Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Gene structure and identification of minimal promoter of Pou2 expressed in spermatogonial cells of rohu carp, Labeo rohita

  • 266 Accesses

  • 20 Citations


Mammalian Pou5f1 is a known transcriptional regulator involving maintenance of embryonic and spermatogonial stem cells. Little is known about teleost Pou2, an ortholog of mammalian Pou5f1. Evidences of discrepancy in expression pattern between fish species were documented. To better understand, we have cloned and characterized Pou2 gene of farmed rohu carp, Labeo rohita. It contained five exons with an open reading frame of 1419 bp long, translatable to 472 aa. A bipartite DNA binding domain termed POU domain, comprising of POU-specific and POU-homeo sub-domains, was identified. Rohu Pou2 is highly conserved with zebrafish counterpart, as evidenced by 92 % overall sequence identity of deduced protein. The POU domain remained highly conserved (showing more than 90 % identities) within fish species. Even though there is a divergence between Pou2 and Pou5f1, the common POU-specific domain remained conserved throughout eukaryotes indicating their possible involvements in common trans-activation pathway(s) between mammals and non-mammals. In support, we have provided evidence that Pou2 is indeed abundantly expressed in proliferating rohu spermatogonial cells and hence participates in stem cell maintenance. Its mRNA accumulation in the ovary supported about its maternal transmission with possible regulatory roles during embryogenesis. The 5′-flanking region (~2.7 kb) of rohu Pou2 was sequenced and computational analysis detected several putative regulatory elements. These elements have been conserved among fish species analysed. Luciferase assay identified a mammalian-type ‘TATA-less promoter’ capable of driving Pou2 gene transcription. These findings will help for future studies in elucidating participatory role of fish Pou2 in male germ cell development.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Pesce M, Scholer HR (2001) Oct-4: Gatekeeper in the beginnings of mammalian development. Stem Cells 19:271–278

  2. 2.

    Niwa H (2007) How is pluripotency determined and maintained? Development 134:635–646

  3. 3.

    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676

  4. 4.

    Kehler J, Tolkunova E, Koschorz B, Pesce M, Gentile L, Boiani M, Lomeli H, Nagy A, McLaughlin KJ, Scholer HR, Tomilin A (2004) Oct4 is required for primordial germ cell survival. EMBO Rep 5:1078–1083

  5. 5.

    Pesce M, Wang X, Wolgemuth DJ, Scholer H (1998) Differential expression of the oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 71:89–98

  6. 6.

    Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW, Staudt LM (1990) A pou-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345:686–692

  7. 7.

    Anderson RA, Fulton N, Cowan G, Coutts S, Saunders PT (2007) Conserved and divergent patterns of expression of dazl, vasa and oct4 in the germ cells of the human fetal ovary and testis. BMC Dev Biol 7:136

  8. 8.

    Dann CT, Alvarado AL, Molyneux LA, Denard BS, Garbers DL, Porteus MH (2008) Spermatogonial stem cell self-renewal requires oct4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells 26:2928–2937

  9. 9.

    Belting HG, Hauptmann G, Meyer D, Abdelilah-Seyfried S, Chitnis A, Eschbach C, Soll I, Thisse C, Thisse B, Artinger KB, Lunde K, Driever W (2001) Spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer. Development 128:4165–4176

  10. 10.

    Burgess S, Reim G, Chen W, Hopkins N, Brand M (2002) The zebrafish spiel-ohne-grenzen (spg) gene encodes the pou domain protein pou2 related to mammalian oct4 and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis. Development 129:905–916

  11. 11.

    Lunde K, Belting HG, Driever W (2004) Zebrafish pou5f1/pou2, homolog of mammalian oct4, functions in the endoderm specification cascade. Curr Biol 14:48–55

  12. 12.

    Reim G, Mizoguchi T, Stainier DY, Kikuchi Y, Brand M (2004) The pou domain protein spg (pou2/oct4) is essential for endoderm formation in cooperation with the hmg domain protein casanova. Dev Cell 6:91–101

  13. 13.

    Reim G, Brand M (2006) Maternal control of vertebrate dorsoventral axis formation and epiboly by the pou domain protein spg/pou2/oct4. Development 133:2757–2770

  14. 14.

    Sanchez-Sanchez AV, Camp E, Garcia-Espana A, Leal-Tassias A, Mullor JL (2010) Medaka oct4 is expressed during early embryo development, and in primordial germ cells and adult gonads. Dev Dyn 239:672–679

  15. 15.

    Hauptmann G, Gerster T (1995) Pou-2–a zebrafish gene active during cleavage stages and in the early hindbrain. Mech Dev 51:127–138

  16. 16.

    Lindeman LC, Winata CL, Aanes H, Mathavan S, Alestrom P, Collas P (2010) Chromatin states of developmentally-regulated genes revealed by DNA and histone methylation patterns in zebrafish embryos. Int J Dev Biol 54:803–813

  17. 17.

    Takeda H, Matsuzaki T, Oki T, Miyagawa T, Amanuma H (1994) A novel pou domain gene, zebrafish pou2: Expression and roles of two alternatively spliced twin products in early development. Genes Dev 8:45–59

  18. 18.

    Marandel L, Labbe C, Bobe J, Jammes H, Lareyre JJ, Le Bail PY (2012) Do not put all teleosts in one net: Focus on the sox2 and pou2 genes. Comp Biochem Physiol B Biochem Mol Biol 164:69–79

  19. 19.

    Ye H, Du H, Chen XH, Cao H, Liu T, Li CJ (2012) Identification of a pou2 ortholog in chinese sturgeon, acipenser sinensis and its expression patterns in tissues, immature individuals and during embryogenesis. Fish Physiol Biochem 38:929–942

  20. 20.

    Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, Chen T, Deng J, Gui J (2004) Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proc Natl Acad Sci USA 101:8011–8016

  21. 21.

    Panda RP, Barman HK, Mohapatra C (2011) Isolation of enriched carp spermatogonial stem cells from labeo rohita testis for in vitro propagation. Theriogenol. 76:241–251

  22. 22.

    Medvedev SP, Shevchenko AI, Elisaphenko EA, Nesterova TB, Brockdorff N, Zakian SM (2008) Structure and expression pattern of oct4 gene are conserved in vole microtus rossiaemeridionalis. BMC Genomics 9:162

  23. 23.

    Nordhoff V, Hubner K, Bauer A, Orlova I, Malapetsa A, Scholer HR (2001) Comparative analysis of human, bovine, and murine oct-4 upstream promoter sequences. Mamm Genome 12:309–317

  24. 24.

    Parvin MS, Okuyama N, Inoue F, Islam ME, Kawakami A, Takeda H, Yamasu K (2008) Autoregulatory loop and retinoic acid repression regulate pou2/pou5f1 gene expression in the zebrafish embryonic brain. Dev Dyn 237:1373–1388

  25. 25.

    Barman HK, Barat A, Yadav BM, Banerjee S, Meher PK, Reddy PVGK, Jana RK (2003) Genetic variation between four species of indian major carps as revealed by random amplified polymorphic DNA assay. Aquaculture 217:115–123

  26. 26.

    Mohapatra C, Barman HK, Panda RP, Kumar S, Das V, Mohanta R, Mohapatra SD, Jayasankar P (2010) Cloning of cdna and prediction of peptide structure of plzf expressed in the spermatogonial cells of labeo rohita. Mar. Genomics 3:157–163

  27. 27.

    Barman HK, Das V, Mohanta R, Mohapatra C, Panda RP, Jayasankar P (2010) Expression analysis of β-actin promoter of rohu (labeo rohita) by direct injection into muscle. Curr Sci 99:1030–1032

  28. 28.

    Barman HK, Patra SK, Das V, Mohapatra SD, Jayasankar P, Mohapatra C, Mohanta R, Panda RP, Rath SN (2012) Identification and characterization of differentially expressed transcripts in the gills of freshwater prawn (macrobrachium rosenbergii) under salt stress. Sci World J 2012:149361

  29. 29.

    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

  30. 30.

    Mount SM (1982) A catalogue of splice junction sequences. Nucleic Acids Res 10:459–472

  31. 31.

    Kozak M (1991) An analysis of vertebrate mrna sequences: Intimations of translational control. J Cell Biol 115:887–903

  32. 32.

    Brehm A, Ohbo K, Scholer H (1997) The carboxy-terminal transactivation domain of oct-4 acquires cell specificity through the pou domain. Mol Cell Biol 17:154–162

  33. 33.

    Phillips K, Luisi B (2000) The virtuoso of versatility: Pou proteins that flex to fit. J Mol Biol 302:1023–1039

  34. 34.

    Ross SA, McCaffery PJ, Drager UC, De Luca LM (2000) Retinoids in embryonal development. Physiol Rev 80:1021–1054

  35. 35.

    Phelan JD, Shroyer NF, Cook T, Gebelein B, Grimes HL (2010) Gfi1-cells and circuits: Unraveling transcriptional networks of development and disease. Curr Opin Hematol 17:300–307

  36. 36.

    Takeda J, Seino S, Bell GI (1992) Human oct3 gene family: Cdna sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Nucl Acids Res 20:4613–4620

  37. 37.

    Yeom YI, Ha HS, Balling R, Scholer HR, Artzt K (1991) Structure, expression and chromosomal location of the oct-4 gene. Mech Dev 35:171–179

  38. 38.

    Wang X, Dai J (2010) Concise review: Isoforms of oct4 contribute to the confusing diversity in stem cell biology. Stem Cells 28:885–893

  39. 39.

    Zhang W, Wang X, Xiao Z, Liu W, Chen B, Dai J (2010) Mapping of the minimal internal ribosome entry site element in the human embryonic stem cell gene oct4b mrna. Biochem Biophys Res Commun 394:750–754

  40. 40.

    Bhartiya D, Kasiviswanathan S, Unni SK, Pethe P, Dhabalia JV, Patwardhan S, Tongaonkar HB (2010) Newer insights into premeiotic development of germ cells in adult human testis using oct-4 as a stem cell marker. J Histochem Cytochem 58:1093–1106

  41. 41.

    Frankenberg S, Pask A, Renfree MB (2010) The evolution of class v pou domain transcription factors in vertebrates and their characterisation in a marsupial. Dev Biol 337:162–170

  42. 42.

    Niwa H, Sekita Y, Tsend-Ayush E, Grutzner F (2008) Platypus pou5f1 reveals the first steps in the evolution of trophectoderm differentiation and pluripotency in mammals. Evol Dev 10:671–682

  43. 43.

    Pesce M, Marin Gomez M, Philipsen S, Scholer HR (1999) Binding of sp1 and sp3 transcription factors to the oct-4 gene promoter. Cell Mol Biol (Noisy-le-grand) 45:709–716

  44. 44.

    Sylvester I, Scholer HR (1994) Regulation of the oct-4 gene by nuclear receptors. Nucl Acids Res 22:901–911

  45. 45.

    Emami KH, Burke TW, Smale ST (1998) Sp1 activation of a tata-less promoter requires a species-specific interaction involving transcription factor iid. Nucl Acids Res 26:839–846

  46. 46.

    Pugh BF, Tjian R (1991) Transcription from a tata-less promoter requires a multisubunit tfiid complex. Genes Dev 5:1935–1945

Download references


This study was partly funded by Department of Biotechnology (DBT), Ministry of Science & Technology, Government of India, New Delhi, India.

Author information

Correspondence to Hirak Kumar Barman.

Additional information

Chinmayee Mohapatra and Swagat Kumar Patra have equally contributed to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 64 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mohapatra, C., Patra, S.K., Panda, R.P. et al. Gene structure and identification of minimal promoter of Pou2 expressed in spermatogonial cells of rohu carp, Labeo rohita . Mol Biol Rep 41, 4123–4132 (2014). https://doi.org/10.1007/s11033-014-3283-6

Download citation


  • Pou2
  • Gene Promoter
  • Gene expression
  • Spermatogonial cell
  • Labeo rohita
  • Rohu