Environmental Science and Pollution Research

, Volume 25, Issue 34, pp 34403–34413 | Cite as

Stage-specific testicular protein levels of the oestrogen receptors (ERα and ERβ) and Cyp19 and association with oestrogenic contamination in the lambari Astyanax rivularis (Pisces: Characidae)

  • André Alberto Weber
  • Davidson Peruci Moreira
  • Rafael Magno Costa Melo
  • Augusto Bicalho Cruz Vieira
  • Nilo Bazzoli
  • Elizete RizzoEmail author
Research Article


Oestrogens participate in various biological processes such as oogenesis, vitellogenesis and testicular development, but studies regarding the distribution and protein levels of oestrogen receptors (ERα and ERβ) and aromatase (Cyp19) in testis are rarely investigated in fish species. The aim of the present study was to analyse the expression pattern of ERα, ERβ and Cyp19 in testis of Astyanax rivularis and, in addition, to verify if oestrogenic contamination interferes in the expression levels of these proteins. Quarterly, field samplings were carried out during a reproductive cycle in a stream of the Upper Velhas River with a good conservation status (site S1). In the gonadal maturation peak (June), when ripe stage was most abundant, fish collection was made in three streams: S1, reference site, and S2 and S3, sites contaminated by untreated sewage. The results of immunohistochemistry demonstrated labelling of Cyp19 in Leydig cells and acidophilic granulocytes, but spermatogonia, Sertoli cells, spermatids and spermatozoa were also labelled. ERα was more widely distributed than ERβ being found in all developmental germ cell phases. On the other hand, ERβ was found only in spermatogonia and spermatocytes. During testicular maturation, ELISA levels for Cyp19, ERα and ERβ followed the gonadosomatic index (GSI) with significant higher values in the ripe stage. Regarding to endocrine disruption, the males exposed to domestic sewage presented significant higher expression of Cyp19 and ERα when compared to the non-exposed fish. Together, our results demonstrate expression patterns of Cyp19, ERα and ERβ in the testis of A. rivularis. In addition, we indicate ERα and Cyp19 as sensitive biomarkers for monitoring of oestrogenic contamination in freshwater environments.


Testis Oestrogenic pathway Aromatase Neotropical fish Endocrine disruption Pollution 



The authors would like to thank Bruna Toledo Maria for preparing the histological slides, Dr. Priscila Divina Diniz Alves for helping in immunofluorescence procedures and also to Stephen Latham for the valuable suggestions with the English language.

Funding information

The work was supported by Brazilian funding agencies: Conselho Nacional Científico e Tecnológico (CNPq 14/2012) and Fundação de Amparo a Pesquisa no Estado de Minas Gerais (FAPEMIG 01/2015 - CVZ-APQ-00898-15).

Supplementary material

11356_2018_3392_MOESM1_ESM.docx (1.2 mb)
ESM 1 (DOCX 1266 kb)


  1. Alves CBM, Pompeu PS (2005) Historical changes in the Rio das Velhas Fish Fauna—Brazil. Am Fish Soc Symp 45:587–602Google Scholar
  2. Aquila S, Sisci D, Gentile M, Middea E, Catalano S, Carpino A, Rago V, Andò S (2004) Estrogen receptor (ER)α and ERβ are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway. J Clin Endocrinol Metab 89:1443–1451. CrossRefGoogle Scholar
  3. Bahamonde PA, Tetreault GR, McMaster ME et al (2014) Molecular signatures in rainbow darter (Etheostoma caeruleum) inhabiting an urbanized river reach receiving wastewater effluents. Aquat Toxicol 148:211–220. CrossRefGoogle Scholar
  4. Bahamonde PA, Fuzzen ML, Bennett CJ, Tetreault GR, McMaster ME, Servos MR, Martyniuk CJ, Munkittrick KR (2015) Whole organism responses and intersex severity in rainbow darter (Etheostoma caeruleum) following exposures to municipal wastewater in the Grand River basin, ON, Canada. Part A. Aquat Toxicol 159:290–301CrossRefGoogle Scholar
  5. Benninghoff AD, Williams DE (2008) Identification of a transcriptional fingerprint of estrogen exposure in rainbow trout liver. Toxicol Sci 101:65–80. CrossRefGoogle Scholar
  6. Bilińska B, Schmalz-Frczek B, Kotula M, Carreau S (2001) Photoperiod-dependent capability of androgen aromatization and the role of estrogens in the bank vole testis visualized by means of immunohistochemistry. Mol Cell Endocrinol 178:189–198. CrossRefGoogle Scholar
  7. Björnström L, Sjöberg M (2005) Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol 19:833–842. CrossRefGoogle Scholar
  8. Burgos-Aceves MA, Cohen A, Smith Y, Faggio C (2016) Estrogen regulation of gene expression in the teleost fish immune system. Fish Shellfish Immunol 58:42–49. CrossRefGoogle Scholar
  9. Caneguim BH, da Luz JS, Valentini SR et al (2013) Immunoexpression of aromatase and estrogen receptors β in stem spermatogonia of bullfrogs indicates a role of estrogen in the seasonal spermatogonial mitotic activity. Gen Comp Endocrinol 182:65–72CrossRefGoogle Scholar
  10. Carreau S, Hess RA (2010) Oestrogens and spermatogenesis. Philos Trans R Soc Lond Ser B Biol Sci 365:1517–1535. CrossRefGoogle Scholar
  11. Carreau S, Bouraima-Lelong H, Delalande C (2011) Estrogens: new players in spermatogenesis. Reprod Biol 11:174–193. CrossRefGoogle Scholar
  12. Carvalho PA, Paschoalini AL, Santos GB et al (2009) Reproductive biology of Astyanax fasciatus (Pisces: Characiformes) in a reservoir in southeastern Brazil. J Appl Ichthyol 25:306–313. CrossRefGoogle Scholar
  13. Delalande C, Goupil AS, Lareyre JJ, Le Gac F (2015) Differential expression patterns of three aromatase genes and of four estrogen receptors genes in the testes of trout (Oncorhynchus mykiss). Mol Reprod Dev 82:694–708. CrossRefGoogle Scholar
  14. Denslow N, Sepúlveda M (2007) Ecotoxicological effects of endocrine disrupting compounds on fish reproduction. In: Babin PJ, Cerdà J, Lubzens E (eds) The fish oocyte: from basic studies to biotechnological applications. Springer, Dordrecht, pp 255–322CrossRefGoogle Scholar
  15. Esterhuyse MM, Helbing CC, van Wyk JH (2010) Isolation and characterization of three estrogen receptor transcripts in Oreochromis mossambicus (Peters). J Steroid Biochem Mol Biol 119:26–34. CrossRefGoogle Scholar
  16. Griffin LB, January KE, Ho KW, Cotter KA, Callard GV (2013) Morpholino-mediated knockdown of ERα, ERβa, and ERβb mRNAs in zebrafish (Danio rerio) embryos reveals differential regulation of estrogen-inducible genes. Endocrinology 154:4158–4169. CrossRefGoogle Scholar
  17. Gustafsson JÅ (2003) What pharmacologists can learn from recent advances in estrogen signalling. Trends Pharmacol Sci 24:479–485. CrossRefGoogle Scholar
  18. He C-L, Du J-L, Lee Y-H et al (2003) Differential messenger RNA transcription of androgen receptor and estrogen receptor in gonad in relation to the sex change in protandrous black porgy, Acanthopagrus schlegeli. Biol Reprod 69:455–461. CrossRefGoogle Scholar
  19. Hess RA (2003) Estrogen in the adult male reproductive tract: a review. Reprod Biol Endocrinol 1:52. CrossRefGoogle Scholar
  20. Hinfray N, Nóbrega RH, Caulier M, Baudiffier D, Maillot-Maréchal E, Chadili E, Palluel O, Porcher JM, Schulz R, Brion F (2013) Cyp17a1 and cyp19a1 in the zebrafish testis are differentially affected by oestradiol. J Endocrinol 216:375–388. CrossRefGoogle Scholar
  21. Ibor OR, Adeogun AO, Fagbohun OA, Arukwe A (2016) Gonado-histopathological changes, intersex and endocrine disruptor responses in relation to contaminant burden in Tilapia species from Ogun River, Nigeria. Chemosphere 164:248–262CrossRefGoogle Scholar
  22. Kloas W, Schrag B, Ehnes C, Segner H (2000) Binding of xenobiotics to hepatic estrogen receptor and plasma sex steroid binding protein in the teleost fish, the common carp (Cyprinus carpio). Gen Comp Endocrinol 119:287–299. CrossRefGoogle Scholar
  23. Kotula-Balak M, Gancarczyk M, Sadowska J, Bilinskai B (2005) The expression of aromatase, estrogen receptor alpha and estrogen receptor beta in mouse Leydig cells in vitro that derived from cryptorchid males. Eur J Histochem 49:59–62CrossRefGoogle Scholar
  24. Lambard S, Galeraud-Denis I, Saunders PTK, Carreau S (2004) Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors. J Mol Endocrinol 32:279–289. CrossRefGoogle Scholar
  25. Lange A, Katsu Y, Ichikawa R, Paull GC, Chidgey LL, Coe TS, Iguchi T, Tyler CR (2008) Altered sexual development in roach (Rutilus rutilus) exposed to environmental concentrations of the pharmaceutical 17α-ethinylestradiol and associated expression dynamics of aromatases and estrogen receptors. Toxicol Sci 106:113–123. CrossRefGoogle Scholar
  26. Lee Pow CSD, Yost EE, Aday DD, Kullman SW (2016) Sharing the roles: an assessment of Japanese Medaka estrogen receptors in vitellogenin induction. Environ Sci Technol 50:8886–8895. CrossRefGoogle Scholar
  27. Liarte S, Chaves-Pozo E, García-Alcazar A, Mulero V, Meseguer J, García-Ayala A (2007) Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration. Reprod Biol Endocrinol 5:20. CrossRefGoogle Scholar
  28. Liarte S, Chaves-Pozo E, Abellán E, Meseguer J, Mulero V, García-Ayala A (2011) 17β-Estradiol regulates gilthead seabream professional phagocyte responses through macrophage activation. Dev Comp Immunol 35:19–27. CrossRefGoogle Scholar
  29. Lima FCT, Malabarba LR, Buckup PA et al (2003) Genera Incertae Sedis in Characidae. In: Reis RE, Kullander SO, Ferraris-Jr CJ (eds) Check list of the freshwater fishes of South and Central America. EDIPUCRS, Porto Alegre, pp 106–168Google Scholar
  30. Lishman L, Smyth SA, Sarafin K, Kleywegt S, Toito J, Peart T, Lee B, Servos M, Beland M, Seto P (2006) Occurrence and reductions of pharmaceuticals and personal care products and estrogens by municipal wastewater treatment plants in Ontario, Canada. Sci Total Environ 367:544–558. CrossRefGoogle Scholar
  31. Lucas TFG, Siu ER, Esteves CA et al (2008) 17beta-estradiol induces the translocation of the estrogen receptors ESR1 and ESR2 to the cell membrane, MAPK3/1 phosphorylation and proliferation of cultured immature rat Sertoli cells. Biol Reprod 78:101–114. CrossRefGoogle Scholar
  32. Martins-Santos E, Pimenta CG, Campos PRN, Franco MB, Gomes DA, Mahecha GAB, Oliveira CA (2017) Persistent testicular structural and functional alterations after exposure of adult rats to atrazine. Reprod Toxicol 73:201–213. CrossRefGoogle Scholar
  33. Miura T, Miura C, Ohta T, Nader MR, Todo T, Yamauchi K (1999) Estradiol-17beta stimulates the renewal of spermatogonial stem cells in males. Biochem Biophys Res Commun 264:230–234. CrossRefGoogle Scholar
  34. Miura T, Ohta T, Miura CI, Yamauchi K (2003) Complementary deoxyribonucleic acid cloning of spermatogonial stem cell renewal factor. Endocrinology 144:5504–5510. CrossRefGoogle Scholar
  35. Morais RDVS, Thomé RG, Santos HB, Bazzoli N, Rizzo E (2016) Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary. Theriogenology 85:1118–1131. CrossRefGoogle Scholar
  36. Moreira M, Aquino S, Coutrim M, Silva J, Afonso R (2011) Determination of endocrine disrupting compounds in waters from Rio das Velhas, Brazil, by liquid chromatography/high resolution mass spectrometry (ESI-LC-IT-TOF/MS). Environ Technol 32:1409–1417. CrossRefGoogle Scholar
  37. Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403:853–858. CrossRefGoogle Scholar
  38. Naderi M, Wong MYL, Gholami F (2014) Developmental exposure of zebrafish (Danio rerio) to bisphenol-S impairs subsequent reproduction potential and hormonal balance in adults. Aquat Toxicol 148:195–203CrossRefGoogle Scholar
  39. 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. CrossRefGoogle Scholar
  40. Nelson ER, Habibi HR (2013) Estrogen receptor function and regulation in fish and other vertebrates. Gen Comp Endocrinol 192:15–24. CrossRefGoogle Scholar
  41. Oliveira AG, Dornas RAP, Mahecha GAB, Oliveira CA (2011) Occurrence and cellular distribution of estrogen receptors ERα and ERβ in the testis and epididymal region of roosters. Gen Comp Endocrinol 170:597–603CrossRefGoogle Scholar
  42. Oliveira RL, Nogueira JC, Mahecha GAB, Oliveira CA (2012) Seasonal variation in estrogen receptor ERα, but not ERβ, androgen receptor and aromatase, in the efferent ductules and epididymis of the big fruit-eating bat Artibeus lituratus. Gen Comp Endocrinol 179:1–13. CrossRefGoogle Scholar
  43. Paulino MG, Benze TP, Sadauskas-Henrique H, Sakuragui MM, Fernandes JB, Fernandes MN (2014) The impact of organochlorines and metals on wild fish living in a tropical hydroelectric reservoir: bioaccumulation and histopathological biomarkers. Sci Total Environ 497–498:293–306. CrossRefGoogle Scholar
  44. Prado PS, Souza CC, Bazzoli N, Rizzo E (2011) Reproductive disruption in lambari Astyanax fasciatus from a Southeastern Brazilian reservoir. Ecotoxicol Environ Saf 74:1879–1887. CrossRefGoogle Scholar
  45. Prado PS, Pinheiro APB, Bazzoli N, Rizzo E (2014) Reproductive biomarkers responses induced by xenoestrogens in the characid fish Astyanax fasciatus inhabiting a South American reservoir: an integrated field and laboratory approach. Environ Res 131:165–173. CrossRefGoogle Scholar
  46. Randak T, Zlabek V, Pulkrabova J, Kolarova J, Kroupova H, Siroka Z, Velisek J, Svobodova Z, Hajslova J (2009) Effects of pollution on chub in the River Elbe, Czech Republic. Ecotoxicol Environ Saf 72:737–746. CrossRefGoogle Scholar
  47. Ropero AB, Alonso-Magdalena P, Ripoll C, Fuentes E, Nadal A (2006) Rapid endocrine disruption: environmental estrogen actions triggered outside the nucleus. J Steroid Biochem Mol Biol 102:163–169. CrossRefGoogle Scholar
  48. Schulz RW, França LR, Lareyre JJ et al (2010) Spermatogenesis in fish. Gen Comp Endocrinol 165:390–411. CrossRefGoogle Scholar
  49. Silva P, Rocha MJ, Cruzeiro C, Malhão F, Reis B, Urbatzka R, Monteiro RAF, Rocha E (2012) Testing the effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens as found in the Douro River (Portugal) on the maturation of fish gonads-a stereological study using the zebrafish (Danio rerio) as model. Aquat Toxicol 124–125:1–10. CrossRefGoogle Scholar
  50. Solakidi S, Psarra A-MG, Nikolaropoulos S, Sekeris CE (2005) Estrogen receptors alpha and beta (ERalpha and ERbeta) and androgen receptor (AR) in human sperm: localization of ERbeta and AR in mitochondria of the midpiece. Hum Reprod 20:3481–3487. CrossRefGoogle Scholar
  51. Stevens JL, Northcott GL, Stern GA, Tomy GT, Jones KC (2003) PAHs, PCBs, PCNs, organochlorine pesticides, synthetic musks, and polychlorinated n-alkanes in U.K. sewage sludge: survey results and implications. Environ Sci Technol 37:462–467. CrossRefGoogle Scholar
  52. Szwejser E, Verburg-van Kemenade BML, Maciuszek M, Chadzinska M (2017) Estrogen-dependent seasonal adaptations in the immune response of fish. Horm Behav 88:15–24. CrossRefGoogle Scholar
  53. Thomas P (2012) Rapid steroid hormone actions initiated at the cell surface and the receptors that mediate them with an emphasis on recent progress in fish models. Gen Comp Endocrinol 175:367–383. CrossRefGoogle Scholar
  54. Thomas P, Dong J (2006) Binding and activation of the seven-transmembrane estrogen receptor GPR30 by environmental estrogens: a potential novel mechanism of endocrine disruption. J Steroid Biochem Mol Biol 102:175–179. CrossRefGoogle Scholar
  55. Thomé RG, Domingos FFT, Santos HB, Martinelli PM, Sato Y, Rizzo E, Bazzoli N (2012) Apoptosis, cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish. Tissue Cell 44:54–62. CrossRefGoogle Scholar
  56. Tolussi CE, Gomes ADO, Kumar A, Ribeiro CS, Lo Nostro FL, Bain PA, de Souza GB, Cuña RD, Honji RM, Moreira RG (2018) Environmental pollution affects molecular and biochemical responses during gonadal maturation of Astyanax fasciatus (Teleostei: Characiformes: Characidae). Ecotoxicol Environ Saf 147:926–934. CrossRefGoogle Scholar
  57. Veado MARV, De Oliveira AH, Veado JCC et al (2000) Analysis and distribution of metals in the Paraopeba and the Das Velhas Rivers, Brazil. Water SA 26:249–254Google Scholar
  58. Veloso-Júnior VC, Guimarães-Cruz RJ, Barros MDM, Barata RSL, Santos JE (2009) Reproduction of the lambari Astyanax scabripinnis (Jenyns, 1842) (Pisces: Characidae) in a small stream in southeastern Brazil. J Appl Ichthyol 25:314–320. CrossRefGoogle Scholar
  59. Viñas J, Piferrer F (2008) Stage-specific gene expression during fish spermatogenesis as determined by laser-capture microdissection and quantitative-PCR in sea bass (Dicentrarchus labrax) Gonads1. Biol Reprod 79:738–747. CrossRefGoogle Scholar
  60. Weber AA, Moreira DP, Melo RMC, Vieira ABC, Prado PS, da Silva MAN, Bazzoli N, Rizzo E (2017) Reproductive effects of oestrogenic endocrine disrupting chemicals in Astyanax rivularis inhabiting headwaters of the Velhas River, Brazil. Sci Total Environ 592:693–703. CrossRefGoogle Scholar
  61. Wen R, Xie Y, Wan C, Fang Z (2013) Estrogenic and androgenic effects in mosquitofish (Gambusia affinis) from streams contaminated by municipal effluent in Guangzhou, China. Aquat Toxicol 132–133:165–172. CrossRefGoogle Scholar
  62. Zhou Q, Nie R, Prins GS, Saunders PT, Katzenellenbogen BS, Hess RA (2002) Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J Androl 23:870–881. CrossRefGoogle Scholar
  63. Zhu P, Zhang Y, Zhuo Q, Lu D, Huang J, Liu X, Lin H (2008) Discovery of four estrogen receptors and their expression profiles during testis recrudescence in male Spinibarbus denticulatus. Gen Comp Endocrinol 156:265–276. CrossRefGoogle Scholar

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© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departamento de Morfologia, Instituto de Ciências BiológicasUniversidade Federal de Minas Gerais, UFMGBelo HorizonteBrazil
  2. 2.Programa de Pós-graduação em Zoologia de VertebradosPontifícia Universidade Católica de Minas Gerais, PUC MinasBelo HorizonteBrazil

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