Skip to main content

The expression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of rats with a dihydrotestosterone (DHT) deficiency

Abstract

In our previous studies, we showed that a finasteride-induced DHT deficiency may cause changes in the morphology of the seminiferous epithelium without any morphological alteration of the epididymis. In this study, we demonstrated the constitutive immunoexpression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of Wistar rats treated with finasteride for 28 days (the duration of two cycles of the seminiferous epithelium) and 56 days (the duration of one spermatogenesis). We noted that a 56-day finasteride treatment mainly caused a decrease in the level of circulating DHT, as well as a statistically insignificant decrease in the level of T. The hormone deficiency also led to a change in the iNOS immnoexpression in the testis and epididymis of the finasteride-treated rats. In vitro, DHT did not modify NO production by the epithelial cells of the caput epididymis even when stimulated with LPS and IFNγ, but it did give rise to an increase in NO production by the epithelial cells of the cauda epididymis without the stimulation. DHT did not have a statistically significant influence on estradiol production by cultured, LPS- and IFNγ-stimulated epithelial cells from the caput and cauda epididymis. In conclusion, our data clearly indicates that a finasterideinduced DHT deficiency intensifies the constitutive expression of iNOS in most rat testicular and epididymal cells, so it can be expected that the expression of inducible nitric oxide synthase (iNOS) could be regulated by DHT. On the other hand, the profile of the circulating DHT and T levels strongly suggests that the regulation of constitutive iNOS expression is complex and needs more detailed study.

Abbreviations

5α-red:

5α-reductase

5α-red1:

5α-reductase type 1

5α-red2:

5α-reductase type 2

AR:

androgen receptor

DHT:

dihydrotestosterone

E2 :

estradiol

ELFA:

enzyme linked fluorescent assay

ERα:

estrogen receptor α

ERβ:

estrogen receptor β

IFNγ:

interferon gamma

LC:

Leydig cell

LPS:

lipopolysaccharide

NO:

nitric oxide

NO2− :

nitrite

NOS:

nitric oxide synthase

eNOS:

endothelial nitric oxide synthase

iNOS:

inducible nitric oxide synthase

nNOS:

neuronal nitric oxide synthase

PS:

pachytene spermatocyte

preLS:

preleptotene spermatocyte

RIA:

radioimmunoassay

RT-PCR:

reverse transcriptase-polymerase chain reaction

SC:

Sertoli cell

SD:

spermatid

SG:

spermatogonium

T:

testosterone

References

  1. Moncada, S., Palmer, R.M. and Higgs, E.A. Nitric oxide: physiology, phatophysiology, and pharmacology. Pharmacol. Rev. 43 (1991) 109–134.

    PubMed  CAS  Google Scholar 

  2. Xie, Q. and Nathan, C. The high-output nitric oxide pathway: role and regulation. J. Leukoc. Biol. 56 (1994) 576–582.

    PubMed  CAS  Google Scholar 

  3. Knowles, R.G. and Moncada, S. Nitric oxide synthases in mammals. Biochem. J. 298 (1994) 249–258.

    PubMed  CAS  Google Scholar 

  4. Lowenstein, C.J., Glatt, C.S., Bredt, D.S. and Snyder, S.H. Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Proc. Natl. Acad. Sci. USA. 89 (1992) 6711–6715.

    PubMed  Article  CAS  Google Scholar 

  5. Kroncke, K.D., Fehsel, K. and Kolb-Bachofen, V. Inducible nitric oxide synthase and its product nitric oxide, a small molecule with complex biological activities. Biol. Chem. Hoppe. Seyler. 376 (1995) 327–343.

    PubMed  CAS  Google Scholar 

  6. Burnett, A.L., Ricker, D.D., Chamness, S.L., Maguire, M.P., Crone, J.K., Bredt, D.S., Snyder, S.H. and Chang, T.S. Localization of nitric oxide synthase in the reproductive organs of the male rat. Biol. Reprod. 52 (1995) 1–7.

    PubMed  Article  CAS  Google Scholar 

  7. Ehren, I., Adolfsson, J. and Wiklund, N.P. Nitric oxide synthase activity in the human urogenital tract. Urol. Res. 22 (1994) 287–290.

    PubMed  Article  CAS  Google Scholar 

  8. Stephan, J.P., Guillemois, C., Jegou, B. and Bauche, F. Nitric oxide production by Sertoli cells in response to cytokines and lipopolysaccharide. Biochem. Biophys. Res. Commun. 213 (1995) 218–224.

    PubMed  Article  CAS  Google Scholar 

  9. Tatsumi, N., Fujisawa, M., Kanazaki, M., Okuda, Y., Okada, H., Arakawa, S. and Kamidono, S. Nitric oxide production by cultured rat Leydig cells. Endocrinology 138 (1997) 994–998.

    PubMed  Article  CAS  Google Scholar 

  10. Weissman, B.A., Niu, E., Ge, R., Sottas, C.M., Holmes, M., Hutson, J.C. and Hardy, M.P. Paracrine modulation of androgen synthesis in rat leydig cells by nitric oxide. J. Androl. 26 (2005) 369–378.

    PubMed  Article  CAS  Google Scholar 

  11. Wiszniewska, B., Kurzawa, R., Ciechanowicz, A. and Machaliński, B. Inducible nitric oxide synthase in the epithelial epididymal cells of the rat. Reprod. Fertil. Dev. 9 (1997) 789–794.

    PubMed  Article  CAS  Google Scholar 

  12. Kurzawa, R., Barcew-Wiszniewska, B. and Skowron, J. Rat epididymal epithelial cells produce nitric oxide upon concomitant lipopolysaccharide and interferon-gamma stimulation. Folia Histochem. Cytobiol. 4 (1996) 85–86.

    Google Scholar 

  13. O’Bryan, M.K., Schlatt, S., Gerdprasert, O., Phillips, D.J., de Kretser, D.M. and Hedger, M.P. Inducible nitric oxide synthase in the rat testis: evidence for potential roles in both normal function and inflammationmediated infertility. Biol. Reprod. 63 (2000) 1285–1293.

    PubMed  Article  Google Scholar 

  14. Ha, T.Y., Kim, H.S. and Shin, T. Expression of constitutive endothelial, neuronal and inducible nitric oxide synthase in the testis and epididymis of horse. J. Vet. Med. Sci. 66 (2004) 351–356.

    PubMed  Article  CAS  Google Scholar 

  15. Kim, H.C., Byun, J.S., Lee, T.K., Jeong, C.W., Ahn, M. and Shin, T. Expression of nitric oxide synthase isoform in the testes of pigs. Anat. Histol. Embryol. 36 (2007) 135–138.

    PubMed  Article  CAS  Google Scholar 

  16. Sun, L., Ren, Y.P., Jiang, W., Zhang, M.Y. and Hou, Q.Y. Expression and role of nitric oxide synthase in the testis and epididymis of Macaca fascicularis. Zhongua Nan Ke Xu 12 (2006) 876–878.

    CAS  Google Scholar 

  17. Jin, Y. and Penning, T.M. Steroid 5alpha-reductases and 3alphahydroxysteroid dehydrogenases: key enzymes in androgen metabolism. Best Pract. Res. Clin. Endocrinol. Metab. 15 (2001) 79–94.

    PubMed  Article  CAS  Google Scholar 

  18. Russell, D.W. and Wilson, J.D. Steroid 5α-reductase: two genes/two enzymes. Annu. Rev. Biochem. 63 (1994) 25–61.

    PubMed  CAS  Google Scholar 

  19. Viger, R.S. and Robaire, B. Steady state steroid 5 alpha-reductase messenger ribonucleic acid levels and immunocytochemical localization of the type 1 protein in the rat testis during postnatal development. Endocrinology 136 (1995) 5409–5415.

    PubMed  Article  CAS  Google Scholar 

  20. Pratis, K., O’Donnell, L., Ooi, G.T., McLachlan, R.I. and Robertson, D.M. Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis. J. Steroid. Biochem. Mol. Biol. 75 (2000) 75–82.

    PubMed  Article  CAS  Google Scholar 

  21. Viger, R.S. and Robaire, B. The mRNAs for the steroid 5α-reductase izoenzymes, types 1 and 2, are differentlly regulated in the rat epididymis. J. Androl. 17 (1996) 27–34.

    PubMed  CAS  Google Scholar 

  22. Mahony, M.C., Swanlund, D.J., Billeter, M., Roberts, K.P. and Pryor, J.L. Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the human epididymis. Fertil. Steril. 69 (1998) 1116–1121.

    PubMed  Article  CAS  Google Scholar 

  23. Robaire, B. and Henderson, N.A. Actions of 5alpha-reductase inhibitors on the epididymis. Mol. Cell. Endocrinol. 250 (2006) 190–195.

    PubMed  Article  CAS  Google Scholar 

  24. Metcalf, B.W., Levy, M.A. and Holt, D.A. Inhibitors of steroid 5alphareductase in benign prostatic hyperplasia, male pattern baldness and acne. Trends Pharmacol. Sci. 10 (1989) 491–495.

    PubMed  Article  CAS  Google Scholar 

  25. George, F.W. Androgen metabolism in the prostate of the finasteridetreated, adult rat: a possible explanation for the differential action of testosterone and 5α-dihydrotestosterone during development of male urogenital tract. Endocrinology 138 (1997) 871–877.

    PubMed  Article  CAS  Google Scholar 

  26. Vaughan, E.D. Long-term experience with 5-alpha-reductase inhibitors. Rev. Urol. 5 (2003) S22–S27.

    PubMed  Google Scholar 

  27. Kolasa, A., Marchlewicz, M., Wenda-Różewicka, L. and Wiszniewska, B. Morphology of the testis and the epididymis in rats with dihydrotestosterone (DHT) deficiency. Rocz. Akad. Med. Bialymst. 49 (2004) 117–119.

    PubMed  Google Scholar 

  28. Wiszniewska, B. Steroidogenic characteristics of in vitro cultured epididymal epithelial cells of the rat. Reprod. Biol. 1 (2001) 60–66.

    PubMed  CAS  Google Scholar 

  29. Ding, A.H., Nathan, C.F. and Stuehr, D.J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J. Immunol. 141 (1988) 2407–2412.

    PubMed  CAS  Google Scholar 

  30. Zini, A., Abitbol, J., Girardi, S.K., Schulsinger, D., Goldstein, M. and Schlegel, P.N. Germ cell apoptosis and endothelial nitric oxide synthase (eNOS) expression following ischemia-reperfusion injury to testis. Arch. Androl. 41 (1998) 57–65.

    PubMed  Article  CAS  Google Scholar 

  31. Lee, N.P. and Cheng, C.Y. Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3′,5′-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study. Endocrinology 144 (2003) 3114–3129.

    PubMed  Article  CAS  Google Scholar 

  32. Lue, Y., Sinha-Hikim, A.P., Wang, C., Leung, A. and Swerdloff, R.S. Functional role of inducible nitric oxide synthase in the induction of male germ cell apoptosis, regulation of sperm number and determination of testis size: evidence from null mutant mice. Endocrinology 144 (2003) 3092–3100.

    PubMed  Article  CAS  Google Scholar 

  33. Zini, A., O’Bryan, M.K., Magid, M.S. and Schlegel, P.N. Immunohistochemical localization of endothelial nitric oxide synthase in human testis, epididymis, and vas deferens suggests a possible role for nitric oxide in spermatogenesis, germ maturation, and programmed cell death. Biol. Reprod. 55 (1996) 935–941.

    PubMed  Article  CAS  Google Scholar 

  34. Gerdprasert, O., O’Bryan, M.K., Muir, J.A., Caldwell, A.M., Schlatt, S., de Krester, D.M. and Hedger, M.P. The response of testicular leucocytes to lipopolysaccharide-induced inflammation: further evidence heterogeneity of the testicular macrophage population. Cell. Tissue Res. 308 (2002) 277–285.

    PubMed  Article  CAS  Google Scholar 

  35. Welsh, C., Watson, M.E., Poth, M., Hong, T. and Frrancis, G.L. Evidence to suggest nitric oxide is an interstitial regulator of Leydig Cell steroidogenesis. Metabolism 44 (1995) 234–238.

    Article  Google Scholar 

  36. Wiszniewska, B. Primary culture of the rat epididymal epithelial cells as a source of oestrogen. Andrologia 34 (2002) 180–187.

    PubMed  Article  CAS  Google Scholar 

  37. Chamness, S.L., Ricker, D.D., Crone, J.K., Dembeck, C.L., Maguire, M.P., Burnett, A.L. and Chang, T.S. The effect of androgen on nitric oxide synthase in the male reproductive tract of the rat. Fertil. Steril. 63 (1995) 1101–1107.

    PubMed  CAS  Google Scholar 

  38. Carreau S. The testicular aromatase: from gene to physiological role. Reprod. Biol. 2 (2002) 5–12.

    PubMed  Google Scholar 

  39. Lambard, S., Galeraud-Denis, I., Saunders, P.T. and Carreau, S. Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors. J. Mol. Endocrinol. 32 (2004) 279–289.

    PubMed  Article  CAS  Google Scholar 

  40. Carpino, A., Romeo, F. and Rago, V. Aromatase immunolocalization in human ductuli efferentes and proximal ductus epididymis. J. Anat. 204 (2004) 217–220.

    PubMed  Article  CAS  Google Scholar 

  41. Shayu, D. and Rao, A.J. Expression of functional aromatase in the epididymis: role of androgens and LH in modulation of expression and activity. Mol. Cell. Endocrinol. 249 (2006) 40–50.

    PubMed  CAS  Google Scholar 

  42. Snyder, G.D., Holmes, R.W., Bates, J.N. and Van Voorhis, B.J. Nitric oxide inhibits aromatase activity: mechanism of action. J. Steroid. Biochem. Mol. Biol. 58 (1996) 63–69.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Agnieszka Kolasa or Barbara Wiszniewska.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kolasa, A., Marchlewicz, M., Kurzawa, R. et al. The expression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of rats with a dihydrotestosterone (DHT) deficiency. Cell Mol Biol Lett 14, 511–527 (2009). https://doi.org/10.2478/s11658-009-0019-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-009-0019-z

Key words

  • iNOS immunoexpression
  • DHT-deficiency
  • Testis
  • Epididymis
  • Rat