Molecular and Cellular Biochemistry

, Volume 126, Issue 1, pp 1–7 | Cite as

The antioxidant defense system of isolated guinea pig Leydig cells

  • Mark A. Kukucka
  • Hara P. Misra


Utilization of highly enriched preparations of steroidogenic Leydig cells have proven invaluable for studying the direct effects of various hormones and agents on Leydig cell functionin vitro. However, recent work indicates that isolated Leydig cells are often subjected to oxygen (O2) toxicity when cultured at ambient (19%) oxygen concentrations. Because intracellular antioxidants play an important role in protecting cells against oxygen toxicity, we have investigated the intracellular antioxidant defense system of isolated Leydig cells. The cellular levels of several antioxidants including catalase, glucose-6-phosphate dehydrogenase (G-6-PDH), superoxide dismutase (SOD) of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione reductase and total glutathione were quantitated using enriched populations of Leydig cells isolated from adult male guinea pig testes. Compared to whole testicular homogenates, Leydig cells contained significantly (P<0.01) less G-6-PDH, total SOD, glutathione reductase and total glutathione, but significantly (P<0.001) more glutathione peroxidase. Compared to hepatic values previously reported in the guinea pig, Leydig cells contain nearly 400 times less catalase, about 14 times less glutathione peroxidase and almost 11 times less glutathione reductase. Since G-6-PDH and glutathione reductase are both necessary to regenerate reduced gluthathione (GSH) which couples with glutathione peroxidase to breakdown hydrogen peroxide (H2O2) under normal conditions, it is plausible that the oxygen toxicity observed in isolated Leydig cells is due to the intracellular accumulation of H2O2. Using the dichlorofluorescin diacetate (DCF-DA) assay, we found that Leydig cells incubated in the presence of 19% O2 produced significantly (P<0.001) higher levels of H2O2 with time in culture compared to Leydig cells maintained at 3% O2. These results support the hypothesis that the increased susceptibility of isolated Leydig cells to oxygen toxicity may be due, in part, to decreased amounts of certain antioxidant defenses and an increased production of the reactive oxygen species H2O2.

Key words

Leydig cells testes superoxide dismutase catalase glutathione peroxidase glutathione hydrogen peroxide 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Maddocks A, Setchell BP: The physiology of the endocrine testis. Oxf Rev Reprod Biol 10: 53–123, 1988PubMedGoogle Scholar
  2. 2.
    Ewing LL: The trophic effect of luteinizing hormone on the rat Leydig cell. J Am Coll Toxicol 8 (3): 473–485, 1989Google Scholar
  3. 3.
    Abney TO, Myers RB: The effects of low oxygen and antioxidants on steroidogenic capacity in cultured rat Leydig cells. Adv Exp Med Biol 219: 609–612, 1987PubMedGoogle Scholar
  4. 4.
    Myers RB, Abney TO: The effects of reduced O2 and antioxidants on steroidogenic capacity of cultured rat Leydig cells. J Steroid Biochem 31 (3): 305–309, 1988PubMedGoogle Scholar
  5. 5.
    Hornsby PJ: Regulation of cytochrome P-450-supported 11β-hydroxylation of deoxycortisol by steroids, oxygen, and antioxidants in adrenocortical cell cultures. J Biol Chem 255 (9): 4020–4027, 1980PubMedGoogle Scholar
  6. 6.
    Georgiou M, Perkins LM, Payne AH: Steroid synthesis-dependent, oxygen-mediated damage of mitochondrial and microsomal cytochrome P-450 enzymes in rat Leydig cell cultures. Endocrinology 121 (4): 1390–1399, 1987PubMedGoogle Scholar
  7. 7.
    Klinefelter GR, Ewing LL: Maintenance of testosterone production by purified adult rat Leydig cells for 3 daysin vitro. In Vitro Cell & Dev Biol 25 (3): 283–288, 1989Google Scholar
  8. 8.
    Halliwell B, Gutteridge JMC: Free Radicals in Biology and Medicine. Clarendon Press, Oxford, 1989, pp 86–187Google Scholar
  9. 9.
    Riley JCM, Behrman HR: Oxygen radicals and reactive oxygen species in reproduction. Proc Soc Exp Biol Med 198: 781–791, 1991PubMedGoogle Scholar
  10. 10.
    NIH: Guide for the Care and Use of Laboratory Animals, NIH publication # 86-23, National Academy of Sciences — National Research Council, Bethesda, 1985Google Scholar
  11. 11.
    NRC: Nutrient Requirements of Domestic Animals, Nutrient Requirements of Laboratory Animals, # 10, Third Edition. National Academy of Sciences-National Research Council, Washington, 1978Google Scholar
  12. 12.
    Mather JP, Phillips DM: Primary culture of testicular somatic cells. In: Barnes DW, Sirbasku DA, Sato GH (eds.) Cell Culture Methods for Molecular and Cell Biology, Volume 2. Alan R Liss, Inc., New York, 1984, pp 29–45Google Scholar
  13. 13.
    Browne ES, Bhalla VK: Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis. Steroids 56 (2): 83–90, 1991PubMedGoogle Scholar
  14. 14.
    Dirami G, Poulter LW, Cooke BA: Separation and characterization of Leydig cells and macrophages from rat testes. J Endocrinol 130 (3): 357–365, 1991PubMedGoogle Scholar
  15. 15.
    Phillips HJ: Dye exclusion tests for cell viability. In: Kruse PF Jr, Patterson MK Jr (eds.) Tissue Culture: methods and applications. Academic Press, New York, 1973, pp 406–408Google Scholar
  16. 16.
    Klinefelter GR, Hall PF, Ewing LL: Effect of luteinizing hormone deprivationin situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36: 769–783, 1987PubMedGoogle Scholar
  17. 17.
    Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 (1, 2): 248–254, 1976PubMedGoogle Scholar
  18. 18.
    Beers RF Jr, Sizer IW: A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195 (1): 133–140, 1952PubMedGoogle Scholar
  19. 19.
    Kornberg A, Horecker BL: Glucose-6-phosphate dehydrogenase. In: Colowick SP, Kaplan NO (eds.) Methods in Enzymology, Volume 1. Academic Press, New York, 1955, pp 323–327Google Scholar
  20. 20.
    Lohr GW, Waller HD: Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU (ed.) Methods of Enzymatic analysis, Volume 2. Academic Press Inc., New York, 1974, pp 636–643Google Scholar
  21. 21.
    McCord JM, Fridovich I: Superoxide dismutase: An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244 (22): 6049–6055, 1969PubMedGoogle Scholar
  22. 22.
    Paglia DE, Valentine WN: Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70 (1): 158–169, 1967PubMedGoogle Scholar
  23. 23.
    Tappel AL: Glutathione peroxidase and hydroperoxides. In: Fleischer S, Packer L (eds.) Methods in Enzymology, Volume LII. Academic Press, New York, 1978, pp 506–513Google Scholar
  24. 24.
    Racker E: Glutathione reductase. In: Colowick SP, Kaplan NO (eds.) Methods in Enzymology, Volume II. Academic Press Inc., New York, 1955, pp 722–725Google Scholar
  25. 25.
    Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal Biochem 27 (3): 502–522, 1969PubMedGoogle Scholar
  26. 26.
    Sies H, Akerboom TPM: Glutathione disulfide (GSSG) efflux from cells and tissues. In: Packer L (ed.) Methods in Enzymology, Volume 105. Academic Press Inc., New York, 1984, pp 445–451Google Scholar
  27. 27.
    Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds M, Thomas M: Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J Immunol 130 (4): 1910–1917, 1983PubMedGoogle Scholar
  28. 28.
    Gustafson TL: Epistatistics Software, Round Rock, Texas, Gustafson, 1984Google Scholar
  29. 29.
    Koul A, Khanduja KL, Koul IB, Gupta MP, Majid S, Sharma RR: Effect of ascorbic acid on antioxidant defense systems and lipid peroxidation in guinea pig. J Clin Biochem Nutr 6 (1): 21–27, 1989Google Scholar
  30. 30.
    Quinn PG, Payne AH: Steroid product-induced, oxygen-mediated damage of microsomal cytochrome P-450 enzymes in Leydig cell cultures. J Biol Chem 260 (4): 2092–2099, 1985PubMedGoogle Scholar
  31. 31.
    Free MJ, Van Demark NL: Gas tensions in spermatic and peripheral blood of rams with normal and heat-treated testes. Am J Physiol 214 (4): 863–865, 1968PubMedGoogle Scholar
  32. 32.
    Lehninger AL: Principles of Biochemistry, Worth Publishers, Inc., New York, 1982, p 216Google Scholar
  33. 33.
    Barman TE: Enzyme Handbook, Supplement I, Springer-Verlag, New York, 1974, pp 136–137Google Scholar
  34. 34.
    Mendis-Handagama SMLC: Mitosis in normal adult guinea pig Leydig cells. J Androl 12 (4): 240–243, 1991PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Mark A. Kukucka
    • 1
  • Hara P. Misra
    • 1
  1. 1.Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary MedicineVirginia Polytechnic Institute & State UniversityBlacksburgUSA

Personalised recommendations