Changes in membrane lipid order with capacitation in rhesus macaque (Macaca mulatta) spermatozoa

Lipophilic fluorescent dye merocyanine 540 is believed to stain cell membranes with increasing affinity as the lipid components become more disordered and has been associated with changes in membrane fluidity. The aim of this study was to determine whether membrane lipid disorder is associated with capacitation of macaque spermatozoa. To induce capacitation, spermatozoa from 5 rhesus macaques were incubated at 37 degrees C (5% CO2 in air) for 2 hours in a modified Biggers-Whitten-Whittingham medium containing 30 mg/mL bovine serum albumin and 36 mmol/L NaHCO3. Caffeine (1 mmol/L) and dbcAMP (1.2 mmol/L) were added to the medium, and incubation was performed for an additional 30 minutes. Sperm motility was determined by computer-assisted sperm analysis, and membrane lipid order and sperm viability was determined by flow cytometry with merocyanine (2.7 mu mol/L) and Yo-Pro-1 (25 nmol/L), respectively. Tyrosine phosphorylation of proteins in sperm tail was immunohistochernically examined by means of anti-phosphotyrosi ne (alpha-PY; clone 4G10). Capacitation resulted in a significant increase in the amplitude of lateral head displacement and beat cross frequency (P < .005) and a significant decrease in linearity and straightness in capacitated spermatozoa (P < .005), compared with control spermatozoa, which suggests the expression of hyperactivated motility. Animals in which capacitation was induced had a significant increase in the number of spermatozoa showing tyrosine phosphorylation of tail proteins (P < .0001) and a significant increase in the intensity of merocyanine fluorescence (P < .0001), compared with control animals. The observed decrease in membrane lipid order after capacitation was induced was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with annexin V-Alexa Fluor 488. Merocyanine may be a useful tool for investigating the role of the plasma membrane on capacitation and other cytotoxic events in macaque spermatozoa.