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Mass Spectrometric Detection of Cholesterol Oxidation in Bovine Sperm

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We report on the presence and formation of cholesterol oxidation products (oxysterols) in bovine sperm. Although cholesterol is the most abundant molecule in the membrane of mammalian cells and is easily oxidized, this is the first report on cholesterol oxidation in sperm membranes as investigated by state of the art liquid chromatographic and mass spectrometric methods. Firstly, oxysterols are already present in fresh semen samples, showing that lipid peroxidation is part of normal sperm physiology. After chromatographic separation (by HPLC), the detected oxysterol species were identified with atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in multiple reaction monitoring mode that enabled detection in a broad and linear concentration range (0.05-100 pmole for each oxysterol species detected). Secondly, exposure of living sperm cells to oxidative stress does not result in the same level and composition of oxysterols species when compared to oxidative stress imposed on reconstituted vesicles from protein-free sperm lipid extracts. This suggests that living sperm cells protect themselves against elevated oxysterol formation. Thirdly, sperm capacitation induces the formation of oxysterols and that these formed oxysterols are almost completely depleted from the sperm surface by albumin. Fourthly and most importantly, capacitation after freeze/thawing sperm fails to induce both the formation of oxysterols and the subsequent albumin-dependent depletion of oxysterols from the sperm surface. The possible physiological relevance of capacitation-dependent oxysterol formation and depletion at the sperm surface as well as the omission of this after freeze/thawing semen is discussed.

The article is published online in the journal Biology of Reproduction and is free to access.