enzymes P450c17-II (Cyp17a2) and 20beta-hydroxysteroid dehydrogenase (Cbr1). Here, we show that follicular cells associated with primary growth (previtellogenic) oocytes of the gilthead seabream also express cyp17a2 and cbr1, in addition to P450c17-I (cyp17a1) and aromatase (cyp19a1), enzymes required for E2 synthesis. Ovaries containing only oogonia and early primary ovarian follicles had a 60-fold higher concentration of 17,20beta-P than ovaries in the succeeding stages and had a higher expression of cbr1 and Fsh receptor (fshra). Stimulation of explants of primary follicles in vitro with recombinant piscine Fsh (rFsh), which specifically activates the seabream Fshra, promoted a rapid accumulation of 17,20beta-P, and synthesis was sustained by an external supply of 17alpha-hydroxyprogesterone. In the presence of Cbr1 inhibitors, rFsh-mediated 17,20beta-P production was reduced, with a concomitant increase in testosterone and E2 synthesis. In primary explants, rFsh up-regulated cyp17a2 and cbr1 transcription and simultaneously down-regulated cyp17a1 and cyp19a1 steady-state mRNA levels within 24 h. In contrast, in explants containing vitellogenic follicles, rFsh had no effect on cyp17a2 and cbr1 expression, but increased that of cyp17a1 and cyp19a1. These data suggest a functional Fshra-activated Cyp17a2/ Cbr1 steroidogenic pathway in gilthead seabream primary ovarian follicles triggering th production of 17,20beta-P. © 2012 by the Society for the Study of Reproduction, Inc., Supported by the Spanish Ministry of Science and Innovation (MICINN; grant AGL2007-60261/ACU to J.C.), with additional financial support from the CEFAS Seedcorn fund. C.Z. and F.C. were supported by predoctoral (FPI) and postdoctoral (Juan de la Cierva Programme) fellowships, respectively, from MICINN.

The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC 50 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole. © 2012 by the Society for the Study of Reproduction, Inc., Supported by the Spanish Fundación Ramón Areces (J.C.). F.C. was supported by a postdoctoral fellowship from Juan de la Cierva Programme (Spanish Ministry of Education and Science).

Ponencia presentada en el Workshop "The Cultivation of Soles V" celebrado en Faro (Portugal) del 5 al 7 de abil de 2011, The reproduction of Senegalese sole in captivity is not yet controlled. As a consequence, the timing of spawning as well as the viability of eggs and larvae, can not be predicted, which hampers the mass production of alevins of this species. The control of reproduction of captive males, specially of the F1 generation, appears as one major bottleneck, since low or null fertilization rates as well as poor sperm quality are often encountered in hatcheries. The Senegalese sole presents unusual characteristics of testicular development showing spermatogenesis of a semi-cystic type (i.e., spermatocytes and spermatids are released into the lumen of the seminiferous tubules, where they differentiate into spermatozoa) with production of only a few tens of microliters of sperm almost all-year around. Spermatogenesis in this species is thus `asynchronous¿ but the particularities of its hormonal control are poorly understood. This is partially caused by the absence of proper methods to determine the mode of action of the gonadotropins (FSH and LH), the main hormones regulating reproduction, in the gonad of this species. It is clear therefore that a deeper understanding of the endocrine regulation of spermatogenesis in Senegalese sole is urgently needed. During the past few years, we have made significant advances in the knowledge of the reproductive biology of Senegalese sole by isolating the cDNAs coding for the gonadotropin subunits and their gonad receptors. In addition, we determined their structural and functional features, and their expression patterns in the pituitary and testis, respectively, during spermatogenesis. These studies reported, for the first time in any teleost, the expression of the LH receptor in spermatids in addition to Leydig cells. This finding suggests that LH in Senegalese sole might regulate the elongation of spermatids and transition to spermatozoa, thus allowing the recruitment of successive batches of spermatids, present in the testis of adult fish almost all year around, into spermiogenesis. The availability of the Senegalese sole cDNAs for gonadotropins and receptors has made it possible recently to produce single-chain recombinant gonadotropins (FSHsc and LHsc) through biotechnological methods. The sole FSHsc and LHsc may show improved stability and long half-life in vivo as it has been described for analogous hormones in mammals. Preliminary data indicate that sole FSHsc and LHsc can activate their cognate receptors in vitro, and therefore they may be useful tools to unravel the endocrine regulation of spermatogenesis in Senegalese sole, and to control gametogenesis and sexual maturation of captive animals., Supported by Fundación Ramón Areces to JC. Participation of FC is funded by Juan de la Cierva Program (Spanish Ministry of Science and Innovation).

XIII Congreso Nacional de Acuicultura. En equilibrio con el medio ambiente. 21-24 noviembre 2011, Barcelona, The aquaculture of Senegalese sole is hampered by the absence of methods to control reproduction, particularly of male fish reared in captivity. Teleost reproduction is regulated by the pituitary gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), but the mode of action of these hormones during Senegalese sole spermatogenesis is poorly known. Here, we aimed at producing Senegalese sole single-chain recombinant Fsh (rFshsc) and Lh (rLhsc). The cDNAs encoding the recombinant gonadotropins were introduced into Chinese hamster ovary (CHO) cells, and stably expressing clones were selected for their production efficiency. The rFshsc and rLhsc were found to be glycosylated when released in the culture medium. Transactivation assays using Senegalese sole gonadotropin receptors indicated that rFshsc and rLhsc are active on their cognate receptors but also some cross reaction is observed. This preliminary study demonstrates that CHO cells are useful systems for producing functional recombinant gonadotropins, and offers new tools for the aquaculture of Senegalese sole and the investigation of its reproductive endocrinology., Financiado por la Fundación Ramón Areces. F.C. está contratado en el Subprograma Juan de la Cierva (MICINN).

9th International Congress on the Biology of Fish. 5-9 Julio de 2010, Barcelona, In the gilthead seabream aquaporin-1b (Aqp1b) mediates oocyte hydration during meiotic maturation. However, although aqp1b mRNA is present in primary growth oocytes, the synthesis of polypeptides does not occur until the onset of vitellogenesis, suggesting that oocyte Aqp1b is regulated independently by transcriptional and post-transcriptional mechanisms. To investigate these processes, a 1.6-kb DNA fragment of the 5’ flanking region of the seabream aqp1b gene was isolated, sequenced and functionally analyzed in HEK293T, MCF7 and Caco-2 mammalian cells lines. Transient transfection assays of a series of 5’ to 3’ deletion constructs coupled to luciferase indicated that different promoter sequences were active in each cell line. Interestingly, MCF7 cells transfected with the aqp1b 1.6-kb fragment showed a 3.5-fold increase in luciferase activity in response to progesterone but not estradiol, which was abolished with the progesterone receptor (PR) inhibitor RU-486. Further deletion experiments indicated two putative PR binding sites in the aqp1b promoter which were active in MCF7 cells. These data suggest that the seabream aqp1b gene might be under PR regulation in previtellogenic primary growth oocytes

9th International Congress on the Biology of Fish. 5-9 Julio de 2010, Barcelona, The Senegalese sole is a flatfish of increasing commercial interest but its reproduction in captivity, particularly of males, is not yet controlled. To investigate the endocrine basis of sole reproduction, we have isolated the full-length cDNAs of the pituitary gonadotropin subunits (Fshb, Lhb, and the common glycoprotein alpha subunit). We further isolated their gonad receptors (Fshr and Lhr), as they are key regulators of gonad development and maturation. Functional characterization suggests that sole Fshr responds to piscine Fsh and Lh, whereas Lhr is preferentially activated by its cognate ligand. As expected, the expression of gonadotropin subunits and their receptors in the pituitary and testis is transiently elevated in winter when spermatogenesis is stimulated. However, the Lhr transcripts, which are expressed in Leydig cells as well as in spermatids, remain elevated in spring suggesting an additional role of this receptor during the transformation of spermatids into spermatozoa. These data provide the basis for the production of Senegalese sole homologous recombinant gonadotropins, which will be of assistance to unravel the hormonal control of gametogenesis and to develop methods for the control of reproduction in captivity

9th International Congress on the Biology of Fish. 5-9 Julio de 2010, Barcelona, The role of aquaporins during fish embryo development is largely unknown. Here, we investigated the expression pattern and cell localization of aquaporin-3 (Aqp3) in embryos of zebrafish and killifish. In both species, transcripts encoding two Aqp3 paralogs (Aqp3a and/or -3b) are transiently The role of aquaporins during fish embryo development is largely unknown. Here, we investigated the expression pattern and cell localization of aquaporin-3 (Aqp3) in embryos of zebrafish and killifish. In both species, transcripts encoding two Aqp3 paralogs (Aqp3a and/or -3b) are transiently accumulated in migrating blastomers during gastrulation. In killifish, immunolocalization experiments revealed the presence of Aqp3a in the basolateral membrane of the cells of the enveloping layer (EVL) as well as in the membrane of ingressing blastomers at the marginal region of the blastoderm. Killifish embryos have the ability to accelerate development in response to environmental desiccation, and under these conditions aqp3a and protein levels are down-regulated. However, reduction of Aqp3a polypetides is only seen in the EVL, whereas the migrating blastomers retain the protein in the plasma membrane. These observations suggest a role of Aqp3 in cell migration as recently proposed in mammals, which is being investigated by using zebrafish embryo explant cultures and wound-healing assays

15 p., il., y bibliografía, Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/ choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucinerich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.

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International audience; Much of the present information on muscle differentiation in fish concerns the early embryonic stages. To learn more about the maturation and the diversification of the fish myotomal fibres in later stages of ontogeny, we investigated, by means of in situ hybridisation, the developmental expression of a large repertoire of muscle-specific genes in trout larvae from hatching to yolk resorption. At hatching, transcripts for fast and slow muscle protein isoforms, namely myosins, tropomyosins, troponins and myosin binding protein C were present in the deep fast and the superficial slow areas of the myotome, respectively. During myotome expansion that follows hatching, the expression of fast isoforms became progressively confined to the borders of the fast muscle mass, whereas, in contrast, slow muscle isoform transcripts were uniformly expressed in all the slow fibres. Transcripts for several enzymes involved in oxidative metabolism such as citrate synthase, cytochrome oxidase component IV and succinate dehydrogenase, were present throughout the whole myotome of hatching embryos but in later stages became concentrated in slow fibre as well as in lateral fast fibres. Surprisingly, the slow fibres that are added externally to the single superficial layer of the embryonic (original) slow muscle fibres expressed not only slow twitch muscle isoforms but also, transiently, a subset of fast twitch muscle isoforms including MyLC1, MyLC3, MyHC and myosin binding protein C. Taken together these observations show that the growth of the myotome of the fish larvae is associated with complex patterns of muscular gene expression and demonstrate the unexpected presence of fast muscle isoform-expressing fibres in the most superficial part of the slow muscle.

Diplôme : Dr. d'Universite; La musculature squelettique des poissons est composée d'un muscle lent périphérique et d'un muscle rapide profond. Les fibres lentes dérivent de précurseurs somitiques adaxiaux migrant en périphérie du myotome et les précurseurs somitiques latéraux se différencient sur place en fibres rapides. Une modalité particulière de la croissance musculaire des poissons est la formation continue de nouvelles fibres, recrutées en périphérie du myotome chez la larve (hyperplasie stratifiée) et dans tout le myotome chez les poissons de grande taille finale (hyperplasie mosaïque). Nos travaux visaient à décrire chez la truite les régulations transcriptionnelles sous-tendant la différenciation contractile et métabolique des fibres musculaires mises en place au cours du développement, par l'examen systématique par hybridation in situ des patrons d'expression de marqueurs musculaires. Nous montrons que la différenciation des fibres musculaires chez le poisson inclue un programme myogénique commun, observé transitoirement dans toutes les fibres embryonnaires et dans les néofibres lentes formées par hyperplasie stratifiée. Par ailleurs, nous montrons que l'IGF1 et le FGF2 sont de bons candidats impliqués dans le maintien de l'hyperplasie chez le poisson.

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