Separation of germ cell apoptosis from toxin-induced cell death by necrosis using in situ end-labeling histochemistry...

In situ end-labeling of fragmented DNA is routinely being used to detect apoptotic cells in various tissues including the testis. In this study, we examined the influence of various fixatives (neutral buffered formalin, paraformaldehyde, and glutaraldehyde) on the testicular structural integrity and immunoreactivity of fragmented DNA in apoptotic germ cells of the adult rat. Accelerated apoptosis of germ cells was induced in the adult rate by gonadotropin deprivation. Visualization of apoptotic DNA fragmentation in individual germ cells was achieved by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Glutaraldehyde fixation significantly improved the in situ detection of apoptotic germ cells while maintaining excellent morphological preservation. The labeling is also specific for apoptosis since necrotic germ cells show no specific signals. Fixed tissues could be processed for electron microscopy for further characterization of germ cell death using morphological criteria. Thus, glutaraldehyde fixation is advantageous for recognition of apoptotic germ cells with high sensitivity and specificity on a cell-by-cell basis. It should also be applicable to detect apoptosis in other cells and tissues.