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A critical investigation of NADPH oxidase activity in human spermatozoa.
Mol Hum Reprod 2001; 7(3):237-44MH

Abstract

It has been suggested that human spermatozoa contain an NADPH oxidase that could generate reactive oxygen species involved in signalling pathways to promote fertility. The proposal depends on observations that the addition of NADPH to purified human spermatozoa stimulates chemiluminescence by the superoxide (O2-) probe, lucigenin. We confirmed these observations, but demonstrated that lucigenin increases NADPH consumption by spermatozoa and stimulates artefactual O2- production via a diphenyleneiodonium (DPI) sensitive flavoprotein. In the absence of cytochrome c, DPI-inhibitable NADPH oxidation by permeabilized spermatozoa was 8 times too small to account for the rate of NADPH-stimulated cytochrome c reduction. Thus NADPH can directly reduce cytochrome c by a flavoprotein dependent mechanism making this O2- assay also unreliable in sperm suspensions. We were unable to observe O2- production by 40 x 10(6) spermatozoa/ml using electron paramagnetic resonance spectroscopy but could identify O(2)(-) generation from 2000 4beta-phorbol-12-myristate-13-actetate (PMA)-stimulated leukocytes. Using spectrophotometry, we did not detect the reduced cytochrome b(558) component of the neutrophil NADPH oxidase in human spermatozoa. No hydrogen peroxide generation was observed using a sensitive Amplex Red assay. We conclude that human spermatozoa do not possess significant NADPH oxidase activity and that the mechanism by which NADPH promotes capacitation must be re-evaluated.

Authors+Show Affiliations

University of Bristol, Division of Obstetrics & Gynaecology, St Michael's Hospital, Southwell Street, Bristol BS2 8EG, UK.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11228243

Citation

Richer, S C., and W C. Ford. "A Critical Investigation of NADPH Oxidase Activity in Human Spermatozoa." Molecular Human Reproduction, vol. 7, no. 3, 2001, pp. 237-44.
Richer SC, Ford WC. A critical investigation of NADPH oxidase activity in human spermatozoa. Mol Hum Reprod. 2001;7(3):237-44.
Richer, S. C., & Ford, W. C. (2001). A critical investigation of NADPH oxidase activity in human spermatozoa. Molecular Human Reproduction, 7(3), pp. 237-44.
Richer SC, Ford WC. A Critical Investigation of NADPH Oxidase Activity in Human Spermatozoa. Mol Hum Reprod. 2001;7(3):237-44. PubMed PMID: 11228243.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A critical investigation of NADPH oxidase activity in human spermatozoa. AU - Richer,S C, AU - Ford,W C, PY - 2001/3/3/pubmed PY - 2001/6/29/medline PY - 2001/3/3/entrez SP - 237 EP - 44 JF - Molecular human reproduction JO - Mol. Hum. Reprod. VL - 7 IS - 3 N2 - It has been suggested that human spermatozoa contain an NADPH oxidase that could generate reactive oxygen species involved in signalling pathways to promote fertility. The proposal depends on observations that the addition of NADPH to purified human spermatozoa stimulates chemiluminescence by the superoxide (O2-) probe, lucigenin. We confirmed these observations, but demonstrated that lucigenin increases NADPH consumption by spermatozoa and stimulates artefactual O2- production via a diphenyleneiodonium (DPI) sensitive flavoprotein. In the absence of cytochrome c, DPI-inhibitable NADPH oxidation by permeabilized spermatozoa was 8 times too small to account for the rate of NADPH-stimulated cytochrome c reduction. Thus NADPH can directly reduce cytochrome c by a flavoprotein dependent mechanism making this O2- assay also unreliable in sperm suspensions. We were unable to observe O2- production by 40 x 10(6) spermatozoa/ml using electron paramagnetic resonance spectroscopy but could identify O(2)(-) generation from 2000 4beta-phorbol-12-myristate-13-actetate (PMA)-stimulated leukocytes. Using spectrophotometry, we did not detect the reduced cytochrome b(558) component of the neutrophil NADPH oxidase in human spermatozoa. No hydrogen peroxide generation was observed using a sensitive Amplex Red assay. We conclude that human spermatozoa do not possess significant NADPH oxidase activity and that the mechanism by which NADPH promotes capacitation must be re-evaluated. SN - 1360-9947 UR - https://www.unboundmedicine.com/medline/citation/11228243/A_critical_investigation_of_NADPH_oxidase_activity_in_human_spermatozoa_ L2 - https://academic.oup.com/molehr/article-lookup/doi/10.1093/molehr/7.3.237 DB - PRIME DP - Unbound Medicine ER -