A new technique to evaluate the ability of cryoprotectors to prevent premature acrosome reaction in human spermatozoa

Abstract

Acrosome reaction (AR) induced by low temperature has been used to evaluate sperm function; it correlates adequately with the fertilization percentages in vitro. In this study, the technique of AR induction by low temperature was used to evaluate the effect in the protection of the acrosome by cryopreservatives normally used in human semen cryopreservation. Donor sperm selected by use of the migration sedimentation technique was incubated in human tubal fluid medium, added to dimethyl sulphoxide 1 m, ethylene glycol 0.75 m, glycerol 1 m, incubated at 4 degrees C and 20 degrees C (as a control) for 18 h, and then for 3 h at 37 degrees C in a cell incubator. The AR was evaluated by triple stain in 100 viable spermatozoa. The effect of cryopreservatives on acrosome preservation in samples incubated for 18 h at 4 degrees C was as follows: 78% intact acrosome for glycerol, 77.8% intact acrosome for dimethyl sulphoxide and 96.2% intact acrosome for ethylene glycol (P < 0.0025 compared with glycerol and dimethyl-sulphoxide). The sperm samples incubated with cryopreservatives for 18 h at 20 degrees C did not show an increase in the percentage of AR in samples incubated with glycerol and ethylene glycol, while a significant variation was observed in the sample incubated with dimethyl sulphoxide (P < 0.001). Additional incubation for 3 h at 37 degrees C significantly increased the AR only in the sample incubated with glycerol (P < 0.001). Acrosome preservation is essential in the fertilization process and the evaluation of acrosome reaction induction by low temperature test was satisfactory. This test proves that ethylene glycol presents a greater protective effect on the acrosome preservation of human spermatozoa.

The cryobiology of spermatozoa

“…Most modern protocols now rely on concentrations of 5% to 10% glycerol (vol/vol) (approximately 0.6 to approximately 1.2 mol/kg) in complex buffer systems. Recently, EG has been shown to be more permeable to human sperm, and is a superior cryoprotectant in place of glycerol [141,142]. Other protective substances were also proposed, such as DMSO and 1,2-propanediol [142,143].…”

“…Recently, EG has been shown to be more permeable to human sperm, and is a superior cryoprotectant in place of glycerol [141,142]. Other protective substances were also proposed, such as DMSO and 1,2-propanediol [142,143]. Although DMSO has been shown as ineffective when compared with glycerol and EG for sperm suspension cryopreservation [142], the recent studies indicate that DMSO is exceptionally effective when compared with glycerol or 1,2-propanediol in maintaining the structure of testicular tissue, especially spermatogonia, after cryopreservation [144,145].…”

Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate

“…We also recognize that DMSO is not used for routine cryopreservation of human ejaculate sperm, and that a replicate study without DMSO would be warranted. In other studies, DMSO has been used for cryopreservation of human and nonhuman primate testicular tissue (25,26), for the study of sperm membrane permeability to cryoprotectants (27,28), and for the study of premature acrosome reactions in human sperm that can be induced by low temperature storage (29).…”

Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis)

“…The acrosome reaction is an irreversible, calcium‐dependent event that modifies the acrosomal cap membrane, exposing the components needed for sperm‐egg fusion (Lybaert, Danguy, Leleux, Meuris, & Lebrun, ); however, premature exocytosis of the acrosome accelerates the degradation of spermatozoan activity. Therefore, assessment of the acrosome status of spermatozoa is a reasonable measure of fertilizing capacity (Ortloff, Deppe, Schill, & Sanchez, ). The association between recombinant RGN in semen extender and a higher percentage of acrosome‐intact post‐thaw spermatozoa suggest that the calcium‐binding properties of RGN (Harikrishna, Thomas, Shende, & Bhure, ) might reduce the abundance of free Ca 2+ in the extender that would otherwise facilitate a premature acrosome reaction.…”