Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm

Steckler, Daniela; Stout, T.A.E.; Durandt, Chrisna; Nöthling, J.O.

doi:10.1016/j.theriogenology.2015.01.019

Cited by 25 publications

(18 citation statements)

References 46 publications

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“…Additionally, we also observed a significant increase in tyrosine phosphorylation levels under these conditions both in Whitten's and HTF media. Merocyanine 540 has been previously used in boar (Harrison & Miller, ), dog (Steckler et al, ) and bovine sperm (Aguila et al, ) as an indicator of plasma membrane destabilization, because it binds to the segments of the plasma membrane that are in a higher state of lipid disorder (Langner & Hui, ; Leahy & Gadella, ; Williamson et al, ), indicating that changes in the membrane architecture related to sperm capacitation have initiated. These changes apparently result from cholesterol efflux supporting the addition of cholesterol acceptors such as BSA, which are normally included in the capacitation media.…”

Section: Discussionmentioning

confidence: 99%

“…Membrane fluidity, as an early capacitation event, was evaluated using the merocyanine assay (MC540, M6390). MC540 is a hydrophobic dye that stains cell membranes more intensely if their lipid components are in a higher state of disorder (Rathi et al, ) and has been used in dog (Steckler, Stout, Durandt, & Nothling, ), bovine (Aguila, Zambrano, Arias, & Felmer, ) and equine sperm (Ortgies et al, ; Rathi et al, ). Briefly, 5 μl of MC540 (270 μM stock solution) and 2 μl of SYTOX green (20 μM stock solution, Molecular Probes, Eugene, OR, USA) were added to the sperm suspension (2 × 10 6 sperm/ml) and incubated for 30 min at 38.5°C in air and darkness ( n = 3 replicates for each stallion).…”

Section: Methodsmentioning

confidence: 99%

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Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Arroyo-Salvo

Sanhueza

Fuentes

et al. 2018

Reprod Domestic Animals

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Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Arroyo-Salvo

Sanhueza

Fuentes

et al. 2018

Reprod Domestic Animals

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“…Gentle pressure was applied to the cover slide, with the aid of absorbent paper to eliminate the excess liquid. Percentages of cells showing either of the Merocyanine patterns, opaque (low fluidity) or brilliant (high fluidity -high-binding cells), were calculated after counting 200 spermatozoa under fluorescence microscopy (Leica DMLS) using the 100x objective (Steckler et al 2015).…”

Section: Semen Assessmentmentioning

confidence: 99%

The effect of non-traditional cooling on dog sperm cryosurvival and ability to perform the acrosome reaction

Ortega-Morales¹,

Alcantar-Rodriguez²,

Espejel³

et al. 2019

Austral j. vet. sci.

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The objective of this study was to assess cryosurvival, plasma membrane fluidity, and capability of cryopreserved dog (Canis lupus familiaris) spermatozoa, cooled to -5 °C before freezing, to perform the acrosome reaction under the effect of progesterone and calcium ionophore. In the first experiment, fresh spermatozoa diluted in Tyrode's medium plus albumin, lactate, and pyruvate (TALP) were incubated at 38 °C in 5% CO 2 in air, with progesterone or calcium ionophore added at 2, 4, and 6 h after incubation and sampled 30 min later to assess the acrosome reaction. In the second experiment, diluted sperm were packaged in plastic straws, cooled to either +5 °C or -5 °C and cryopreserved. Progressive motility, plasma membrane integrity and fluidity, capacitation status and acrosome integrity were assessed before and after freeze-thawing. After thawing, sperm were assessed, resuspended in TALP and incubated to assess the acrosome reaction. Parameters for sperm cryosurvival were similar in sperm cooled to either +5 °C or -5 °C, except in the percentage of hyper-fluid membranes which was lower (P<0.05) in sperm cooled to -5 °C. There were no differences in the percentages of frozen-thawed spermatozoa with acrosome reaction, induced by progesterone or calcium ionophore, between cooling treatments. In conclusion, cooling of dog spermatozoa to -5 °C did not improve sperm cryosurvival but had a positive effect on plasma membrane fluidity.

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“…In addition, Harrison and Miller (2000) observed in pig sperm, activated with bicarbonate, 35 and 52% of MC540 high-binding cells after 9 and 25 min of incubation, respectively. In contrast, Steckler et al (2015) reported 75% of viable dog sperm with destabilized membranes after 2 hr of incubation. On the other hand, Aboagla and Terada (2003) reported that 51% of goat spermatozoa, diluted in a trehalose freezing extender, showed high merocyanine 540 fluorescence after cryopreservation.…”

Section: Discussionmentioning

confidence: 92%

“…In contrast, Steckler et al. () reported 75% of viable dog sperm with destabilized membranes after 2 hr of incubation. On the other hand, Aboagla and Terada () reported that 51% of goat spermatozoa, diluted in a trehalose freezing extender, showed high merocyanine 540 fluorescence after cryopreservation.…”

Section: Discussionmentioning

confidence: 92%

The effect of cooling to different subzero temperatures on dog sperm cryosurvival

Alcantar-Rodriguez

Medrano

2017

Reprod Domestic Animals

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The objective was to assess the effect of cooling to different subzero temperatures around ice formation (-5°C) on dog sperm cryosurvival and plasma membrane fluidity. Semen was centrifuged, and sperm were resuspended in a Tris-egg yolk medium (3% glycerol). Diluted sperm were cooled from 22 to 5°C, and then, a Tris-egg yolk medium containing 7% glycerol was added (final concentration of 5% glycerol and 200 × 10 cells/ml). Sperm were packaged in 0.5-ml plastic straws, and equilibration was done 16 hr at 5°C before freezing. I. Straws (n = 47) at 5°C were exposed to nitrogen vapours to determine the freezing point. II. Other straws (from different ejaculates) processed as mentioned, were further cooled to -3, -5 or -7°C and immediately rewarmed in a water bath at 37°C. Motility, plasma membrane functionality and acrosome integrity were assessed. III. Other straws (from different ejaculates) processed as mentioned were further cooled to -3 or -5°C, frozen over nitrogen vapours and stored in liquid nitrogen for one month. Straws were thawed in a water bath at 38°C for 30 s. Motility, plasma membrane functionality, plasma membrane integrity, acrosome integrity, capacitation status and plasma membrane fluidity were assessed. Ice nucleation temperature was -14.3 ± 2.05°C (mean ± SD); cooling to +5, -3, -5 and -7°C, without freezing, produces no differences on sperm quality between target temperatures; cooling to +5, -3, and -5°C produced no differences on sperm survival and plasma membrane fluidity after freeze-thawing. In conclusion, cooling of dog spermatozoa to different subzero temperatures did not improve sperm cryosurvival and had no effect on plasma membrane fluidity after thawing.

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Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm

Cited by 25 publications

References 46 publications

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

The effect of non-traditional cooling on dog sperm cryosurvival and ability to perform the acrosome reaction

The effect of cooling to different subzero temperatures on dog sperm cryosurvival

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