Flagellar movement of intact and demembranated, reactivated ram spermatozoa

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Abstract
The flagellar movement of intact ejaculated ram sperm, and of demembranated models reactivated with ATP, has been studied using high-speed, high-resolution video microscopy. Intact sperm attached to the coverslip by their heads had an average beat frequency of 20.9 Hz and an average wave amplitude of 20.2 μm. There was little difference in the beat frequency or waveform of these sperm and sperm swimming freely near the coverslip or captured by their heads with a micropipette and held far from the coverslip, inducationg that the flagellar waveform of ram sperm is relatively resistant to distorition as a result of immobilization of the head or proximity to a surface. The beat envelope was nearly planar as determined by observations of free-swimming sperm and sperm captured by their head and oriented so they were beating either parallel or perpendicular to the plane of focus. The effect of various conditions for demembranation and reactivation of the sperm were examined. Treatment of sperm with 0.2 % Triton X-100 removed most of their plasma membrane. Under optimal conditions, nearly 100 % of the demembranted sperm reactivated at MgATP2− concentrations ranging from ∼4 μM to ∼20 mM. From ∼ 1 mM to ∼ 10 mM MgATP2−, their beat pattern closely resembled that of intact sperm; beat frequency depended on MgATP2− concentration. Percent motility was maximal between pH 7.5 and 8.0 and decreased sharply below pH 7.0 and avove pH 8.5. The addition of 50 μM cAMP to the reactivation medium had no effect on percent motility or the beat pattern and did not accelerate the initiation of movement.