Molecular Biology Reports

, Volume 38, Issue 1, pp 275–282 | Cite as

Molecular analysis shows differential expression of R-spondin1 in zebrafish (Danio rerio) gonads

  • Yanmei Zhang
  • Fei Li
  • Dongchang Sun
  • Jiangdong Liu
  • Na Liu
  • Qixing Yu
Article

Abstract

R-spondin1 (RSPO1) is a potential female-determining gene in human (Homo sapiens) and mouse (Mus musculus). Its differential expression in these mammals is correlated with signaling for sex determination. As a way of studying sex determination in fish we cloned and analyzed a RSPO1 gene in zebrafish (Danio rerio). Using real-time PCR, we observed that RSPO1 is expressed more strongly in ovaries than in testes, suggesting that RSPO1 may have a role in gonad differentiation. High RSPO1 expression was detected in some non-gonadal organs like muscle and kidneys. In situ hybridization results demonstrate that RSPO1 is expressed in premature germ cells, in oogonia and primary oocytes in ovaries and in spermatogonia and spermatocytes in testes. It is also expressed in gonad somatic cells during gonadal development: in granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, and in Leydig cells in testes. This differential expression may indicate that RSPO1 has a role(s) in zebrafish gonad development and differentiation. By fusing zebrafish RSPO1 with a green fluorescent protein gene, we found that RSPO1 is located in the cytosol and Golgi apparatus but not the nucleus of fish epithelioma papulosum cyprinid (EPC) cells. These preliminary findings suggest some aspects of RSPO1 like differential expression linked to sex determination may be conserved in fish while other aspects like subcellular localization differ from the mammalian RSPO1.

Keywords

R-spondin1 Differential gene expression Sex determination In situ hybridization Zebrafish 

Notes

Acknowledgements

We are very grateful to Prof. Fengjiao Deng, Prof. Yunhan Hong and Dr. Meisheng Yi for their helpful suggestions and to Yaping Zhou and Yao Liu for technical help with digoxygenin labeling. We also thank Yunjun Mei for analyzing real-time PCR data. This study was supported by the National Nature Science Foundation of China (Grant No. 30370772 and No. 30400044).

Supplementary material

11033_2010_105_MOESM1_ESM.doc (79 kb)
Supplementary material 1 (DOC 79 kb)

References

  1. 1.
    Cai H, Hu J, Song P, Gong W (2006) PSM2, a novel protein similar to MCAF2, is involved in the mouse embryonic and adult male gonad development. Mol Biol Rep 33:159–166CrossRefPubMedGoogle Scholar
  2. 2.
    DeFalco T, Capel B (2009) Gonad morphogenesis in vertebrates: divergent means to a convergent end. Annu Rev Cell Dev Biol 25:457–482CrossRefPubMedGoogle Scholar
  3. 3.
    Yang X, Zheng J, Xu G, Qu L, Chen S, Li J, Yang N (2009) Exogenous cMHM regulates the expression of DMRT1 and ERα in avian testes. Mol Biol Rep. doi: 10.1007/s11033-009-9619-y
  4. 4.
    Cao J, Chen J, Wu T, Gan X, Luo Y (2009) Molecular cloning and sexually dimorphic expression of DMRT4 gene in Oreochromis aureus. Mol Biol Rep. doi: 10.1007/s11033-009-9820-z
  5. 5.
    Peng J, Yu L, Horiuchi M, Zhang P, Huang X, Zhang Y, Li D, Jalil MA, Zhao S (2002) Identification of human CDV-1R and mouse CDV-1R, two novel proteins with putative signal peptides, especially highly expressed in testis and increased with the male sex maturation. Mol Biol Rep 29:353–362CrossRefPubMedGoogle Scholar
  6. 6.
    Wilhelm D, Palmer S, Koopman P (2007) Sex determination and gonadal development in mammals. Physiol Rev 87:1–28CrossRefPubMedGoogle Scholar
  7. 7.
    Chassot AA, Gregoire EP, Magliano M, Lavery R, Chaboissier MC (2008) Genetics of ovarian differentiation: Rspo1, a major player. Sex Dev 2:219–227CrossRefPubMedGoogle Scholar
  8. 8.
    Chassot AA, Ranc F, Gregoire EP, Roepers-Gajadien HL, Taketo MM, Camerino G, Rooij DG, Schedl A, Chaboissier MC (2008) Activation of β-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Hum Mol Genet 17:1264–1277CrossRefPubMedGoogle Scholar
  9. 9.
    Parma P, Radi O, Vidal V, Chaboissier MC, Dellambra E, Valentini S, Guerra L, Schedl A, Camerino G (2006) R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet 38:1304–1309CrossRefPubMedGoogle Scholar
  10. 10.
    Smith CA, Shoemaker CM, Roeszler KN, Queen J, Crews D, Sinclair AH (2008) Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development. BMC Dev Biol 8:72CrossRefPubMedGoogle Scholar
  11. 11.
    Tomizuka K, Horikoshi K, Kitada R, Sugawara Y, Iba Y, Kojima A, Yoshitome A, Yamawaki K, Amagai M, Inoue A, Oshima T, Kakitani M (2008) R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. Hum Mol Genet 17:1278–1291CrossRefPubMedGoogle Scholar
  12. 12.
    Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351:117–121CrossRefPubMedGoogle Scholar
  13. 13.
    Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M (2002) DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417:559–563CrossRefPubMedGoogle Scholar
  14. 14.
    Nanda I, Kondo M, Hornung U, Asakawa S, Winkler C, Shimizu A, Shan Z, Haaf T, Shimizu N, Shima A, Schmid M, Schartl M (2002) A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. Proc Natl Acad Sci USA 99:11778–11783CrossRefPubMedGoogle Scholar
  15. 15.
    Kocer A, Pinheiro I, Pannetier M, Renault L, Parma P, Radi O, Kim KA, Camerino G, Pailhoux E (2008) R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation. BMC Dev Biol 8:36CrossRefPubMedGoogle Scholar
  16. 16.
    Capel B (2006) R-spondin1 tips the balance in sex determination. Nat Genet 38:1233–1234CrossRefPubMedGoogle Scholar
  17. 17.
    Blaydon DC, Philpott MP, Kelsell DP (2007) R-spondins in cutaneous biology: nails and cancer. Cell Cycle 6:895–897PubMedGoogle Scholar
  18. 18.
    Kim KA, Kakitani M, Zhao J, Oshima T, Tang T, Binnerts M, Liu Y, Boyle B, Park E, Emtage P, Funk WD, Tomizuka K (2005) Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309:1256–1259CrossRefPubMedGoogle Scholar
  19. 19.
    Zhao J, Kim KA, De Vera J, Palencia S, Wagle M, Abo A (2009) R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/β-catenin pathway. Proc Natl Acad Sci USA 106:2331–2336CrossRefPubMedGoogle Scholar
  20. 20.
    Westerfield M (1994) The zebrafish book: a guide for the laboratory use of the zebrafish (Danio rerio). University of Oregon, Eugene, ORGoogle Scholar
  21. 21.
    Tang R, Dodd A, Lai D, McNabb WC, Love DR (2007) Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin (Shanghai) 39:384–390CrossRefGoogle Scholar
  22. 22.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034Google Scholar
  23. 23.
    Trant JM, Gavasso S, Ackers J, Chung BC, Place AR (2001) Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio). J Exp Zool 290:475–483CrossRefPubMedGoogle Scholar
  24. 24.
    Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36CrossRefPubMedGoogle Scholar
  25. 25.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2T−ΔΔC method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  26. 26.
    Jordan BK, Mohammed M, Ching ST, Delot E, Chen XN, Dewing P, Swain A, Rao PN, Elejalde BR, Vilain E (2001) Up-regulation of WNT-4 signaling and dosage-sensitive sex reversal in humans. Am J Hum Genet 68:1102–1109CrossRefPubMedGoogle Scholar
  27. 27.
    DiNapoli L, Capel B (2008) SRY and the standoff in sex determination. Mol Endocrinol 22:1–9CrossRefPubMedGoogle Scholar
  28. 28.
    Buscara L, Montazer-Torbati F, Chadi S, Auguste A, Laubier J, Chassot AA, Renault L, Passet B, Costa J, Pannetier M, Vilotte M, Chaboissier MC, Vilotte JL, Pailhoux E, Le Provost F (2009) Goat RSPO1 over-expression rescues sex-reversal in Rspo1-knockout XX mice but does not perturb testis differentiation in XY or sex-reversed XX mice. Transgenic Res 18:649–654CrossRefPubMedGoogle Scholar
  29. 29.
    Maack G, Segner H (2004) Life-stage-dependent sensitivity of zebraish (Danio rerio) to estrogen exposure. Comp Biochem Physiol C-Toxicol Pharmacol 139:47–55CrossRefPubMedGoogle Scholar
  30. 30.
    Takahashi H (1977) Juvenile hermaphroditism in the zebrafish, Brachydanio rerio. Bull Fac Fish Hokkaido Univ 28:57–65Google Scholar
  31. 31.
    Siegfried KR, Nusslein-Volhard C (2008) Germ line control of female sex determination in zebrafish. Dev Biol 324:277–287CrossRefPubMedGoogle Scholar
  32. 32.
    Uchida D, Yamashita M, Kitano T, Iguchi T (2002) Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. J Exp Biol 205:711–718PubMedGoogle Scholar
  33. 33.
    Maack G, Segner H (2003) Morphological development of the gonads in zebrafish. J Fish Biol 62:895–906CrossRefGoogle Scholar
  34. 34.
    Braat AK, Speksnijder JE, Zivkovic D (1999) Germ line development in fishes. Int J Dev Biol 43:745–760PubMedGoogle Scholar
  35. 35.
    Çakıcı Ö, Üçüncü Sİ (2007) Oocyte Development in the Zebrafish, Danio rerio (Teleostei: Cyprinidae). EU J Fish Aqua Sci 24:137–141Google Scholar
  36. 36.
    Yang Q, Hu J, Ye D, Zhao C, Song S, Gong W, Tan Z, Song P (2009) Identification and expression analysis of two zebrafish E2F5 genes during oogenesis and development. Mol Biol Rep. doi: 10.1007/s11033-009-9605-4

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Yanmei Zhang
    • 1
  • Fei Li
    • 1
  • Dongchang Sun
    • 2
  • Jiangdong Liu
    • 1
  • Na Liu
    • 1
  • Qixing Yu
    • 1
  1. 1.College of Life SciencesWuhan UniversityWuhanPeople’s Republic of China
  2. 2.Zhejiang Academy of Agricultural SciencesHangzhouPeople’s Republic of China

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