Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa

Geoffry N. De Iuliis, Jordana K. Wingate, Adam J. Koppers, Eileen A. McLaughlin, R. John Aitken

Research output: Contribution to journalArticle

95 Citations (Scopus)

Abstract

Context: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved. Objective: The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function. Method: DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells. Results: HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage. Conclusions: These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.

Original languageEnglish
Pages (from-to)1968-1975
Number of pages8
JournalJournal of Clinical Endocrinology and Metabolism
Volume91
Issue number5
DOIs
Publication statusPublished - 1 May 2006
Externally publishedYes

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Superoxides
Spermatozoa
Vitamin K 3
Oxidative stress
Germ Cells
DNA Damage
Oxidative Stress
High Pressure Liquid Chromatography
Flow cytometry
DNA
Cell membranes
Mitochondrial Genome
Sperm Motility
Unsaturated Fatty Acids
Fluorescence Spectrometry
Spontaneous Abortion
Nuclear magnetic resonance spectroscopy
Free Radicals
Mass spectrometry
Embryonic Development

Cite this

De Iuliis, Geoffry N. ; Wingate, Jordana K. ; Koppers, Adam J. ; McLaughlin, Eileen A. ; Aitken, R. John. / Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa. In: Journal of Clinical Endocrinology and Metabolism. 2006 ; Vol. 91, No. 5. pp. 1968-1975.
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Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa. / De Iuliis, Geoffry N.; Wingate, Jordana K.; Koppers, Adam J.; McLaughlin, Eileen A.; Aitken, R. John.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 91, No. 5, 01.05.2006, p. 1968-1975.

Research output: Contribution to journalArticle

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T1 - Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa

AU - De Iuliis, Geoffry N.

AU - Wingate, Jordana K.

AU - Koppers, Adam J.

AU - McLaughlin, Eileen A.

AU - Aitken, R. John

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N2 - Context: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved. Objective: The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function. Method: DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells. Results: HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage. Conclusions: These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.

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SN - 0021-972X

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