The Histochemical Journal

, Volume 27, Issue 6, pp 431–439 | Cite as

Ultrastructural localization of PGP 9.5 and ubiquitin immunoreactivities in rat ductus epididymidis epithelium

  • R. Martín
  • L. Santamaría
  • B. Fraile
  • R. Paniagua
  • J. M. Polak


The distribution of protein gene product 9.5 (PGP) and ubiquitin in the spermatozoa and epithelial cells in the different regions of the rat duetus epididymidis (proximal caput, distal caput, corpus and cauda) was studied by Western blotting analyses and electron microscopical immunogold labelling. Western blotting analyses showed that the PGP immunoreactive band was very intense in the caput and cauda epididymidis and almost irrelevant in the corpus, while the ubiquitin immunoreactive band was intense in the distal caput and cauda. No ubiquitin immunoreactive band was observed in the proximal caput and only a very weak band was seen in the corpus. The results of electron microscopical immunogold labelling varied from one epididymal region to another. The proximal caput epididymidis presented immunoreaction to PGP in the rough endoplasmic reticulum, cytosol, mitochondria and microvilli of most principal cells, and in the cytosol, rough endoplasmic reticulum and mitochondria of most basal cells. No ubiquitin immunoreaction was observed in this epididymal region. In the distal caput epididymidis, PGP immunoreactivity was detected in some principal and basal cells in the same intracellular locations as described in the proximal caput. In this region, ubiquitin immunoreactivity appears in the apical cytosol and mitochondria of principal cells. The corpus epididymidis showed no immunoreaction to PGP or ubiquitin. In the cauda epididymidis, immunostaining to PGP was observed in most clear cells and in isolated principal cells. The intracellular location of PGP in both cell types was the cytosol, mitochondria and microvilli. Ubiquitin immunoreactivity was detected in the perinuclear cytosol and mitochondria — but not in the digestive vacuoles — of some clear cells. Scanty ubiquitin immunolabelling was also found in the microvilli, cytosol and mitochondria of some principal cells. The head of the spermatozoa present in the ductal lumen in all epididymal regions immunoreacted intensely to PGP. Ubiquitin was detected in the intermediate piece and residual cytoplasm of intraluminal spermatozoa present in the corpus and cauda epididymidis. These findings suggest that a non-ubiquitinated PGP irnrnunoreactive protein is secreted by the principal cells in caput epididymidis and binds the spermatozoon heads. It is possible that the clear cells of the cauda epididymidis secrete the ubiquitin that binds to spermatozoon tail.


Cytosol Clear Cell Rough Endoplasmic Reticulum Intracellular Location Principal Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Agell,N. &Mezquita,C. (1988) Cellular content of ubiquitin and formation of ubiquitin conjugates during chicken spermatogenesis.Biochem. J. 250, 883–9.PubMedGoogle Scholar
  2. Anderson,V. E. R.,Hajimohammadreza,I.,Gallo,J. M.,Anderson,B. H.,Uney,J.,Brown,A. W.,Nolan,C. C.,Cavanagh,J. B. &Leigh,P. N. (1992) Ubiquitin, PGP 9.5 and dense body formation in trimethyltin intoxication: differential neuronal responses to chemically induced cell damage.Neuropathol. Appl. Neurobiol. 1, 360–75.Google Scholar
  3. Cooper,T. G. (1990) Secretory proteins from the epididymis and their clinical relevance.Andrologia (suppl)22, 155–65.Google Scholar
  4. Crooke,S. T.,Mirabelli,C. K.,Ecker,D. J.,Butt,T. R.,Jonnalagadda,S.,Dixon,S.,Müller,L.,Brown,F.,Weber,P. &Monia,B. P. (1990) Studies on the structure and function of ubiquitin. InProtein Design and the Development of New Therapeutics and Vaccines (edited byHook,J. B. &Poste,G.), pp. 425–45. New York: Plenum Press.Google Scholar
  5. Doran,J. F.,Jackson,P.,Kynoch,P. A. M. &Thompson,R. J. (1983) Isolation of PGP 9.5, a new human neuron-specific protein detected by high resolution of two-dimensional electrophoresis.J. Neurochem. 40, 1542–7.PubMedGoogle Scholar
  6. Francavilla,S.,DeMartino,C.,Barcelona,P. S. &Natali,P. G. (1983) Ultrastructural and immunohistochemical studies of rat epididymis.Cell Tissue Res. 233, 523–37.CrossRefPubMedGoogle Scholar
  7. Fried,V.,Smith,H. T.,Hildebrandt,E. &Weiner,K. (1987) Ubiquitin has intrinsic proteolytic activity: implication for cellular regulation.Proc. Natl Acad. Sci. USA 84, 3685–9.PubMedGoogle Scholar
  8. Goldstein,G.,Scheid,M.,Hammerling,U.,Boyse,E. A.,Schlesinger,D. H. &Niall,H. D. (1975) Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells.Proc. Natl Acad. Sci. USA 72, 11–5.PubMedGoogle Scholar
  9. Gulbenkian,S.,Wharton,J. &Polar,J. M. (1987) The visualisation of cardiovascular innervation in the guinea pig using an antiserum to protein gene product 9.5 (PGP 9.5).J. Autonom. Nerv. Syst. 18, 235–47.CrossRefGoogle Scholar
  10. Hoffer,A. P.,Hamilton,D. W. &Fawcett,D. W. (1973) The ultrastructure of the principal cells and intraepithelial leukocytes in the initial segment of the rat epididymis.Anat. Rec. 175, 169–202.PubMedGoogle Scholar
  11. Jackson,P.,Thompson,V. M. &Thompson,R. J. (1985) A comparison of the evolutionary distribution of the two neuroendocrine markers, neuron-specific enolase and protein gene product 9.5.J. Neurochem. 45, 185–90.PubMedGoogle Scholar
  12. Kent,C. &Clarke,P. J. (1991) The immunolocalisation of the neuroendocrine specific protein PGP 9.5 during neurogenesis in the rat.Dev. Brain Res. 58, 147–50.CrossRefGoogle Scholar
  13. Kent,C. &Rowe,H. L. (1992) The immunolocalisation of ubiquitin carboxyl-terminal hydrolase (PGP 9.5) in developing paraneurons in the rat.Dev. Brain Res. 68, 241–6.CrossRefGoogle Scholar
  14. Laemmli,U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.CrossRefPubMedGoogle Scholar
  15. Laszlo,L.,Doherty,F. J.,Watson,A.,Self,T.,Landon,N.,Lowe,J. &Mayer,J. (1991) Immunogold localization of ubiquitin-protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils.FEBS Lett. 279, 175–8.PubMedGoogle Scholar
  16. Lippert, T. H., Seeger, H., Schieferstein, G. & Voelter, W. (1993) Immunoreactive ubiquitin in human seminal plasma.J. Androl. 14, 130–1.PubMedGoogle Scholar
  17. Lowe,J.,Blanchard,A.,Morrell,K.,Lennox,G.,Reynolds,L.,Billett,M.,Landon,M. &Mayer,J. (1988) Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man including those of Parkinson's disease, Pick's disease, and ALzheimer's disease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease.J. Pathol. 155, 9–15.CrossRefPubMedGoogle Scholar
  18. Manetto,V.,Abdul-karim,F. W.,Pery,G.,Tabton,M. &Autilio-gambetti,P. (1989) Selective presence of ubiquitin in intracellular inclusions.Am. J. Pathol. 134, 505–13.PubMedGoogle Scholar
  19. Mayer,R. J.,Lowe,J.,Landon,M.,Mcdermott,H.,Tuckwell,J.,Doherty,F. &Laszlo,L. (1991) Ubiquitin and lysosome system: molecular immunopathology reveals connection.Biomed. Biochim. Acta 50, 4–6.Google Scholar
  20. Moore,H. D. M. &Bedford,J. M. (1979) The differential absorptive activity of epithelial cells of the rat epididymis before and after castration.Anat. Rec. 193, 313–28.PubMedGoogle Scholar
  21. Murti,K. G.,Smith,H. T. &Fried,V. A. (1988) Ubiquitin is a component of the microtubule network.Proc. Natl Acad. Sci. USA 85, 3019–23.PubMedGoogle Scholar
  22. Ohta,M.,Marceau,N.,Perry,G.,Manetto,V.,Gambetti,P.,Autilio-gambetti,L.,Metuzls,J.,Kawahara,H.,Cadrin,M. &French,S. W. (1988) Ubiquitin is present in cytokeratin intermediate filaments and Mallory bodies of hepatocytes.Lab. Invest. 59, 848–56.PubMedGoogle Scholar
  23. Properzi,G.,Cordeschi,G. &Francavilla,S. (1992) Post-natal development and distribution of peptide containing nerves in the genital system of the male rat. An immunohistochemical study.Histochemistry 97, 61–9.CrossRefPubMedGoogle Scholar
  24. Rode,J.,Dhillon,A. P.,Doran,J. J.,Jackson,P. &Thompson,R. J. (1985) PGP 9.5 a new marker for human neuroendocrine tumors.Histopathology 9, 147–58.PubMedGoogle Scholar
  25. Santamaría,L.,Martín,R.,Paniagua,R.,Fraile,B.,Nistal,M.,Terenghi,G. &Polak,J. M. (1993) Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium.Histochemistry 100, 131–8.CrossRefPubMedGoogle Scholar
  26. Schwartz,A. L.,Ciechanover,A.,Brandt,R. A. &Greuze,R. J. (1988) Immunoelectron microscopic localization of ubiquitin in hepatoma cells.EMBO J. 7, 2961–6.PubMedGoogle Scholar
  27. Towbin,H.,Staehlin,T. &Gordon,J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl Acad. Sci. USA 76, 4350–4.PubMedGoogle Scholar
  28. Tuner,T. T. (1991) Spermatozoa are exposed to a complex microenvironment as they traverse the epididymis. InThe Male Germ Cell. Spermatogonium to Fertilization. (edited byRobarie,E.), pp. 364–87. New York: Nyas.Google Scholar
  29. Wilkinson,K. D.,Lee,K.,Deshpande,S.,Duerken-Hughes,P.,Boss,J. M. &Pohl,J. (1989) The neuronspecific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase.Science 246, 670–3.PubMedGoogle Scholar
  30. Wilkinson,K. D.,Deshpande,S. &Larsen,C. N. (1992) Comparisons of neuronal (PGP 9.5) and non-neuronal ubiquitin C-terminal hydrolases.Biochem. Soc. Trans. 20, 631–7.PubMedGoogle Scholar

Copyright information

© Chapman & Hall 1995

Authors and Affiliations

  • R. Martín
    • 1
    • 2
  • L. Santamaría
    • 1
  • B. Fraile
    • 3
  • R. Paniagua
    • 3
  • J. M. Polak
    • 4
  1. 1.Department of Morphology (Histology)School of Medicine, Autonomous UniversityMadridSpain
  2. 2.Department of Histopathology (Hospital de la Princesa)School of Medicine, Autonomous University of MadridSpain
  3. 3.Department of Cell Biology and GeneticsUniversity of Alcalá de HenaresMadridSpain
  4. 4.Department of HistochemistryRoyal Postgraduate Medical SchoolLondonUK

Personalised recommendations