In silico and in situ characterization of the zebrafish (Danio rerio) gnrh3 (sGnRH) gene

BACKGROUND: Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio). RESULTS: We have characterized a zebrafish [Trp(7), Leu(8)] or salmon (s) GnRH variant, gnrh3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish. CONCLUSIONS: The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III

Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

Background: The Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. Methodology/Principal Findings: The zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO) knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf) as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genomewide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. Conclusions/Significance: The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerativ

Gnrh receptor gnrhr2bbα is expressed exclusively in lhb-expressing cells in Atlantic salmon male parr

Gonadotropin-releasing hormone (Gnrh) plays a major role in the regulation of physiological and behavioural processes related to reproduction. In the pituitary, it stimulates gonadotropin synthesis and release via activation of Gnrh receptors (Gnrhr), belonging to the G protein-coupled receptor superfamily. Evidence suggests that differential regulation of the two gonadotropins (Fsh and Lh) is achieved through activation of distinct intracellular pathways and, probably, through the action of distinct receptors. However, the roles of the different Gnrhr isoforms in teleosts are still not well understood. This study investigates the gene expression of Gnrhr in the pituitary gland of precociously maturing Atlantic salmon (Salmo salar) male parr. A total of six Gnrhr paralogs were identified in the Atlantic salmon genome and named according to phylogenetic relationship; gnrhr1caα, gnrhr1caβ, gnrhr1cbα, gnrhr1cbβ, gnrhr2bbα, gnrhr2bbβ. All paralogs, except gnrhr1caα, were expressed in male parr pituitary during gonadal maturation as evidenced by qPCR analysis. Only one gene, gnrhr2bbα, was differentially expressed depending on maturational stage (yearly cycle), with high expression levels in maturing fish, increasing in parallel with gonadotropin subunit gene expression. Additionally, a correlation in daily expression levels was detected between gnrhr2bbα and lhb (daily cycle) in immature fish in mid-April. Double fluorescence in situ hybridization showed that gnrhr2bbα was expressed exclusively in lhb gonadotropes in the pituitary, with no expression detected in fshb cells. These results suggest the involvement of receptor paralog gnrhr2bbα in the regulation of lhb cells, and not fshb cells, in sexually maturing Atlantic salmon male parr.publishedVersio

The regulation of aromatase and androgen receptor expression during gonad development in male and female European eel

[EN] This research investigated the regulation of aromatase and androgen receptor gene expression in the brain–pituitary– gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa-cyp19a1) and partial androgen receptor a and b (aa-ara and aa-arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa-arb was expressed at higher levels than aa-ara in the pituitary and gonad in both sexes, suggesting aa-arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa-ara and an increase in aa-cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa-arb and aa-cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa-arb and an increase in aa-cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa-cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex-dependent differences in the regulation of the expression of aa-ara, aa-arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturationThis work was funded by the European Community's 7th Framework Programme under the Theme 2 'Food, Agriculture and Fisheries, and Biotechnology', grant agreement no 245257 (PRO-EEL). D. S. P. received a postdoc grant from UPV (CEI-01-10), a mobility grant from UPV (PAID-00-11) and has also been supported by a contract cofinanced by MICINN and UPV (PTA2011-4948-I). V. G. and I. M. received predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN) and Generalitat Valenciana, respectively. F.-A. W. received funding from the Norwegian School of Veterinary Science. The fish farm Valenciana de Acuicultura, S. A. supplied the male eels used in the experiments. The English revision was carried out by Professor Lucy Robertson (Lucy Robertson Writing Services, Norway).Peñaranda, D.; Mazzeo, I.; Gallego Albiach, V.; Hildahl, J.; Nourizadeh-Lillabadi, R.; Pérez Igualada, LM.; Weltzien, FA.... (2014). The regulation of aromatase and androgen receptor expression during gonad development in male and female European eel. Reproduction in Domestic Animals. 49(3):512-521. https://doi.org/10.1111/rda.12321512521493Aroua, S., Weltzien, F.-A., Belle, N. L., & Dufour, S. (2007). Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids. General and Comparative Endocrinology, 153(1-3), 333-343. doi:10.1016/j.ygcen.2007.02.027Blázquez, M., & Piferrer, F. (2004). Cloning, sequence analysis, tissue distribution, and sex-specific expression of the neural form of P450 aromatase in juvenile sea bass (Dicentrarchus labrax). Molecular and Cellular Endocrinology, 219(1-2), 83-94. doi:10.1016/j.mce.2004.01.006Blázquez, M., & Piferrer, F. (2005). Sea bass (Dicentrarchus labrax) androgen receptor: cDNA cloning, tissue-specific expression, and mRNA levels during early development and sex differentiation. Molecular and Cellular Endocrinology, 237(1-2), 37-48. doi:10.1016/j.mce.2005.04.001Borg, B. (1994). Androgens in teleost fishes. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 109(3), 219-245. doi:10.1016/0742-8413(94)00063-gChang, X. T., Kobayashi, T., Kajiura, H., Nakamura, M., & Nagahama, Y. (1997). Isolation and characterization of the cDNA encoding the tilapia (Oreochromis niloticus) cytochrome P450 aromatase (P450arom): changes in P450arom mRNA, protein and enzyme activity in ovarian follicles during oogenesis. Journal of Molecular Endocrinology, 18(1), 57-66. doi:10.1677/jme.0.0180057Choi, J. Y., Park, J. G., Jeong, H. B., Lee, Y. D., Takemura, A., & Kim, S. J. (2005). Molecular cloning of cytochrome P450 aromatases in the protogynous wrasse, Halichoeres tenuispinis. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 141(1), 49-59. doi:10.1016/j.cbpc.2005.01.009Diotel, N., Page, Y. L., Mouriec, K., Tong, S.-K., Pellegrini, E., Vaillant, C., … Kah, O. (2010). Aromatase in the brain of teleost fish: Expression, regulation and putative functions. Frontiers in Neuroendocrinology, 31(2), 172-192. doi:10.1016/j.yfrne.2010.01.003Dufour, 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)90090-1Dufour, S., Sebert, M.-E., Weltzien, F.-A., Rousseau, K., & Pasqualini, C. (2010). Neuroendocrine control by dopamine of teleost reproduction. Journal of Fish Biology, 76(1), 129-160. doi:10.1111/j.1095-8649.2009.02499.xGelinas, D., A. Pitoc, G., & V. Callard, G. (1998). Isolation of a goldfish brain cytochrome P450 aromatase cDNA: Molecular and Cellular Endocrinology, 138(1-2), 81-93. doi:10.1016/s0303-7207(98)00015-xHarada, N. (1988). Cloning of a complete cDNA encoding human aromatase : Immunochemical identification and sequence analysis. Biochemical and Biophysical Research Communications, 156(2), 725-732. doi:10.1016/s0006-291x(88)80903-3Harbott, L. K., Burmeister, S. S., White, R. B., Vagell, M., & Fernald, R. D. (2007). Androgen receptors in a cichlid fish,Astatotilapia burtoni: Structure, localization, and expression levels. The Journal of Comparative Neurology, 504(1), 57-73. doi:10.1002/cne.21435Henkel, 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.0032231Hildahl, J., Sandvik, G. K., Edvardsen, R. B., Fagernes, C., Norberg, B., Haug, T. M., & Weltzien, F.-A. (2011). Identification and gene expression analysis of three GnRH genes in female Atlantic cod during puberty provides insight into GnRH variant gene loss in fish. General and Comparative Endocrinology, 172(3), 458-467. doi:10.1016/j.ygcen.2011.04.010Ijiri, S., Kazeto, Y., Mark Lokman, P., Adachi, S., & Yamauchi, K. (2003). Characterization of a cDNA Encoding P-450 aromatase (CYP19) from Japanese eel ovary and its expression in ovarian follicles during induced ovarian development. General and Comparative Endocrinology, 130(2), 193-203. doi:10.1016/s0016-6480(02)00589-0Ikeuchi, T., Todo, T., Kobayashi, T., & Nagahama, Y. (1999). cDNA Cloning of a Novel Androgen Receptor Subtype. Journal of Biological Chemistry, 274(36), 25205-25209. doi:10.1074/jbc.274.36.25205Ikeuchi, T., Todo, T., Kobayashi, T., & Nagahama, Y. (2001). Two subtypes of androgen and progestogen receptors in fish testes. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 129(2-3), 449-455. doi:10.1016/s1096-4959(01)00375-xJeng, S.-R., Dufour, S., & Chang, C.-F. (2005). Differential expression of neural and gonadal aromatase enzymatic activities in relation to gonadal development in Japanese eel,Anguilla japonica. Journal of Experimental Zoology Part A: Comparative Experimental Biology, 303A(9), 802-812. doi:10.1002/jez.a.194Kazeto, Y., Tosaka, R., Matsubara, H., Ijiri, S., & Adachi, S. (2011). Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel. The Journal of Steroid Biochemistry and Molecular Biology, 127(3-5), 149-154. doi:10.1016/j.jsbmb.2011.03.013Khan, I. A., Lopez, E., & Leloup-Hâtey, J. (1987). Induction of spermatogenesis and spermiation by a single injection of human chorionic gonadotropin in intact and hypophysectomized immature European eel (Anguilla anguilla L.). General and Comparative Endocrinology, 68(1), 91-103. doi:10.1016/0016-6480(87)90064-5Kishida, M., & Callard, G. V. (2001). Distinct Cytochrome P450 Aromatase Isoforms in Zebrafish (Danio rerio) Brain and Ovary Are Differentially Programmed and Estrogen Regulated during Early Development**This research was supported by grants from the National Science Foundation (IBN-96-05053) and the NIH (P42 ES-07381). The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL Data Bank with accession numbers AF226619 and AF226620. Endocrinology, 142(2), 740-750. doi:10.1210/endo.142.2.7928Kwon, J. Y., McAndrew, B. J., & Penman, D. J. (2001). Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapiaOreochromis niloticus. Molecular Reproduction and Development, 59(4), 359-370. doi:10.1002/mrd.1042Levavi-Sivan, B., Biran, J., & Fireman, E. (2006). Sex Steroids Are Involved in the Regulation of Gonadotropin-Releasing Hormone and Dopamine D2 Receptors in Female Tilapia Pituitary1. Biology of Reproduction, 75(4), 642-650. doi:10.1095/biolreprod.106.051540Lin, C.-J., Wu, G.-C., Lee, M.-F., Lau, E.-L., Dufour, S., & Chang, C.-F. (2010). Regulation of two forms of gonadotropin-releasing hormone receptor gene expression in the protandrous black porgy fish, Acanthopagrus schlegeli. Molecular and Cellular Endocrinology, 323(2), 137-146. doi:10.1016/j.mce.2010.04.003Liu, X., Su, H., Zhu, P., Zhang, Y., Huang, J., & Lin, H. (2009). Molecular cloning, characterization and expression pattern of androgen receptor in Spinibarbus denticulatus. General and Comparative Endocrinology, 160(1), 93-101. doi:10.1016/j.ygcen.2008.10.026Lokman, P. M., George, K. A. N., & Young, G. (2003). Effects of steroid and peptide hormones on in vitro growth of previtellogenic oocytes from eel, Anguilla australis. Fish Physiology and Biochemistry, 28(1-4), 283-285. doi:10.1023/b:fish.0000030556.34592.41Lokman, P. M., George, K. A. N., Divers, S. L., Algie, M., & Young, G. (2007). 11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro. Reproduction, 133(5), 955-967. doi:10.1530/rep-06-0229MATSUBARA, H., KAZETO, Y., IJIRI, S., HIRAI, T., ADACHI, S., & YAMAUCHI, K. (2003). Changes in mRNA levels of ovarian steroidogenic enzymes during artificial maturation of Japanese eel Anguilla japonica. Fisheries Science, 69(5), 979-988. doi:10.1046/j.1444-2906.2003.00716.xMatsubara, M., Lokman, P. M., Senaha, A., Kazeto, Y., Ijiri, S., Kambegawa, A., … Yamauchi, K. (2003). Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.). Fish Physiology and Biochemistry, 28(1-4), 353-354. doi:10.1023/b:fish.0000030585.22093.7aMazzeo, I., Peñaranda, D. S., Gallego, V., Hildahl, J., Nourizadeh-Lillabadi, R., Asturiano, J. F., … Weltzien, F.-A. (2012). Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation. General and Comparative Endocrinology, 178(2), 338-346. doi:10.1016/j.ygcen.2012.06.003Miura, T., Yamauchi, K., Takahashi, H., & Nagahama, Y. (1991). Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proceedings of the National Academy of Sciences, 88(13), 5774-5778. doi:10.1073/pnas.88.13.5774Miura, T., Miura, C., Ohta, T., Nader, M. R., Todo, T., & Yamauchi, K. (1999). Estradiol-17β Stimulates the Renewal of Spermatogonial Stem Cells in Males. Biochemical and Biophysical Research Communications, 264(1), 230-234. doi:10.1006/bbrc.1999.1494Montero, M., Le Belle, N., King, J. A., Millar, R. P., & Dufour, S. (1995). Differential Regulation of the Two Forms of Gonadotropin-Releasing Hormone (mGnRH and cGnRH-II) by Sex Steroids in the European Female Silver Eel (Anguilla anguilla). Neuroendocrinology, 61(5), 525-535. doi:10.1159/000126876Montero, M., Le Belle, N., Vidal, B., & Dufour, S. (1996). Primary Cultures of Dispersed Pituitary Cells from Estradiol-Pretreated Female Silver Eels (Anguilla anguillaL.): Immunocytochemical Characterization of Gonadotropic Cells and Stimulation of Gonadotropin Release. General and Comparative Endocrinology, 104(1), 103-115. doi:10.1006/gcen.1996.0146Rout, U. K., Saed, G. M., & Diamond, M. P. (2005). Reproductive Biology and Endocrinology, 3(1), 1. doi:10.1186/1477-7827-3-1Pasqualini, C., Weltzien, F.-A., Vidal, B., Baloche, S., Rouget, C., Gilles, N., … Dufour, S. (2009). Two Distinct Dopamine D2 Receptor Genes in the European Eel: Molecular Characterization, Tissue-Specific Transcription, and Regulation by Sex Steroids. Endocrinology, 150(3), 1377-1392. doi:10.1210/en.2008-0578Peñaranda, D. S., Pérez, L., Gallego, V., Jover, M., Tveiten, H., Baloche, S., … Asturiano, J. F. (2010). Molecular and physiological study of the artificial maturation process in European eel males: From brain to testis. General and Comparative Endocrinology, 166(1), 160-171. doi:10.1016/j.ygcen.2009.08.006Peñaranda, D. S., Mazzeo, I., Hildahl, J., Gallego, V., Nourizadeh-Lillabadi, R., Pérez, L., … Weltzien, F.-A. (2013). Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: Tissue-distribution and changes in transcript abundance during artificially induced sexual development. Molecular and Cellular Endocrinology, 369(1-2), 1-14. doi:10.1016/j.mce.2013.01.025Perez, 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., Peñaranda, D. S., Dufour, S., Baloche, S., Palstra, A. P., Van Den Thillart, G. E. E. J. M., & Asturiano, J. F. (2011). Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females. General and Comparative Endocrinology, 174(1), 51-59. doi:10.1016/j.ygcen.2011.08.009Pfaffl, M. W., Tichopad, A., Prgomet, C., & Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnology Letters, 26(6), 509-515. doi:10.1023/b:bile.0000019559.84305.47Sperry, T. S., & Thomas, P. (1999). Characterization of Two Nuclear Androgen Receptors in Atlantic Croaker: Comparison of Their Biochemical Properties and Binding Specificities*. Endocrinology, 140(4), 1602-1611. doi:10.1210/endo.140.4.6631Sperry, T. S., & Thomas, P. (1999). Identification of Two Nuclear Androgen Receptors in Kelp Bass (Paralabrax clathratus) and Their Binding Affinities for Xenobiotics: Comparison with Atlantic Croaker (Micropogonias undulatus) Androgen Receptors1. Biology of Reproduction, 61(4), 1152-1161. doi:10.1095/biolreprod61.4.1152Todo, T., Ikeuchi, T., Kobayashi, T., & Nagahama, Y. (1999). Fish Androgen Receptor: cDNA Cloning, Steroid Activation of Transcription in Transfected Mammalian Cells, and Tissue mRNA Levels. Biochemical and Biophysical Research Communications, 254(2), 378-383. doi:10.1006/bbrc.1998.9919Tosaka, R., Todo, T., Kazeto, Y., Mark Lokman, P., Ijiri, S., Adachi, S., & Yamauchi, K. (2010). Expression of androgen receptor mRNA in the ovary of Japanese eel, Anguilla japonica, during artificially induced ovarian development. General and Comparative Endocrinology, 168(3), 424-430. doi:10.1016/j.ygcen.2010.05.005Trudeau, V. L., Peter, R. E., & Sloley, B. D. (1991). Testosterone and Estradiol Potentiate the Serum Gonadotropin Response to Gonadotropin-Releasing Hormone in Goldfish1. Biology of Reproduction, 44(6), 951-960. doi:10.1095/biolreprod44.6.951Tzchori, I., Degani, G., Hurvitz, A., & Moav, B. (2004). Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel (Anguilla anguilla). General and Comparative Endocrinology, 138(3), 271-280. doi:10.1016/j.ygcen.2004.06.007Vidal, B., Pasqualini, C., Le Belle, N., Holland, M. C. H., Sbaihi, M., Vernier, P., … Dufour, S. (2004). Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty1. Biology of Reproduction, 71(5), 1491-1500. doi:10.1095/biolreprod.104.030627Weltzien, F.-A., Pasqualini, C., Vernier, P., & Dufour, S. (2005). 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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

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

Cortisol differentially affects cell viability and reproduction-related gene expression in Atlantic cod pituitary cultures dependent on stage of sexual maturation

Through the action of cortisol, stress can affect reproductive biology with behavioural and physiological alterations. Using mixed sex primary pituitary cultures from Atlantic cod (Gadus morhua), the present study aimed to investigate potential direct effects of basal and stress level cortisol on the pituitary in terms of cell viability and reproduction-related gene expression at different stages of sexual maturity. Stress level of cortisol stimulated cell viability in cells derived from sexually maturing and mature fish. In cells from spent fish, high cortisol levels did not affect cell viability in terms of metabolic activity, but did stimulate viability in terms of membrane integrity. Basal cortisol levels did not affect cell viability. Ethanol, used as solvent for cortisol, decreased cell viability at all maturity stages, but did generally not affect gene expression. Genes investigated were fshb, lhb and two Gnrh receptors expressed in cod gonadotropes (gnrhr1b and gnrhr2a). Cortisol had dual effects on fshb expression; stimulating expression in cells from mature fish at stress dose, while inhibiting expression in cells from spent fish at both doses. In contrast, cortisol had no direct effect on lhb expression. While gnrhr2a transcript levels largely increased following cortisol treatment, gnrhr1b expression decreased in cells from spent fish and was unaffected at other maturity stages. These findings demonstrate that cortisol can act directly and differentially at the pituitary level in Atlantic cod and that factors facilitating these actions are dose-dependently activated and vary with level of sexual maturity.publishedVersio

Effects of microgravity simulation on zebrafish transcriptomes and bone physiology; exposure starting at 5 days post-fertilization.

Physiological modifications in near weightlessness, as experienced by astronauts during space flight, have been the subject of numerous studies. Various animal models have been used on space missions or in microgravity simulation on ground to understand the effects of gravity on living animals. Here, we used the zebrafish larvae as a model to study the effect of microgravity simulation on bone formation and whole genome gene expression. To simulate microgravity (sim-mu g), we used two-dimensional (2D) clinorotation starting at 5 days post fertilization to assess skeletal formation after 5 days of treatment. To assess early, regulatory effects on gene expression, a single day clinorotation was performed. Clinorotation for 5 days caused a significant decrease of bone formation, as shown by staining for cartilage and bone structures. This effect was not due to stress, as assessed by measuring cortisol levels in treated larvae. Gene expression results indicate that 1-day simulated microgravity affected musculoskeletal, cardiovascular, and nuclear receptor systems. With free-swimming model organisms such as zebrafish larvae, the 2D clinorotation setup appears to be a very appropriate approach to sim-mu g. We provide evidence for alterations in bone formation and other important biological functions; in addition several affected genes and pathways involved in bone, muscle or cardiovascular development are identified

In Vitro Effects of Bisphenol A and Tetrabromobisphenol A on Cell Viability and Reproduction-Related Gene Expression in Pituitaries from Sexually Maturing Atlantic Cod (Gadus morhua L.)

Bisphenol A (BPA) and tetrabromobisphenol A (TBBPA) are widely used industrial chemicals, ubiquitously present in the environment. While BPA is a well-known endocrine disruptor and able to affect all levels of the teleost reproductive axis, information regarding TBBPA on this subject is very limited. Using primary cultures from Atlantic cod (Gadus morhua), the present study was aimed at investigating potential direct effects of acute (72 h) BPA and TBBPA exposure on cell viability and the expression of reproductive-relevant genes in the pituitary. The results revealed that both bisphenols stimulate cell viability in terms of metabolic activity and membrane integrity at environmentally relevant concentrations. BPA had no direct effects on gonadotropin gene expression, but enhanced the expression of gonadotropin-releasing hormone (GnRH) receptor 2a, the main gonadotropin modulator in Atlantic cod. In contrast, TBBPA increased gonadotropin transcript levels but had no effect on GnRH receptor mRNA. In conclusion, both anthropogenic compounds display endocrine disruptive properties and are able to directly interfere with gene expression related to reproductive function in cod pituitary cells at environmentally relevant concentrations in vitro