Equine Sperm Membrane Phase Behavior: The Effects of Lipid-Based Cryoprotectants1

Abstract

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.

Sperm surface changes and physiological consequences induced by sperm handling and storage

“…Direct associations between the lipids of the egg yolk and the sperm membrane have been reported (Pace & Graham 1974, Ollero et al 1998, Ricker et al 2006, and it is believed that these have a stabilising effect, minimising lateral phase separations involved in cold shock deterioration (Pettitt & Buhr 1998).…”

Protective effects of exogenous gangliosides on ROS-induced changes in human spermatozoa

“…The most important role of phospholipids could be coating the cell membrane, resulting in reinforcement during cryostorage. 80 Because the freezing/thawing process results in the loss of sperm surface hydrophobicity, 81 one of the proposed mechanisms of sperm resistance to cryostorage-induced stress is its ability to shed hydrophilic lipids, thus increasing the hydrophobic properties of the membrane and enhancing sperm tolerance to cryopreservation. 82 The exogenous addition of specific lipid compounds to the cell suspension can therefore be hypothesized to improve cell hydrophobicity and protect from cryopreservation-induced stress.…”

The role of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in comparison with whole egg yolk for sperm cryopreservation in rhesus monkeys

“…9 Because LDL consists of 86%-89% lipids and 12.5% proteins, 19 lipids have been the main focus for the study of the protective role of egg yolk. 20,21 One major source of injury during cryopreservation is membrane destabilization that occurs as temperature decreases and the fluid phase transitions into the gel phase. 22 Some of this destabilization and damage can be prevented by the addition of lipids (reviewed by Ref.…”

“…When samples were frozen without permeable cryoprotectant, LDL in a concentration range of 10%-30% yielded similar post-thaw motility (33%-40%) and AMI (17)(18)(19)(20)(21)(22)(23)(24) as that of samples frozen in 20% egg yolk alone (39%67% for motility and 2469 for AMI). However, LDL at concentrations lower than 8% reduced post-thaw motility and AMI when compared with controls at 20% egg yolk (P,0.05; Figure 2).…”

“…Sperm suspensions were washed twice with a modified Tyrode's medium supplemented with 3 mg ml 21 bovine serum albumin (TL-BSA, 290 mOsmol kg 21 ) at 300 g for 10 min, and resuspended to 2310 8 cells ml 21 of total motile sperm (sperm density3 initial motility) with TEST solution (43.25 g TES, 10.265 g Tris, 10 g glucose in one liter distilled water, pH 7.4, 350 mOsmol kg 21 ) before subject to various treatments. Sperm were frozen at a final concentration of 5310 7 cells ml 21 . Unless specified otherwise, all chemicals used for preparation of solutions were of reagent grade (Sigma Chemical Corporation, St Louis, MO, USA).…”