QUANTIFICATION OF APOPTOSIS (PROGRAMMED CELL DEATH) IN MAMMALIAN TESTIS BY DNA-FRAGMENTATION ELISA

摘要

Apoptosis (programmed cell death) could contribute to fluctuations in sperm production and involution of testis in dependence on seasonal, genetic, environmental or individual factors. Investigations of such factors require a reliable quantitative examination of apoptotic processes. Therefore, a standardized procedure was developed for quantification of apoptosis in samples of testicular parenchyma in bull. This test is based on a highly sensitive DNA-fragmentation ELISA which was used originally for somatic cells in culture. Aliquots of testicular parenchyma were minced and homogenized by freezing/thawing and subsequent sonification at 4 degrees C. In comparison, aliquots were lysed in original buffer from the cell death detection ELISA-kit. Nucleosomes from the cell cytoplasm were obtained in supernatant of homogenate or lysate after centrifugation. The absorbance was linear over the range of sample concentrations from 5 to 20 mu g testis equivalent/100 mu l solution. Therefore, samples were standardized to a final concentration of 10 mu g testis equivalent/100 mu l. The recorded values were expressed in units per mg tissue (U/mg). The method was used to study testicular apoptotic processes in the guinea pig and roe deer.