Cell and Tissue Research

, Volume 328, Issue 2, pp 411–419 | Cite as

Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress, BAX, and caspase 2

  • Jeffrey J. Lysiak
  • Shuqiu Zheng
  • Robin Woodson
  • Terry T. Turner
Regular Article


Ischemia-reperfusion (IR) of the testis results in germ-cell-specific apoptosis (GCA) and a reduction in daily sperm production. This has been correlated with and is dependent upon neutrophil recruitment to the testis. In a rat model of testicular IR, this has also been correlated with an increase in reactive oxygen species (ROS). We have investigated ROS in the mouse testis after IR and determined whether the observed GCA is mediated via a mitochondrial caspase-9-dependent pathway involving the upstream mediators caspase 2 and BAX. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. An accumulation of 8-isoprostane, a marker of oxidative stress, occurred 4 h after reperfusion. Activation of a mitochondrial dependent pathway to GCA after testicular IR was determined based on the observations that both BAX and caspase 2 translocated to the mitochondria, and that an increase occurred in cytoplasmic cytochrome c. Moreover, microinfusion of a specific caspase 9 inhibitor significantly reduced active caspase 3 after testicular IR and the number of apoptotic germ cells. These results suggest that oxidative stress products accumulate in the testis following IR and demonstrate that the observed GCA is stimulated through a mitochondrial caspase-9-dependent pathway. The identification of the germ-cell apoptotic pathway induced after testicular IR, including the key players in the pathway subsequent to ROS (BAX, caspase 9, and caspase 2), aids our understanding of IR injury in the testis and provides a wider background for the development of therapeutic interventions to rescue testis function.


Germ cell Apoptosis Ischemia-reperfusion Testis Caspase Mouse (C57BL/6) 


  1. Ambrosio G, Tritto I (1999) Reperfusion injury. Experimental evidence and clinical implications. Am Heart J 138:S69–S75PubMedCrossRefGoogle Scholar
  2. Armstrong JS (2006) Mitochondrial membrane permeabilization: the sine qua non for cell death. BioEssays 28:253–260PubMedCrossRefGoogle Scholar
  3. Baker LA, Turner TT (1995) Leydig cell function after experimental testicular torsion despite loss of spermatogenesis. J Androl 16:12–16PubMedGoogle Scholar
  4. Beumer TL, Roepers-Gajadien HL, Gademan IS, Lock TMTW, Kal HB, De Rooij DG (2000) Apoptosis regulation in the testis: involvement of Bcl-2 family members. Mol Reprod Dev 56:353–359PubMedCrossRefGoogle Scholar
  5. Celik-Ozenci C, Sahin Z, Ustunel I, Akkoyunlu G, Erdogru T, Korgun ET, Baykara M, Demir R (2006) The Fas system may have a role in male reproduction. Fertil Steril 85:1168–1178PubMedCrossRefGoogle Scholar
  6. Fontana L, Giagulli C, Minuz P, Lechi A, Laudanna C (2001) 8-Iso-PGF2 alpha induces beta 2-integrin-mediated rapid adhesion of human polymorphonuclear neutrophils: a link between oxidative stress and ischemia/reperfusion injury. Arterioscler Throm Vas Biol 21:55–60Google Scholar
  7. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL (1994) Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94:1621–1628PubMedCrossRefGoogle Scholar
  8. Jassem W, Fuggle SV, Rela M, Koo DDH, Heaton ND (2002) The role of mitochondria in ischemia/reperfusion injury. Transplantation 73:493–499PubMedCrossRefGoogle Scholar
  9. Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ (1995) BAX-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270:96–99PubMedCrossRefGoogle Scholar
  10. Lassus P, Opitz-Araya X, Lazebnik Y (2002) Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 297:1352–1354PubMedCrossRefGoogle Scholar
  11. Lee J, Richburg JH, Younkin SC, Boekelheide K (1997) The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 138:2081–2088PubMedCrossRefGoogle Scholar
  12. Li C, Jackson RM (2002) Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol 282:C227–C241Google Scholar
  13. Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431–1568PubMedGoogle Scholar
  14. Lysiak JJ, Turner SD, Turner TT (2000) Molecular pathway of germ cell apoptosis following acute ischemia in the rat testis. Biol Reprod 63:1475–1482Google Scholar
  15. Lysiak JJ, Turner SD, Nguyen QAT, Singbartl K, Ley K, Turner TT (2001) Essential role of neutrophils in germ cell-specific apoptosis following ischemia/reperfusion of the mouse testis. Biol Reprod 65:718–725PubMedCrossRefGoogle Scholar
  16. Lysiak JJ, Nguyen QAT, Turner TT (2002) Peptide and nonpeptide reactive oxygen scavengers provide partial rescue of the testis after torsion. J Androl 23:400–409PubMedGoogle Scholar
  17. Lysiak JJ, Nguyen QAT, Kirby JL, Turner TT (2003) Ischemia-reperfusion of the murine testis stimulates the expression of proinflammatory cytokines and activation of c-jun N-terminal kinase in a pathway to E-selectin expression. Biol Reprod 69:202–210PubMedCrossRefGoogle Scholar
  18. Prillaman HM, Turner TT (1997) Rescue of testicular function after acute experimental torsion. J Urol 157:340–345PubMedCrossRefGoogle Scholar
  19. Rabb H, Postler G (1998) Leucocyte adhesion molecules in ischemic renal injury: kidney specific paradigms? Clin Exp Pharmacol Physiol 25:286–291PubMedGoogle Scholar
  20. Robertson JD, Enoksson M, Suomela M, Zhivotovsky B, Orrenius S (2002) Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J Biol Chem 277:29803–29809PubMedCrossRefGoogle Scholar
  21. Sheu SS, Nauduri D, Anders MW (2006) Targeting antioxidants to mitochondria: a new therapeutic direction. Biochem Biophys Acta 1762:256–265PubMedGoogle Scholar
  22. Starkov AA (2006) Protein-mediated energy-dissipating pathways in mitochondria. Chem Biol Interact 161:57–68PubMedCrossRefGoogle Scholar
  23. Turner TT, Miller DW (1997) On the synthesis and secretion of rat seminiferous tubule proteins in vivo after ischemia and germ cell loss. Biol Reprod 57:1275–1284PubMedCrossRefGoogle Scholar
  24. Turner TT, Tung KSK, Tomomasa H, Wilson LW (1997) Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 57:1267–1274PubMedCrossRefGoogle Scholar
  25. Vera Y, Diaz-Romero M, Rodriguez S, Lue Y, Wang C, Swerdloff RS, Sinha Hikim AP (2004) Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice. Biol Reprod 70:1534–1540PubMedCrossRefGoogle Scholar
  26. Williamson RNC (1985) The continuing conundrum of testicular torsion. Br J Surg 72:509–510PubMedCrossRefGoogle Scholar
  27. Wolf BB, Green DR (1999) Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 274:20049–20052PubMedCrossRefGoogle Scholar
  28. Zheng S, Turner TT, Lysiak JJ (2006) Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis. Biol Reprod 74:1026–1033PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Jeffrey J. Lysiak
    • 1
  • Shuqiu Zheng
    • 1
  • Robin Woodson
    • 1
  • Terry T. Turner
    • 1
    • 2
  1. 1.Department of UrologyUniversity of Virginia Health Science SystemCharlottesvilleUSA
  2. 2.Department of Cell BiologyUniversity of Virginia Health Science SystemCharlottesvilleUSA

Personalised recommendations