326 COMPARING CHANGES IN MEMBRANE LIPID ORDER IN EPIDIDYMAL AND EJACULATED BOAR SPERMATOZOA

Abstract

The diffusion of lipids in the plasma membrane of ejaculated spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium (Wolfe et al. 2001 Mol. Reprod. Dev. 59, 306–313). Merocyanine 540 (M540) is a hydrophobic dye that has been shown to stain cell membranes more intensely if their lipid components are in a higher state of disorder, as is the case of capacitated spermatozoa. It is believed that the membrane fluidity changes detected by M540 precede the calcium influx, making M540 a method for evaluating the early events of capacitation. The aim of this study was to determine if there are differences in the dynamics of lipid disorder in the plasma membrane of ejaculated and epididymal boar spermatozoa under different conditions of capacitation. The sperm capacitation treatments were: washed in Delbucco's PBS supplemented with 0.1 % BSA (PBS-BSA), washed on a Percoll gradient (PG), and unwashed (UW: Control). During measurement, the samples were kept at 38°C and 5 % CO₂ to maintain constant incubation conditions. Membrane lipid order and sperm viability were determined by flow cytometry with M540 (2.7 µM) and Yo-Pro-1 (25 nM), respectively. Samples were analyzed on a Coulter Epics XL flow cytometer (Beckman Coulter Co., Inc., Fullerton, CA, USA). A total of 10 000 gated events were collected per sample, with sample running rates of approximately 600 events/s. Data were analyzed by analysis of variance (ANOVA). For the epidydimal vs. ejaculated results, the percentage of low lipid disorder spermatozoa was higher in the epididymal (19.23%) than in the ejaculated (5.84%) groups, and the proportion of high disorder (42.85%) and dead cells (48.59%) was higher in the ejaculated group. In relation to sperm treatment (UW, PBS-BSA, and PG), the percentage of high disorder was similar in all of the treatment groups (UW: 44.62 %; PBS-BSA: 43.08%; PG: 43.41%). Finally, the percentage of low disorder was lower in the PBS-BSA and PERCOLL (10.68% and 12.83%, respectively) groups, and the highest was obtained for the UW group (14.09%). In conclusion, the staining with M540 revealed that the lipid disorder was affected by the source of the sperm and the sperm treatment. A significant increase in membrane lipid low disorder and decrease in high disorder and dead cells were detected when epididymal sperm were compared with ejaculated sperm, so the seminal plasma and the sperm treatment to eliminate disorder have an important effect in the lipid membrane order.

Supported by MEC (AGL2006-03495/GAN) and Fundación Séneca (03018/PI/05).