Spontaneous lipid peroxidation in washed human spermatozoa was induced by aerobic incubation at 32 C and measured by malonaldehyde production; loss of motility during the incubation was determined simultaneously. Malonaldehyde production at the point of complete loss of motility, defined as the lipoperoxidative lethal end‐point (LLE), was 0.10 ± 0.03 nmol/108 cells (X̄ ± SD, n = 40), and was independent of the time to complete loss of motility. Human spermatozoa produced both H2O2 and O2−. during aerobic incubation. Inhibition of superoxide dismutase in these cells with KCN showed that all the H2O2 production is due to action of the dismutase. The superoxide dismutase activity of individual human sperm samples varied between 1 and 10 U/108 cells, variations between samples from a single donor being nearly as great as those between different donors. The time to complete motility loss (tL) showed equal variation of 1 to 10 hours among samples. The rate of spontaneous lipid peroxidation, calculated as LLE/tL, for a given sperm sample and the superoxide dismutase activity of the same sample, determined prior to aerobic incubation, gave a good linear correlation (r = 0.97). Glutathione reductase, glutathione peroxidase, and glutathione were found to be present in human spermatozoa, but showed little variation among samples. These results suggest that superoxide dismutase plays the major role in protecting human spermatozoa against lipid peroxidation. In addition, the superoxide dismutase activity of a fresh sperm sample appears to be a good predictor of the lifetime (up to the complete loss of motility) of that particular sample, and so may prove useful in semen analysis.

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These data indicate that alterations in fatty acids similar to those in cystic fibrosis-knockout mice are present in CFTR-expressing tissue from subjects with cystic fibrosis.

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsaturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k1 = 0.003 hr-1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k1 = 0.145 and 0.162 hr-1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37 degrees C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A2 may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation.

A deficiency in essential fatty acid metabolism has been reported in plasma from patients with cystic fibrosis (CF). However, its etiology and role in the expression of disease is unknown. The objective of this study was to determine whether alterations in fatty acid metabolism are specific to CF-regulated organs and whether they play a role in the expression of disease. A membrane lipid imbalance was found in ileum, pancreas, and lung from cftr ؊͞؊ mice characterized by an increase in phospholipid-bound arachidonic acid and a decrease in phospholipid-bound docosahexaenoic acid (DHA). This lipid imbalance was observed in organs pathologically affected by CF including lung, pancreas, and ileum and was not secondary to impaired intestinal absorption or hepatic biosynthesis of DHA. As proof of concept, oral administration of DHA to cftr ؊͞؊ mice corrected this lipid imbalance and reversed the observed pathological manifestations. These results strongly suggest that certain phenotypic manifestations of CF may result from remediable alterations in phospholipid-bound arachidonic acid and DHA levels.docosahexaenoic acid ͉ arachidonic acid ͉ fish oil ͉ pancreas ͉ lung C ystic fibrosis (CF) is the most prevalent lethal autosomal recessive disorder in the Caucasian population, affecting 1 in 2,500 newborns (1). Patients with CF express a typical phenotype characterized by pancreatic insufficiency, ileal hypertrophy, and recurrent pulmonary infections that ultimately lead to pulmonary failure and death. In 1989, the gene whose mutation results in CF was identified and cloned (2, 3). The product of the gene, the CF transmembrane conductance regulator (CFTR), was characterized as an ATP-gated chloride channel that is regulated by cAMP-dependent protein kinase phosphorylation (4).Despite the significant advances made in CF research in recent years, the mechanism by which a mutation in the CFTR gene leads to the manifestations of this disease remains unclear. Although a decrease in apical membrane CFTR-dependent chloride conductance might explain some of the pathological manifestations observed in CF, e.g., viscous secretions, it explains neither the increased inflammation in the lung nor the membrane-recycling defects observed in CF (5-7).Arachidonic acid (AA), an agonist of inflammatory pathways and a stimulant of mucus secretion, is elevated in the phospholipid fraction from bronchial alveolar lavage fluid in CF patients (5). However, the increased inflammation and elevated AA levels observed in CF have long been thought to be secondary to infection (8). This conclusion has been challenged recently by Heeckeren et al. (9), who demonstrated that instillation of agarose beads coated with Pseudomonas into the lungs of cftr Ϫ͞Ϫ mice resulted in increased inflammation and mortality compared with that observed in wild-type mice. These findings suggest that the lungs of cftr Ϫ͞Ϫ mice are primed for inflammation and that the increase in AA and inflammation observed in cftr Ϫ͞Ϫ mice may be a primary event and not secondary to infect...

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

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