Molecular approaches related to the European eel (Anguilla anguilla) reproductive process

Tesis por compendio[EN] The European eel (Anguilla anguilla, L., 1758) population is in dramatic decline, so much so that this species has been listed as "Critically Endangered" on the Red List of Threatened Species, by the International Union for Conservation of Nature (IUCN). The European eel has a complex life cycle, with sexual maturation blocked in the absence of the reproductive oceanic migration, and an inability to mature in captivity without the administration of hormonal treatments. Even though experimental maturation induces gamete production of both sexes, the fertilization results in infertile eggs, unviable embryos and larvae, which die within a few days of hatching. Therefore, understanding the eel reproductive physiology during maturation is very important if we want to recover the wild eel population. Furthermore, due to its phylogenetic position, representative of a basal group of teleosts, the Elopomorphs, the Anguilla species may provide insights into ancestral regulatory physiology processes of reproduction in teleosts, the largest group of vertebrates. In this thesis, characterization, phylogeny and synteny analyses have given us new insight into the evolutionary history of the reproductive process in vertebrates. The European eel possesses five membrane (mPRs) and two nuclear (nPR or pgrs) progestin receptors. Eel mPRs clustered in two major monophyletic groups. Phylogeny analysis of vertebrate nPRs and PLCz1 (sperm specific protein) places both eel PLCz1 and nPR sequences at the base of the teleost clade, which is consistent with the basal position of elopomorphs in the phylogeny of teleosts. To further resolve the origin of the duplicated eel nPRs, synteny analyses of the nPR neighboring genes in several vertebrate genomes were performed. Phylogeny and synteny analyses allowed us to propose the hypothesis that eel duplicated nPRs originated from the 3R. In order to gain a better understanding of the role of the genes implicated in eel reproduction, analyses of their regulation during experimental maturation were carried out. The change in salinity induced parallel increases in E2 plasma and nuclear estrogen receptor expression levels, revealing a stimulatory effect of salinity on the E2 signalling pathway along the BPG axis, leading to a control of spermatogonial stem cell renewal. Brain and pituitary estrogen receptors may then mediate the stimulation of androgens and steroidogenic enzymes linked to androgen synthesis. Androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This is consistent with our studies on estrogen and progestin receptors. In the testis, progestin seems to regulate meiosis through membrane and nuclear progestin receptors, and final sperm maturation seems to be controlled by both estrogen and progestin through the estrogen and progestin membrane receptors. Finally, eel sperm-specific PLCz1 seems to have an important function in spermatozoa by inducing egg activation and temperature may play a role in its regulation, especially during the process of spermiogenesis. This thesis attempts to evaluate the physiological function of the genes involved in eel reproduction during spermatogenesis, and demonstrates that salinity and temperature play crucial roles in the sexual maturation of the male European eel.[ES] La anguila europea (Anguilla anguilla, L., 1758) está sufriendo un declive dramático y ha sido incluida en la categoría de especies "En peligro crítico" en la Lista Roja de Especies Amenazadas, por la International Union for Conservation of Nature (IUCN). La anguila europea tiene un ciclo de vida complejo, con un bloqueo de la maduración sexual hasta que se produce la migración reproductiva, y no madura en cautividad sin la aplicación de tratamientos hormonales. La inducción de la maduración sexual conlleva la producción de gametos de ambos sexos, pero los resultados de la fertilización son huevos no fértiles, embriones no viables, o larvas que mueren pocos días después de la eclosión. Por tanto, la comprensión de la fisiología reproductiva de la anguila durante la maduración es imprescindible para recuperar sus poblaciones naturales. Además, dada su posición filogenética, como representantes de un grupo basal de los teleósteos, los elopomorfos, las especies del género Anguilla podrían proporcionar nuevas perspectivas sobre los procesos ancestrales de regulación de la fisiología de la reproducción de los teleósteos, el mayor grupo de vertebrados. En esta tesis, los resultados de caracterización, análisis de filogenia y sintenia ofrecen nuevas perspectivas de la historia evolutiva del proceso reproductivo de los vertebrados. La anguila europea posee cinco receptores de progestágenos de membrana (mPRs) y dos nucleares (nPR o pgrs). Los mPRs de la anguila se engloban en dos grandes grupos monofiléticos. Las filogenias de los nPRs y de la PLCz1 (una proteína específica del esperma) sitúan a las secuencias de la anguila de PLCz1 y de nPRs en la base del grupo de los teleósteos, lo que coincide con la posición basal de los elopomorfos en la filogenia de los teleósteos. Para resolver el origen de la duplicidad de los nPRs de anguila, se realizaron análisis de sintenia de los genes próximos a los nPRs. Los análisis de filogenia y sintenia nos permitieron formular la hipótesis de que los nPRs duplicados de la anguila se originaron en la 3° duplicación del genoma que se produjo en teleósteos. Para entender mejor el papel de los genes implicados en la reproducción de la anguila, se hicieron análisis de su regulación durante la maduración experimental. El cambio de salinidad indujo aumentos paralelos del nivel plasmático de E2 y de la expresión de los receptores nucleares de estrógenos, que refleja un efecto estimulador de la salinidad sobre la ruta de señalización del E2 dentro del eje cerebro-hipófisis-gónada, que conlleva el control de la renovación de las espermatogonias indiferenciadas. Los receptores de estrógeno en el eje cerebro-hipófisis-gónada podrían mediar la estimulación de la síntesis de andrógenos y de los enzimas esteroidogénicos unidos a ella. Esa síntesis de andrógenos no depende de la temperatura, pero la continuación del proceso de maduración requiere de temperaturas más altas para inducir un cambio en las rutas esteroidogénicas hacia la síntesis de estrógenos y progestágenos. Esto coincide con nuestros estudios sobre receptores de estrógenos y de progestágenos. En el testículo, los progestágenos parecen regular la meiosis mediante la participación de dos mPRs y un nPR, y la maduración final del esperma parece estar controlada tanto por estrógenos como por progestágenos mediante los receptores de estrógenos y de progestágenos de membrana. Finalmente, la PLCz1 de anguila podría tener una importante función en la activación del huevo inducida por el espermatozoide, y la temperatura podría jugar un papel en su regulación, especialmente durante el proceso de espermiogénesis. Esta tesis intentó evaluar la función fisiológica de los genes implicados en la reproducción de la anguila durante la espermatogénesis, y demuestra que la salinidad y la temperatura juegan papeles cruciales en la maduración sexual de los machos de anguila[CA] La població d'anguila europea (Anguilla anguilla, L., 1758) està sofrint un declivi dramàtic, i aquesta espècie ha estat inclosa en la categoria d'espècies "En perill crític" en la Llista Roja d'Espècies Amenaçades per la International Union for Conservation of Nature (IUCN). L'anguila europea té un cicle de vida complex, amb un bloqueig de la maduració sexual que es manté fins que es produeix la migració reproductiva, i no madura en captivitat sense l'aplicació de tractaments hormonals. Però, fins i tot quan la inducció de la maduració sexual comporta la producció de gàmetes d'ambdós sexes, els resultats de la fertilització son ous no fèrtils, embrions no viables o larves que moren pocs dies després de l'eclosió. Per això, la comprensió de la fisiologia reproductiva de l'anguila durant la maduració és imprescindible per aconseguir la recuperació de les poblacions naturals d'anguila. A més, donada la seua posició filogenètica com a representant d'un grup basal de teleostis, els elopomorfos, les espècies del gènere Anguilla podrien proporcionar noves perspectives al voltant dels processos ancestrals de regulació de la fisiologia de la reproducció dels teleostis, el grup més nombrós dels vertebrats. En aquesta tesi, els resultats de caracterització i l'anàlisi de la filogènia i la sintènia ofereixen noves perspectives de la història evolutiva del procés reproductiu dels vertebrats. L'anguila europea posseeix cinc receptors de progestàgens de membrana (mPRs) i dos nuclears (nPR o pgrs). Els mPRs de l'anguila s'engloben en dos grans grups monofilètics. L'anàlisi filogenètic dels nPRs i de la PLCz1 (una proteïna específica de l'esperma) de l'anguila respecte a les de la resta de vertebrats situa a les seqüències d'aquestes proteïnes en la base dels grups dels teleostis, la qual cosa coincideix amb la posició basals dels elopomorfos en la filogènia dels teleostis. Per tal de resoldre l'origen de la duplicitat dels nPRs de l'anguila, es realitzaren anàlisis de sintènia dels gens pròxims als dels nPRs en els genomes de diversos vertebrats. Aquests anàlisis ens permeteren formular la hipòtesi de que els nPRS duplicats de l'anguila es van originar en la tercera duplicació del genoma que es va produir en teleostis. Per arribar a entendre millor el paper dels gens implicats en la reproducció de l'anguila, s'analitzà la seua regulació durant la maduració experimental. Els canvis en la salinitat induïren augments en paral·lel del nivell plasmàtic d'E2 i de l'expressió dels receptors nuclears d'estrògens, reflectint un efecte estimulador de la salinitat sobre la ruta de senyalització d'E2 en l'eix cervell-hipòfisi-gònada, que comportaria el control de la renovació dels espermatogonis indiferenciats. Els receptors d'estrògens en l'eix cervell-hipòfisi-gònada podrien, d'aquesta forma, intervindre en l'estimulació de la síntesi d'andrògens i dels enzims esteroidogènics units a la síntesi d'andrògens. Aquesta síntesi d'andrògens no depén de la temperatura, però la continuació del procés de maduració requereix de temperatures més altes per induir un canvi en les rutes esteroidogènics cap a la síntesi d'estrògens i progestàgens. En els testicles, els progestàgens pareixen regular la meiosi mitjançant la participació dels receptors de progestàgens de membrana i nuclears, i la maduració final de l'esperma sembla estar controlada tant pels estrògens com per progestàgens de membrana. Finalment, la PLCz1 específica de l'esperma de l'anguila podria tindre una funció de rellevància en l'activació dels ous induïda pels espermatozoides, i la temperatura podria tindre el seu paper en la regulació d'aquesta, especialment durant el procés de l'espermiogènesi. Aquesta tesi ha avaluat la funció fisiològica dels gens implicats en al reproducció de l'anguila durant l'espermatogènesi, i ha demostrat que la salinitat i la temperatura tenen papers clauMorini, MAM. (2016). Molecular approaches related to the European eel (Anguilla anguilla) reproductive process [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68513TESISPremios Extraordinarios de tesis doctoralesCompendi

Palliation with Oesophageal Metal Stent of Pseudoachalasia from Gastric Carcinoma at the Cardia: A Case Report

We present an 82-year-old woman with a 3-month history of progressive dysphagia and a normal initial upper gastrointestinal endoscopy. The diagnosis of pseudoachalasia was suspected by oesophageal manometric and barium swallow studies, and confirmed by biopsies revealing an intestinal type carcinoma of the stomach at a repeated endoscopy. In view of the history of heart disease, diabetes, and old age, this patient was treated by a partially covered Ultraflex self-expanding metal stent (Boston Scientific, Natick, MA, USA) placed into the oesophageal body with no direct complications and obtaining the relief from dysphagia. During the 11-month follow-up she was treated for an iron deficiency anaemia due to reflux oesophagitis with ulcerations in the oesophageal body and died from myocardial infarction. According to the localization of the cancer, the old age, and the presence of comorbidities, we should recommend the insertion of a partially covered self-expanding metal stent as a reasonable palliative treatment in selected subjects with pseudoachalasia

Identification and stable expression of vitellogenin receptor (VTGR) through vitellogenesis in the European eel

[EN] In teleosts, vitellogenin (Vtg) is a phospholipoglycoprotein synthesized by the liver, released into the blood circulation and incorporated into the oocytes via endocytosis mediated by the Vtg receptor (VTGR) to form the yolk granules. The VTGR is crucial for oocyte growth in egg-laying animals but is also present in non-oviparous vertebrates, such as human. The VTGR belongs to the low-density lipoprotein receptor superfamily (LDLR) and is also named very-low-density lipoprotein receptor (VLDLR). In this study, we identified and phylogenetically positioned the VTGR of a basal teleost, the European eel, Anguilla anguilla. We developed quantitative real-time PCR (qRT-PCR) and investigated the tissue distribution of vtgr transcripts. We compared by qRT-PCR the ovarian expression levels of vtgr in juvenile yellow eels and pre-pubertal silver eels. We also analyzed the regulation of ovarian vtgr expression throughout vitellogenesis in experimentally matured eels. The Vtg plasma level was measured by homologous ELISA experimental maturation. Our in silico search and phylogenetical analysis revealed a single vtgr in the European eel, orthologous to other vertebrate vtgr. The qRT-PCR studies revealed that vtgr is mainly expressed in the ovary and also detected in various other tissues such as brain, pituitary, gill, fat, heart, and testis, suggesting some extra-ovarian functions of VTGR. We showed that vtgr is expressed in ovaries of juvenile yellow eels with no higher expression in pre-pubertal silver eels nor in experimentally matured eels. This suggests that vtgr transcription already occurs during early pre-vitellogenesis of immature eels and is not further activated in vitellogenic oocytes. European eel Vtg plasma level increased throughout experimental maturation in agreement with previous studies. Taken together, these results suggest that vtgr transcript levels may not be a limiting step for the uptake of Vtg by the oocyte in the European eel.M.M., A.G.L. and S.D. were granted with Short Term Scientific Missions by the COST Office (COST Action FA1205: AQUAGAMETE). S.D. was also awarded with a grant from the UPV's School of Doctorate (Accion para la Internacionalizacion de los Programas de Doctorado) in 2017. We thank E. Feunteun and colleagues, MNHN, Dinard, for the ovarian samples from eels of the Fremur, France.Morini, M.; Lafont, AG.; Maugars, G.; Baloche, S.; Dufour, S.; Asturiano, JF.; Pérez Igualada, LM. (2020). Identification and stable expression of vitellogenin receptor (VTGR) through vitellogenesis in the European eel. Animal. 14(6):1213-1222. https://doi.org/10.1017/S1751731119003355S12131222146Abascal, F., Zardoya, R., & Posada, D. (2005). ProtTest: selection of best-fit models of protein evolution. Bioinformatics, 21(9), 2104-2105. doi:10.1093/bioinformatics/bti263Ali, B. R., Silhavy, J. L., Gleeson, M. J., Gleeson, J. G., & Al-Gazali, L. (2012). A missense founder mutation in VLDLR is associated with Dysequilibrium Syndrome without quadrupedal locomotion. BMC Medical Genetics, 13(1). doi:10.1186/1471-2350-13-80Andersen, Ø., Xu, C., Timmerhaus, G., Kirste, K. H., Naeve, I., Mommens, M., & Tveiten, H. (2017). Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar ): Ancient origin of the phosvitin-less VtgC in chondrichthyean fishes. Molecular Reproduction and Development, 84(11), 1191-1202. doi:10.1002/mrd.22881Bidwell, C., & Carlson, D. (1995). Characterization of vitellogenin from white sturgeon, Acipenser transmontanus. Journal of Molecular Evolution, 41(1). doi:10.1007/bf00174046Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., … Schneider, W. J. (1994). Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family. The EMBO Journal, 13(21), 5165-5175. doi:10.1002/j.1460-2075.1994.tb06847.xBurzawa-Gerard, E., & Dumas-Vidal, A. (1991). Effects of 17β-estradiol and carp gonadotropin on vitellogenesis in normal and hypophysectomized European silver female eel (Anguilla anguilla L.) employing a homologous radioimmunoassay for vitellogenin. General and Comparative Endocrinology, 84(2), 264-276. doi:10.1016/0016-6480(91)90049-cChen, J.-N., López, J. A., Lavoué, S., Miya, M., & Chen, W.-J. (2014). Phylogeny of the Elopomorpha (Teleostei): Evidence from six nuclear and mitochondrial markers. Molecular Phylogenetics and Evolution, 70, 152-161. doi:10.1016/j.ympev.2013.09.002Clelland, E. S., & Kelly, S. P. (2010). Tight junction proteins in zebrafish ovarian follicles: Stage specific mRNA abundance and response to 17β-estradiol, human chorionic gonadotropin, and maturation inducing hormone. General and Comparative Endocrinology, 168(3), 388-400. doi:10.1016/j.ygcen.2010.05.011Damsteegt, E. L., Mizuta, H., Hiramatsu, N., & Lokman, P. M. (2015). How do eggs get fat? Insights into ovarian fatty acid accumulation in the shortfinned eel, Anguilla australis. General and Comparative Endocrinology, 221, 94-100. doi:10.1016/j.ygcen.2014.12.019Dufour S, Burzawa-Gerard E, Le Belle N, Sbaihi M and Vidal B 2003. Reproductive endocrinology of the European eel, Anguilla anguilla. In Eel biology (eds. K Aida, K Tsukamoto and K Yamauchi ), pp 373–383. Springer-Verlag, Tokyo, Japan.Dufour, S., Lopez, E., Le Menn, F., Le Belle, N., Baloche, S., & Fontaine, Y. A. (1988). Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol. General and Comparative Endocrinology, 70(1), 20-30. doi:10.1016/0016-6480(88)90090-1Henkel, C. V., Burgerhout, E., de Wijze, D. L., Dirks, R. P., Minegishi, Y., Jansen, H. J., … van den Thillart, G. E. E. J. M. (2012). Primitive Duplicate Hox Clusters in the European Eel’s Genome. PLoS ONE, 7(2), e32231. doi:10.1371/journal.pone.0032231Henkel, C. V., Dirks, R. P., de Wijze, D. L., Minegishi, Y., Aoyama, J., Jansen, H. J., … van den Thillart, G. E. E. J. M. (2012). First draft genome sequence of the Japanese eel, Anguilla japonica. Gene, 511(2), 195-201. doi:10.1016/j.gene.2012.09.064Herz, J., & Bock, H. H. (2002). Lipoprotein Receptors in the Nervous System. Annual Review of Biochemistry, 71(1), 405-434. doi:10.1146/annurev.biochem.71.110601.135342Hiramatsu, N., Luo, W., Reading, B. J., Sullivan, C. V., Mizuta, H., Ryu, Y.-W., … Hara, A. (2012). Multiple ovarian lipoprotein receptors in teleosts. Fish Physiology and Biochemistry, 39(1), 29-32. doi:10.1007/s10695-012-9612-6Hiramatsu, N., Todo, T., Sullivan, C. V., Schilling, J., Reading, B. J., Matsubara, T., … Hara, A. (2015). Ovarian yolk formation in fishes: Molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins. General and Comparative Endocrinology, 221, 9-15. doi:10.1016/j.ygcen.2015.01.025Hummel, S., Lynn, E. G., Osanger, A., Hirayama, S., Nimpf, J., & Schneider, W. J. (2003). Molecular characterization of the first avian LDL receptor. Journal of Lipid Research, 44(9), 1633-1642. doi:10.1194/jlr.m300014-jlr200Jéhannet P, Kruijt L, Damsteegt EL, Swinkels W, Heinsbroek LTN, Lokman PM and Palstra AP. A mechanistic model for studying the initiation of anguillid vitellogenesis by comparing the European eel (Anguilla anguilla) and the shortfinned eel (A. australis). General and Comparative Endocrinology (in press). https://doi.org/10.1016/j.ygcen.2019.02.018Lafont, A.-G., Rousseau, K., Tomkiewicz, J., & Dufour, S. (2016). Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation. General and Comparative Endocrinology, 235, 177-191. doi:10.1016/j.ygcen.2015.11.021Mazzeo, I., Peñaranda, D. S., Gallego, V., Baloche, S., Nourizadeh-Lillabadi, R., Tveiten, H., … Pérez, L. (2014). Temperature modulates the progression of vitellogenesis in the European eel. Aquaculture, 434, 38-47. doi:10.1016/j.aquaculture.2014.07.020Mizuta, H., Luo, W., Ito, Y., Mushirobira, Y., Todo, T., Hara, A., … Hiramatsu, N. (2013). Ovarian expression and localization of a vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 166(1), 81-90. doi:10.1016/j.cbpb.2013.07.005Mizuta, H., Mushirobira, Y., Nagata, J., Todo, T., Hara, A., Reading, B. J., … Hiramatsu, N. (2017). Ovarian expression and localization of clathrin (Cltc) components in cutthroat trout, Oncorhynchus clarki: Evidence for Cltc involvement in endocytosis of vitellogenin during oocyte growth. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 212, 24-34. doi:10.1016/j.cbpa.2017.06.021Morini, M., Peñaranda, D. S., Vílchez, M. C., Gallego, V., Nourizadeh-Lillabadi, R., Asturiano, J. F., … Pérez, L. (2015). Transcript levels of the soluble sperm factor protein phospholipase C zeta 1 (PLCζ1) increase through induced spermatogenesis in European eel. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 187, 168-176. doi:10.1016/j.cbpa.2015.05.028Morini, M., Peñaranda, D. S., Vílchez, M. C., Nourizadeh-Lillabadi, R., Lafont, A.-G., Dufour, S., … Pérez, L. (2017). Nuclear and membrane progestin receptors in the European eel: Characterization and expression in vivo through spermatogenesis. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 207, 79-92. doi:10.1016/j.cbpa.2017.02.009Nader, N., Dib, M., Courjaret, R., Hodeify, R., Machaca, R., Graumann, J., & Machaca, K. (2018). VLDL receptor regulates membrane progesterone receptor trafficking and non-genomic signaling. Journal of Cell Science. doi:10.1242/jcs.212522Okabayashi, K., Shoji, H., Nakamura, T., Hashimoto, O., Asashima, M., & Sugino, H. (1996). cDNA Cloning and Expression of theXenopus laevisVitellogenin Receptor. Biochemical and Biophysical Research Communications, 224(2), 406-413. doi:10.1006/bbrc.1996.1040Palstra, A. P., & van den Thillart, G. E. E. J. M. (2010). Swimming physiology of European silver eels (Anguilla anguilla L.): energetic costs and effects on sexual maturation and reproduction. Fish Physiology and Biochemistry, 36(3), 297-322. doi:10.1007/s10695-010-9397-4Pankhurst, N. W. (1982). Relation of visual changes to the onset of sexual maturation in the European eel Anguilla anguilla (L.). Journal of Fish Biology, 21(2), 127-140. doi:10.1111/j.1095-8649.1982.tb03994.xPasquier, J., Lafont, A.-G., Jeng, S.-R., Morini, M., Dirks, R., van den Thillart, G., … Dufour, S. (2012). Multiple Kisspeptin Receptors in Early Osteichthyans Provide New Insights into the Evolution of This Receptor Family. PLoS ONE, 7(11), e48931. doi:10.1371/journal.pone.0048931Perez, L., Aturiano, J. F., Tomas, A., Zegrari, S., Barrera, R., Espinos, F. J., … Jover, M. (2000). Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. Journal of Fish Biology, 57(6), 1488-1504. doi:10.1111/j.1095-8649.2000.tb02227.xPérez L, Vílchez MC, Gallego V, Mazzeo I, Peñaranda DS, Weltzien FA, Dufour S and Asturiano JF 2012. Trying to reproduce the European eel (Anguilla anguilla) under captivity: experiments with females. In Proceeding of the Domestication in Finfish Aquaculture, October 2012, Olsztyn, Poland, pp. 11–15.Prat, F., Coward, K., Sumpter, J. P., & Tyler, C. R. (1998). Molecular Characterization and Expression of two Ovarian Lipoprotein Receptors in the Rainbow Trout, Oncorhynchus mykiss 1. Biology of Reproduction, 58(5), 1146-1153. doi:10.1095/biolreprod58.5.1146Reading, B. J., Hiramatsu, N., Schilling, J., Molloy, K. T., Glassbrook, N., Mizuta, H., … Sullivan, C. V. (2014). Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes. Journal of Lipid Research, 55(11), 2287-2295. doi:10.1194/jlr.m050286Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9), 1312-1313. doi:10.1093/bioinformatics/btu033Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., & Yamamoto, T. (1992). Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity. Proceedings of the National Academy of Sciences, 89(19), 9252-9256. doi:10.1073/pnas.89.19.9252Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673-4680. doi:10.1093/nar/22.22.4673Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., … Herz, J. (1999). Reeler/Disabled-like Disruption of Neuronal Migration in Knockout Mice Lacking the VLDL Receptor and ApoE Receptor 2. Cell, 97(6), 689-701. doi:10.1016/s0092-8674(00)80782-5Tyler, C. R., Pottinger, T. G., Coward, K., Prat, F., Beresford, N., & Maddix, S. (1997). Salmonid Follicle-Stimulating Hormone (GtH I) Mediates Vitellogenic Development of Oocytes in the Rainbow Trout, Oncorhynchus mykiss 1. Biology of Reproduction, 57(5), 1238-1244. doi:10.1095/biolreprod57.5.1238Weltzien, F.-A., Pasqualini, C., Vernier, P., & Dufour, S. (2005). A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. General and Comparative Endocrinology, 142(1-2), 134-142. doi:10.1016/j.ygcen.2004.12.01

Recombinant vs purified mammal gonadotropins as maturation hormonal treatments of European eel males

[EN] In the past three decades the European eel Anguilla anguilla experienced up to 99% decline in recruitment in some parts of its distribution range, thus breeding in captivity is nowadays considered key in order to save this species. With this in mind, obtaining high quality gametes is fundamental, as is the ongoing study of new hormonal treatments in order to improve current methods. Therefore, the aim of this research study was i) to assess the effect of two hormonal treatments (OVI, a recombinant alpha-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine) on the reproductive performance of European eel males, and, after choosing the best hormone, ii) to compare the effects of three doses in order to cut the costs of artificial maturation. Our results indicated that the type of hormone used (recombinant vs purified gonadotropins) significantly affected the progression of spermiation in European eel males, and that the recombinant hormone (OVI) produced better results in terms of sperm quantity and quality in most of the weeks of the treatment, remaining thus an effective treatment to induce spermiation in this species. On the other hand, in terms of the doses experiment, our results showed that from the lowest to the highest dose (0.25 to 1.5 IU/g fish) all the treatments were able to induce the whole spermiation process. However, a weekly dose of 1.5 IU/g fish of recombinant hormone (OVI) was necessary in order to provide a notable amount (volume and density) of high quality (motility and velocity) samples throughout the treatment. Finally, the economic analysis demonstrated that the recombinant hormone (OVI, 1.5 IU/g fish) had a greater profitability than the other treatments, making it possible to obtain high-quality sperm for a lower price. In this context, and considering the fact that in the first few weeks of any hormonal treatment there is no high-quality sperm production, long-term hormonal therapies are necessary in order to lessen the cost of high-quality European eel sperm.This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 642893 (ETN IMPRESS). VG has a postdoc grant from the UPV (PAID-10-16).Herranz-Jusdado, JG.; Rozenfeld, C.; Morini, M.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Gallego Albiach, V. (2019). Recombinant vs purified mammal gonadotropins as maturation hormonal treatments of European eel males. Aquaculture. 501:527-536. https://doi.org/10.1016/j.aquaculture.2018.12.015S52753650

European eel sperm storage: optimization of short-term protocols and cryopreservation of large volumes

[EN] Maturation in captivity of European eel (Anguilla anguilla) requires long and costly hormonal treatments that often lead to asynchronic maturation between sexes. Therefore, optimization of sperm short-term storage methods and cryopreservation protocols can be a key factor for successful artificial fertilization. Two experiments were carried out to optimize the existing protocols. For the short-term storage experiment, sperm was diluted in P1 extender and then stored at different dilution ratios (1,9 and 1,49). The best outcome was then tested at different temperatures (4 and 20¿°C) and in constant agitation or still. In the cryopreservation experiments, large sperm volumes (cryotubes of 2 and 5¿ml), different cooling rates (freezing tubes 1 or 3¿cm above liquid nitrogen during 15 and 20¿min), and different extender compositions (methanol 10% was used as cryoprotectant, and complemented with FBS 20%, BSA 5% or egg yolk 5%) were tested. Sperm kinetic parameters were analyzed with a CASA-Mot system both in fresh and short- or long-term stored samples. In the short-term storage trial, sperm quality did not show significant differences in the first 24¿h after sperm collection between the different storage conditions tested. For longer time, 1:49 dilution ratio showed significantly better results than 1:9, and low temperature (4¿°C) was better for sperm preservation after 3¿days. Cryopreserved sperm samples showed good motility results when they were frozen in cryotubes of 2 and 5¿ml, with no significant differences compared to samples cryopreserved in lower volumes (straws of 0.5¿mL). Furthermore, the combination of methanol (10%) and egg yolk (5%) as freezing medium, induced significant higher post-thawing motility values (over 50%) than the control (methanol 10%), whereas the addition of FBS (20%) and BSA (5%) led to a significant reduction of the sperm motility. The establishment of these storage and cryopreservation protocols will be important for the improvement of European eel artificial reproduction programs.Funded by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement N° 642893 (IMPRESS), including the JGHJ and CR predoctoral contracts. VG has a postdoc grant from the UPV (PAID-10-16).Herranz-Jusdado, JG.; Gallego Albiach, V.; Rozenfeld, C.; Morini, M.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2019). European eel sperm storage: optimization of short-term protocols and cryopreservation of large volumes. Aquaculture. 506:42-50. https://doi.org/10.1016/j.aquaculture.2019.03.019S425050

Role of calcium on the initiation of sperm motility in the European eel

[EN] Sperm from European eel males treated with hCG(rec), was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCI + EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca2+](i) fluorescence (and in [Na+](i) in some cases) were evaluated by flow cytometry. After sperm motility was activated in a medium containing Ca2+ (seawater, SW) the intracellular fluorescence emitted by Ca2+ increased 4-6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCI + EDTA) resulted in a percentage of motility similar to seawater, the [Ca2+](i) levels did not increase at all. This result strongly suggests that increasing [Ca2+](i) is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca2+ has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca2+](i) concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm. (c) 2015 Elsevier Inc All rights reserved.Funded from the SPERMOT project (Spanish Ministry of Science and Innovation, MICINN; AGL2010-16009). M.C. Vilchez has a predoctoral grant from UPV PAID Program (2011-S2-02-6521), Marina Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia, GRISOLIA/2012/006), Victor Gallego has a postdoctoral contract from UPV (PAID-10-14), and David S. Penaranda was supported by MICINN (PTA2011-4948-I) and UPV (PTA2011-4948-I). Grants to attend meetings were received from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).Pérez Igualada, LM.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Morini, M.; Peñaranda, D.; Asturiano Nemesio, JF. (2016). Role of calcium on the initiation of sperm motility in the European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 191:98-106. https://doi.org/10.1016/j.cbpa.2015.10.009S9810619

There are differences in hematological and biochemical parameters between carriers and not carriers of experimental model of GRMD (Golden Retriever Muscular Dystrophy)?

A proposta deste estudo foi avaliar se existem alterações nos padrões hematológicos e bioquímicos de cadelas da raça Golden Retriever portadoras do gene da distrofia muscular progressiva em comparação aos valores obtidos em cadelas não portadoras de mesma raça e idade. Foram analisados 33 animais, distribuídos em dois grupos, um composto por 19 cadelas Golden Retrievers não portadoras (GRNP) e outro composto por 14 cadelas Golden Retrievers portadoras do gene da distrofia muscular (GRP). Os dois grupos foram submetidos aos mesmos testes hematológicos e bioquímicos, com a mesma frequência e durante o mesmo intervalo de tempo. Apesar de existir diferença estatisticamente significativa entre os grupos para alguns parâmetros hematológicos avaliados, todos os resultados obtidos estavam de acordo com os valores de referência utilizados. Na avaliação dos parâmetros bioquímicos séricos a dosagem de ALT no grupo GRNP ficou levemente acima da média, porém sem grandes significados clínicos A CK também apresentou níveis elevados no grupo GRP, devido à degeneração e necrose muscular característicos da doença, as alterações encontradas nessa análise já eram esperadas. Os demais parâmetros não se alteraram.The purpose of this study was to evaluate whether there are alterations in hematological and biochemical patterns of female Golden Retriever dogs carrying the gene for progressive muscular dystrophy compared to not carriers dogs of the same breed, age and gender. We analyzed 33 animals, divided into two groups; one consisting of 13 not carriers dogs Golden Retrievers (GRNP) and the other composed of 14 dogs Golden Retrievers carrying the gene for muscular dystrophy (GRP). Animals of both groups underwent biochemical and hematological tests with the same frequency and at the same time interval. Although there is a statistically significant difference between groups for some hematological parameters evaluated, all results were in line with the benchmarks used. In the assessment of serum biochemical parameters in the determination of ALT GRS group was slightly above average, but without major clinical significance CK also showed high levels in GRP group, due to muscle degeneration and necrosis characteristic of the disease, the changes found in this analysis were already expected. The other parameters did not change

Eel sperm cryopreservation: an overview

[EN] The eels are teleost fishes from the order Anguilliformes that includes several species with high commercial value. Due to the high interest for aquaculture production of some eel species and for the need to restore eel species that are endangered, several research groups have directed their research toward developing protocols to cryopreserve the spermatozoa of Japanese eel (Anguilla japonica) and European eel (Anguilla anguilla). In this review, we provide an overview on the different protocols that have been developed so far. The first developed protocols used DMSO as cryoprotectant in both species with good success, obtaining sperm motilities of over 45% in Japanese eel and over 35% in European eel. Moreover, sperm cryopreserved using DMSO was successfully used in fertilization trials, although with low fertilization rates. However, recent studies show that DMSO produce epigenetic changes in eel sperm and therefore, the last developed protocols used methanol as cryoprotectant instead. Cryopreservation protocols using methanol as cryoprotectant, showed improved motility values in both Japanese and European eel. In addition, the latest protocols have been adapted to cryopreserve larger volumes of sperm of up to 5¿mL, which is useful for larger scale fertilization trials. The present study introduces the state of the art and future perspectives of the eel sperm cryopreservation to be applied in aquaculture and biological conservation programs.Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement N 642893 (IMPRESS), including the JGHJ and CR pre-doctoral contracts. MM has a postdoc grant from the UPV (PAID -10-18). VG has a postdoc grant from the MICIU (Juan de la CiervaIncorporacion; IJCI-2017-34200). This research was supported by the Higher Education Institutional Excellence Program (1783-3/2018/FEKUTSRAT) awarded by the Ministry of Human Capacities of Hungary within the framework of water related researches of Szent Istvan University as well as the EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union and the European Social Fund.Herranz-Jusdado, JG.; Gallego Albiach, V.; Morini, M.; Rozenfeld, C.; Pérez Igualada, LM.; Müller, T.; Horváth, Á.... (2019). Eel sperm cryopreservation: an overview. Theriogenology. 133:210-215. https://doi.org/10.1016/j.theriogenology.2019.03.033S21021513

Nuclear and membrane progestin receptors in the European eel: characterization and expression in vivo through spermatogenesis

[EN] Characterization of all the progestin receptor genes (PRs) found in the European eel has been performed. There were five membrane PRs (mPRs): mPR alpha (alpha), mPRAL1 (alpha-likel), mPRAL2 (alpha-like2), mPRy (gamma), mPR delta (delta) and two nuclear PRs (nPRs or PGRs): pgr1 and pgr2. In silico studies showed that the C and E(F) domains of Pgr are well conserved among vertebrates whereas the A/B domain is not. Phylogeny and synteny analyses suggest that eel duplicated pgr (pgr1 and pgr2) originated from the teleost-specific third whole genome duplication (3R). mPR phylogeny placed three eel mPRs together with the mPRce Glade, being termed mPRet, mPRAL1 and mPRAL2, while the other two eel mPRs clustered with mPRy and mPRS clades, respectively. The in vivo study showed differential expression patterns along the brain-pituitary-gonad axis. An increase in nPR transcripts was observed in brain (in pgrl) and pituitary (in pgrl and pgr2) through the spermatogenesis, from the spermatogonia B/spermatocyte stage to the spermiation stage. In the testis, mPRy, mPRS and pgr2 transcripts showed the highest levels in testis with A spermatogonia as dominant germ cell, while the highest mPRce, mPRAL1 and mPRAL2 transcripts were observed in testis from spermiating males, where the dominant germ cell were spermatozoa. Further studies should elucidate the role of both nuclear and membrane progestin receptors on eel spermatogenesis.This work was supported by the Spanish Ministry of Science and Innovation (SPERMOT project; AGL2010-16009; REPRO-TEMP project, AGL2013-41646-R), and IMPRESS (Marie Sklodowska Curie Actions Innovative Training Network; Grant agreement no: 642893). M.C. Vilchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), M. Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia), D.S. Penaranda was supported by MICINN and UPV (PTA2011-4948-I). Grants to attend meetings were funded by COST Office (COST Action FA1205: AQUAGAMETE).Morini, M.; Peñaranda, D.; Vilchez Olivencia, MC.; Nourizadeh-Lillabadi, R.; Lafont, A.; Dufour, S.; Asturiano Nemesio, JF.... (2017). Nuclear and membrane progestin receptors in the European eel: characterization and expression in vivo through spermatogenesis. Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology. 207:79-92. https://doi.org/10.1016/j.cbpa.2017.02.009S799220

Transcript levels of the soluble sperm factor protein phospholipase C zeta 1 (PLCZ1) increase through induced spermatogenesis in European eel

[EN] Activation at fertilization of the vertebrate egg is triggered by Ca2+ waves. Recent studies suggest the phospholipase C zeta (PLC zeta), a sperm-specific protein, triggers egg activation by an 1P3-mediated Ca2+ release and allow Ca2+ waves at fertilization. In the present study we cloned, characterized, and phylogenetically positioned the European eel PLC zeta (PLC zeta 1). It is 1521bp long, with 10 exons encoding an open reading frame of 506 amino acids. The amino acid sequence contains an EF-hand domain, X and Y catalytic domains, and a carboxy-terminal C2 domain, all typical of other PLC zeta orthologous. The tissue distribution was studied, and the gene expression was determined in testis during induced sexual maturation at three different thermal regimes. Also, brain and pituitary expression was studied through sex maturation at constant temperature. plc zeta was expressed in brain of male and female, in testis but not in ovaries. By first time in vertebrates, it is reported plc zeta 1 expression in the pituitary gland. Testis plc zeta 1 expression increased through spermatogenesis under all the thermal regimes, but being significantly elevated at lower temperatures. It was very low when testis contained only spermatogonia or spermatocytes, while maximum expression was found during spermiogenesis. These results support the hypothesis for an eel sperm-specific PLC zeta inducing egg activation, similarly to mammals and some teleosts, but different from some other teleost species, which express this protein in ovaries, but not in testes. (C) 2015 Elsevier Inc. All rights reserved.Funded by the SPERMOT project (Spanish Ministry of Science and Innovation, MICINN; AGL2010-16009). M.C. Vílchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), Marina Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolía, GRISOLIA/2012/006), Victor Gallego has a postdoctoral grant (UPV; PAID-10-14), and David S. Peñaranda was a contract cofinanced by MICINN and UPV (PTA2011-4948-I). Grants to attend meetings from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).Morini, MAM.; Peñaranda, D.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Nourizadeh-Lillabadi, R.; Asturiano Nemesio, JF.; Weltzien, F.... (2015). Transcript levels of the soluble sperm factor protein phospholipase C zeta 1 (PLCZ1) increase through induced spermatogenesis in European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 187:168-176. https://doi.org/10.1016/j.cpba.2015.05.028S16817618