Fish Physiology and Biochemistry

, Volume 44, Issue 3, pp 895–910 | Cite as

Cloning, characterization, and molecular expression of gonadotropin receptors in European hake (Merluccius merluccius), a multiple-spawning species

  • Michela Candelma
  • Luisa Dalla Valle
  • Sabrina Colella
  • Alberto Santojanni
  • Oliana Carnevali


Teleosts have many spawning strategies and the hormonal control of gametogenesis is not well defined among the species or even, between sexes. To increase the knowledge of gonadotropin hormones, we studied the trend by gene expression of gonadotropin receptors in the follicles and testis at different maturity stages in the European hake (Merluccius merluccius), a multiple-spawning species. With this aim, fshr and lhr were sequenced, characterized, and their gene expression was quantified in oocytes and in testes at different maturity stages. The deduced amino acid sequences were used to phylogenetic studies and evidenced that both receptors are phylogenetically closed to other gadoid species. The gene expression of both receptors was poorly expressed in primary follicles, increased in vitellogenic follicles and to later decrease in hydrated oocytes. In testis, highest levels of lhr were detected during spermiation, while levels of fshr were constant. For the first time, a histological analysis was performed in European hake testes showing an unrestricted lobular testis. To better elucidate the mechanisms involved in the oogenesis of the European hake, the expression of estrogen receptor and cyp19a was also investigated displaying high levels in all classes of follicles. All these data allow to increase the knowledge on reproductive physiology of an important socioeconomical species and it seeks to shed more light on the role of the receptors here studied during gametogenesis of multiple-spawning fish.


Merluccius merluccius Follicle-stimulating hormone receptor Luteinizing hormone receptor Multiple-spawning fish Alpha estrogen receptor cyt P450arom 



The authors wish to thank the Dr. Filippo Domenichetti and Dr. Camilla Croci of Marine Science Institute—CNR and the Captain Giordano and crew of “Orizzonte” vessel for their support in sampling.

Funding information

This work was partially supported by the Italian Ministry of Agricultural, Food, and Forestry Policies (Mipaaf) and European Union (Italian National Programs 2011–2013 and 2014–2016, in the ambit of Data Collection Framework).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.


  1. Asturiano JF, Pérez L, Tomás A, Zegrari S, Espinós FJ, Jover M (2002a) Inducción hormonal de la maduración gonadal y la puesta en hembras de anguila europea Anguilla anguilla L., 1758: cambios morfológicos y desarrollo oocitario. Boletín del Inst Español Oceanogr 18:127–137Google Scholar
  2. Asturiano JF, Sorbera LA, Ramos J, Kime DE, Carrillo M, Zanuy S (2002b) Group-synchronous ovarian development, spawning and spermiation in the European sea bass (Dicentrarchus labrax L.) could be regulated by shifts in gonadal steroidogenesis. Sci Mar 66:273–282. CrossRefGoogle Scholar
  3. Bobe J, Maugars G, Nguyen T, Rime H, Jalabert B (2003) Rainbow trout follicular maturational competence acquisition is associated with an increased expression of follicle stimulating hormone receptor and insulin-like growth factor 2 messenger RNAs. Mol Reprod Dev 66:46–53. CrossRefPubMedGoogle Scholar
  4. Brown-Peterson NJ, Wyanski DM, Saborido-Rey F, Macewicz BJ, Lowerre-Barbieri SK (2011) A standardized terminology for describing reproductive development in fishes. Mar Coast Fish 3:52–70. CrossRefGoogle Scholar
  5. Candelma M, Fontaine R, Colella S, Santojanni A, Weltzien FA, Carnevali O (2017) Gonadotropin characterization, localization and expression in European hake (Merluccius merluccius). Reproduction 153:1–10.
  6. Cao H, Zhou L, Zhang YZ, Wei QW, Chen XH, Gui JF (2009) Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals. Mol Cell Endocrinol 303:34–42. CrossRefPubMedGoogle Scholar
  7. Gen K, Okuzawa K, Kumakura N, Yamaguchi S, Kagawa H (2001) Correlation between messenger RNA expression of cytochrome P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major). Biol Reprod 65:1186–1194CrossRefPubMedGoogle Scholar
  8. Hickey GJ, Chen S, Besman MJ, Shively JE, Hall PF, Gaddy-Kurten D (1988) Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis. Endocrinology 122:1426–1436. CrossRefPubMedGoogle Scholar
  9. Kagawa H, Gen K, Okuzawa K, Tanaka H (2003) Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream, Pagrus major. Biol Reprod 68:1562–1568. CrossRefPubMedGoogle Scholar
  10. Kajimura S, Yoshiura Y, Suzuki M, Aida K (2001) cDNA cloning of two gonadotropin beta subunits (GTH-Ibeta and -IIbeta) and their expression profiles during gametogenesis in the Japanese flounder (Paralichthys olivaceus). Gen Comp Endocrinol 122:117–129. CrossRefPubMedGoogle Scholar
  11. Kobayashi T, Andersen Ø (2008) The gonadotropin receptors FSH-R and LH-R of Atlantic halibut (Hippoglossus hippoglossus). 1: isolation of multiple transcripts encoding full-length and truncated variants of FSH-R. Gen Comp Endocrinol 156:584–594. CrossRefPubMedGoogle Scholar
  12. Kobayashi T, Pakarinen P, Torgersen J, Huhtaniemi I, Andersen Ø (2008) The gonadotropin receptors FSH-R and LH-R of Atlantic halibut (Hippoglossus hippoglossus)—2. Differential follicle expression and asynchronous oogenesis. Gen Comp Endocrinol 156:595–602. CrossRefPubMedGoogle Scholar
  13. Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11:725–732. CrossRefPubMedGoogle Scholar
  14. Kumar RS, Ijiri S, Trant JM (2000) Changes in the expression of genes encoding steroidogenic enzymes in the channel catfish (Ictalurus punctatus) ovary throughout a reproductive cycle. Biol Reprod 63:1676–1682CrossRefGoogle Scholar
  15. Kumar RS, Ijiri S, Trant JM (2001) Molecular biology of the channel catfish gonadotropin receptors: 2. Complementary DNA cloning, functional expression, and seasonal gene expression of the follicle-stimulating hormone receptor 1. Biol Reprod 65:710–717CrossRefPubMedGoogle Scholar
  16. Kwok H, So W, Wang Y, Ge W (2005) Zebrafish gonadotropins and their receptors: I. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone receptors—evidence for their distinct functions in follicle development. Biol Reprod 72:1370–1381. CrossRefPubMedGoogle Scholar
  17. Levavi-Sivan B, Bogerd J, Mañanos EL, Gomez A, Lareyre JJ (2010) Perspectives on fish gonadotropins and their receptors. Gen Comp Endocrinol 165:412–437. CrossRefPubMedGoogle Scholar
  18. Luckenbach JA, Dickey JT, Swanson P (2011) Follicle-stimulating hormone regulation of ovarian transcripts for steroidogenesis-related proteins and cell survival, growth and differentiation factors in vitro during early secondary oocyte growth in coho salmon. Gen Comp Endocrinol 171:52–63. CrossRefPubMedGoogle Scholar
  19. Mateos J, Mañanos E, Martinez-Rodriguez G, Carrillo M, Querat B, Zanuy S (2003) Molecular characterization of sea bass gonadotropin subunits (α, FSHβ, and LHβ) and their expression during the reproductive cycle. Gen Comp Endocrinol 133:216–232. CrossRefPubMedGoogle Scholar
  20. Mittelholzer C, Andersson E, Consten D, Hirai T, Nagahama Y, Norberg B (2007) 20 beta-hydroxysteroid dehydrogenase and CYP19A1 are differentially expressed during maturation in Atlantic cod (Gadus morhua). J Mol Endocrinol 39:319–328. CrossRefPubMedGoogle Scholar
  21. Mittelholzer C, Andersson E, Taranger GL, Consten D, Hirai T, Senthilkumaran B, Nagahama Y, Norberg B (2009) Molecular characterization and quantification of the gonadotropin receptors FSH-R and LH-R from Atlantic cod (Gadus morhua). Gen Comp Endocrinol 160:47–58. CrossRefPubMedGoogle Scholar
  22. Montserrat N, González A, Méndez E, Piferrer F, Planas JV (2004) Effects of follicle stimulating hormone on estradiol-17β production and P-450 aromatase (CYP19) activity and mRNA expression in brown trout vitellogenic ovarian follicles in vitro. Gen Comp Endocrinol 137:123–131. CrossRefPubMedGoogle Scholar
  23. Morini M, Peñaranda DS, Vílchez MC, Tveiten H, Lafont AG, Dufour S, Pérez L, Asturiano JF (2017) The expression of nuclear andmembrane estrogen receptors in the European eel throughout spermatogenesis. Comp Biochem Physiol, Part A 203:91–99. CrossRefGoogle Scholar
  24. Murua H, Saborido-Rey F (2003) Female reproductive strategies of marine fish species of the North Atlantic. J Northwest Atl Fish Sci 33:23–31. CrossRefGoogle Scholar
  25. Murua H, Motos L, Lucio P (1998) Reproductive modality and batch fecundity of the European hake (Merluccius merluccius L.) in the Bay of Biscay. Calif Coop Ocean Fish Investig Reports 39:196–203Google Scholar
  26. Nagahama Y (1994) Endocrine regulation of gametogenesis in fish. Int J Dev Biol 38:217–229 . doi: doi=7981031Google Scholar
  27. Nagasawa K, Presslauer C, Kirtiklis L, Babiak I, Fernandes JMO (2014) Sexually dimorphic transcription of estrogen receptors in cod gonads throughout a reproductive cycle. J Mol Endocrinol 52:357–371. CrossRefPubMedGoogle Scholar
  28. Nyuji M, Kitano H, Shimizu A, Lee JM, Kusakabe T, Yamaguchi A, Matsuyama M (2013) Characterization, localization, and stage-dependent gene expression of gonadotropin receptors in chub mackerel (Scomber japonicus) ovarian follicles. Biol Reprod 88:148. CrossRefPubMedGoogle Scholar
  29. Oba Y, Hirai T, Yoshiura Y, Yoshikuni M, Kawauchi H, Nagahama Y (2001) Fish gonadotropin and thyrotropin receptors: the evolution of glycoprotein hormone receptors in vertebrates. Comp Biochem Physiol - B Biochem Mol Biol 129:441–448. CrossRefPubMedGoogle Scholar
  30. Peñaranda DS, Pérez L, Gallego V, Jover M, Tveiten H, Baloche S, Dufour S, Asturiano JF (2010) Molecular and physiological study of the artificial maturation process in European eel males: from brain to testis. Gen Comp Endocrinol 166:160–171. CrossRefPubMedGoogle Scholar
  31. Pérez L, Peñaranda DS, Dufour S, Baloche S, Palstra AP, Van Den Thillart GEEJM, Asturiano JF (2011) Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females. Gen Comp Endocrinol 174:51–59. CrossRefPubMedGoogle Scholar
  32. Petersen TN, Brunak S, Von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. CrossRefPubMedGoogle Scholar
  33. Planas JV, Swanson P (1995) Maturation-associated changes in the response of the salmon testis to the steroidogenic actions of gonadotropins (GTH I and GTH II) in vitro. Biol Reprod 52:697–704. CrossRefPubMedGoogle Scholar
  34. Raingeard D, Bilbao E, Sáez-Morquecho C, de Cerio OD, Orbea A, Cancio I (2009) Cloning and transcription of nuclear receptors and other toxicologically relevant genes, and exposure biomarkers in European hake (Merluccius merluccius) after the Prestige oil spill. Mar Genomics 2:201–213. CrossRefPubMedGoogle Scholar
  35. Rasheeda MK, Sridevi P, Senthilkumaran B (2010) Cytochrome P450 aromatases: impact on gonadal development, recrudescence and effect of hCG in the catfish, Clarias gariepinus. Gen Comp Endocrinol 167:234–245. CrossRefPubMedGoogle Scholar
  36. Richards JS, Fitzpatrick SL, Clemens JW, Morris JK, Alliston T, Sirois J (1995) Ovarian cell differentiation: a cascade of multiple hormones, cellular signals, and regulated genes. Recent Prog Horm Res 50:223–254PubMedGoogle Scholar
  37. Rocha A, Gomez A, Zanuy S, Cerda-Reverter JM, Carrillo M (2007) Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity. Mol Cell Endocrinol 272:63–76. CrossRefPubMedGoogle Scholar
  38. Rocha A, Zanuy S, Carrillo M, Gomez A (2009) Seasonal changes in gonadal expression of gonadotropin receptors, steroidogenic acute regulatory protein and steroidogenic enzymes in the European sea bass. Gen Comp Endocrinol 162:265–275. CrossRefPubMedGoogle Scholar
  39. Sambroni E, Le Gac F, Breton B, Lareyre J-J (2007) Functional specificity of the rainbow trout (Oncorhynchus mykiss) gonadotropin receptors as assayed in a mammalian cell line. J Endocrinol 195:213–228. CrossRefPubMedGoogle Scholar
  40. Schmitz M, Aroua S, Vidal B, Le Belle N, Elie P, Dufour S (2005) Differential regulation of luteinizing hormone and follicle-stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel. Neuroendocrinology 81:107–119. CrossRefPubMedGoogle Scholar
  41. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Sohn YC, Yoshiura Y, Kobayashi M, Aida K (1999) Seasonal changes in mRNA levels of gonadotropin and thyrotropin subunits in the goldfish, Carassius auratus. Gen Comp Endocrinol 113:436–444. CrossRefPubMedGoogle Scholar
  43. Swanson P, Suzuki K, Kawauchi H, Dickhoff WW (1991) Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II. Biol Reprod 44:29–38. CrossRefPubMedGoogle Scholar
  44. Swanson P, Dickey JT, Campbell B (2003) Biochemistry and physiology of fish gonadotropins. Fish Physiol Biochem 28:53–59. CrossRefGoogle Scholar
  45. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Tyler CR, Sumpter JP (1996) Oocyte growth and development in teleosts. Rev Fish Biol Fish 6:287–318. CrossRefGoogle Scholar
  47. Vassart G, Pardo L, Costagliola S (2004) A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 29:119–126. CrossRefPubMedGoogle Scholar
  48. Yoshiura Y, Suetake H, Aida K (1999) Duality of gonadotropin in a primitive teleost, Japanese eel (Anguilla japonica). Gen Comp Endocrinol 114:121–131 . doi: S0016–6480(98)97242–2 [pii]\r

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory of Developmental and Reproductive Biology, DiSVAUniversità Politecnica delle MarcheAnconaItaly
  2. 2.Department of BiologyUniversity of PaduaPaduaItaly
  3. 3.CNR-National Research Council of ItalyISMAR-Marine Sciences InstituteAnconaItaly

Personalised recommendations