Organization of lipids in the artificial outer membrane of bull spermatozoa reconstructed at the air–water interface

• Sphingomyelin undergoes a gel-to-fluid transition in the temperature range 40–5 °C.
• The miscibility of lipids is controlled by temperature.
• Cholesterol and sphingomyelin organize in condensed domains, like lipid rafts.
• Plasmalogen and phosphatidylcholine are not miscible at low temperatures.
• The organization of membrane lipids is a reliable parameter for sperm quality.

Cryopreservation is widely used to preserve the quality of bull spermatozoa over time. Sequestration of seminal plasma proteins by low density lipoproteins and formation of a protective film around the spermatozoa membrane by low density lipoproteins were the main mechanisms proposed. However, the organization of lipids in the outer leaflet of the spermatozoa membrane has been never considered as a possible parameter. This study evaluated whether a change in the organization of the outer leaflet of the spermotozoa membrane could occur during cooling down. The organization of the main components of the spermatozoa membrane's outer layer at the liquid–gas interface was analysed. Cryopreservative media (at 8° and 34 °C) were used to study the miscibility of the spermatozoa membrane lipids using epifluorescence imaging and by tensiometry on Langmuir films. The results show that the four lipids: sphingomyelin, cholesterol, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC) and plasmalogen 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (P-PC) were not fully miscible and their organization was controlled by temperature. Cholesterol and sphingomyelin form condensed domains surrounded by a mixture of PC and P-PC at 34 °C while these condensed domains are surrounded by separated domains of pure PC and pure P-PC at 8 °C. The organization of the outer membrane lipids, in particular the separation of PC and P-PC lipids during cooling down, must be considered to fully understand preservation of membrane integrity during cryopreservation.

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