Isolation of Plasmalemmae-Free, Mitochondria-Free Bull Sperm Flagella

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Abstract
A rapid, reproducible, and relatively simple method for preparing and isolating bull sperm flagella free of their heads, plasmalemmae and mitochondrial sheaths is reported. Ejaculated bull sperm, washed free of semen and extender, were incubated in dithiothreitol/collagenase B in hyposmolar buffer. After being washed in isosmolar buffer, the sperm were resuspended in hyposmolar buffer and sonicated for 10 seconds to remove the heads and plasmalemmae and to initiate fragmentation of the mitochondrial sheath. Various fractions of sperm produced during sonication were separated on a discontinuous sucrose gradient and washed individually. The 5000 G sediment, containing flagella and long flagellar segments, was resuspended in hyposmolar buffer at alkaline pH, and the disrupted mitochondrial sheath was removed from these flagella by incubation in dithiothreitol/Triton X-100 in alkaline hyposmolar buffer and washing in alkaline hyposmolar buffer. Alterations in sperm morphology, effected by each treatment, were monitored by brightfield light microscopy, and the degree of mitochondrial removal was confirmed at higher resolution by transmission electron microscopy of negatively stained samples.