Assessment of different membrane fluidity patterns in thawed Australian Merino ram spermatozoa by means of Merocyanine 540 and fluorescence microscopy

VENTURINO, A.; AMBROSI, C.P.; AISEN, E.G.; LÓPEZ ARMENGOL, M.F.

Nouzilly

Congreso; ICAR: 18th International Congress on Animal Reproduction; 2016

Fluidity of sperm head plasma membrane (PM) ismodified stepwise during maturation, capacitation and acrosomal reaction (AR).It increases at the acrosomal caput region during capacitation. After AR, thefluidity of the PM overlying the equatorial segment also increases, and PMbecomes fusogenic. Capacitation and ??cryocapacitation?? are not equivalentprocesses, but it is well known that the proportion of cells exhibiting PMlipid disorder and phospholipid scrambling increases with both capacitation andcryopreservation [1]. Trehalose (Tre) stabilizes the PM by interacting with thepolar head groups of phospholipids, thus increasing its fluidity after thawing[2]. Spermine (Spm) is a polyamine with antioxidant and stabilizing properties[3]. The aim of this study was to evaluate the effect of Tre combined with Spmon PM fluidity after cryopreservation. Ejaculates from five Australian Merinorams were pooled. Semen samples were diluted with four different solutions:Tris-citric acid-glucose-glycerol-egg yolk extender (B), B + Spm 1 mM (B1), B +Tre 100 mM (T), and T+ Spm 1 mM (T1). After freezing-thawing, PM fluiditypatterns were assessed by means of Merocyanine 540 stain (M540) usingepifluorescence microscopy. Different M540 staining patterns at sperm head PMwere observed, indicating different regions of increased fluidity: acrosomal PM(AC), whole head PM (WH) and equatorial PM (EQ). The parameters were analyzedin a 2 x 2 factorial design by ANOVA, with Fisher ?LSD post hoc tests, usingthe software StatSoft, Inc. (2007). No interactions between Tre or Spm additionwere found for the M540 patterns (p>0.05). At AC region; extenders withoutTre showed more fluidity (p<0.05) than those with Tre (53.5±2.0% and46.8±2.4% stained cells, respectively). The presence of Spm showed a tendencyto decrease PM fluidity, obtaining the lowest value at this region (44.5±3.6%).The same occurred at EQ domain: extenders without Tre showed more spermatozoawith PM fluidity (p<0.03) than those with Tre (27.9+2.2% and 20.0+2.2respectively). The presence of Spm shows a tendency to improve EQ stabilitywhen Tre is absent. Fluidity at WH was lower (p<0.05) without Tre than withit (37.0+3.4% and 47.2+2.7, respectively). In conclusion, M540 patternsdetermined by epifluorescence microscopy are an important tool for studying theeffect of different agents with PM stabilization properties. In this sense, Trehas demonstrated the highest effect on PM fluidity after cryopreservation. [1]Thomas AD, Meyers MA, Ballet BA. Capacitation-like changes in equinespermatozoa following cryopreservation. Theriogenology 2006,65:1531?50. [2]Eiman M, Aboagla E, Terada T. Trehalose-enhanced fluidity of the goat spermmembrane and its protection during freezing. Biol Reprod 2003;69:1245-50. [3]Rubinstein S, Breitbart H. Cellular localization of polyamines: cytochemicaland ultrastructural methods providing new clues to polyamine function in ramspermatozoa. Biol Cell 1994;81:177-83.