PCNA indexing as a preclinical immunohistochemical biomarker for testicular toxicity

It is well known that toxicants such as cyclophosphamide and ethanol can have deleterious effects on normal spermatogenesis. End points such as testis weight and sperm counts have been used widely to assess gross structural and functional changes in testes resulting from toxicant exposure. Histopathological assessments are more sensitive measures of testicular health, but generally they are neither quantitative nor sensitive enough to detect early toxicity. Recently, immunolabeling cells with proliferating cell nuclear antigen (PCNA) has been used to identify proliferating spermatogonia; however, there have been no systematic attempts to quantify these changes. We have developed a sensitive, reliable and quantitative assay using immunohistochemistry on formalin fixed, paraffin embedded rat testes to assess the degree of proliferation-related toxicity. An indexing scheme was derived based on the determination of radially positioned PCNA-positive cells within similarly staged seminiferous tubules presenting a single layer of PCNA-positive cells along the basement membrane of the basal tubular compartment. An average of 60 tubules in the testes were counted per animal. Our results show significant decreases in the PCNA index in rats treated with an experimental compound that has been shown to produce testicular histopathology. The analysis provides a quick, reliable, sensitive, and quantitative means for assessing early testicular toxicity. The assay has potential utility as an in vivo biomarker for detecting early testicular toxicity of experimental compounds in preclinical development as well as for refining follow-up compounds with reduced testicular toxicity.