Treatment of human sperm with serine protease during density gradient centrifugation

Abstract

Purpose Seminal pathogens can bind specifically or nonspecifically to spermatozoa, rendering semen decontamination procedures ineffective, whereby vertical or horizontal transmission of the infection could occur. Serine proteases have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could negatively impact on sperm parameters. This study investigated the effect of the addition of a recombinant, human-sequence protease (rhProtease) on sperm parameters during density gradient centrifugation. Methods (i) Pooled semen samples (n09) were split and processed by density gradient centrifugation, with the top density layers supplemented, or non-supplemented with rhProtease at three different concentrations (diluted 2, 10 and 20 times). Sperm parameters were then analysed by flow cytometry and computer-assisted semen analyses.(ii) Semen samples (n05) were split and similarly processed using PureSperm® Pro, with rhProtease in the 40 % density gradient layer, or standard PureSperm® not supplemented with rhProtease (Nidacon, International) respectively. The Hemizona assay was then utilized to compare sperm-zona binding post processing.Results Evaluation of sperm parameters indicated that rhProtease did not, at any of the tested concentrations, have an impact on (i) mitochondrial membrane potential, vitality, motility, or (ii) zona binding potential. Conclusion We report that the addition of rhProtease to density gradients is a non-detrimental approach that could improve the effectiveness of semen processing for the elimination of seminal pathogens, and benefit assisted reproduction outcome.

Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling

“…This method has proven effective in leaving the most robust sperm cells intact but also tends to solubilize sperm-membrane structures. Another broadly accepted method used to purify sperm cells away from other contaminants is by gradient centrifugation using Percoll or clinical-grade reagents like PureSperm, an isotonic salt solution containing silane-coated silica particles [Fourie et al 2012; Johnson et al 2011; Nicolas, et al 2012; Ostermeier, et al 2002]. In this case, a density gradient is used to separate the sperm cells from the larger and less dense somatic cells during centrifugation.…”

Bovine herpes virus-1: comparison of methods for removal of a commercially important pathogen from cattle sperm, oocytes and pre-implantation embryos

“…However, the effectiveness of trypsin washing for decontamination of in vitro-produced bovine embryos is controversial. 16 In support of this contention, while several studies demonstrated that trypsin treatment is very effective for the removal or inactivation of BoHV-1, 7,10,17 other investigators found that the inclusion of trypsin solution in the washing procedure may be sufficient to strip BoHV-1 from the surface of the embryo but has no such effect on virus particles once inside. 14,18,19 Bielanski and Lalonde demonstrated that embryo cryopreservation may be regarded as a safety measure to decrease the potential risk or even mitigate disease transmission by embryo transfer.…”