Ram sperm mitochondrial DNA

Abstract

Circular DNA was isolated from mitochondrial fractions of ram spermatozoa by SDS treatment followed by convex sucrose gradient centrifugation. The DNA had a contour length of 5.0μm. Its buoyant density was 1.6983 g cm −3, which was smaller than two nuclear DNA components with buoyant densities of 1.6999 and 1.7156 g cm −3, found in ram spermatozoa. The Tm of the mitochondrial DNA was 69.7 °C. The mole fraction G + C calculated from the buoyant density and melting temperature was 39.1% and 38.6%, respectively. During mammalian spermiogenesis, mitochondria elongate and undergo major internal reorganization ( Phillips, 1975). They assume an end-to-end arrangement and form a helix around the fibrous element of the tail, maintained largely by s-s bridges ( Bartoov and Messer, 1976). Premkumar and Bhargava (1972) have shown that mature bovine spermatozoa transcribe in vitro 23 and 16S mitochondrial rRNA resembling bacterial rRNA. Thus, sperm mitochondrial DNA transscribes rRNA which differs from rRNA of mitochondria from somatic animal cells, known to sediment at about 16 and 12S ( Bartoov et al. , 1970; Borst, 1972). The question arises if mammalian sperm mitochondrial DNA has the properties typical to animal mitochondrial DNA. Our method, described earlier ( Bartoov and Messer, 1976), for isolation of ejaculated ram sperm mitochondria enabled us to isolate mitochondrial DNA directly from a mitochondrial fraction, with a minimum of nuclear DNA contamination. Mitochondrial fraction from fresh ram semen, containing about 10 10 cells, was isolated after dithiothreitol (DTT) treatment, suspended in 0.4 ml of a solution containing 0.3M sucrose, 2mM Tris-HCl, pH 7.4 at 0 °C and subjected to a two min treatment with an equal volume of 10% SDS, 10mM EDTA solution at 27 °C. The lysed.