Skip to main content

Advertisement

SpringerLink
Log in

Molecular cloning of Foxl2 gene and the effects of endocrine-disrupting chemicals on its mRNA level in rare minnow, Gobiocypris rarus

  • Published:
Fish Physiology and Biochemistry Aims and scope Submit manuscript

Abstract

Endocrine-disrupting chemicals (EDCs) can affect normal sexual differentiation in fish. Foxl2, one forkhead transcription factor, plays an important role in ovarian differentiation in the early development of the female gonad in mammals and fish. How EDCs affect Foxl2 expression is little known. In this study, we isolated a Foxl2 cDNA from the ovary of rare minnow Gobiocypris rarus and examined its expression during early development stages and in different adult tissues. Then, we analyzed Foxl2 expression in G. rarus juvenile following 3-day exposure to 17α- ethinylestradiol (EE2), 4-n-nonylphenol (NP), and bisphenol A (BPA). Alignment of known Foxl2 sequences among vertebrates showed high identity in forkhead domain and C-terminal region with other vertebrate proteins. Quantitative RT-PCR analysis showed that Foxl2 expression was linear decrease and cyp19a1a, the downstream target gene of Foxl2, had no correlation with Foxl2 from 18 to 50 days post fertilization (dpf). Among different adult tissues, Foxl2 is mainly expressed in ovary, brain, gill, eye, and male spleen. In the 3-day exposure, the juvenile fish to EDCs, 0.1 nM EE2, and 1 nM BPA significantly up-regulated the expression of Foxl2 gene, while NP had no effect on Foxl2 expression. Altogether, these results provide basic data for further study on how Foxl2 mediates EDCs impact on the sexual differentiation in G. rarus.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Alam MA, Horiguchi R, Kobayashi Y, Hirai T, Nakamura M (2008) Molecular cloning and quantitative expression of sexually dimorphic markers Dmrt1 and Foxl2 during female-to-male sex change in Epinephelus merra. Gen Comp Endocrinol 157:75–85

    Article  PubMed  CAS  Google Scholar 

  • Atkinson S, Atkinson MJ, Tarrant AM (2003) Estrogens from sewage in coastal marine environments. Environ Health Perspect 111:531–535

    Article  PubMed  CAS  Google Scholar 

  • Baron D, Cocquet J, Xia X, Fellous M, Guiguen Y, Veitia RA (2004) An evolutionary and functional analysis of FoxL2 in rainbow trout gonad differentiation. J Mol Endocrinol 33:705–715

    Article  PubMed  CAS  Google Scholar 

  • Baron D, Batista F, Chaffaux S, Cocquet J, Cotinot C, Cribiu E, De Baere E, Guiguen Y, Jaubert F, Pailhoux E, Pannetier M, Vaiman D, Vigier B, Veitia R, Fellous M (2005) Foxl2 gene and the development of the ovary: a story about goat, mouse, fish and woman. Reprod Nutr Dev 45:377–382

    Article  PubMed  CAS  Google Scholar 

  • Belaid B, Richard-Mercier N, Pieau C, Dorizzi M (2001) Sex reversal and aromatase in the European pond turtle: treatment with letrozole after the thermosensitive period for sex determination. J Exp Zool 290:490–497

    Article  PubMed  CAS  Google Scholar 

  • Biles JE, McNeal TP, Begley TH, Hollifield HC (1997) Determination of bisphenol-A in reusable polycarbonate food-contact plastics and migration to food-stimulating liquids. J Agric Food Chem 45:3541–3544

    Article  CAS  Google Scholar 

  • Bjerselius R, Lundstedt-Enkel K, Olsén H, Mayer I, Dimberg K (2001) Male goldfish reproductive behavior and physiology are severely affected by exogenous exposure to 17β-estradiol. Aquat Toxicol 53:139–152

    Article  PubMed  CAS  Google Scholar 

  • Blázquez M, Gonzalez A, Papadaki M, Mylonas C, Piferrer F (2008) Sex-related changes in estrogen receptors and aromatase gene expression and enzymatic activity during early development and sex differentiation in the European sea bass (Dicentrarchus labrax). Gen Comp Endocrinol 158:95–101

    Article  PubMed  Google Scholar 

  • Brotons JA, Olea-Serrano MF, Villalobos M, Pedraza V, Olea N (1995) Xenoestrogens released from lacquer coating in food cans. Environ Health Perspect 103:608–612

    Article  PubMed  CAS  Google Scholar 

  • Chardard D, Dournon C (1999) Sex reversal by aromatase inhibitor treatment in the newt Pleurodeles waltl. J Exp Zool 283:43–50

    Article  PubMed  CAS  Google Scholar 

  • Cheshenko K, Pakdel F, Segner H, Kah O, Eggen RI (2008) Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen Comp Endocrinol 155:31–62

    Article  PubMed  CAS  Google Scholar 

  • Cocquet J, Pailhoux E, Jaubert F, Servel N, Xia X, Pannetier M, De Baere E, Messiaen L, Cotinot C, Fellous M, Veitia RA (2002) Evolution and expression of FOXL2. J Med Genet 39:916–922

    Article  PubMed  CAS  Google Scholar 

  • Conley A, Hinshelwood M (2001) Mammalian aromatases. Reproduction 121:685–695

    Article  PubMed  CAS  Google Scholar 

  • Crisponi L, Deiana M, Loi A, Chiappe F, Uda M, Amati P, Bisceglia L, Zelante L, Nagaraja R, Porcu S, Ristaldi MS, Marzella R, Rocchi M, Nicolino M, Lienhardt-Roussie A, Nivelon A, Verloes A, Schlessinger D, Gasparini P, Bonneau D, Cao A, Pilia G (2001) The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat Genet 27:159–166

    Article  PubMed  CAS  Google Scholar 

  • Elbrecht A, Smith RG (1992) Aromatase enzyme activity and sex determination in chickens. Science 255:467–470

    Article  PubMed  CAS  Google Scholar 

  • Ellsworth BS, Burns AT, Escudero KW, Duval DL, Nelson SE, Clay CM (2003) The gonadotropin releasing hormone (GnRH) receptor activating sequence (GRAS) is a composite regulatory element that interacts with multiple classes of transcription factors including Smads, AP-1 and a forkhead DNA binding protein. Mol Cell Endocrinol 206:93–111

    Article  PubMed  CAS  Google Scholar 

  • Esterhuyse MM, Helbing CC, van Wyk JH (2008) Temporal expression of two Cytochrome P450 Aromatase isoforms during development in Oreochromis mossambicus, in association with histological development. Comp Biochem Physiol D 3:297–306

    CAS  Google Scholar 

  • Fenske M, Segner H (2004) Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio). Aquat Toxicol 67:105–126

    Article  PubMed  CAS  Google Scholar 

  • Galay-Burgos M, Gealy C, Navarro-Martin L, Piferrer F, Zanuy S, Sweeney GE (2006) Cloning of the promoter from the gonadal aromatase gene of the European sea bass and identification of single nucleotide polymorphisms. Comp Biochem Physiol A 145:47–53

    Article  Google Scholar 

  • Govoroun MS, Pannetier M, Pailhoux E, Cocquet J, Brillard JP, Couty I, Batellier F, Cotinot C (2004) Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development. Dev Dyn 231:859–870

    Article  PubMed  CAS  Google Scholar 

  • Guiguen Y, Baroiller JF, Ricordel MJ, Iseki K, Mcmeel OM, Martin SA, Fostier A (1999) Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus). Mol Reprod Dev 54:154–162

    Article  PubMed  CAS  Google Scholar 

  • Hill RL Jr, Janz DM (2003) Developmental estrogenic exposure in zebrafish (Danio rerio): I. Effects on sex ratio and breeding success. Aquat Toxicol 63:417–429

    Article  PubMed  CAS  Google Scholar 

  • Hudson QJ, Smith CA, Sinclair AH (2005) Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary. Dev Dyn 233:1052–1055

    Article  PubMed  CAS  Google Scholar 

  • Ijiri S, Kaneko H, Kobayashi T, Wang DS, Sakai F, Paul-Prasanth B, Nakamura M, Nagahama Y (2008) Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus. Biol Reprod 78:333–341

    Article  PubMed  CAS  Google Scholar 

  • Kanda H, Okubo T, Omori N, Niihara H, Matsumoto N, Yamada K, Yoshimoto S, Ito M, Yamashita S, Shiba T, Takamatsu N (2006) Transcriptional regulation of the rainbow trout CYP19a gene by FTZ-F1 homologue. J. Steroid Biochem. Mol Biol 99:85–92

    Article  PubMed  CAS  Google Scholar 

  • Kaufmann E, Knochel W (1996) Five years on the wings of fork head. Mech Dev 57:3–20

    Article  PubMed  CAS  Google Scholar 

  • Kazeto Y, Place AR, Trant JM (2004) Effects of endocrine disrupting chemicals on the expression of CYP19 genes in zebrafish (Danio rerio) juveniles. Aquat Toxicol 69:25–34

    Article  PubMed  CAS  Google Scholar 

  • Khim JS, Lee KT, Kannan K, Villeneuve DL, Giesy JP, Koh CH (2001) Trace organic contaminants in sediment and water from Ulsan Bay and its vicnity, Kerea. Arch Environ Contam Toxicol 40:141–150

    Article  PubMed  CAS  Google Scholar 

  • Kobayashi T, Kajiura-Kobayashi H, Nagahama Y (2003) Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia. Cytogenet Genome Res 101:289–294

    Article  PubMed  CAS  Google Scholar 

  • Lange R, Hutchinson TH, Croudace CP, Siegmund F, Scheweinfurth H, Hampe P, Panter GH, Sumpter JP (2001) Effects of the synthetic estrogen 17α- ethinylestradiol on the life cycle of the fathead minnow (Pimephales promelas). Environ Toxicol Chem 20:1216–1227

    PubMed  CAS  Google Scholar 

  • Lee YM, Seo JS, Kim IC, Yoon YD, Lee JS (2006) Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol, 4-tert-octylphenol) modulate expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus. Biochem Biophys Res Commun 345:894–903

    Article  PubMed  CAS  Google Scholar 

  • Liao T, Guo QL, Jin SW, Cheng W, Xu Y (2009) Comparative responses in rare minnow exposed to 17β-estradiol during different life stages. Fish Physiol Biochem 35:341–349

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Loffler KA, Zarkower D, Koopman P (2003) Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: Foxl2 is a conserved, early-acting gene in vertebrate ovarian development. Endocrinology 144:3237–3243

    Article  PubMed  CAS  Google Scholar 

  • Miyata S, Kubo T (2000) In vitro effects of estradiol and aromatase inhibitor treatment on sex differentiation in Xenopus laevis gonads. Gen Comp Endocrinol 119:105–110

    Article  PubMed  CAS  Google Scholar 

  • Monteiro PR, Reis-Henriques MA, Coimbra J (2000) Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons. Mar Environ Res 49:453–467

    Article  PubMed  CAS  Google Scholar 

  • Nagahama Y, Nakamura M, Kitano T, Tokumoto T (2004) Sexual plasticity in fish: a possible target of endocrine disruptor action. Environ Sci 11:73–82

    PubMed  CAS  Google Scholar 

  • Naimi A, Martinez AS, Specq ML, Diss B, Mathieu M, Sourdaine P (2009) Molecular cloning and gene expression of Cg-Foxl2 during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas. Comp Biochem Physiol B 154:134–142

    Article  PubMed  Google Scholar 

  • Nakamoto M, Matsuda M, Wang DS, Nagahama Y, Shibata N (2006) Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes. Biochem Biophys Res Commun 344:353–361

    Article  PubMed  CAS  Google Scholar 

  • Noaksson E, Linderoth M, Bosveld ATC, Balka L (2003) Altered steroid metabolism in several teleost species exposed to endocrine disrupting substances in refuse dump leachate. Gen Comp Endocrinol 134:273–284

    Article  PubMed  CAS  Google Scholar 

  • Olea N, Pulger R, Perez P, Olea-Serrano MF, Rivas A, NovilloFertrell A, Pedraza V, Soto AM, Sonnenschein C (1996) Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect 104:298–305

    Article  PubMed  CAS  Google Scholar 

  • Orn S, Holbech H, Madsen TH, Norrgren L, Petersen GI (2003) Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone. Aquat Toxicol 65:397–411

    Article  PubMed  CAS  Google Scholar 

  • Oshima Y, Uno Y, Matsuda Y, Kobayashi T, Nakamura M (2008) Molecular cloning and gene expression of Foxl2 in the frog Rana rugosa. Gen Comp Endocrinol 159:170–177

    Article  PubMed  CAS  Google Scholar 

  • Pannetier M, Fabre S, Batista F, Kocer A, Renault L, Jolivet G, Mandon-Pepin B, Cotinot C, Veitia R, Pailhoux E (2006) FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development. J Mol Endocrinol 36:399–413

    Article  PubMed  CAS  Google Scholar 

  • Patil JG, Gunasekera RM (2008) Tissue and sexually dimorphic expression of ovarian and brain aromatase mRNA in the Japanese medaka (Oryzias latipes): implications for their preferential roles in ovarian and neural differentiation and development. Gen Comp Endocrinol 158:131–137

    Article  PubMed  CAS  Google Scholar 

  • Piferrer F (2001) Endocrine sex control strategies for the feminization of teleost fish. Aquaculture 197:229–281

    Article  CAS  Google Scholar 

  • Piferrer F, Blázquez M (2005) Aromatase distribution and regulation in fish. Fish Physiol Biochem 31:215–226

    Article  PubMed  CAS  Google Scholar 

  • Rempel MA, Schlenk D (2008) Effects of environmental estrogens and antiandrogens on endocrine function, gene regulation, and health in fish. In: Jeon KW (ed) International review of cell and molecular biology, vol 267. Elsevier, Amsterdam, pp 251–296

    Google Scholar 

  • Richard-Mercier N, Dorizzi M, Desvages G, Girondot M, Pieau C (1995) Endocrine sex reversal of gonads by the aromatase inhibitor Letrozole (CGS 20267) in Emys orbicularis, a turtle with temperature dependent sex determination. Gen Comp Endocrinol 100:314–326

    Article  PubMed  CAS  Google Scholar 

  • Sudhakumari CC, Senthilkumaran B, Kobayashi T, Kajiura-Kobayashi H, Wang DS, Yoshikuni M, Nagahama Y (2005) Ontogenic expression patterns of several nuclear receptors and cytochrome P450 aromatases in brain and gonads of the Nile tilapia Oreochromis niloticus suggests their involvement in sex differentiation. Fish Physiol Biochem 31:129–135

    Article  PubMed  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  • Tashiro Y, Takemura A, Fujii H, Takahira K, Nakanishi Y (2003) Livestock wastes as a source of estrogens and their effects on wildlife of Manko tidal flat, Okinawa. Mar Pollut Bull 47:143–147

    Article  PubMed  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  PubMed  CAS  Google Scholar 

  • Vaillant S, Guemene D, Dorizzi M, Pieau C, Richard-Mercier N, Brillard JP (2003) Degree of sex reversal as related to plasma steroid levels in genetic female chickens (Gallus domesticus) treated with Fadrozole. Mol Reprod Dev 65:420–428

    Article  PubMed  CAS  Google Scholar 

  • Vizziano D, Baron D, Randuineau G, Mahe S, Cauty C, Guiguen Y (2008) Rainbow trout gonadal masculinization induced by inhibition of estrogen synthesis is more physiological than masculinization induced by androgen supplementation. Biol Reprod 78:939–946

    Article  PubMed  CAS  Google Scholar 

  • Vizziano-Cantonnet D, Baron D, Mahe S, Cauty C, Fostier A, Guiguen Y (2008) Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation. J Mol Endocrinol 41:277–288

    Article  PubMed  CAS  Google Scholar 

  • Wang JW (1999) Spawning performance and development of oocytes in Gobiocypris rarus. Acta Hydrobiol Sin 23:161–166

    CAS  Google Scholar 

  • Wang D, Kobayashi T, Zhou L, Nagahama Y (2004) Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus. Biochem Biophys Res Commun 320:83–89

    Article  PubMed  CAS  Google Scholar 

  • Wang DS, Kobayashi T, Zhou LY, Paul-Prasanth B, Ijiri S, Sakai F, Okubo K, Morohashi K, Nagahama Y (2007) Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1. Mol Endocrinol 21:712–725

    Article  PubMed  CAS  Google Scholar 

  • Wang J, Liu X, Wang H, Wu T, Hu X, Qin F, Wang Z (2010) Expression of two cytochrome P450 aromatase genes is regulated by endocrine disrupting chemicals in rare minnow Gobiocypris rarus juveniles. Comp Biochem Physiol C 152:313–320

    Google Scholar 

  • Wang H, Wang J, Wu T, Qin F, Hu X, Wang L, Wang Z (2011) Molecular characterization of estrogen receptor genes in Gobiocypris rarus and their expression upon endocrine disrupting chemicals exposure in juveniles. Aquat Toxicol 101:276–287

    Article  PubMed  CAS  Google Scholar 

  • Wartenberg H, Lenz E, Schweikert HU (1992) Sexual differentiation and the germ cell in sex reversed gonads after aromatase inhibition in the chicken embryo. Andrologia 24:1–6

    Article  PubMed  CAS  Google Scholar 

  • Wibbels T, Crews D (1994) Putative aromatase inhibitor induces male sex determination in a female unisexual lizard and in a turtle with temperature-dependent sex determination. J Endocrinol 141:295–299

    Article  PubMed  CAS  Google Scholar 

  • Wong TT, Shigeho I, Zohar Y (2006) Molecular biology of ovarian aromatase in sex reversal: complementary DNA and 50-flanking region isolation and differential expression of ovarian aromatase in the gilthead seabream (Sparus aurata). Biol Reprod 74:857–864

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi T, Yamaguchi S, Hirai T, Kitano T (2007) Follicle-stimulating hormone signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during gonadal sex differentiation in Japanese flounder, Paralichthys olivaceus. Biochem Biophys Res Commun 359:935–940

    Article  PubMed  CAS  Google Scholar 

  • Zha J, Wang Z, Wang N, Ingersoll C (2007) Histological alternation and vitellogenin induction in adult rare minnow (Gobiocypris rarus) after exposure to ethynylestradiol and nonylphenol. Chemosphere 66:488–495

    Article  PubMed  CAS  Google Scholar 

  • Zha J, Sun L, Spear PA, Wang Z (2008a) Comparison of ethinylestradiol and nonylphenol effects on reproduction of Chinese rare minnows (Gobiocypris rarus). Ecotoxicol Environ Saf 71:390–399

    Article  PubMed  CAS  Google Scholar 

  • Zha J, Sun L, Zhou Y, Spear PA, Ma M, Wang Z (2008b) Assessment of 17α-ethinylestradiol effects and underlying mechanisms in a continuous, multigeneration exposure of the Chinese rare minnow. Toxicol Appl Pharmacol 226:298–308

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The work has been carried out with the financial support of Northwest A&F University. We are grateful to Mr. Yi Luo and Ms. Jingjing Wang for their technical help in the gene isolation.

Author information

Authors and Affiliations

  1. Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, 22 Xinong Road, Yangling, 712100, Shaanxi, China

    Houpeng Wang, Tingting Wu, Fang Qin & Zaizhao Wang

  2. The Affiliated Hospital, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Lihong Wang

Authors
  1. Houpeng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tingting Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fang Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lihong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zaizhao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zaizhao Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, H., Wu, T., Qin, F. et al. Molecular cloning of Foxl2 gene and the effects of endocrine-disrupting chemicals on its mRNA level in rare minnow, Gobiocypris rarus . Fish Physiol Biochem 38, 653–664 (2012). https://doi.org/10.1007/s10695-011-9548-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10695-011-9548-2

Keywords

Search

Navigation