Changes in deoxyribonucleoprotein during spermiogenesis in the bull: Sensitivity of DNA to heat denaturation

Abstract

The thermal stability of DNA at various stages of spermiogenesis in bulls was studied by new cytochemical techniques. Fixed smears of testicular cells were heated to various temperatures in the presence of formaldehyde. After cooling, the extent of single-stranded DNA regions was measured by microspectrophotometric determination of the increment in UV-absorbancy at 265 nm or by microfluorimetry after acridine orange (AO) staining. The extent to which DNA had become denatured into a single-stranded state was determined from the magnitude of a shift in the fluorescence emission spectrum. Although UV-microspectrophotometry and acridine orange microfluorimetry measure different physical phenomena, the present results show that it is possible to follow the denaturation of DNA (“melting profile”) in intact cell nuclei by both these techniques. Since both techniques were used in parallel throughout the present investigation it has been possible to compare the two techniques relative to each other. The main biological conclusion from the present work is that during spermiogenesis the DNA becomes progressively more resistant to heat denaturation. This is thought to be due to changes in the state and composition of the deoxyribonucleoprotein. The relationship of these DNP changes to genome inactivation and chromatin condensation is briefly discussed.