Analysis of lipid peroxidation in human spermatozoa using BODIPY C11.
Lipid peroxidation is known to be a major factor in the aetiology of defective sperm function. Although biochemical assays for this process exist, they are relatively insensitive and require large numbers of spermatozoa; a condition that cannot be met with many infertility specimens. Recently, a new approach for monitoring peroxidative damage has been introduced, involving the probe BODIPY (581/591) C(11), which readily incorporates into cells and undergoes a spectral emission shift when attacked by reactive oxygen metabolites. We have examined the applicability of this probe as an indicator of oxidative stress in human sperm populations using flow cytometry as an end point. The measurement of peroxidation with BODIPY C(11) demonstrated significant dependence on the presence of a ferrous ion promoter (P < 0.001), which was significantly enhanced in sperm recovered from low-density Percoll fractions (P < 0.05) and was particularly damaging to the sperm midpiece. Iron-induced radical formation was suppressed by ascorbate in a dose-dependent manner (P < 0.001) and could only be promoted by Fe(II) and Cu(II); nickel, zinc and Fe(III) were ineffective. The Fe(II)-promoted BODIPY C(11) signal was significantly correlated with the measurement of reactive oxygen species generation with dihydroethidium. We conclude that BODIPY C(11) is an extremely useful probe for indexing peroxidative damage in human spermatozoa.
Aitken
R John
RJ
ARC Centre of Excellence in Biotechnology and Development, Discipline of Biological Sciences, University of Newcastle, NSW, Australia. jaitken@mail.newcastle.edu.au
Wingate
Jordana K
JK
De Iuliis
Geoffry N
GN
McLaughlin
Eileen A
EA
D000975:Antioxidants / Q000737:chemistry; D001205:Ascorbic Acid / Q000737:chemistry; D001372:Aza Compounds / Q000737:chemistry*; D002498:Centrifugation / Q000379:methods; D003300:Copper / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry*; D005296:Ferrous Compounds / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D015227:Lipid Peroxidation; D008297:Male; D008956:Models, Chemical; D015335:Molecular Probes / Q000737:chemistry*; D009566:Nitrates / Q000737:chemistry; D018384:Oxidative Stress; D011205:Povidone; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism; D012822:Silicon Dioxide; D013094:Spermatozoa / Q000378:metabolism*; D013997:Time Factors
D000975:Antioxidants; D001372:Aza Compounds; C463799:BODIPY581; D005227:Fatty Acids; D005296:Ferrous Compounds; D015335:Molecular Probes; D009566:Nitrates; D017382:Reactive Oxygen Species; C020748:ferrous sulfate; C016039:Percoll; D012822:Silicon Dioxide; D003300:Copper; C516433:copper(II) nitrate; D011205:Povidone; D001205:Ascorbic Acid