Paz Martínez

Membrane lipid dynamics during human sperm capacitation.

Sperm membranes have an unusual lipidic composition which is distinct from those of mammalian somatic cells. They have high levels of plasmalogens, a kind of ether-linked lipids, and a high content of polyunsaturated fatty acyl groups. Plasmalogens may form non-diffusible membrane regions or domains, whereas polyunsaturated ethanolamine plasmalogens are known to destabilize the lipidic bilayer. During transit of sperm through the female reproductive tract, sperm-coating proteins bind to heparin-like glycosaminoglycans. An essential feature of capacitation is the removal of cholesterol from the acrosomal membrane of sperm. Albumin and high-density lipoproteins present in the uterine and follicular fluid act as cholesterol acceptors. Plasma membrane of sperm organize in large non-diffusible lipid domains. This regionalization affects the distribution of both lipids and proteins. A barrier to lateral diffusion of lipids and proteins in the equatorial segment has been reported and contributes to the formation of macrodomains. Lateral separation into cholesterol-rich and cholesterol-depleted microdomains could also be created. Cone-shaped phospholipids induce the formation of non-bilayer phases and might facilitate membrane fusion. This review will discuss the removal of coating proteins, cholesterol efflux, domain organization, relocalization of lipids and proteins and the role of fusogenic lipids during capacitation.

The presence of antibodies to oxidative modified proteins in serum from polycystic ovary syndrome patients

Polycystic 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.

Cholesterol Efflux Promotes Acrosome Reaction in Goat Spermatozoa

Abstract Cholesterol efflux and membrane destabilization play an important role in sperm capacitation and membrane fusion in the acrosome reaction (AR). In this study we establish the effect of cholesterol removal from spermatozoa on acrosomal responsiveness. Mature goat spermatozoa were incubated in BSA-free medium in the presence of β-cyclodextrin (βCD) as cholesterol acceptor. After incubation with 8 mM βCD, 50–60% of cholesterol was released from sperm membranes with no loss in the phospholipid content, and 35% of AR was induced. However, when 30% of cholesterol was lost, this moderate cholesterol decrease was unable to initiate AR. Cholesterol desorption was very rapid, following an exponential kinetics with a half-time of around 10 min, which is in contrast with the slow sigmoidal kinetics of acrosomal responsiveness: around 2 h was required for maximal AR. Our results suggest that cholesterol efflux has a direct influence on the onset of the AR, that is, merely removing cholesterol would trigger the AR.

Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages

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.

Oxidative stress and autoimmune response in the infertile woman.

There is convincing evidence that the establishment of a chronic inflammatory response, together with the presence of a local oxidative environment, could play an important role in the etiology and the progression of several human diseases. In the reproductive system, pathologies such as endometriosis, polycystic ovary syndrome, tubal obstruction, preeclampsia and recurrent abortions are related to the presence of inflammatory cytokines (TNF-alpha, IFN-gamma, IL-1) and to high levels of free radicals that may damage biological molecules, such as lipids, proteins or DNA. Membrane lipids become oxidized and some of their products (malondialdehyde, acetaldehyde, hydroxynonenal) chemically modify proteins. These modified proteins consequently can change their function, antigenicity and therefore become implicated in immunological deleterious reactions associated with inflammatory and/or autoimmune injury. An altered protein function and the presence of circulating autoantibodies to new epitopes, such as malondialdehyde bound to proteins, could block some membrane surface antigens with a receptor function in the reproductive system. This explains how sperm capacitation, oocyte fertilization, or embryo implantation may be inhibited as a consequence of oxidative stress and chronic inflammatory conditions.

An ELISA for five glycolipids from the cell wall of Mycobacterium tuberculosis:: Tween 20 interference in the assay

Mycobacterium tuberculosis cell wall contains antigenic glycolipids: phenol-glycolipid (PGL), diacyltrehalose (DAT), triacyltrehalose (TAT), cord-factor (CF), and sulpholipid-I (SL-I). In the last decade, the usefulness of these antigens for the serodiagnosis of tuberculosis has been evaluated mainly using enzyme-linked immunosorbent assays (ELISA). Currently, there are no conclusive results about the utility of these glycolipidic antigens, because the results obtained by different groups are discrepant. In order to explain these discrepancies, we have investigated the methodological variations in the ELISAs used previously. Specifically, we have studied the following: the coating solvent, the optimum amount of glycolipid coated per well, the blocking agent, and the use of detergent (Tween 20) in the washing buffer. The most significant finding was that Tween 20 detaches PGL, DAT, TAT and SL-I from microtitre wells. However, Tween 20 does not remove CF from the wells. In addition, we have found that the best solvent for coating is n-hexane, that the optimum antigen coating concentration is 1000 ng/well, and that BSA and gelatin are equally effective blocking agents. We can therefore conclude that the use of Tween 20 as a detergent, and the lower antigen coating concentrations (100-200 ng/well), may well explain some of the discrepancies in previous studies.

Autoimmune Response in Women with Endometriosis

PROBLEM: The aim of this study was to investigate the humoral immune response to the female reproductive tissues associated with endometriosis (grades I–III) (n=52), compared with a group of healthy fertile women (n=6).
 METHOD OF STUDY: An ELISA with cultured endometrial cell lines in monolayer was used to determine the presence of anti‐endometrial antibodies (AEA). For anti‐zona pellucida antibodies (AZPA) assessment a conventional ELISA was employed. The presence of antibodies to human sperm (ASA) was performed by the tray agglutination test (TAT).
 RESULTS: Endometriosis grade III was associated with AEA in serum in the 45.4% of patients. The presence of AEA in serum is correlated to endometriosis severity. The 8.7% of women with endometriosis showed ASA, and the 10.9% of them were positive for AZPA. Antibodies specific for endometrial cells do not show reaction to any gamete antigen (sperm or oocyte), suggesting that they are not cross reactive.
 CONCLUSIONS: Severity of endometriosis is correlated with high titers of AEA.

β-Galactomannan and Saccharomyces cerevisiae var. boulardii Modulate the Immune Response against Salmonella enterica Serovar Typhimurium in Porcine Intestinal Epithelial and Dendritic Cells

ABSTRACT Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes inflammation, necrosis, and diarrhea in pigs, as well as being an important source of food-borne diseases in humans. Probiotics and prebiotics are promising alternatives to antibiotics to control and prevent intestinal infections. The present work investigated a recently developed β-galactomannan (βGM) prebiotic compared to the proven probiotic Saccharomyces cerevisiae var. boulardii on porcine ileum intestinal epithelial cells (IECs) of the IPI-2I line and monocyte-derived dendritic cells (DCs) cocultured in vitro with Salmonella. We observed that both S. cerevisiae var. boulardii and βGM inhibited the association of Salmonella with IECs in vitro. Our data indicated that βGM has a higher ability than S. cerevisiae var. boulardii to inhibit Salmonella-induced proinflammatory mRNA (cytokines tumor necrosis factor alpha [TNF-α], interleukin-1α [IL-1α], IL-6, and granulocyte-macrophage colony-stimulating factor [GM-CSF] and chemokines CCL2, CCL20, and CXCL8) and at protein levels (IL-6 and CXCL8). Additionally, βGM and S. cerevisiae var. boulardii induced some effects on DCs that were not observed on IECs: βGM and S. cerevisiae var. boulardii showed slight upregulation of mRNA for TNF-α, GM-CSF, and CCR7 receptor on porcine monocyte-derived dendritic cells (DCs). Indeed, the addition of βGM or S. cerevisiae var. boulardii on DCs cocultured with Salmonella showed higher gene expression (mRNA) for TNF-α, GM-CSF, and CXCL8 compared to that of the control with Salmonella. In conclusion, the addition of βGM inhibits Salmonella-induced proinflammatory profiles in IECs but may promote DC activation, although associated molecular mechanisms remain to be elucidated.

Anti-Endometrial Autoantibodies in Women with a Diagnosis of Infertility

PROBLEM: The purpose of this study was to investigate the frecuency of anti‐endometrial antibodies (AEA) in infertile women.

Phosphorylation of Shc Proteins in Human Sperm in Response to Capacitation and Progesterone Treatment

Several authors have demonstrated the involvement of tyrosine kinases during sperm capacitation and acrosome reaction. Shc proteins (p46Shc, p52Shc, and p66Shc) are cytoplasmic substrates of activated tyrosine kinases and are widely expressed in mammalian somatic tissues. Experiments were designed to demonstrate the presence of Shc in spermatozoa and to study its involvement in the signal transduction events leading to acrosome reaction. Anti‐Shc antibodies strongly reacted with the acrosomal region of methanol‐fixed human sperm. Only one Shc isoform (p52Shc) was detected on Western blot. To study the degree of phosphorylation of Shc during capacitation and acrosome reaction, sperm samples were divided into two groups: noncapacitated and capacitated/progesterone treated. Lysates from both groups were immunoprecipitated with anti‐phosphotyrosine antibodies and the precipitated (ie, phosphorylated) proteins were tested with anti‐Shc antibodies. The intensity of p52Shc was clearly increased in capacitated/progesterone‐stimulated cells. Mol. Reprod. Dev. 50:113–120, 1998.