Reproductive Sciences

, Volume 19, Issue 2, pp 190–201 | Cite as

Interaction Between Smad1 and p97/VCP in Rat Testis and Epididymis During the Postnatal Development

  • Sevil CayliEmail author
  • Fikret Erdemir
  • Seda Ocaklı
  • Bahadir Ungor
  • Hakan Kesici
  • Tamer Yener
  • Huseyin Aslan
Original Articles


Members of the bone morphogenetic proteins (BMPs) superfamily are expressed in the testis and epididymis and are believed to have different biological functions during testicular and epididymal development. Smad1 is one of the signal transducers of BMP signaling and binds to several proteins involved in ubiquitin–proteasome system (UPS). Valosin-containing protein (p97/VCP) is required for the degradation of some UPS substrates. Although p97/VCP has been indicated in different cellular pathways, its association with BMP signaling in male reproductive system has not been elucidated. The aim of the present study was to investigate the cellular localization of Smad1, phospho-Smad1, and p97/VCP and the interaction of proteins in the postnatal rat testis and epididymis. Testicular and epididymal tissues from 5-, 15- and 60-day-old rats were examined by immunohistochemistry, immunofluorescence, Western blotting, and immunoprecipitation techniques. In 5-day-old rat testis, Smad1, phospho-Smad1, and p97/VCP were mainly expressed in gonocytes. In 15- and 60-day-old rat testis, proteins were overlapped in spermatogonia, Sertoli cells, and spermatocytes. Expression of proteins in the epithelial cells of epididymis was gradually increased from 5 to 15 days of age. Smad1 and phospho-Smad1 expressions showed uniformity in the different regions of epididymis, however p97/VCP immunoreactivity was higher only in caput epididymis compared to corpus and cauda epididymis in 15- and 60-day-old rat epididymis. Co-immunoprecipitation experiments further confirmed the Smad1-p97/VCP and p-Smad1-p97/VCP interactions. The overlap between Smad1 and p97/VCP expressions in the postnatal rat testis and epididymis suggests that p97/VCP may play important roles in mediating BMP signaling during spermatogenesis.


Smad1 phospho-Smad1 p97/VCP rat testis epididymis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Massague J. Transforming growth factor-alpha. A model for membrane-anchored growth factors. J Biol Chem. 1990;265(35): 21393–21396.Google Scholar
  2. 2.
    Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors. 2004;22(4):233–241.Google Scholar
  3. 3.
    Lawrence DA. Transforming growth factor-beta: a general review. Eur Cytokine Netw. 1996;7(3):363–374.Google Scholar
  4. 4.
    Brandes ME, Mai UE, Ohura K, Wahl SM. Type I transforming growth factor-beta receptors on neutrophils mediate chemotaxis to transforming growth factor-beta. J Immunol. 1991;147(5): 1600–1606.Google Scholar
  5. 5.
    Zhu HJ, Burgess AW. Regulation of transforming growth factor-beta signaling. Mol Cell Biol Res Commun. 2001;4(6):321–330.Google Scholar
  6. 6.
    Mather JP, Attie KM, Woodruff TK, Rice GC, Phillips DM. Activin stimulates spermatogonial proliferation in germ-Sertoli cell cocultures from immature rat testis. Endocrinology. 1990; 127(6):3206–3214.Google Scholar
  7. 7.
    Matzuk MM, Kumar TR, Shou W, et al. Transgenic models to study the roles of inhibins and activins in reproduction, oncogenesis, and development. Recent Prog Horm Res. 1996;51:123–154; discussion 55–57.Google Scholar
  8. 8.
    Zhao GQ, Deng K, Labosky PA, Liaw L, Hogan BL. The gene encoding bone morphogenetic protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse. Genes Dev. 1996;10(13):1657–1669.Google Scholar
  9. 9.
    Narula A, Kilen S, Ma E, Kroeger J, Goldberg E, Woodruff TK. Smad4 overexpression causes germ cell ablation and leydig cell hyperplasia in transgenic mice. Am J Pathol. 2002;161(5): 1723–1734.Google Scholar
  10. 10.
    Dunker N, Krieglstein K. Targeted mutations of transforming growth factor-beta genes reveal important roles in mouse development and adult homeostasis. Eur J Biochem. 2000;267(24): 6982–6988.Google Scholar
  11. 11.
    Chang H, Lau AL, Matzuk MM. Studying TGF-beta superfamily signaling by knockouts and knockins. Mol Cell Endocrinol. 2001; 180(1–2):39–46.Google Scholar
  12. 12.
    Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–791.Google Scholar
  13. 13.
    Itoh S, Itoh F, Goumans MJ, Ten Dijke P. Signaling of transforming growth factor-beta family members through Smad proteins. Eur J Biochem. 2000;267(24):6954–6967.Google Scholar
  14. 14.
    ten Dijke P, Hill CS. New insights into TGF-beta-Smad signalling. Trends Biochem Sci. 2004;29(5):265–273.Google Scholar
  15. 15.
    Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003;113(6):685–700.Google Scholar
  16. 16.
    Itman C, Loveland KL. SMAD expression in the testis: an insight into BMP regulation of spermatogenesis. Dev Dyn. 2008;237(1): 97–111.Google Scholar
  17. 17.
    Hu J, Zhang YQ, Liu XP, Wang RA, Jin Y, Xu RJ. Expression and localization of Smad1, Smad2 and Smad4 proteins in rat testis during postnatal development. Asian J Androl. 2003;5(1):51–55.Google Scholar
  18. 18.
    Xu J, Beyer AR, Walker WH, McGee EA. Developmental and stage-specific expression of Smad2 and Smad3 in rat testis. J Androl. 2003;24(2):192–200.Google Scholar
  19. 19.
    Pellegrini M, Grimaldi P, Rossi P, Geremia R, Dolci S. Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation. J Cell Sci. 2003;116(pt 16):3363–3372.Google Scholar
  20. 20.
    Luukko K, Ylikorkala A, Makela TP. Developmentally regulated expression of Smad3, Smad4, Smad6, and Smad7 involved in TGF-beta signaling. Mech Dev. 2001;101(1–2):209–212.Google Scholar
  21. 21.
    Adams J. The proteasome: structure, function, and role in the cell. Cancer Treat Rev. 2003;29(suppl 1):3–9.Google Scholar
  22. 22.
    Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol. 1995;7(2): 215–223.Google Scholar
  23. 23.
    Inoue Y, Imamura T. Regulation of TGF-beta family signaling by E3 ubiquitin ligases. Cancer Sci. 2008;99(11):2107–2112.Google Scholar
  24. 24.
    Zhang S, Fei T, Zhang L, et al. Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation. Mol Cell Biol. 2007; 27(12):4488–4499.Google Scholar
  25. 25.
    Murakami G, Watabe T, Takaoka K, Miyazono K, Imamura T. Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads. Mol Biol Cell. 2003;14(7): 2809–2817.Google Scholar
  26. 26.
    Kavsak P, Rasmussen RK, Causing CG, et al. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell. 2000;6(6):1365–1375.Google Scholar
  27. 27.
    Dai RM, Li CC. Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat Cell Biol. 2001;3(8):740–744.Google Scholar
  28. 28.
    Wojcik C, Yano M, DeMartino GN. RNA interference of Valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis. J Cell Sci. 2004;117(pt 2):281–292.Google Scholar
  29. 29.
    Wojcik C, Rowicka M, Kudlicki A, et al. Valosin-containing protein (p97) is a regulator of endoplasmic reticulum stress and of the degradation of N-end rule and ubiquitin-fusion degradation pathway substrates in mammalian cells. Mol Biol Cell. 2006;17(11): 4606–4618.Google Scholar
  30. 30.
    Dai RM, Chen E, Longo DL, Gorbea CM, Li CC. Involvement of Valosin-containing protein, an ATPase Co-purified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkappaBalpha. J Biol Chem. 1998;273(6): 3562–3573.PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Meusser B, Hirsch C, Jarosch E, Sommer T. ERAD: the long road to destruction. Nat Cell Biol. 2005;7(8):766–772.Google Scholar
  32. 32.
    Hetzer M, Meyer HH, Walther TC, Bilbao-Cortes D, Warren G, Mattaj IW. Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly. Nat Cell Biol. 2001;3(12): 1086–1091.PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Sati L, Seval-Celik Y, Demir R. Lung surfactant proteins in the early human placenta. Histochem Cell Biol. 133(1):85–93.PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Itman C, Mendis S, Barakat B, Loveland KL. All in the family: TGF-beta family action in testis development. Reproduction. 2006;132(2):233–246.Google Scholar
  35. 35.
    Moreno SG, Attali M, Allemand I, et al. TGFbeta signaling in male germ cells regulates gonocyte quiescence and fertility in mice. Dev Biol. 342(1):74–84.Google Scholar
  36. 36.
    Sipila P, Jalkanen J, Huhtaniemi IT, Poutanen M. Novel epididymal proteins as targets for the development of posttesticular male contraception. Reproduction. 2009;137(3): 379–389.Google Scholar
  37. 37.
    Patil AA, Cai Y, Sang Y, Blecha F, Zhang G. Cross-species analysis of the mammalian beta-defensin gene family: presence of syntenic gene clusters and preferential expression in the male reproductive tract. Physiol Genomics. 2005;23(1):5–17.Google Scholar
  38. 38.
    Ellerman DA, Busso D, Maldera JA, Cuasnicú PS. Immunocontraceptive properties of recombinant sperm protein DE: implications for the development of novel contraceptives. Fertil Steril. 2008;89(1):199–205.Google Scholar
  39. 39.
    Zhou CX, Zhang YL, Xiao L, et al. An epididymis-specific beta-defensin is important for the initiation of sperm maturation. Nat Cell Biol. 2004;6(5):458–464.Google Scholar
  40. 40.
    Wotton D, Lo RS, Lee S, Massagué J. A Smad transcriptional corepressor. Cell. 1999;97(1):29–39.Google Scholar
  41. 41.
    Lin X, Liang M, Feng XH. Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling. J Biol Chem. 2000;275(47): 36818–36822.Google Scholar
  42. 42.
    Ebisawa T, Fukuchi M, Murakami G, et al. Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J Biol Chem. 2001;276(16): 12477–12480.Google Scholar
  43. 43.
    Gruendler C, Lin Y, Farley J, Wang T. Proteasomal degradation of Smad1 induced by bone morphogenetic proteins. J Biol Chem. 2001;276(49):46533–46543.Google Scholar
  44. 44.
    Vij N. AAA ATPase p97/VCP: cellular functions, disease and therapeutic potential. J Cell Mol Med. 2008;12(6A): 2511–2518.PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Kim BC, Lee HJ, Park SH, et al. Jab1/CSN5, a component of the COP9 signalosome, regulates transforming growth factor beta signaling by binding to Smad7 and promoting its degradation. Mol Cell Biol. 2004;24(6):2251–2262.Google Scholar
  46. 46.
    Wan M, Cao X, Wu Y, et al. Jab1 antagonizes TGF-beta signaling by inducing Smad4 degradation. EMBO Rep. 2002;3(2):171–176.Google Scholar

Copyright information

© Society for Reproductive Investigation 2012

Authors and Affiliations

  • Sevil Cayli
    • 1
    Email author
  • Fikret Erdemir
    • 2
  • Seda Ocaklı
    • 1
  • Bahadir Ungor
    • 3
  • Hakan Kesici
    • 1
  • Tamer Yener
    • 4
  • Huseyin Aslan
    • 1
  1. 1.Department of Histology and EmbryologyGaziosmanpasa UniversityTokatTurkey
  2. 2.Department of UrologyGaziosmanpasa UniversityTokatTurkey
  3. 3.Department of AnatomyGaziosmanpasa UniversityTokatTurkey
  4. 4.Experimental Animal CenterGaziosmanpasa UniversityTokatTurkey

Personalised recommendations