Fertilization induces a transient exposure of phosphatidylserine in mouse eggs

Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca2+ concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca2+ spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca2+ release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca2+ ionophore, suggesting that the Ca2+ source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca2+ rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.Fil: Curia, Claudio Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Ernesto, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Stein, Paula. University of Pennsylvania; Estados UnidosFil: Busso, Dolores. Pontificia Universidad Católica de Chile; ChileFil: Schultz, Richard. University of Pennsylvania; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Pharmacological Analyses of Protein Kinases Regulating Egg Maturation in Marine Nemertean Worms: A Review and Comparison with Mammalian Eggs

For development to proceed normally, animal eggs must undergo a maturation process that ultimately depends on phosphorylations of key regulatory proteins. To analyze the kinases that mediate these phosphorylations, eggs of marine nemertean worms have been treated with pharmacological modulators of intracellular signaling pathways and subsequently probed with immunoblots employing phospho-specific antibodies. This article both reviews such analyses and compares them with those conducted on mammals, while focusing on how egg maturation in nemerteans is affected by signaling pathways involving cAMP, mitogen-activated protein kinases, Src-family kinases, protein kinase C isotypes, AMP-activated kinase, and the Cdc2 kinase of maturation-promoting factor

Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

BACKGROUND: Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. METHODS: An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated. RESULTS: The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P < 0.05, Student-Newman-Keuls Method). When the peptide was applied to the oocytes at these concentrations, a dose-dependent increase of PKC activity was observed, in association with a loss of cortical granules ranging from 38+/-2.5 % to 52+/-5.4 %. Evaluation of meiotic status revealed that cyclic RGD peptide was ineffective in inducing meiosis resumption under conditions used in the present study. CONCLUSION: The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for a full oocyte activation response. This study may contribute to the understanding of possible negative effects of skipping gamete interaction in IVF techniques

Echocardiography practice, training and accreditation in the intensive care: document for the World Interactive Network Focused on Critical Ultrasound (WINFOCUS)

Echocardiography is increasingly used in the management of the critically ill patient as a non-invasive diagnostic and monitoring tool. Whilst in few countries specialized national training schemes for intensive care unit (ICU) echocardiography have been developed, specific guidelines for ICU physicians wishing to incorporate echocardiography into their clinical practice are lacking. Further, existing echocardiography accreditation does not reflect the requirements of the ICU practitioner. The WINFOCUS (World Interactive Network Focused On Critical UltraSound) ECHO-ICU Group drew up a document aimed at providing guidance to individual physicians, trainers and the relevant societies of the requirements for the development of skills in echocardiography in the ICU setting. The document is based on recommendations published by the Royal College of Radiologists, British Society of Echocardiography, European Association of Echocardiography and American Society of Echocardiography, together with international input from established practitioners of ICU echocardiography. The recommendations contained in this document are concerned with theoretical basis of ultrasonography, the practical aspects of building an ICU-based echocardiography service as well as the key components of standard adult TTE and TEE studies to be performed on the ICU. Specific issues regarding echocardiography in different ICU clinical scenarios are then described

The Association between Sarcoidosis and Ischemic Heart Disease—A Healthcare Analysis of a Large Israeli Population

(1) Background: Inflammation plays a pivotal role in atherosclerosis, and the association between chronic inflammatory states and ischemic heart disease (IHD) has been shown in several rheumatic diseases. Persistent inflammation might also be a risk factor for IHD in sarcoidosis patients. (2) Methods: Demographic and clinical data of 3750 sarcoidosis patients and 18,139 age- and sex-matched controls were retrieved from the database of Clalit Health Services, Israel’s largest healthcare organization. Variables associated with IHD were assessed by a logistic regression model. To assess for variables that were related to increased risk of all-cause mortality, the Cox proportional hazards method was used, and a log-rank test was performed for survival analysis. (3) Results: Both groups were composed of 64% females with a median age of 56 years. An association between sarcoidosis and IHD was demonstrated by a multivariate analysis (adjusted odds ratio (OR) 1.5; 95% confidence interval (CI) 1.36–1.66). Long-term follow-up revealed increased mortality among sarcoidosis patients: 561 (15%) deaths compared to 1636 (9%) deaths among controls (p &lt; 0.001). Survival analysis demonstrated that sarcoidosis patients were also at increased risk for all-cause mortality compared to controls (multivariate model, adjusted HR 1.93; 95% CI 1.76–2.13)

A Novel Technique of Preserving Internal Mammary Artery Perforators in Nipple Sparing Breast Reconstruction

Summary: As nipple-sparing mastectomy with implant-based reconstruction has increased, attention must be paid to the viability of the nipple-areolar complex. This article describes the use of preoperative Doppler ultrasound to identify the internal mammary artery perforators. Preserving the internal mammary artery improves vascular supply to the nipple-areolar complex