Plasma membrane is one of the preferential targets of reactive oxygen species which cause lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and function. The aim of this study is to evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages. These cells were oxidized with H(2)O(2) at different concentrations, and plasma membrane fluidity was analyzed by two-photon microscopy in combination with the environment-sensitive probe Laurdan. Results show a significant H(2)O(2) concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. A novel statistical analysis evaluated changes in size and number of lipid raft domains under oxidative stress conditions, as lipid rafts are platforms aiding cell signaling and are thought to have relevant roles in macrophage functions. It is shown that H(2)O(2) causes an increase in the number, but not the size, of raft domains. As macrophages are highly resistant to H(2)O(2), these new raft domains might be involved in cell survival pathways.
SummaryPolycystic ovary syndrome (PCOS) affects 5-10% of women of reproductive age. Free radicals, as a product of oxidative stress, impair cells and tissue properties related to human fertility. These free radicals, together with the oxidized molecules, may have a cytotoxic or deleterious effects on sperm and oocytes, on early embryo development or on the endometrium. Aldehyde-modified proteins are highly immunogenic and circulating autoantibodies to new epitopes, such as malondialdehyde (MDA), may affect the reproductive system. Autoantibodies or elevated reactive oxygen species (ROS) in serum are often associated with inflammatory response. The purpose of this work is to investigate whether PCOS women show increased levels of oxidized proteins (protein-MDA) and anti-endometrial antibodies (AEA) in their sera, compared with control patients, and to determine whether AEA specificity is related to oxidized protein derivatives. Sera from 31 women [10 patients with PCOS (PCOS group) and 21 women with male factor of infertility (control group)] were chosen from patients attending for infertility. Anti-endometrial antibodies were determined by enzyme-linked immunosorbent assay (ELISA) with an endometrial cell line (RL-95). Antibodies against MDA modified human serum albumin (HSA-MDA) were also determined by ELISA. Oxidized proteins (protein-MDA) in serum were determined by a colorimetric assay. Patients with PCOS have significantly higher levels of AEA and anti-HSA-MDA, as well as oxidized proteins (protein-MDA) in serum than control patients. For the first time, we describe an autoimmune response in PCOS patients, in terms of AEA. The evidence of protein-MDA in the serum of these patients, together with the increased antibody reactivity to MDA-modified proteins (HSA-MDA) in vitro , supports the conclusion that oxidative stress may be one of the important causes for abnormal endometrial environment with poor embryo receptivity in PCOS patients.
The effects of known inducers of liver metallothionein (MT) synthesis on MT concentrations in the rat brain have been determined using antibodies that are specific for MT I and II and do not cross-react with MT III. There were substantial differences in the MT concentrations in different areas of the brain. Dexamethasone increased MT levels after 24 h in the frontal cortex, cortex, medulla oblongata plus pons, midbrain, striatum, hippocampus, and cerebellum but not in the hypothalamus. Corticosterone produced similar results except in the hippocampus. Long-lasting adrenocorticotropic hormone increased MT concentrations after 12 h in midbrain and striatum but not in the liver. Adrenalectomy decreased MT concentrations after 6 days in the medulla oblongata plus pons, striatum, hippocampus, and hypothalamus but increased concentrations in the liver and kidneys; these effects were reversed by corticosterone. The role of glucocorticoids in the regulation of MT levels therefore differs between tissues and within specific areas of the brain. Injection of zinc or copper intracerebroventricularly and the use of a zinc-deficient diet increased and decreased MT levels, respectively, in some but not all brain areas. Endotoxin increased liver MT but not brain MT I levels after 8 h.
Spermadhesin AWN is a major protein of boar seminal plasma and a sperm surface-associated lectin. AWN binds to beta-galactosides and to porcine zona pellucida glycoproteins, suggesting a role for this protein in primary gamete interaction. However, because capacitation induces remodelling of the sperm surface and AWN is peripherally bound to the plasma membrane, the present study sought to investigate whether AWN is present or absent in the subpopulation of spermatozoa that reaches the ovulated oocyte at the period of fertilization in vivo. Therefore, tubal tissues and oocytes from sows mated with a fertile boar were collected 6-8 h after ovulation. Tissues and oocyte sperm complexes were fixed, immunolabelled with anti-AWN monoclonal antibodies, and examined by means of light and scanning electron microscopy. The results show that spermadhesin AWN is present in spermatozoa seen along the genital tract of the natural mated sow as well as on plasmalemmal remnants of spermatozoa bound to the zona pellucida in vivo.
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