Intracellular pH regulation and sperm motility in the European eel

[EN] Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pH(i)) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4-7.6. The natural pH(i) range of sperm samples in the quiescent stage was 7.4-8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pH(e)), as well as between the pH(i) and the pH of the diluent. The change post-activation in seawater (ASW) depended on the initial pH(e) of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pH(e) 8, and no pH(i) variation when pH(e) was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pH(i) changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation. (C) 2020 Elsevier Inc. All rights reserved.Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS). Victor Gallego has a postdoc grant from the MICIU (Juan de la Cierva-Incorporacion; IJCI-2017-34200Pérez Igualada, LM.; Gallego Albiach, V.; Asturiano, JF. (2020). Intracellular pH regulation and sperm motility in the European eel. Theriogenology. 145:48-58. https://doi.org/10.1016/j.theriogenology.2020.01.026S4858145Nishigaki, T., José, O., González-Cota, A. L., Romero, F., Treviño, C. L., & Darszon, A. (2014). Intracellular pH in sperm physiology. Biochemical and Biophysical Research Communications, 450(3), 1149-1158. doi:10.1016/j.bbrc.2014.05.100Ohta, H., Ikeda, K., & Izawa, T. (1997). Increases in concentrations of potassium and bicarbonate ions promote acquisition of motility in vitro by Japanese eel spermatozoa. The Journal of Experimental Zoology, 277(2), 171-180. doi:10.1002/(sici)1097-010x(19970201)277:23.0.co;2-mOda, S., & Morisawa, M. (1993). Rises of intracellular Ca2+ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts. Cell Motility and the Cytoskeleton, 25(2), 171-178. doi:10.1002/cm.970250206TANAKA, S., UTOH, T., YAMADA, Y., HORIE, N., OKAMURA, A., AKAZAWA, A., … KUROKURA, H. (2004). Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel. Fisheries Science, 70(5), 780-787. doi:10.1111/j.1444-2906.2004.00871.xGallego, V., Martínez-Pastor, F., Mazzeo, I., Peñaranda, D. S., Herráez, M. P., Asturiano, J. F., & Pérez, L. (2014). Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results. Aquaculture, 418-419, 155-158. doi:10.1016/j.aquaculture.2013.10.022Vílchez, M. C., Morini, M., Peñaranda, D. S., Gallego, V., Asturiano, J. F., & Pérez, L. (2017). Role of potassium and pH on the initiation of sperm motility in the European eel. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 203, 210-219. doi:10.1016/j.cbpa.2016.09.024M�ri�n, T., Krasznai, Z., Balkay, L., Emri, M., & Tr�n, L. (1997). Role of extracellular and intracellular pH in carp sperm motility and modifications by hyperosmosis of regulation of the Na+/H+ exchanger. Cytometry, 27(4), 374-382. doi:10.1002/(sici)1097-0320(19970401)27:43.0.co;2-cPerez, 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.xGallego, V., Carneiro, P. C. F., Mazzeo, I., Vílchez, M. C., Peñaranda, D. S., Soler, C., … Asturiano, J. F. (2013). Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology, 79(7), 1034-1040. doi:10.1016/j.theriogenology.2013.01.019Mylonas, C. C., Duncan, N. J., & Asturiano, J. F. (2017). Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality. Aquaculture, 472, 21-44. doi:10.1016/j.aquaculture.2016.04.021Asturiano, J. F., Pérez, L., Garzón, D. L., Marco-Jiménez, F., Peñaranda, D. S., Vicente, J. S., & Jover, M. (2004). Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm. Fish Physiology and Biochemistry, 30(3-4), 283-293. doi:10.1007/s10695-005-1553-xThomas, J. A., Buchsbaum, R. N., Zimniak, A., & Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry, 18(11), 2210-2218. doi:10.1021/bi00578a012Krasznai, Z. (2003). Role of ion channels and membrane potential in the initiation of carp sperm motility. Aquatic Living Resources, 16(5), 445-449. doi:10.1016/s0990-7440(03)00054-8Miura, T., & Miura, C. I. (2003). Molecular control mechanisms of fish spermatogenesis. Fish Physiology and Biochemistry, 28(1-4), 181-186. doi:10.1023/b:fish.0000030522.71779.47Fechner, S., Alvarez, L., Bönigk, W., Müller, A., Berger, T. K., Pascal, R., … Kaupp, U. B. (2015). A K+-selective CNG channel orchestrates Ca2+ signalling in zebrafish sperm. eLife, 4. doi:10.7554/elife.07624Peñaranda, D., Pérez, L., Gallego, V., Barrera, R., Jover, M., & Asturiano, J. (2008). European Eel Sperm Diluent for Short-term Storage. Reproduction in Domestic Animals, 45(3), 407-415. doi:10.1111/j.1439-0531.2008.01206.xChauvaud, L., Cosson, J., Suquet, M., & Billard, R. (1995). Sperm motility in turbot, Scophthalmus marimus: initiation of movement and changes with time of swimming characteristics. Environmental Biology of Fishes, 43(4), 341-349. doi:10.1007/bf00001167Billard, R., Cosson, J., & Crim, L. W. (1993). Motility of fresh and aged halibut sperm. Aquatic Living Resources, 6(1), 67-75. doi:10.1051/alr:1993008Effer, B., Figueroa, E., Augsburger, A., & Valdebenito, I. (2013). Sperm biology of Merluccius australis: Sperm structure, semen characteristics and effects of pH, temperature and osmolality on sperm motility. Aquaculture, 408-409, 147-151. doi:10.1016/j.aquaculture.2013.05.040Inaba, K., Dréanno, C., & Cosson, J. (2003). Control of flatfish sperm motility by CO2and carbonic anhydrase. Cell Motility and the Cytoskeleton, 55(3), 174-187. doi:10.1002/cm.10119Lee, H. cheung, Johnson, C., & Epel, D. (1983). Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm. Developmental Biology, 95(1), 31-45. doi:10.1016/0012-1606(83)90004-0Babcock, D. F., Rufo, G. A., & Lardy, H. A. (1983). Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proceedings of the National Academy of Sciences, 80(5), 1327-1331. doi:10.1073/pnas.80.5.132

Determinación del desarrollo sexual de peces por medio de preparaciones histológicas

En este artículo vamos a aprender a diferenciar los estados de desarrollo sexual de un pez por medio de láminas con fotografías, siempre que tengamos preparaciones histológicas de su ovario o testículo. Se describen brevemente las diferentes etapas de desarrollo sexual femenino y masculino, así como la utilidad que puede tener este conocimiento.Gallego Albiach, V.; Pérez Igualada, LM. (2021). Determinación del desarrollo sexual de peces por medio de preparaciones histológicas. http://hdl.handle.net/10251/16937

Estimación de la densidad celular mediante el uso de cámaras de recuento

En este artículo vamos a explicar cómo estimar la densidad de células y/o partículas en una suspensión mediante el uso de diferentes tipos de cámaras de recuento. El conteo de partículas es una técnica ampliamente utilizada en una gran cantidad de ramas como la biotecnología, biomedicina, producción animal, producción vegetal, etc. por lo que la comprensión y el desarrollo de este proceso permitirá al alumno poder aplicarla en diferentes etapas y procedimientos.Gallego Albiach, V.; Pérez Igualada, LM. (2021). Estimación de la densidad celular mediante el uso de cámaras de recuento. http://hdl.handle.net/10251/169378DE

Ionic control of sperm motility and trials for the improvement of pufferfish (Takifugu alboplumbeus) sperm extenders

[EN] Seminal plasma characteristics, such as ionic composition and pH, are important for sperm maturation and further motility. In this work, a first batch of experiments evaluated the effect of the absence of several ions (Na+, K+, Mg2+) in the artificial seminal plasma, as well as the effect of artificial seminal plasma pH and seawater pH, on the sperm activation in the pufferfish (Takifugu alboplumbeus, named before Takifugu niphobles). In all the experiments, total motility, progressive motility, curvilinear velocity, straight line velocity, and average path velocity were measured. The absence of Na+, K+ or Mg2+ ions did not reduce the sperm motility in comparison with the control. In contrast, artificial seminal plasma pH had a strong effect on sperm motility. Samples diluted in artificial seminal plasma at pH 6.5 showed a high reduction of sperm motility in comparison with higher pHs (7.5, 8.5 and 9.5), and this inhibition was reversible. Where the effect of the pH of artificial seawater in sperm motility (used as activation medium) was concerned, there were no significant differences between the control (pH 8.2) and pH 6.5, indicating that pufferfish spermatozoa can swim in acidic environments. Finally, nigericin (that make equal the intracellular pH and extracellular pH) was added, and the highest sperm motility was found at pH 7.0, which suggested that the optimal intracellular pH of pufferfish for sperm motility is 7.0. Our second batch of experiments was developed to optimize the pufferfish sperm extenders to achieve longer preservation under refrigeration, assessing the addition of several concentrations of magnesium and NaHCO3 (used in extenders for other fish species), as well as different pHs. From the extender experiments it was observed that bicarbonate reduced the sperm motility after 7 days of incubation, and it can be concluded that the use of magnesium 2 mM as part of the extender composition, in combination with the pH measured in the pufferfish sperm (7.5), seems to be the best option for the short-medium term storage of the sperm of this species.Generalitat Valenciana funded the stay in Japan of JFA, LPI and VG (BEST 2018/093,/112 and/124, respectively). VG has a postdoctoral grant from the Spanish Ministry of Science, Innovation and Universities (MICIU; Programa Juan de la Cierva-Incorporacion; IJCI-2017-34200).Pérez Igualada, LM.; Asturiano, JF.; Yoshida, M.; Gallego Albiach, V. (2022). Ionic control of sperm motility and trials for the improvement of pufferfish (Takifugu alboplumbeus) sperm extenders. Aquaculture. 554:1-11. https://doi.org/10.1016/j.aquaculture.2022.73814611155

Sperm quality parameters of Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica): new conservation tools from a gamete perspective

[EN] The sensitive state of conservation of several endemic fish species such as Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica) has led governments to consider the implementation of conservation measures to preserve their populations. However, limited knowledge about the reproductive biology of these species makes it necessary to investigate different aspects of their reproductive cycle. In this sense, the main objectives of this work were i) to advance knowledge of the breeding biology of both species, and ii) to develop protocols for the conservation of gametes for the future management and conservation. During the spring of 2019 a temporal series of samplings were carried out in different places in the Comunitat Valenciana. Sperm samples were collected and sperm motion parameters were assessed for the first time in both species. Kinetic patterns were similar showing high motility and velocity values during the first 30 s, and a rapid decrease from that point. At the same time, an in-depth morphometric analysis was carried out using computer-assisted sperm analysis software. Spermatozoa from A. iberus and V. hispanica showed similar sizes and shapes to other external fertilizers belonging to Cyprinodontiformes, with small spherical heads, uniflagellated and without acrosomes. In addition, a new cryopreservation protocol was designed for cryobanking the sperm of these threatened species. Cryopreserved samples showed lower motility than fresh samples but reaching acceptable percentages of motile cells after thawing of around 20 and 25% (A. iberus and V. hispanica, respectively). This study is the first of its kind to successfully achieve gamete cryopreservation of these two endemic and endangered species from the Iberian Peninsula, providing new and useful tools to complement the management and conservation programs that are being developed for both species.This project has received funding from the Generalitat Valenciana (GV; Valencia, Spain) under the "Subvenciones para la realizacion de proyectos de I+D+I desarrollados por grupos de investigacion emergentes (GV/2019/130)". VG has a postdoc grant from the Ministerio de Economia, Industria y Competitividad (MINECO; Madrid, Spain) under the project IJCI-2017-34200. We would like to thank all the technicians of the CCEDCV for their work and effort during the samplings, specially to J. Velazquez, J. Hernandez and A. Pradillo.Blanes-García, M.; Risueño, P.; Pérez Igualada, LM.; Asturiano, JF.; Gallego Albiach, V. (2021). Sperm quality parameters of Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica): new conservation tools from a gamete perspective. Aquaculture. 530:1-9. https://doi.org/10.1016/j.aquaculture.2020.735819S1953

Subjective and objective assessment of fish sperm motility: when the technique and technicians matter

[EN] Fish sperm motility is nowadays considered the best sperm quality biomarker in fish, and can be evaluated both by subjective and computerized methods. With the aim to compare the precision and accuracy of both techniques, fish sperm samples were assessed by subjective methods and by a computerassisted sperm analysis (CASA-Mot) system, and simultaneously by three different technicians with different degrees of expertise on the sperm quality analysis. Statistical dispersion parameters (CV, coefficient of variation; and RG, range) were estimated in order to determine the precision and accuracy of the techniques and the influence of laboratory staff on sperm motion assessments. Concerning precision, there were not much significant differences between the technical support staff (high, medium, and low experimented technician), and statistical dispersion parameters were quite similar between them independent of the technique used and the sperm motility class analyzed. However, concerning accuracy, experimented technician reported subjective motility values very closed to the values provided by the CASA-Mot system, only 10 percentage points away from the data provided by a CASA-Mot system. However, medium and low experimented technicians often overestimate the CASA-Mot values, and amplitudes up to 30 percentage points were detected in several sperm assessments. To sum up, both the technique (subjective or objective) and the technician (degree of expertise) became key factors in order to reach accurate motility estimations, so the use of both qualified staff and novel CASA-Mot systems seems to be a critical requirement for obtaining satisfying results in fish species with similar motility patterns.This study is funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 642893 (IMPRESS) and the COST Office (COST Action FA1205: AQUAGAMETE). VG has a postdoc grant from the UPV (PAID-10-16).Gallego Albiach, V.; Herranz-Jusdado, JG.; Rozenfeld, C.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2018). Subjective and objective assessment of fish sperm motility: when the technique and technicians matter. Fish Physiology and Biochemistry. 1-11. https://doi.org/10.1007/s10695-018-0505-1S111Boryshpolets S, Kowalski RK, Dietrich GJ, Dzyuba B, Ciereszko A (2013) Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters. Theriogenology 80:758–765. https://doi.org/10.1016/j.theriogenology.2013.06.019Bozkurt Y, Secer S (2006) Relationship between spermatozoa motility, egg size, fecundity and fertilization success in brown trout (Salmo trutta fario). Pakistan J Biol Sci 9:2141–2144. https://doi.org/10.3923/pjbs.2006.2141.2144Cabrita E, Martínez-Páramo S, Gavaia PJ, Riesco MF, Valcarce DG, Sarasquete C, Herráez MP, Robles V (2014) Factors enhancing fish sperm quality and emerging tools for sperm analysis. Aquaculture 432:389–401. https://doi.org/10.1016/j.aquaculture.2014.04.034Castellini C, Dal Bosco A, Ruggeri S, Collodel G (2011) What is the best frame rate for evaluation of sperm motility in different species by computer-assisted sperm analysis? Fertil Steril 96(1):24–27Fauvel C, Suquet M, Cosson J (2010) Evaluation of fish sperm quality. J Appl Ichthyol 26:636–643. https://doi.org/10.1111/j.1439-0426.2010.01529.xGage MJG, Macfarlane CP, Yeates S, Ward RG, Searle JB, Parker GA (2004) Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success. Curr Biol 14:44–47. https://doi.org/10.1016/S0960-9822(03)00939-4Gallego V, Asturiano JF (2018) Sperm motility in fish: technical applications and perspectives through computer-aided sperm analysis (CASA) systems. Reprod Fertil Dev ( http://www.publish.csiro.au/RD/justaccepted/RD17460 )Gallego V, Carneiro PCF, Mazzeo I, Vílchez MC, Peñaranda DS, Soler C, Pérez L, Asturiano JF (2013a) Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology 79:1034–1040. https://doi.org/10.1016/j.theriogenology.2013.01.019Gallego V, Cavalcante SS, Fujimoto RY, Carneiro PCF, Azevedo HC, Maria AN (2017) Fish sperm subpopulations: changes after cryopreservation process and relationship with fertilization success in tambaqui (Colossoma macropomum). Theriogenology 87:16–24. https://doi.org/10.1016/j.theriogenology.2016.08.001Gallego V, Pérez L, Asturiano JF, Yoshida M (2013b) Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles). Aquaculture 416–417:238–243. https://doi.org/10.1016/j.aquaculture.2013.08.035Gasparini C, Simmons LW, Beveridge M, Evans JP (2010) Sperm swimming velocity predicts competitive fertilization success in the green swordtail Xiphophorus helleri. PLoS One 5:e12146. https://doi.org/10.1371/journal.pone.0012146Hala DN, VanLook K, Holt WV, Jobling S (2009) Validation of a method for measuring sperm quality and quantity in reproductive toxicity tests with pair-breeding male fathead minnows (Pimephales promelas). ILAR J 50:e1–e10Kime DE, VanLook KJW, McAllister BG et al (2001) Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish. Comp Biochem Physiol - C Toxicol Pharmacol 130:425–433. https://doi.org/10.1016/S1532-0456(01)00270-8Komatireddy R r, Madishetti R (2017) Coefficient of variation assessment for seminal traits evaluated by computer assisted semen analysis (CASA). Int J Sci Environ Technol 5:3452–3456Liu QH, Li J, Xiao ZZ, Ding FH, Yu DD, Xu XZ (2007) Use of computer-assisted sperm analysis (CASA) to evaluate the quality of cryopreserved sperm in red seabream (Pagrus major). Aquaculture 263:20–25. https://doi.org/10.1016/j.aquaculture.2006.11.017McAuliffe RE (2015) Coefficient of variation. In: Wiley encyclopedia of management. John Wiley & Sons, Ltd, Chichester, pp 1–1Peñaranda DS, Pérez L, Gallego V, Barrera R, Jover M, Asturiano JF (2010) European eel sperm diluent for short-term storage. Reprod Domest Anim 45:407–415. https://doi.org/10.1111/j.1439-0531.2008.01206.xPepper-Yowell AR (2011) The use of computer assisted semen analysis to predict fertility in Holstein bulls. Colorado State University. Doctoral ThesisPérez L, Asturiano JF, Tomás A et al (2000) Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. J Fish Biol 57:1488–1504. https://doi.org/10.1006/jfbi.2000.1411Reicks DL, Center SV, St Peter MN (2012) Capturing value of CASA-Mot systemsRosenthal H, Asturiano JF, Linhart O, Horváth Á (2010) On the biology of fish gametes: summary and recommendations of the Second International Workshop, Valencia, Spain, 2009. J Appl Ichthyol 26:621–622. https://doi.org/10.1111/j.1439-0426.2010.01550.xRudolfsen G, Figenschou L, Folstad I, Kleven O (2008) Sperm velocity influence paternity in the Atlantic cod (Gadus morhua L.). Aquac Res 39:212–216. https://doi.org/10.1111/j.1365-2109.2007.01863.xRurangwa E, Kime DE, Ollevier F, Nash JP (2004) The measurement of sperm motility and factors affecting sperm quality in cultured fish. Aquaculture 234:1–28. https://doi.org/10.1016/j.aquaculture.2003.12.006Verstegen J, Iguer-Ouada M, Onclin K (2002) Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 57:149–179. https://doi.org/10.1016/S0093-691X(01)00664-1Walker JS, Winet H, Freund M (1982) A comparison of subjective and objective sperm motility evaluation. J Androl 3:184–192. https://doi.org/10.1002/j.1939-4640.1982.tb00667.xWorld Health Organization (2010). WHO laboratory manual for the examination and processing of human semen (5th edition). Printed in Switzerland (286 pp.

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

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

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

Intracellular changes in Ca2 +, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results

P. 155-158Although it is widely accepted that osmolality and ion fluxes are the main factors triggering sperm motility in fish, a complex universal mechanism for sperm motility activation does not exist in fish, and studies of marine fish species are even more scarce. Therefore, the main goal of this study was to estimate the intracellular variations in the main ions involved in sperm activation for the first time in European eel, in order to provide additional new data about this little-known process. It was observed that levels of intracellular Ca2 + and K+ sperm ions increased significantly 30 s after the hyperosmotic shock compared to baseline levels, and remained at this level until 120 s post-activation. In contrast, the intracellular pH remained constant during the first 30 s, and decreased gradually at 60 and 120 s post-activation. Our data agree with the current main theory for explaining motility activation in marine fish, in which internal fluctuations of Ca2 + and K+ seem to participate in sperm activation. In addition, fluorescent images showed that both Ca2 + and K+ were concentrated in the apical area of the sperm head, which corresponds to the location of the eel sperm mitochondria, suggesting this organelle plays an important role in sperm motility activation.S