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Regular Article |
a Departments of Urology and Cell Biology,
b The University of Virginia Health Science Center, Charlottesville, Virginia 22908
ABSTRACT
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-XL 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-XL and Bcl-XS 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.
First decision: 28 March 2000.
1 Supported by NIH R01-DK-53072.
2 Correspondence: Jeffrey J. Lysiak, Department of Urology, Box 422, University of Virginia Health Science Center, Charlottesville, VA 22908. FAX: 804 924 8311; jl6n{at}virginia.edu
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