Quantification of apoptosis (programmed cell death) in mammalian testis by DNA-fragmentation ELISA

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 microg testis equivalent/100 microl solution. Therefore, samples were standardized to a final concentration of 10 microg testis equivalent/100 microl. 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. The results showed that apoptosis can be detected in testicular homogenate prepared from 0.01 mg testis parenchyma within 24 h after recovery of testes without significant variations. Detectable apoptosis levels showed differences among sexually active guinea pig (7.08 +/- 1.95 U/mg), roe deer (16.32 +/- 3.45 U/mg), and cattle (29.0 +/- 7.1 U/mg). This species-specificity suggests different cross reactivity of the monoclonal antibody used in the ELISA. A significantly higher amount of testicular apoptosis was detected in a population of guinea pigs with increased inbreeding coefficients (f = 0.785 - 0.998) than in outbred animals (11.41 +/- 3.50 U/mg and 7.08 +/- 1.95 U/mg, respectively). The inverse relationship of testicular apoptosis and proliferation in these two populations was significant (r = -0.531; P < 0.05). In conclusion, the relative simplicity and high sensitivity of this nonradioactive method provides a useful approach to investigate spermatogenesis under different conditions. Results in the guinea pig showed that apoptosis plays an important role in the regulation of gonadal efficiency.