Merocyanine 540

Merocyanine 540 (Sigma) was used to assess membrane fluidity of human spermatozoa. Following capacitation, aliquots of human sperm from each treatment were diluted to 1 × 10 6 cells/ml and incubated in SYTOX green vitality stain at 378C for 10 min. This preparation was washed once in BWW and then incubated in 2.7 mM Merocyanine 540 at 378C for 10 min. After re-suspending cells in BWW, 200 cells from each treatment were scored on a Zeiss fluorescence microscope at excitation wavelengths 590 nm (Merocyanine 540), 470 nm (SYTOX green) (Carl Zeiss, Thornwood, NY, USA). Merocyanine positive sperm were identified through bright red fluorescence over the entire head and the absence of SYTOX green staining. The localization of membrane raft marker, G M1 ganglioside, was visualized in human spermatozoa by staining with Alexa Fluor 488-labelled B subunit of cholera toxin (CTB) as previously described (Nixon et al., 2011) . For each treatment, 200 cells were classified into two fluorescent patterns (head and tail or head only labelling) and representative images were taken.

Dulbecco's Modified Eagle's Medium/Ham's F12 (DMEM/F12) was from Gibco, (Thermo Fisher Scientific, Inc., MA, USA); trypsin, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Hoechst 33258 fluorochrome, Merocyanine 540, 2′,7′-dichlorodihydrofluorescein diacetate (DCDHF-DA), rotenone and ECL detection reagents (luminol and p-coumaric acid) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Natocor (Córdoba, Argentina). Streptomycin, penicillin and amphotericin B were from Richet (Buenos Aires, Argentina). Lysotracker Red DND-99 and MitoSOX Red were from Molecular Probes (Eugene, OR, USA). Final concentration of dimethyl sulfoxide (DMSO) did not exceed 0.25%. DMSO added to the samples did not affect neither cell viability, morphology nor others parameters tested in this study. All others chemicals used were of the highest purity commercially available.

Soybean phosphatidylcholine (SPC) was purchased from Avanti® Polar Lipids. 5FU was an injectable solution from Laboratorios Filaxis (Buenos Aires, Argentina); sodium chol a t e ( N a C h o l ) , 1 , 2 -d i m y r i s t o y l -s n -g l y c e r o -3phosphoethanolamine-N-(Lissamine™ rhodamine B sulfonyl), fluorescein isothiocyanate, merocyanine 540, and sephadex G-50 were purchased from Sigma-Aldrich (Buenos Aires, Argentina). Sodium carboxymethylcellulose was from Fluka-BioChemika (Sigma-Aldrich, Argentina). 6-Dodecanoyl-2-dimethylaminonaphthalene was purchased from Thermo Fisher Scientific (Buenos Aires, Argentina). Crystal violet, neutral red (NR), ethanol, and methanol were from BioPack (Buenos Aires, Argentina). Chloroform was from J.T.Baker®. 3-[4,5-Dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide salt was obtained from Life Technologies™ (Thermo Fisher Scientific Inc., Argentina). Minimum essential medium, RPMI 1640 medium, fetal bovine serum, glutamine, pyruvate, penicillin, and streptomycin were obtained from Gibco (Waltham, MA, USA). Brine shrimp (Artemia persimilis) was from Biosima SRL (Buenos Aires, Argentina). All other reagents used were from analytical grade.