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Lipid Diffusion in the Plasma Membrane of Mouse Spermatozoa

Changes During Epididymal Maturation, Effects of pH, Osmotic Pressure, and Knockout of the c-ros Gene

YONKA CHRISTOVA^*,

TREVOR G. COOPER

From the ^* Gamete Signalling Laboratory, The Babraham Institute, Cambridge, United Kingdom; the Department of Biochemistry, Sofia University, Sofia, Bulgaria; and the Institute of Reproductive Medicine of the University, Münster, Germany.

Correspondence to: Dr Roy Jones, Gamete Signalling Laboratory, The Babraham Institute, Cambridge CB3 0NQ, United Kingdom (e-mail: roy.jones{at}bbsrc.ac.uk ).

It is well known that the plasma membranes of mammalian spermatozoa undergo extensive remodeling during maturation in the epididymal duct. In this investigation, we have used fluorescence recovery after photobleaching (FRAP) techniques to: 1) measure rates of lipid diffusion in the plasma membrane of mouse spermatozoa at different stages of maturation; 2) examine the effects of varying external conditions found in the epididymal duct (pH, temperature, and osmotic pressure) on lipid diffusion in mature sperm; and 3) investigate the effects of the c-ros null mutation that causes tail angulation in cauda spermatozoa after ejaculation as a result of cell swelling due to altered membrane function. Our results show that lipid diffusion (as measured using reporter probes 5-(N-octadecanoyl)aminofluorescein [ODAF] and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine [NBD-C₆-PC]) is several times faster across the membrane on the sperm head than on the tail and that it increases significantly during passage from caput to cauda. Temperature variations between 20°C and 37°C have a substantial effect on diffusion coefficients, with the sperm head being more responsive than the tail. Changes in external pH (6.5-8.5) or osmotic pressure (202-389 mOsm/kg), however, have little relative effect on lipid diffusion on any region of the sperm. The rate of diffusion of 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3ßol (NBD-cholesterol) is 10-fold higher across the head plasma membrane than across the tail and does not change significantly during epididymal maturation. Similarly, lipid diffusion in hairpin-shaped cauda sperm from c-ros (-/-) males is not significantly different from (+/+) controls. These results suggest that temperature and compositional changes are 2 of the important factors that regulate the dynamics of lipid molecules in the mouse sperm plasma membrane.

     Key words: Photobleaching analysis, lipid dynamics, fertilizing capacity, germ cells

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