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Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa¹

Leibniz-Institut für Zoo-und Wildtierforschung im Forschungsverbund Berlin e.V.,³ 0315 Berlin, Germany Humboldt-Universität zu Berlin,⁴ Institut für Biologie, 10115 Berlin, Germany Institut National de la Recherche Agronomique,⁵ IFR 140, UR1037, 35000 Rennes, France Institut für Fortpflanzung landwirtschaftlicher Nutztiere Schönow e.V.,⁶ 6321 Bernau, Germany

ABSTRACT

During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.

electron spin resonance, fluidity, gamete biology, methyl beta-cyclodextrin, permeability, phospholipid translocation, sperm

Karin Müller, Leibniz-Institut für Zoo-und Wildtierforschung im Forschungsverbund Berlin e.V., Alfred-Kowalke-Strasse 17, 10315 Berlin, Germany; e-mail: mueller{at}izw-berlin.de

¹Collaboration between authors was supported by EU fellowship PROCOPE.

Correspondence: ²Catherine Labbe, INRA SCRIBE, Campus de Beaulieu, 35000 Rennes, France; e-mail: catherine.labbe{at}rennes.inra.fr