Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis.
The present study investigates the molecular apoptotic pathway in germ cells following acute ischemia of the rat testis. Rats were subjected to ischemia-inducing torsion and testes were harvested after reperfusion. Apoptotic cells were identified with an antibody to single-stranded DNA. Seminiferous tubule RNA was examined by RNase protection assay or by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence and regulation of apoptotic molecules. Proteins from seminiferous tubules were used for Western blot analysis of cytochrome c. Germ cell apoptosis was maximal at 24 h after repair of torsion. Germ cells in stages II-III of the seminiferous epithelium cycle were the predominant early responders. The RNase protection assays revealed that Bcl-X(L) was the prominent mRNA species. Caspases 1, 2, 3, and Bax mRNA were consistently upregulated; however, the time of upregulation after torsion was variable. The Bcl-X(L) and Bcl-X(S) mRNAs were less consistently upregulated and there was no evidence for upregulation of Fas or Bcl-2. Fas ligand (FasL) was not detected by RNase protection assay, but RT-PCR revealed a significant increase in FasL expression 4 h after the repair of torsion. Western blot analysis for cytochrome c release demonstrated a significant increase 4 h after the repair of torsion. Results suggest that germ cell apoptosis following ischemia/reperfusion of the rat testis is initiated through the mitochondria-associated molecule Bax as well as Fas-FasL interactions.
Lysiak
J J
JJ
Departments of Urology and Cell Biology, The University of Virginia Health Science Center, Charlottesville, Virginia 22908, USA. jl6n@virginia.edu
Turner
S D
SD
Turner
T T
TT
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D005854:Germ Cells / Q000502:physiology*; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D023282:Nuclease Protection Assays; D051381:Rats; D017207:Rats, Sprague-Dawley; D012039:Regional Blood Flow / Q000502:physiology; D015427:Reperfusion Injury / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013086:Spermatic Cord Torsion / Q000473:pathology; D013737:Testis / Q000098:blood supply / Q000502:physiology*