Semen samples were obtained from 20 non-smoking healthy volunteers (22–30 years old) after three days of sexual abstinence. This study was approved by Ethics Committee of the McGill University Health Centre as we previously described (Lee et al., 2017) and followed the suggested guidelines for human semen studies (Sanchez-Pozo et al., 2013; Bjordahl et al., 2016). Donors who participated in this study were from our cohort of healthy donors, recruited at the Royal Victoria Hospital, Montreal, Quebec, Canada. The exclusion criteria were the presence of illness or whether the taking of any medication. After collection, semen samples were incubated at 37°C for 30 min to induce liquefaction and sperm motility was analyzed using a computer-assisted semen analysis system (CASA) (Sperm vision HR software v1.01, Penetrating Innovations, Ingersoll, Ontario, Canada) (Suppl. Table S1). Samples were centrifuged on a 4-layer Percoll gradient (95–65% to 40–20%) to select the highly motile spermatozoa. This procedure has been used to select highly motile spermatozoa without increasing ROS levels (Iwasaki and Gagnon, 1992). Spermatozoa were recovered from the 95% layer and the 65–95% interface and concentration was determined using a Neubauer chamber. Samples were then diluted to 50 × 106 cell/ml in Biggers, Whitten and Whittingham (BWW, pH 7.8) medium (Biggers et al., 1971) and used for experimentation. For all experiments, 1 million cells were incubated for 2 h at 37°C with or without the inhibitors described in Table I. We then determine the impact of these inhibitors on sperm viability, ROS production and DNA oxidation as described below.

Table I
Open in new tab

Description of the inhibitors used in this study to inhibit the PRDXs systems.

InhibitorChemical nameTargetReferences
Conoidin A2,3-bis(bromomethyl)-quinoxaline 1,4-dioxide2-Cys PRDXsLiu et al. (2010), Brizuela et al. (2014), Ryu et al. (2017)
AuranofinS-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosideThioredoxin reductaseGromer et al. (1998), Rigobello et al. (2005), Cox et al. (2008), Hwang-Bo et al. (2017)
DHEADehydroepiandrosteroneGlucose-6-phosphate dehydrogenaseRaineri and Levy (1970), Heffner and Milam (1990), Miraglia et al. (2010)
Oxalomalic Acid3-Carboxy-3-deoxy-2-pentulosaric acidNADP-dependent isocitrate dehydrogenaseO’Flaherty et al. (2006b), Breininger et al. (2017)
3BP3 BromopyruvateMalic enzymeChang and Hsu (1973), Satterlee and Hsu (1991), Singh et al. (2008)
Ezatiostat(2R)-L-γ-glutamyl-S-(phenylmethyl)-L-cysteinyl-2-phenyl-glycine-1,3-diethyl esterGlutathione S-transferasePiLiu et al. (2014), Mahadevan and Sutton (2015), Crawford and Weerapana (2016)
S-HexylGluthathione(2S)-2-Amino-5-[[(2R)-1-(carboxymethylamino)-3-hexylsulfanyl-1-oxopropan-2-yl]amino]-5-oxopentanoic acidMitochondrial membrane-bound glutathione transferase(s) (mtMGST1).Erhardt and Dirr (1996), Gopalakrishnan et al. (1998), Ulziikhishig et al. (2010)
Ethacrynic acid(2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxy)acetic acidGluthathione adductPloemen et al. (1993), Gopalakrishnan et al. (1998)
MJ331-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithiumPRDX6 Ca2+-iPLA2 activityFisher et al. (1992), Moawad et al. (2017)
InhibitorChemical nameTargetReferences
Conoidin A2,3-bis(bromomethyl)-quinoxaline 1,4-dioxide2-Cys PRDXsLiu et al. (2010), Brizuela et al. (2014), Ryu et al. (2017)
AuranofinS-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosideThioredoxin reductaseGromer et al. (1998), Rigobello et al. (2005), Cox et al. (2008), Hwang-Bo et al. (2017)
DHEADehydroepiandrosteroneGlucose-6-phosphate dehydrogenaseRaineri and Levy (1970), Heffner and Milam (1990), Miraglia et al. (2010)
Oxalomalic Acid3-Carboxy-3-deoxy-2-pentulosaric acidNADP-dependent isocitrate dehydrogenaseO’Flaherty et al. (2006b), Breininger et al. (2017)
3BP3 BromopyruvateMalic enzymeChang and Hsu (1973), Satterlee and Hsu (1991), Singh et al. (2008)
Ezatiostat(2R)-L-γ-glutamyl-S-(phenylmethyl)-L-cysteinyl-2-phenyl-glycine-1,3-diethyl esterGlutathione S-transferasePiLiu et al. (2014), Mahadevan and Sutton (2015), Crawford and Weerapana (2016)
S-HexylGluthathione(2S)-2-Amino-5-[[(2R)-1-(carboxymethylamino)-3-hexylsulfanyl-1-oxopropan-2-yl]amino]-5-oxopentanoic acidMitochondrial membrane-bound glutathione transferase(s) (mtMGST1).Erhardt and Dirr (1996), Gopalakrishnan et al. (1998), Ulziikhishig et al. (2010)
Ethacrynic acid(2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxy)acetic acidGluthathione adductPloemen et al. (1993), Gopalakrishnan et al. (1998)
MJ331-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithiumPRDX6 Ca2+-iPLA2 activityFisher et al. (1992), Moawad et al. (2017)
Table I
Open in new tab

Description of the inhibitors used in this study to inhibit the PRDXs systems.

InhibitorChemical nameTargetReferences
Conoidin A2,3-bis(bromomethyl)-quinoxaline 1,4-dioxide2-Cys PRDXsLiu et al. (2010), Brizuela et al. (2014), Ryu et al. (2017)
AuranofinS-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosideThioredoxin reductaseGromer et al. (1998), Rigobello et al. (2005), Cox et al. (2008), Hwang-Bo et al. (2017)
DHEADehydroepiandrosteroneGlucose-6-phosphate dehydrogenaseRaineri and Levy (1970), Heffner and Milam (1990), Miraglia et al. (2010)
Oxalomalic Acid3-Carboxy-3-deoxy-2-pentulosaric acidNADP-dependent isocitrate dehydrogenaseO’Flaherty et al. (2006b), Breininger et al. (2017)
3BP3 BromopyruvateMalic enzymeChang and Hsu (1973), Satterlee and Hsu (1991), Singh et al. (2008)
Ezatiostat(2R)-L-γ-glutamyl-S-(phenylmethyl)-L-cysteinyl-2-phenyl-glycine-1,3-diethyl esterGlutathione S-transferasePiLiu et al. (2014), Mahadevan and Sutton (2015), Crawford and Weerapana (2016)
S-HexylGluthathione(2S)-2-Amino-5-[[(2R)-1-(carboxymethylamino)-3-hexylsulfanyl-1-oxopropan-2-yl]amino]-5-oxopentanoic acidMitochondrial membrane-bound glutathione transferase(s) (mtMGST1).Erhardt and Dirr (1996), Gopalakrishnan et al. (1998), Ulziikhishig et al. (2010)
Ethacrynic acid(2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxy)acetic acidGluthathione adductPloemen et al. (1993), Gopalakrishnan et al. (1998)
MJ331-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithiumPRDX6 Ca2+-iPLA2 activityFisher et al. (1992), Moawad et al. (2017)
InhibitorChemical nameTargetReferences
Conoidin A2,3-bis(bromomethyl)-quinoxaline 1,4-dioxide2-Cys PRDXsLiu et al. (2010), Brizuela et al. (2014), Ryu et al. (2017)
AuranofinS-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosideThioredoxin reductaseGromer et al. (1998), Rigobello et al. (2005), Cox et al. (2008), Hwang-Bo et al. (2017)
DHEADehydroepiandrosteroneGlucose-6-phosphate dehydrogenaseRaineri and Levy (1970), Heffner and Milam (1990), Miraglia et al. (2010)
Oxalomalic Acid3-Carboxy-3-deoxy-2-pentulosaric acidNADP-dependent isocitrate dehydrogenaseO’Flaherty et al. (2006b), Breininger et al. (2017)
3BP3 BromopyruvateMalic enzymeChang and Hsu (1973), Satterlee and Hsu (1991), Singh et al. (2008)
Ezatiostat(2R)-L-γ-glutamyl-S-(phenylmethyl)-L-cysteinyl-2-phenyl-glycine-1,3-diethyl esterGlutathione S-transferasePiLiu et al. (2014), Mahadevan and Sutton (2015), Crawford and Weerapana (2016)
S-HexylGluthathione(2S)-2-Amino-5-[[(2R)-1-(carboxymethylamino)-3-hexylsulfanyl-1-oxopropan-2-yl]amino]-5-oxopentanoic acidMitochondrial membrane-bound glutathione transferase(s) (mtMGST1).Erhardt and Dirr (1996), Gopalakrishnan et al. (1998), Ulziikhishig et al. (2010)
Ethacrynic acid(2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxy)acetic acidGluthathione adductPloemen et al. (1993), Gopalakrishnan et al. (1998)
MJ331-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithiumPRDX6 Ca2+-iPLA2 activityFisher et al. (1992), Moawad et al. (2017)

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