Increase in membrane thickness during development compensates for eggshell thinning due to calcium uptake by the embryo in falcons

NASA Astrophysics Data System (ADS)

Castilla, Aurora M.; van Dongen, Stefan; Herrel, Anthony; Francesch, Amadeu; Martínez de Aragón, Juan; Malone, Jim; José Negro, Juan

2010-02-01

We compared membrane thickness of fully developed eggs with those of non-developed eggs in different endangered falcon taxa. To our knowledge, membrane thickness variation during development has never been examined before in falcons or any other wild bird. Yet, the egg membrane constitutes an important protective barrier for the developing embryo. Because eggshell thinning is a general process that occurs during bird development, caused by calcium uptake by the embryo, eggs are expected to be less protected and vulnerable to breakage near the end of development. Thus, egg membranes could play an important protective role in the later stages of development by getting relatively thicker. We used linear mixed models to explore the variation in membrane thickness ( n = 378 eggs) in relation to developmental stage, taxon, female age, mass and identity (73 females), egg-laying sequence (105 clutches) and the study zone. Our results are consistent with the prediction that egg membranes are thicker in fully developed eggs than in non-developed eggs, suggesting that the increase in membrane thickness during development may compensate for eggshell thinning. In addition, our data shown that thicker membranes are associated with larger, heavier and relatively wider eggs, as well as with eggs that had thinner eggshells. Egg-laying sequence, female age and the study zone did not explain the observed variation of membrane thickness in the falcon taxa studied. As we provide quantitative data on membrane thickness variation during development in falcons not subjected to contamination or food limitation (i.e. bred under captive conditions), our data may be used as a reference for studies on eggs from natural populations. Considering the large variation in membrane thickness and the multiple factors affecting on it and its importance in the protection of the embryo, we encourage other researchers to include measurements on membranes in studies exploring eggshell thickness variation.

A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants.

PubMed

Antoine, A F; Faure, J E; Cordeiro, S; Dumas, C; Rougier, M; Feijó, J A

2000-09-12

In this paper, we report direct measurement of an influx of extracellular Ca(2+) induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca(2+)-selective vibrating probe. Ca(2+) influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca(2+) influx in the vicinity of the sperm entry site with a delay of 1.8 +/- 0.6 sec. The Ca(2+) influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 micrometer.(-1). Once established, Ca(2+) influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol .cm(-2).(-1) and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca(2+) ((c)[Ca(2+)]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca(2+) ionophores mimicked some aspects of egg activation. Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca(2+) influx. In turn, (c)[Ca(2+)] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation.

Localized accumulation of cytosolic calcium near the fused sperm is associated with the calcium- and voltage-dependent block of sperm entry in the sea urchin egg.

PubMed

Ivonnet, Pedro I; Mohri, Tatsuma; McCulloh, David H

2017-10-01

Interaction of the sperm and egg depolarizes the egg membrane, allowing the sperm to enter; however, if the egg membrane is not allowed to depolarize from its resting potential (e.g., by voltage-clamp), the sperm will not enter. Previous studies demonstrated that sperm entry into sea urchin eggs that are voltage-clamped at negative membrane potentials is regulated both by the egg's membrane potential and a voltage-dependent influx of calcium into the egg. In these cases, electrical or cytoplasmic continuity (sperm-egg membrane fusion) occurs at negative membrane potentials, but subsequent loss of cytoplasmic continuity results in failure of sperm entry (unfusion). The work presented herein examined where, in relation to the sperm, and when, in relation to the sperm-induced electrophysiological events, the egg's calcium influx occurs, and how these events relate to successful or failed sperm entry. When sperm entered the egg, elevation of intracellular calcium concentration ([Ca 2+ ] i ) began near the fused sperm on average 5.9 s after sperm-egg membrane fusion. Conversely, when sperm failed to enter the egg, [Ca 2+ ] i elevated near the site of sperm-egg fusion on average 0.7 s after sperm-egg membrane fusion, which is significantly earlier than in eggs for which sperm entered. Therefore, the accumulation of calcium near the site of sperm-egg fusion is spatially and temporally consistent with the mechanism that may be responsible for loss of cytoplasmic continuity and failure of sperm entry. © 2017 Wiley Periodicals, Inc.

Elastic-mathematical theory of cells and mitochondria in swelling process. II. Effect of temperature upon modulus of elasticity of membranous material of egg cells of sea urchin, Strongylocentrotus purpuratus, and of oyster, Crassostrea virginica.

PubMed

Mela, M J

1968-01-01

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13 degrees C the modulus of elasticity (E) is constant, independent of the stress (sigma) and strain (epsilon(nu)) which exist at the cell wall; the membranous material follows Hooke's law, and E approximately 3 x 10(7) dyn/cm(2) for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13 degrees C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function E(sigma) = E(sigma) approaches saturation. The corresponding stress-strain diagrams, sigma = sigma(epsilon(nu)), and the graphs, E(sigma) = E(sigma) and E(sigma) = E(t) are given. The cyto-elastic phenomena at the membrane are discussed.

Lateral mobility of plasma membrane proteins in dividing eggs of the loach (Misgurnus fossilis): Regional differences and changes during the cell cycle.

PubMed

Bozhkova, V P; Budayova, M; Kvasnicka, P; Cigankova, N; Chorvat, D

1994-12-01

Regional differences in lateral diffusion rates of fluorescence-labeled proteins have been studied in the plasma membrane of dividing eggs of the loach (Misgurnus fossilis) by fluorescence recovery after photobleaching (FRAP). Apparent animal-vegetal differences in fluorescence intensity, lateral diffusion coefficients, and fractions of mobile proteins have been found, with all these quantities being higher in the animal pole region than in the yolk region. Cyclic changes in protein diffusion coefficients and mobile fractions during the first few cell cycles have also been recorded. Soon after the end of a cleavage, the diffusion coefficient reaches its minimal value and increases rapidly before the next cleavage.

Comparative evaluation of air cell and eggshell temperature measurement methodologies used in broiler hatching eggs during late incubation.

PubMed

Peebles, E D; Zhai, W; Gerard, P D

2012-07-01

The current study was conducted to compare and contrast the uses of 2 devices (temperature transponder or infrared thermometer) and their locations (inner air cell membrane or outer eggshell surface) in Ross × Ross 708 broiler hatching eggs. The air cells of 14 embryonated and 10 nonembryonated eggs were implanted with temperature transponders on d 13.5 of incubation. Likewise, for these same eggs, eggshell surface temperature was detected with the use of transponders and an infrared thermometer. Temperatures were recorded every 12 h between 14.5 and 18 d of incubation, and graphs and corresponding regression values were used to track the temperatures over these time periods. The temperature readings using all methods in embryonated and nonembryonated eggs were positively correlated. In nonembryonated eggs, temperatures in the air cell and on the eggshell surface using transponders were higher than those on the eggshell surface using an infrared thermometer. Mean air cell temperature readings of embryonated eggs using transponders were higher than those of the eggshell, as determined with the use of transponders or an infrared thermometer. Furthermore, the differences in air cell temperature using transponders and eggshell temperature using an infrared thermometer in embryonated eggs increased with embryonic age. These readings confirmed increased embryo heat production during the incubational period examined. It was further concluded that when compared with actual embryo body temperatures determined in previous studies, the use of transponders in the air cells of broiler hatching eggs detected a higher and closer temperature than eggshell surface temperature. It is suggested that the air cell transponders in embryonated eggs circumvented the confounding effects of the thermal barrier properties of the eggshell and the flow of air across its surface.

Difference in cellular damage and cell death in thermal death time disks and high hydrostatic pressure treated Salmonella Enteritidis (ATCC13076) in liquid whole egg

USDA-ARS?s Scientific Manuscript database

Differences in membrane damage including leakage of intracellular UV-materials and loss of viability of Salmonella Enteritidis (ATCC13076) in liquid whole egg (LWE) following thermal-death-time (TDT) disk and high hydrostatic pressure treatments were examined. Salmonella enteritidis was inoculated ...

Chick chorioallantoic membrane assay as an in vivo model to study the effect of nanoparticle-based anticancer drugs in ovarian cancer.

PubMed

Vu, Binh Thanh; Shahin, Sophia Allaf; Croissant, Jonas; Fatieiev, Yevhen; Matsumoto, Kotaro; Le-Hoang Doan, Tan; Yik, Tammy; Simargi, Shirleen; Conteras, Altagracia; Ratliff, Laura; Jimenez, Chiara Mauriello; Raehm, Laurence; Khashab, Niveen; Durand, Jean-Olivier; Glackin, Carlotta; Tamanoi, Fuyuhiko

2018-06-04

New therapy development is critically needed for ovarian cancer. We used the chicken egg CAM assay to evaluate efficacy of anticancer drug delivery using recently developed biodegradable PMO (periodic mesoporous organosilica) nanoparticles. Human ovarian cancer cells were transplanted onto the CAM membrane of fertilized eggs, resulting in rapid tumor formation. The tumor closely resembles cancer patient tumor and contains extracellular matrix as well as stromal cells and extensive vasculature. PMO nanoparticles loaded with doxorubicin were injected intravenously into the chicken egg resulting in elimination of the tumor. No significant damage to various organs in the chicken embryo occurred. In contrast, injection of free doxorubicin caused widespread organ damage, even when less amount was administered. The lack of toxic effect of nanoparticle loaded doxorubicin was associated with specific delivery of doxorubicin to the tumor. Furthermore, we observed excellent tumor accumulation of the nanoparticles. Lastly, a tumor could be established in the egg using tumor samples from ovarian cancer patients and that our nanoparticles were effective in eliminating the tumor. These results point to the remarkable efficacy of our nanoparticle based drug delivery system and suggests the value of the chicken egg tumor model for testing novel therapies for ovarian cancer.

Membrane lipid layers vs. polarized water dominated by fixed ions: a comparative study of the effects of three macrocyclic ionophores on the K+ permeability of frog skeletal muscle, frog ovarian eggs, and human erythrocytes.

PubMed

Ling, G N; Ochsenfeld, M M

1986-01-01

The effects of 10(-7) M valinomycin, nonactin, and monactin on human erythrocytes, frog sartorius muscle, and frog ovarian oocytes in the presence of varying external K+ concentration were studied. The results showed essentially a consistent but relatively modest increase of the K+ permeability constant in cm/sec with all three antibiotics on human erythrocytes. No change in response to any one of the antibiotics was observed in frog muscles or in frog ovarian eggs. These results and reports of similar failure to demonstrate ionophore-mediated increase of K+ permeability in squid axon and inner membrane of the liver mitochondria led to the conclusion that lipid membrane barrier to ionic traffic may be significant in the human erythrocytes but even here one must regard the evidence as tentative. In contrast, for the majority of other cell types studied, the data indicate the primary, if not exclusive route of ion traffic, is via the nonlipid component of the cell membrane. The evidence that these nonlipid paths are the fixed charge-polarized water layer complex and that they cover much of the cell surface of many types of living cells was discussed.

Selective expression of a sec1/munc18 member in sea urchin eggs and embryos.

PubMed

Leguia, Mariana; Wessel, Gary M

2004-10-01

Regulated secretion is mediated by SNAREs (soluble NSF attachment receptors) and their regulators and effectors, which include the SM (sec1/munc18) family of proteins. Homologs of the SNAREs have been identified in sea urchins, associated with cortical granule exocytosis at fertilization, with membranes of the cleavage furrow, and in secretory cells later in development. To contribute to the understanding of regulated secretion in sea urchins we have cloned the single SM protein homolog from two species of sea urchin, Lytechinus variegatus and Strongylocentrotus purpuratus. In oocytes and eggs, we find that it localizes to the plasma membrane and the cortical region of the egg, consistent with a role in one of the steps leading to cortical granule exocytosis. The protein is also expressed throughout development, enriched in membranes of the cleavage furrow in early embryos, and in cells of the gut in advanced embryos. Furthermore, we find that sec1/munc18 co-localizes with its cognate binding partner syntaxin. Finally, our biochemical analysis shows that the protein associates with rab3 in high molecular weight complexes, suggesting that the exocytotic machinery functions as a multi-protein subunit to mediate regulated secretion in sea urchins. These results will be instrumental in the future to functionally test the SNARE regulators associated with multiple membrane fusion events.

The endomembrane requirement for cell surface repair

NASA Technical Reports Server (NTRS)

McNeil, Paul L.; Miyake, Katsuya; Vogel, Steven S.

2003-01-01

The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca(2+)-dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.

Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmalzing, G.; Eckard, P.; Kroener, S.P.

1990-01-01

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by (gamma-32P)ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiographymore » showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from (3H)ouabain bound to the cell surface before maturation could be phosphorylated with inorganic (32P)phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.« less

THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

PubMed Central

Rothschild, Lord

1957-01-01

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions. PMID:13416315

Assessment of Chicken-Egg Membrane as a Dressing for Wound Healing.

PubMed

Guarderas, Fernando; Leavell, Yaowaree; Sengupta, Trisha; Zhukova, Mariya; Megraw, Timothy L

2016-03-01

To examine the efficacy of the folk remedy of chicken-egg membrane dressing on wound healing. Full-thickness excisional wounds were created on 14 male Sprague-Dawley rats in 2 separate trials. Each animal received 2 wounds on the upper back. One wound was untreated, and the other was dressed with chicken-egg membrane to assess its impact on wound healing. Half of the rats received egg membrane treatment on the inferior wound, whereas the other half received egg membrane treatment on the superior wound. Membrane replacement, wound debridement, and imaging were done on days 5, 8, and 10 and then imaging continued on days 12, 14, 16, 18, and 20 of the experiment. Healing rate was measured based on the wound area over the 20 days of the experiment. The wounds dressed with chicken-egg membrane had a significantly (P < .01) faster rate of healing compared with the control at the early stages of healing between days 0 and 5. This group healed 21% faster during this early phase, compared with the control group. Overall, however, wound healing rates were indistinguishable from days 5 to 20. Chicken-egg membrane dressing significantly improves healing of cutaneous wounds in the early stages of wound healing.

Tissue Sculpting by Fibrils.

PubMed

Jayadev, Ranjay; Sherwood, David R

2016-07-11

In this issue of Developmental Cell, Isabella and Horne-Badovinac (2016) show that Rab10 directs site-specific secretion of basement membrane components, which assemble into fibrils that spool out to elongate the Drosophila egg chamber. These findings establish the basement membrane's active role in tissue sculpting. Copyright © 2016 Elsevier Inc. All rights reserved.

Animal-vegetal polarity in the plasma membrane of a molluscan egg: a quantitative freeze-fracture study.

PubMed

Speksnijder, J E; Mulder, M M; Dohmen, M R; Hage, W J; Bluemink, J G

1985-03-01

Using freeze-fracture electron microscopy, the numerical particle distribution in the fertilized Nassarius egg plasma membrane has been analyzed in four areas at different positions along the animal-vegetal axis of the egg. These areas can be distinguished by distinct microvilli patterns and differences in microvilli densities. In all areas, more IMPs (intramembrane particles) are present on the P face than on the corresponding E face. The ratio of the number of IMPs present on E and P face is similar in all areas (0.48-0.55) except for the most animal part of the vegetal hemisphere, where relatively more IMPs remain attached to the exterior half of the fractured membrane (E/P ratio = 0.88). The IMP density at the vegetal pole of the egg is considerably higher than in the animal hemisphere and in the animal part of the vegetal hemisphere. This difference is due to an increased number of IMPs in all size classes (4-18 nm). In the area adjacent to the vegetal pole the density of particles is also higher than in the two more animal areas, but here the difference is exclusively due to the smaller IMP size classes (4-8 nm). Statistical analysis of our data reveals that the area adjacent to the vegetal pole patch is significantly different from the other areas with respect to the distribution of the IMPs over the different IMP size classes. These results demonstrate the polar organization of the Nassarius egg plasma membrane. The possible role of this surface heterogeneity in the spatial organization of the egg cell and the later embryo is discussed.

[Observation on eggs of Oncomelania hupensis hupensis with scanning electron microscope].

PubMed

Xia, Q B; Yuan, Y B; Liu, B; Tan, P P

2001-01-01

To observe the structure of the mud hull packed Oncomelania eggs and the surface structure of colloid membrane called the third grade membrane of eggs. Scanning electron microscopy was used to observe Oncomelania snail eggs with integral mud hull collected from eastern Dongting Lake. The mud hull of eggs was made of unshapen small humification combined with earth granules with a diameter of 2.6-9.2 microns. The mud hull in 60 um thickness was honeycomb-like in shape with many small holes and small folds on the wall. There were many round or irregularly round hollownesses on the inner layer of mud hull that contacts colloid membrane but no hole through mud hull. There were some protein fiber networks covering on the colloid membrane and apophysis. The structure of the mud hull showed that the exchange of matter was maintained between eggs and outside, and the mud hull is of great importance to regulating temperature and moisture for the growth of eggs by preventing hydrosoluble substances from penetrating into eggs. The protein fiber networks act on gluing mud hull and buffering outside power. The dense glue membrane might be a main barricade to prevent pharmaceutical molecules from penetrating into eggs.

The cooling time of fertile chicken eggs at different stages of incubation.

PubMed

Mortola, Jacopo P; Gaonac'h-Lovejoy, Vanda

2016-01-01

We asked whether or not the thermal characteristics of fertile avian eggs changed throughout incubation. The cooling and warming times, expressed by the time constant τ of the egg temperature response to a rapid change in ambient temperature, were measured in fertile chicken eggs at early (E7), intermediate (E11) and late (E20) stages of embryonic development. Same measurements were conducted on eggs emptied of their content and refilled with water by various amounts. The results indicated that (1) the τ of a freshly laid egg was ~50 min; (2) τ decreased linearly with the drop in egg water volume; (3) the dry eggshell had almost no thermal resistance but its wet inner membrane contributed about one-third to the stability of egg temperature; (4) the egg constituents (yolk, albumen and embryonic tissues) and the chorioallantoic circulation had no measurable effect on τ; (5) the presence of an air pocket equivalent in volume to the air cell of fertile eggs reduced τ by about 3 min (E7), 5 min (E11) and 11 min (E20). Hence, in response to warming the egg τ at E20 was slightly shorter than at E7. In response to cooling, the egg τ at E20 was similar to, or longer than, E7 because embryonic thermogenesis (evaluated by measurements of oxygen consumption during cold) offset the reduction in τ introduced by the air cell. In conclusion, until the onset of thermogenesis the thermal behavior of a fertile egg is closely approximated by that of a water-filled egg with an air volume equivalent to the air cell. It is possible to estimate the cooling τ of avian eggs of different species from their weight and incubation time. Copyright © 2015 Elsevier Ltd. All rights reserved.

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism.

PubMed

Cowan, A E; Myles, D G; Koppel, D E

1991-03-01

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

Juno is the egg Izumo receptor and is essential for mammalian fertilisation

PubMed Central

Bianchi, Enrica; Doe, Brendan; Goulding, David; Wright, Gavin J.

2014-01-01

Fertilisation occurs when sperm and egg recognise each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell surface protein, but its egg receptor has remained a mystery. Here, we identify Juno as the receptor for Izumo1 on mouse eggs, and show this interaction is conserved within mammals. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilisation suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives. PMID:24739963

Effect of gamma irradiation on commercial eggs experimentally inoculated with Salmonella enteritidis

NASA Astrophysics Data System (ADS)

Tellez, I. G.; Trejo, R. M.; Sanchez, R. E.; Ceniceros, R. M.; Luna, Q. P.; Zazua, P.; Hargis, B. M.

1995-02-01

Using intact, fresh shell eggs, inoculated with 10 8 colony-forming units (cfu) of S. enteritidis, the effect of three doses of gamma irradiation on bacteriologic population and physical characteristics (Haugh units and yolk color) of the eggs was determinated. Penetration test area was picked at random just off the air cell of each egg. Aluminum cylinders were attached to the egg surface with a rim of molten paraffin, and 10 8S. enteritidis was then applied to inoculate the egg. Eggs were then irradiated within 2 hours using a Cobalt-60 gamma source at either 1, 2, or 3 kGy. A second set of inoculated, non-irradiated was used as controls. Following irradiation, eggs were maintained at 4°C for 42 hours prior culture. Irradiation with 1 kGy resulted in a significant (P < .05), 3.9 log reduction in detectable S. enteritidis in the shell and a higly significant (P < .025) 95% reduction in detectable S. enteritidis in the internal shell membranes. Irradiation of eggs with either 2 or 3 kGy reduced bacterial contamination to non-detectable levels in both the shell and internal membranes. However, irradiation at either 1, 2 or 3 kGy resulted in a significant (P <- .05) decrease (approximately 50%) in Haugh units. Additionally, irradiation of intact shell eggs at 2 or 3 Kgy significantly (P ≤ .05) reduced yolk color regardless of the level of irradiation exposure implemented. This data indicates that gamma irradiation of intact raw eggs is effective in reducing (1 kGy) or eliminating (2 or more kGy) S. enteritidis contamination. However, each of the levels of irradiation used in the present experiments caused marked reduction of selected measures of egg quality.

Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes.

PubMed

Ruan, Guang-Ping; Ma, Li; Meng, Xiao-Jing; Meng, Min-Jie; Wang, Xiao-Ning; Lin, Ying; Wu, Zheng-Qiang; He, Xiao-Wei; Wang, Ju-Fang; Zhu, Yong

2007-01-01

HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.

Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts.

PubMed

Hara, Yuki; Merten, Christoph A

2015-06-08

Nuclear size changes dynamically during development and has long been observed to correlate with the space surrounding the nucleus, as well as with the volume of the cell. Here we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter range, herein referred to as the nuclear domain. Under spatial constraints smaller than this nuclear domain, the size of microtubule-occupied space surrounding the nucleus turned out to be limiting for the accumulation of membranes around the nucleus via the motor protein dynein, therefore determining the speed of nuclear expansion. This mechanism explains how spatial information surrounding the nucleus, such as the positioning of the nucleus inside the cell, can control nuclear expansion. Copyright © 2015 Elsevier Inc. All rights reserved.

Predicting mercury in mallard ducklings from mercury in chorioallantoic membranes

USGS Publications Warehouse

Heinz, G.H.; Hoffman, D.J.

2003-01-01

Methylmercury has been suspected as a cause of impaired reproduction in wild birds, but the confounding effects of other environmental stressors has made it difficult to determine how much mercury in the eggs of these wild species is harmful. Even when a sample egg can be collected from the nest of a wild bird and the mercury concentration in that egg compared to the laboratory-derived thresholds for reproductive impairment, additional information on the mercury levels in other eggs from that nest would be helpful in determining whether harmful levels of mercury were present in the clutch. The measurement of mercury levels in chorioallantoic membranes offers a possible way to estimate how much mercury was in a chick that hatched from an egg, and also in the whole fresh egg itself. While an embryo is developing, wastes are collected in a sac called the chorioallantoic membranes, which often remain inside the eggshell and can be collected for contaminant analysis. We fed methylmercury to captive mallards to generate a broad range of mercury levels in eggs, allowed the eggs to hatch normally, and then compared mercury concentrations in the hatchling versus the chorioallantoic membranes left behind in the eggshell. When the data from eggs laid by mercury- treated females were expressed as common logarithms, a linear equation was created by which the concentration of mercury in a duckling could be predicted from the concentration of mercury in the chorioallantoic membranes from the same egg. Therefore, if it were not possible to collect a sample egg from a clutch of wild bird eggs, the collection of the chorioallantoic membranes could be substituted, and the mercury predicted to be in the chick or whole egg could be compared to the thresholds of mercury that have been shown to cause harm in controlled feeding studies with pheasants, chickens, and mallards.

ELECTRIC IMPEDANCE OF THE FROG EGG

PubMed Central

Cole, Kenneth S.; Guttman, Rita M.

1942-01-01

Electrical impedance measurements were made upon unfertilized and fertilized eggs of the leopard frog, Rana pipiens, over a frequency range of 0.05 to 10 kc. Average values of 170 ohm cm.2 were obtained for the plasma membrane resistance of the egg, 2.0 µf/cm.2 for the plasma membrane capacity, 86° for the phase angle of the membrane, and 570 ohm cm. for the specific resistance of the interior. These values did not change upon fertilization. No spontaneous rhythmical impedance changes such as have been found by Hubbard and Rothschild in the trout egg were found in frog eggs. PMID:19873312

β1 Integrin is an Adhesion Protein for Sperm Binding to Eggs

PubMed Central

Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.

2009-01-01

We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment, but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different. PMID:19338281

The timing of cortical granule fusion, content dispersal, and endocytosis during fertilization of the hamster egg: an electrophysiological and histochemical study.

PubMed

Kline, D; Stewart-Savage, J

1994-03-01

To determine the temporal relationship between cortical granule exocytosis and the repetitive calcium transients, which are characteristic of mammalian fertilization, we monitored membrane addition from exocytosis during fertilization of hamster eggs. Continuous measurement of membrane capacitance by applying a 3.1-nA alternating current at 375 Hz showed addition of cortical granule membrane. Simultaneous measurement of membrane potential revealed each calcium transient by the appearance of transient hyperpolarizing responses due to calcium-activated potassium channels in the egg. The initial membrane capacitance of the eggs averaged 736 +/- 44 pF (mean +/- SD; n = 7) and an increase in capacitance of 61 +/- 19 pF occurred within 4 sec of the start of the first hyperpolarizing response (HR) after fertilization. Immediately after the first increase in capacitance there was a gradual decline in membrane capacitance in all eggs and in five/seven eggs the capacitance returned to the unfertilized level in 7.8 +/- 4.4 min. The gradual decline in capacitance after the first increase indicated endocytosis, which was confirmed by the internalization of fluorescently labeled dextran. Superimposed on the gradual decline in membrane capacitance were smaller increases in capacitance that occurred with the second and later HRs. The total increase in capacitance from the first three events averaged 72 +/- 19 pF, representing an average increase in capacitance of about 10% of the capacitance of the unfertilized egg. By labeling eggs before and after permeabilization with two different fluorochromes attached to Lens culinaris agglutinin, we demonstrate that the dispersal of the cortical granules contents does not occur immediately after exocytosis. Our results demonstrate that cortical granule exocytosis in hamster eggs is closely coupled to the periodic increases in calcium, that the contents of the cortical granules are slow to disperse, and that after exocytosis, the surface area of the egg returns to the unfertilized level because of a period of endocytosis.

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376

Fertilization Induces a Transient Exposure of Phosphatidylserine in Mouse Eggs

PubMed Central

Curia, Claudio A.; Ernesto, Juan I.; Stein, Paula; Busso, Dolores; Schultz, Richard M.; Cuasnicu, Patricia S.; Cohen, Débora J.

2013-01-01

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. PMID:23951277

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo.

PubMed

Speksnijder, J E; Dohmen, M R; Tertoolen, L G; de Laat, S W

1985-07-01

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1'-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient (D) and mobile fraction (MF) are 2.9 +/- 0.2 X 10(-9) cm2/sec and 51 +/- 2%, respectively, while in the four vegetal macromeres D = 5.0 +/- 0.3 X 10(-9) cm2/sec and MF = 78 +/- 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 X 10(-9) cm2/sec in the three cycles measured), while at the end of G2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 X 10(-9) cm2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles (D = 4.3-5.0 X 10(-9) cm2/sec), except for a peak at the S phase of the first cell cycle (D = 6.0 X 10(-9) cm2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animal-vegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.

Arabidopsis thaliana GEX1 has dual functions in gametophyte development and early embryogenesis

USDA-ARS?s Scientific Manuscript database

GEX1 is a plasma membrane protein conserved among plant species, and was previously shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immedi...

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

PubMed Central

Lehto, M T; Sharom, F J

1998-01-01

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane. PMID:9576857

Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

PubMed Central

Terasaki, Mark; Miyake, Katsuya; McNeil, Paul L.

1997-01-01

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events. PMID:9314529

Taspine isolated from Radix et Rhizoma Leonticis inhibits proliferation and migration of endothelial cells as well as chicken chorioallantoic membrane neovascularisation.

PubMed

Zhang, Yanmin; He, Langchong; Meng, Liang; Luo, Wenjuan

2008-01-01

The aim of the present study was to investigate an anti-angiogenic effect of taspine isolated from Radix et Rhizoma Leonticsi. Taspine was screened for the first time, using cell membrane chromatography (CMC). The anti-angiogeneic activity of taspine was tested by using the chicken chorioallantoic membrane (CAM) neovascularisation model in vivo and the HUVEC proliferation and migration models in vitro, respectively. The results showed that taspine could inhibit CAM angiogenesis significantly within the concentration range of 0.5-2 mug/egg, proliferation and migration of endothelial cells in a dose-dependent manner. The CAM histomorphology results indicated that taspine could inhibit blood vessels sprouts and proliferation of vascular endothelial cell. These findings suggest that taspine is a promising candidate for use as an angiogenesis inhibitor.

[Kinematics and ultrastructure of plasmic factor regions in the egg of Wachtliella persicariae L. (Diptera) : II. The behaviour of ooplasmic partial systems after centrifugation of eggs in the stage of four cleavage nuclei].

PubMed

Wolf, Rainer

1969-03-01

Kinematics and ultrastructure of centrifuged and untreated eggs fromWachtliella persicariae were investigated for the micromorphological properties of ooplasmic factor regions and their role in early developmental processes by means of time-lapse motion pictures and electron microscopic analysis (see part I).After centrifugation the eggs show up to five different layers, among them a pole of fatty yolk with lipid droplets, a region of clear plasm (rich in ground plasm) which itself may become subdivided into a centripetal region with nuclei and endoplasmic reticulum, followed by a centrifugal part with mitochondria and ribosomes, another region containing orange clods of proteid yolk and finally a cup of glycogen. Displacement of pole plasm from the posterior pole always is accompanied by dislocation of the basophilic oosome material contained therein. At sufficient r.p.m. both of them enter the centripetal area of clear plasm. Structures of "di-polar density" type are orientated by centrifugation. The initial phase till the centrifuge reaches its final r.p.m. may act decicively upon the site of certain egg components after centrifugation as upon the nuclei, and thus may essentially influence the experimental results. In case centrifugation coincides with certain dividing phases of energides, the nuclear envelope becomes fragmented. The fragments then may appear piled up to form annulated membranes which have been recognized as pathological structures in centrifuged eggs. Besides lamellar cytosomes are often found. In centrifuged as well as in untreated eggs the nuclear envelope either consists of two layers as usual or may be of the complex multi-layered type (see part I). As for the movement of nuclei, the possible role of the complex nuclear envelope is not yet clear. The pigment halo of cleavage nuclei does not play an active part in nuclear migration. In centrifuged eggs yolk nuclei are of the usual type i.e. either roundish, horse-shoe-shaped or multi-lobed. They mostly appear in parts of the entoplasm which are poor in yolk. A surrounding rich in yolk does not seem to be essential for transforming normal cleavage nuclei into vitellophagues. For their changing into the multi-lobed type yolk nuclei must be surrounded by a sufficient amount of ground plasm.Pole cells have been found in the posterior pole region only. Their formation requires an abundant amount of ground plasm, the presence of cleavage energides, as well as pole plasm and oosome material, if not either of the two latter systems. Since in centrifuged eggs pole plasm and basophilic oosome material are always shifted together into the region of clear plasm, contrary to the opinion of other authors (p. 42; part I, p. 124) the technique of centrifugation does not permit any decision as to which of both ooplasmic systems controls the karyotic differentiation of the germ line or the formation of pole cells, respectively, or whether both systems are essential to promote these processes.The oolemma may also become invaginated to form cell membranes when no nuclei are present ("pseudoblastoderm"), the formation occurring in regions with sufficient amounts of ground plasm only. For that reason the formation of pole cells is restricted to the posterior pole rich in ground plasm, whereas blastoderm cells exclusively occur in the area of preblastoderm plasm. The ground plasm plays a decisive part in the dynamics of cell membrane formation. As for blastoderm cells, the nuclei seem to be necessary only to control their regular shape. In contradiction to the opinion of other authors (p. 42, part I, p. 124), the periplasm of young eggs cannot range among the essential prerequisites of blastoderm formation. During centrifugation it does not stay at the surface of the egg poles where nevertheless a blastoderm may be formed. Yet blastoderm formation is only possible if, in spite of the compact condition of polar yolk material, the egg poles become covered with preblastoderm plasm from the region of clear plasm, rich in ground plasm, and thus replacing sufficient amounts of periplasm and ground plasm shifted by centrifugation.

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

PubMed

Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

1984-07-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

Protein profiles of hatchery egg shell membrane

USDA-ARS?s Scientific Manuscript database

Background: Eggshells, which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of m...

ELECTRIC IMPEDANCE OF HIPPONOË EGGS

PubMed Central

Cole, Kenneth S.

1935-01-01

Alternating current resistance and capacity measurements have been made from 1.08 103 to 2.32 106 cycles per second on suspensions of unfertilized, fertilized, and swollen unfertilized eggs of the echinoderm Hipponoë esculenta. A simple method has been developed for measuring the volume concentration of eggs in a suspension. The membrane of the unfertilized egg is practically non-conducting at low frequencies and shows a static capacity of 0.87 µf/cm.2 except perhaps at the highest frequencies. The equivalent specific resistance of the egg interior is 11 times that of sea water. The membrane of the fertilized egg is practically non-conducting at low frequencies and shows a static capacity 2.5 times that of the unfertilized egg except at the higher frequencies where another reactive element produces a marked effect. The internal resistance is apparently higher than that of the unfertilized egg. The static capacity per unit area of the membrane decreases as a linear function of the surface area when the eggs are swollen in dilute sea water. In 40 per cent sea water, the capacity falls to about 75 per cent of normal. PMID:19872897

Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

PubMed

Pawlikowska-Pawlęga, Bożena; Misiak, Lucjan E; Jarosz-Wilkołazka, Anna; Zarzyka, Barbara; Paduch, Roman; Gawron, Antoni; Gruszecki, Wieslaw I

2014-08-01

With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Calcium influx is required for endocytotic membrane retrieval

PubMed Central

Vogel, Steven S.; Smith, Robert M.; Baibakov, Boris; Ikebuchi, Yoshihide; Lambert, Nevin A.

1999-01-01

Cells use endocytotic membrane retrieval to compensate for excess surface membrane after exocytosis. Retrieval is thought to be calcium-dependent, but the source of this calcium is not known. We found that, in sea urchin eggs, endocytotic membrane retrieval required extracellular calcium. Inhibitors of P-type calcium channels—cadmium, ω-conotoxin MVIIC, ω-agatoxin IVA, and ω-agatoxin TK—blocked membrane retrieval; selective inhibitors of N-type and L-type channels did not. Treatment with calcium ionophores overcame agatoxin inhibition in a calcium-dependent manner. Cadmium blocked membrane retrieval when applied during the first 5 minutes after fertilization, the period when the membrane potential is depolarized. We conclude that calcium influx through ω-agatoxin-sensitive channels plays a key role in signaling for endocytotic membrane retrieval. PMID:10220411

Antimitotic effect of an extract of the sea anemone Bunodosoma caissarum on sea urchin egg development.

PubMed

Malpezzi, E L; Freitas, J C

1990-01-01

A methanolic extract of the sea anemone Bunodosoma caissarum has an antimitotic effect on sea urchin egg development. The extract produces a dose-dependent inhibition of cell cleavage. When the extract is added together with sperm to unfertilized sea urchin eggs, the ED50 is 0.60 +/- 0.03 mg/ml (mean +/- SEM). When added shortly after fertilization, the extract produces the same kind of progressive inhibition but with an ED50 of 0.98 +/- 0.16 mg/ml. In the first case, detachment of the vitelline layer is inhibited whereas in the second case the extract inhibits cleavage even when the membrane is present.

Multiplication of Salmonella Enteritidis in egg yolks after inoculation outside, on, and inside vitelline membranes and storage at different temperatures

USDA-ARS?s Scientific Manuscript database

Prompt refrigeration to restrict bacterial growth is important for reducing egg-borne transmission of Salmonella enterica serovar Enteritidis (SE). The nutrient-rich yolk interior is a relatively infrequent location for initial SE deposition in eggs, but migration across the vitelline membrane can ...

Transfection of embryonated Muscovy duck eggs with a recombinant plasmid is suitable for rescue of infectious Muscovy duck parvovirus.

PubMed

Wang, Jianye; Huang, Yu; Ling, Jueyi; Wang, Zhixiang; Zhu, Guoqiang

2017-12-01

For members of the family Parvoviridae, rescue of infectious virus from recombinant plasmid is usually done in cultured cells. In this study, the whole genome of the pathogenic Muscovy duck parvovirus (MDPV) strain YY was cloned into the pBluescript II (SK) vector, generating recombinant plasmid pYY. With the aid of a transfection reagent, pYY plasmid was inoculated into 11-day-old embryonated Muscovy duck eggs via the chorioallantoic membrane route, resulting in the successful rescue of infectious virus and death of the embryos. The rescued virus exhibited pathogenicity in Muscovy ducklings similar to that of its parental strain, as evaluated based on the mortality rate. The results demonstrate that plasmid transfection in embryonated Muscovy duck eggs is a convenient and efficacious method for rescue of infectious MDPV in comparison to transfection of primary cells, which is somewhat time-consuming and laborious.

Coplanar PCB distribution between chorioallantoic membranes and eggs of alligators and Loggerhead sea turtles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bargar, T.A.; Cobb, G.P.

1995-12-31

The relative distribution of coplanar polychlorinated biphenyls (PCBs) between chorioallantoic membranes (CAMS) and eggs was investigated in inviable American alligator (Alligator mississippiensis) and Loggerhead sea turtle (Caretta caretra) eggs. Cam and egg extracts were fractionated by HPLC using a porous graphitic column (PGC) and an in line switching valve to separate coplanar from non-coplanar PCBs. The fractions were collected, concentrated by nitrogen evaporation, and injected on GC-ECD (60M DB-5 capillary column) for quantification. Alligator and Loggerhead sea turtle eggs contain toxicologically significant coplanar PCBs. Mono-ortho substituted PCBs were present with greater frequency relative to non-ortho substituted PCBs in both eggsmore » and CAMS. The presence of coplanar PCBs in eggs appears to be correlated to coplanar PCB presence in CAMS. The chorioallantoic membrane could serve as a biomarker of embryo exposure to coplanar PCBs.« less

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization

PubMed Central

Jégou, Antoine; Ziyyat, Ahmed; Barraud-Lange, Virginie; Perez, Eric; Wolf, Jean Philippe; Pincet, Frédéric; Gourier, Christine

2011-01-01

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm–egg fusion is a direct consequence of CD9 controlled sperm–egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs. PMID:21690351

Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis

PubMed Central

Field, Christine M.; Pelletier, James F.; Mitchison, Timothy J.

2017-01-01

We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen, and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ~11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers, and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues. PMID:28065319

Surfing the vegetal pole in a small population: extracellular vertical transmission of an 'intracellular' deep-sea clam symbiont.

PubMed

Ikuta, Tetsuro; Igawa, Kanae; Tame, Akihiro; Kuroiwa, Tsuneyoshi; Kuroiwa, Haruko; Aoki, Yui; Takaki, Yoshihiro; Nagai, Yukiko; Ozawa, Genki; Yamamoto, Masahiro; Deguchi, Ryusaku; Fujikura, Katsunori; Maruyama, Tadashi; Yoshida, Takao

2016-05-01

Symbiont transmission is a key event for understanding the processes underlying symbiotic associations and their evolution. However, our understanding of the mechanisms of symbiont transmission remains still fragmentary. The deep-sea clam Calyptogena okutanii harbours obligate sulfur-oxidizing intracellular symbiotic bacteria in the gill epithelial cells. In this study, we determined the localization of their symbiont associating with the spawned eggs, and the population size of the symbiont transmitted via the eggs. We show that the symbionts are located on the outer surface of the egg plasma membrane at the vegetal pole, and that each egg carries approximately 400 symbiont cells, each of which contains close to 10 genomic copies. The very small population size of the symbiont transmitted via the eggs might narrow the bottleneck and increase genetic drift, while polyploidy and its transient extracellular lifestyle might slow the rate of genome reduction. Additionally, the extracellular localization of the symbiont on the egg surface may increase the chance of symbiont exchange. This new type of extracellular transovarial transmission provides insights into complex interactions between the host and symbiont, development of both host and symbiont, as well as the population dynamics underlying genetic drift and genome evolution in microorganisms.

The Sperm-surface glycoprotein, SGP, is necessary for fertilization in the frog, Xenopus laevis.

PubMed

Nagai, Keita; Ishida, Takuya; Hashimoto, Takafumi; Harada, Yuichirou; Ueno, Shuichi; Ueda, Yasushi; Kubo, Hideo; Iwao, Yasuhiro

2009-06-01

To identify a molecule involved in sperm-egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm-surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti-SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti-SGP antibody recognized large molecules, with molecular masses of 65-150 kDa and minor smaller molecules with masses of 20-28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle-binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm-egg membrane binding and is responsible for the establishment of fertilization in Xenopus.

Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos

NASA Technical Reports Server (NTRS)

Danilchik, Michael V.; Bedrick, Steven D.; Brown, Elizabeth E.; Ray, Kimberly

2003-01-01

In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of microtubules near the surface is sufficient to mobilize them for exocytosis at the end of the cell cycle.

Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

PubMed Central

Székács, Inna; Kaszás, Nóra; Gróf, Pál; Erdélyi, Katalin; Szendrő, István; Mihalik, Balázs; Pataki, Ágnes; Antoni, Ferenc A.; Madarász, Emilia

2013-01-01

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na+ and organic cations through gramicidin channels and detecting the Cl–-channel functions of the (α5β2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline. PMID:24339925

Egg structure and ultrastructure of Paterdecolyus yanbarensis (Insecta, Orthoptera, Anostostomatidae, Anabropsinae).

PubMed

Mashimo, Yuta; Fukui, Makiko; Machida, Ryuichiro

2016-11-01

The egg structure of Paterdecolyus yanbarensis was examined using light, scanning electron and transmission electron microscopy. The egg surface shows a distinct honeycomb pattern formed by exochorionic ridges. Several micropyles are clustered on the ventral side of the egg. The egg membrane is composed of an exochorion penetrated with numerous aeropyles, an endochorion, and an extremely thin vitelline membrane. The endochorion is thickened at the posterior egg pole, probably associated with water absorption. A comparison of egg structure among Orthoptera revealed that the micropylar distribution pattern is conserved in Ensifera and Caelifera and might be regarded as a groundplan feature for each group; in Ensifera, multiple micropyles are clustered on the ventral side of the egg, whereas in Caelifera, micropyles are arranged circularly around the posterior pole of the egg. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture

PubMed Central

Gilhare, Varsha Rani; Hirpurkar, S. D.; Kumar, Ashish; Naik, Surendra Kumar; Sahu, Tarini

2015-01-01

Aim: The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV) in chorioallantoic membrane (CAM) of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF) cell culture. Materials and Methods: Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the virus is confirmed by clinical findings of affected birds, pock morphology and histopathology of infected CAM. In addition one field isolate and vaccine strain of FPV was adapted to CEF cell culture. CEF cell culture was prepared from 9-day-old embryonated chicken eggs. Result: Clinical symptoms observed in affected birds include pox lesion on comb, wattle, eyelids and legs, no internal lesions were observed. All field isolates produced similar findings in CAM. Pocks produced by field isolates ranged from 3 mm to 5 mm at the third passage while initial passages edematous thickening and necrosis of CAM was observed. Pocks formed by lyophilized strain were ranges from 0.5 mm to 2.5 mm in diameter scattered all over the membrane at the first passage. Intra-cytoplasmic inclusion bodies are found on histopathology of CAM. At third passage level, the CEF inoculated with FPV showed characteristic cytopathic effect (CPE) included aggregation of cells, syncytia and plaque formation. Conclusion: FPV field isolates and vaccine strain produced distinct pock lesions on CAMs. Infected CAM showed intracytoplasmic inclusion bodies. The CEF inoculated with FPV field isolate as well as a vaccine strain showed characteristic CPE at third passage level. PMID:27047081

Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture.

PubMed

Gilhare, Varsha Rani; Hirpurkar, S D; Kumar, Ashish; Naik, Surendra Kumar; Sahu, Tarini

2015-03-01

The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV) in chorioallantoic membrane (CAM) of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF) cell culture. Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the virus is confirmed by clinical findings of affected birds, pock morphology and histopathology of infected CAM. In addition one field isolate and vaccine strain of FPV was adapted to CEF cell culture. CEF cell culture was prepared from 9-day-old embryonated chicken eggs. Clinical symptoms observed in affected birds include pox lesion on comb, wattle, eyelids and legs, no internal lesions were observed. All field isolates produced similar findings in CAM. Pocks produced by field isolates ranged from 3 mm to 5 mm at the third passage while initial passages edematous thickening and necrosis of CAM was observed. Pocks formed by lyophilized strain were ranges from 0.5 mm to 2.5 mm in diameter scattered all over the membrane at the first passage. Intra-cytoplasmic inclusion bodies are found on histopathology of CAM. At third passage level, the CEF inoculated with FPV showed characteristic cytopathic effect (CPE) included aggregation of cells, syncytia and plaque formation. FPV field isolates and vaccine strain produced distinct pock lesions on CAMs. Infected CAM showed intracytoplasmic inclusion bodies. The CEF inoculated with FPV field isolate as well as a vaccine strain showed characteristic CPE at third passage level.

CRYPTIC CHOICE OF CONSPECIFIC SPERM CONTROLLED BY THE IMPACT OF OVARIAN FLUID ON SPERM SWIMMING BEHAVIOR

PubMed Central

Yeates, Sarah E; Diamond, Sian E; Einum, Sigurd; Emerson, Brent C; Holt, William V; Gage, Matthew J G

2013-01-01

Despite evidence that variation in male–female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species’ ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species’ identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior. PMID:24299405

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

PubMed Central

1975-01-01

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles. PMID:1174130

SODIUM CHLORIDE AND SELECTIVE DIFFUSION IN LIVING ORGANISMS.

PubMed

Loeb, J

1922-11-20

1. It is shown that NaCl acts like CaCl(2) or LaCl(3) in preventing the diffusion of strong acids through the membrane of the egg of Fundulus with this difference only that a M/8 solution of NaCl acts like a M/1,000 solution of CaCl(2) and like a M/30,000 solution of LaCl(3). 2. It is shown that these salts inhibit the diffusion of non-dissociated weak acid through the membrane of the Fundulus egg but slightly if at all. 3. Both NaCl and CaCl(2) accelerate the diffusion of dissociated strong alkali through the egg membrane of Fundulus and CaCl(2) is more efficient in this respect than NaCl. 4. It is shown that in moderate concentrations NaCl accelerates the rate of diffusion of KCl through the membrane of the egg of Fundulus while CaCl(2) does not.

A single cell penetration system by ultrasonic driving

NASA Astrophysics Data System (ADS)

Zhou, Zhaoying; Xiao, Mingfei; Yang, Xing; Wu, Ting

2008-12-01

The researches of single cell's control and operation are the hotspots in whole world. Among the various technologies, the transmission of ectogenic genetic materials between cell membrane is very significant. Imitating the Chinese traditional acupuncture therapy, a new ultrasonic resonance driving method, is imported to drive a cell's penetration probe. A set of the single cell penetration system was established to perform this function. This system includes four subsystems: driving part, micromanipulation part, observation and measurement part, and actuation part. Some fish egg experiments indicate that this system is workable and effective.

Embryonic integument and "molts" in Manduca sexta (Insecta, Lepidoptera).

PubMed

Ziese, Stefanie; Dorn, August

2003-02-01

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the embryo of M. sexta secretes three covers at approximately the same developmental stage. A marked difference: the second embryonic cover, which in Hemimetabola clearly exhibits a cuticular organization, has instead a membranous, cuticulin-like structure. We see the difference as the result of an evolutionary reductional process promoted by the redundancy of embryonic covers in the egg shell. Embryonic "molts" also occur in noninsect arthropods; their phylogenetical aspects are discussed. Copyright 2002 Wiley-Liss, Inc.

Compositional analysis and structural elucidation of glycosaminoglycans in chicken eggs

PubMed Central

Liu, Zhangguo; Zhang, Fuming; Li, Lingyun; Li, Guoyun; He, Wenqing; Linhardt, Robert J.

2014-01-01

Glycosaminoglycans (GAGs) have numerous applications in the fields of pharmaceuticals, cosmetics, nutraceuticals, and foods. GAGs are also critically important in the developmental biology of all multicellular animals. GAGs were isolated from chicken egg components including yolk, thick egg white, thin egg white, membrane, calcified shell matrix supernatant, and shell matrix deposit. Disaccharide compositional analysis was performed using ultra high-performance liquid chromatography-mass spectrometry. The results of these analyses showed that all four families of GAGs were detected in all egg components. Keratan sulfate was found in egg whites (thick and thin) and shell matrix (calcified shell matrix supernatant and deposit) with high level. Chondroitin sulfates were much more plentiful in both shell matrix components and membrane. Hyaluronan was plentiful in both shell matrix components and membrane, but were only present in a trace of quantities in the yolk. Heparan sulfate was plentiful in the shell matrix deposit but was present in a trace of quantities in the egg content components (yolk, thick and thin egg whites). Most of the chondroitin and heparan sulfate disaccharides were present in the GAGs found in chicken eggs with the exception of chondroitin and heparan sulfate 2,6-disulfated disaccharides. Both CS and HS in the shell matrix deposit contained the most diverse chondroitin and heparan sulfate disaccharide compositions. Eggs might provide a potential new source of GAGs. PMID:25218438

Confocal observation of hydrophilic and lipophilic photosensitizers in endothelial cells, lumen of the vessels, interstitium, and tumor cells using the chicken chorioallantoic membrane

NASA Astrophysics Data System (ADS)

Rueck, Angelika C.; Akguen, Nermin; Heckelsmiller, K.; Beck, Gerd C.; Genze, Felicitas; Steiner, Rudolf W.

1998-05-01

The dynamic behavior of lipophilic and hydrophilic sensitizers in cell cultures and non animal in vivo systems with varying incubation but also during the photodynamic therapy will be summarized within the presentation. As an appropriate in vivo system we used the chorioallantoic membrane (CAM) of fertilized eggs, which served as a substrate for tumor cells. Because the CAM is a transparent membrane it is possible to view individual blood vessels and to examine tumor cells as well as structural changes of the supplying vasculature. To adapt this system to high magnification microscopy, we established a new technique for in vivo observation of the CAM tissue. This technique enables online investigations of alterations at cellular level induced by drugs with confocal laser scanning microscopy. The localization of the drugs with clinical importance was observed after different application times in the lumen of the vessels, the endothelial cells and the tumor cells. In addition light induced subcellular Ca2+-changes were observed and correlated with the photodynamic process.

'Soya milk Tris-based phytoextender reduces apoptosis in cryopreserved buffalo (Bubalus bubalis) spermatozoa'.

PubMed

Mohan, R; Atreja, S K

2014-10-01

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)-based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS-PAGE followed by immunoblotting against caspase-8, caspase-9, caspase-3, poly(ADP-ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC-1 assay) and apoptotic cells (annexin V-FITC/PI assay). The results obtained clearly indicate the significant (p < 0.05) reduction in the expression of caspase-3 (27 kDa), caspase-8 (53 kDa), caspase-9 (50 kDa) precursor and cytochrome c (17 kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non-significant (p > 0.05) reduction in expression of PARP-DNA-binding subunit (24 kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p < 0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non-significant (p > 0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT-based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non-animal origin. © 2014 Blackwell Verlag GmbH.

Experimental parameterisation of principal physics in buoyancy variations of marine teleost eggs.

PubMed

Jung, Kyung-Mi; Folkvord, Arild; Kjesbu, Olav Sigurd; Sundby, Svein

2014-01-01

It is generally accepted that the high buoyancy of pelagic marine eggs is due to substantial influx of water across the cell membrane just before ovulation. Here we further develop the theoretical basis by applying laboratory observations of the various components of the fertilized egg in first-principle equations for egg specific gravity (ρ(egg)) followed by statistical validation. We selected Atlantic cod as a model animal due to the affluent amount of literature on this species, but also undertook additional dedicated experimental works. We found that specific gravity of yolk plus embryo is central in influencing ρ(egg) and thereby the buoyancy. However, our established framework documents the effect on ρ(egg) of the initial deposition of the heavy chorion material in the gonad prior to spawning. Thereafter, we describe the temporal changes in ρ(egg) during incubation: Generally, the eggs showed a slight rise in ρ(egg) from fertilization to mid-gastrulation followed by a gradual decrease until full development of main embryonic organs just before hatching. Ontogenetic changes in ρ(egg) were significantly associated with volume and mass changes of yolk plus embryo. The initial ρ(egg) at fertilization appeared significantly influenced by the chorion volume fraction which is determined by the combination of the final chorion volume of the oocyte and of the degree of swelling (hydrolyzation) prior to spawning. The outlined principles and algorithms are universal in nature and should therefore be applicable to fish eggs in general.

Experimental Parameterisation of Principal Physics in Buoyancy Variations of Marine Teleost Eggs

PubMed Central

Jung, Kyung-Mi; Folkvord, Arild; Kjesbu, Olav Sigurd; Sundby, Svein

2014-01-01

It is generally accepted that the high buoyancy of pelagic marine eggs is due to substantial influx of water across the cell membrane just before ovulation. Here we further develop the theoretical basis by applying laboratory observations of the various components of the fertilized egg in first-principle equations for egg specific gravity (ρegg) followed by statistical validation. We selected Atlantic cod as a model animal due to the affluent amount of literature on this species, but also undertook additional dedicated experimental works. We found that specific gravity of yolk plus embryo is central in influencing ρegg and thereby the buoyancy. However, our established framework documents the effect on ρegg of the initial deposition of the heavy chorion material in the gonad prior to spawning. Thereafter, we describe the temporal changes in ρegg during incubation: Generally, the eggs showed a slight rise in ρegg from fertilization to mid-gastrulation followed by a gradual decrease until full development of main embryonic organs just before hatching. Ontogenetic changes in ρegg were significantly associated with volume and mass changes of yolk plus embryo. The initial ρegg at fertilization appeared significantly influenced by the chorion volume fraction which is determined by the combination of the final chorion volume of the oocyte and of the degree of swelling (hydrolyzation) prior to spawning. The outlined principles and algorithms are universal in nature and should therefore be applicable to fish eggs in general. PMID:25122447

Laminin Levels Regulate Tissue Migration and Anterior-Posterior Polarity during Egg Morphogenesis in Drosophila.

PubMed

Díaz de la Loza, María C; Díaz-Torres, Alfonsa; Zurita, Federico; Rosales-Nieves, Alicia E; Moeendarbary, Emad; Franze, Kristian; Martín-Bermudo, María D; González-Reyes, Acaimo

2017-07-05

Basement membranes (BMs) are specialized extracellular matrices required for tissue organization and organ formation. We study the role of laminin and its integrin receptor in the regulation of tissue migration during Drosophila oogenesis. Egg production in Drosophila involves the collective migration of follicle cells (FCs) over the BM to shape the mature egg. We show that laminin content in the BM increases with time, whereas integrin amounts in FCs do not vary significantly. Manipulation of integrin and laminin levels reveals that a dynamic balance of integrin-laminin amounts determines the onset and speed of FC migration. Thus, the interplay of ligand-receptor levels regulates tissue migration in vivo. Laminin depletion also affects the ultrastructure and biophysical properties of the BM and results in anterior-posterior misorientation of developing follicles. Laminin emerges as a key player in the regulation of collective cell migration, tissue stiffness, and the organization of anterior-posterior polarity in Drosophila. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cortical mechanics and myosin-II abnormalities associated with post-ovulatory aging: implications for functional defects in aged eggs

PubMed Central

Mackenzie, Amelia C.L.; Kyle, Diane D.; McGinnis, Lauren A.; Lee, Hyo J.; Aldana, Nathalia; Robinson, Douglas N.; Evans, Janice P.

2016-01-01

STUDY HYPOTHESIS Cellular aging of the egg following ovulation, also known as post-ovulatory aging, is associated with aberrant cortical mechanics and actomyosin cytoskeleton functions. STUDY FINDING Post-ovulatory aging is associated with dysfunction of non-muscle myosin-II, and pharmacologically induced myosin-II dysfunction produces some of the same deficiencies observed in aged eggs. WHAT IS KNOWN ALREADY Reproductive success is reduced with delayed fertilization and when copulation or insemination occurs at increased times after ovulation. Post-ovulatory aged eggs have several abnormalities in the plasma membrane and cortex, including reduced egg membrane receptivity to sperm, aberrant sperm-induced cortical remodeling and formation of fertilization cones at the site of sperm entry, and reduced ability to establish a membrane block to prevent polyspermic fertilization. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovulated mouse eggs were collected at 21–22 h post-human chorionic gonadotrophin (hCG) (aged eggs) or at 13–14 h post-hCG (young eggs), or young eggs were treated with the myosin light chain kinase (MLCK) inhibitor ML-7, to test the hypothesis that disruption of myosin-II function could mimic some of the effects of post-ovulatory aging. Eggs were subjected to various analyses. Cytoskeletal proteins in eggs and parthenogenesis were assessed using fluorescence microscopy, with further analysis of cytoskeletal proteins in immunoblotting experiments. Cortical tension was measured through micropipette aspiration assays. Egg membrane receptivity to sperm was assessed in in vitro fertilization (IVF) assays. Membrane topography was examined by low-vacuum scanning electron microscopy (SEM). MAIN RESULTS AND THE ROLE OF CHANCE Aged eggs have decreased levels and abnormal localizations of phosphorylated myosin-II regulatory light chain (pMRLC; P = 0.0062). Cortical tension, which is mediated in part by myosin-II, is reduced in aged mouse eggs when compared with young eggs, by ∼40% in the cortical region where the metaphase II spindle is sequestered and by ∼50% in the domain to which sperm bind and fuse (P < 0.0001). Aging-associated parthenogenesis is partly rescued by treating eggs with a zinc ionophore (P = 0.003), as is parthenogenesis induced by inhibition of mitogen-activated kinase (MAPK) 3/1 [also known as extracellular signal-regulated kinase (ERK)1/2] or MLCK. Inhibition of MLCK with ML-7 also results in effects that mimic those of post-ovulatory aging: fertilized ML-7-treated eggs show both impaired fertilization and increased extents of polyspermy, and ML-7-treated young eggs have several membrane abnormalities that are shared by post-ovulatory aged eggs. LIMITATIONS, REASONS FOR CAUTION These studies were done with mouse oocytes, and it remains to be fully determined how these findings from mouse oocytes would compare with other species. For studies using methods not amenable to analysis of large sample sizes and data are limited to what images one can capture (e.g. SEM), data should be interpreted conservatively. WIDER IMPLICATIONS OF THE FINDINGS These data provide insights into causes of reproductive failures at later post-copulatory times. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTEREST(S) This project was supported by R01 HD037696 and R01 HD045671 from the NIH to J.P.E. Cortical tension studies were supported by R01 GM66817 to D.N.R. The authors declare there are no financial conflicts of interest. PMID:26921397

The extraembryonic serosa protects the insect egg against desiccation

PubMed Central

Jacobs, Chris G. C.; Rezende, Gustavo L.; Lamers, Gerda E. M.; van der Zee, Maurijn

2013-01-01

Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle. To experimentally test the protective function of the serosa, we exploit an exceptional possibility to eliminate this membrane by zerknüllt1 RNAi in the beetle Tribolium castaneum. We analyse hatching rates of eggs under a range of humidities and find dramatically decreasing hatching rates with decreasing humidities for serosa-less eggs, but not for control eggs. Furthermore, we show serosal expression of Tc-chitin-synthase1 and demonstrate that its knock-down leads to absence of the serosal cuticle and a reduction in hatching rates at low humidities. These developmental genetic techniques in combination with ecological testing provide experimental evidence for a crucial role of the serosa in desiccation resistance. We propose that the origin of this extraembryonic membrane facilitated the spectacular radiation of insects on land, as did the origin of the amniote egg in the terrestrial invasion of vertebrates. PMID:23782888

First ultrastructural data on the human tapeworm Taenia asiatica eggs by scanning and transmission electron microscopy (SEM, TEM).

PubMed

Galán-Puchades, M Teresa; Yang, Yichao; Marcilla, Antonio; Choe, Seongjun; Park, Hansol; Osuna, Antonio; Eom, Keeseon S

2016-09-01

Humans are definitive hosts of three species of the Taenia genus, namely Taenia solium, Taenia saginata and Taenia asiatica. The relative novelty of the latter explains the lack of knowledge concerning certain relevant aspects related to this parasite, such as its definite geographical distribution and whether its eggs can infect humans or not. So far, only the eggs of T. solium are known to be infective for humans, producing cysticercosis. Although eggs contain the infective stage, the oncosphere, there is a lack of research on the ultrastructure of eggs of human taeniids. We show, for the first time, the ultrastructure of eggs of T. asiatica by means of SEM and TEM analyses. We detected all the envelopes, namely the egg shell, vitelline layer, outer embryophoric membrane, embryophore, granular layer, basal membrane, oncospheral membrane and oncospheral tegument. Hooks surrounded by myofibrils and glycogen-like particles, the two types of secretory granules of the penetration glands, as well as several nuclei and mitochondria were also revealed in the oncospheres. In addition to the already known structures in eggs from other Taenia species, the presence of two types of small vesicles is described herein, possibly corresponding to exosomes and ectosomes because of their shape and size, which could participate in the host/parasite intercellular communication.

Chicken egg shell membrane associated proteins and peptides

USDA-ARS?s Scientific Manuscript database

Egg shells are poultry industry byproducts with potential for use in various biological and agricultural applications. We have been interested in the membranes underlying the calcareous shell, as a feed supplement which showed potential to t improve immunity and performance of post hatch poultry. ...

Sexing of chicken eggs by fluorescence and Raman spectroscopy through the shell membrane

PubMed Central

Preusse, Grit; Schnabel, Christian; Bartels, Thomas; Cramer, Kerstin; Krautwald-Junghanns, Maria-Elisabeth; Koch, Edmund; Steiner, Gerald

2018-01-01

In order to provide an alternative to day-old chick culling in the layer hatcheries, a noninvasive method for egg sexing is required at an early stage of incubation before onset of embryo sensitivity. Fluorescence and Raman spectroscopy of blood offers the potential for precise and contactless in ovo sex determination of the domestic chicken (Gallus gallus f. dom.) eggs already during the fourth incubation day. However, such kind of optical spectroscopy requires a window in the egg shell, is thus invasive to the embryo and leads to decreased hatching rates. Here, we show that near infrared Raman and fluorescence spectroscopy can be performed on perfused extraembryonic vessels while leaving the inner egg shell membrane intact. Sparing the shell membrane makes the measurement minimally invasive, so that the sexing procedure does not affect hatching rates. We analyze the effect of the membrane above the vessels on fluorescence signal intensity and on Raman spectrum of blood, and propose a correction method to compensate for it. After compensation, we attain a correct sexing rate above 90% by applying supervised classification of spectra. Therefore, this approach offers the best premises towards practical deployment in the hatcheries. PMID:29474445

Cryptic choice of conspecific sperm controlled by the impact of ovarian fluid on sperm swimming behavior.

PubMed

Yeates, Sarah E; Diamond, Sian E; Einum, Sigurd; Emerson, Brent C; Holt, William V; Gage, Matthew J G

2013-12-01

Despite evidence that variation in male-female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species' ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species' identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior. © 2013 The Authors. Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.

Engineering Tough Materials: Biomimetic Eggshell

DTIC Science & Technology

2016-08-29

known that this domain interacts with CO32- ions. Eggshell collagen membrane (Figure 1) was extracted by dissolving the shell of a chicken egg in 1 M...Therefore, an experiment was designed and executed with the aim of trying to elucidate whether the shell membrane has a mechanical role. Eggs 8 were...obtained from the local supermarket and holes were punctured at the equator in order to remove the liquid contents (both yolk and white). Eggs were tested in

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bleil, J.D.; Wassarman, P.M.

1986-04-01

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetratemore » the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only background levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.« less

Elastic-Mathematical Theory of Cells and Mitochondria in Swelling Process

PubMed Central

Mela, M. J.

1968-01-01

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13°C the modulus of elasticity (E) is constant, independent of the stress (σ) and strain (εν) which exist at the cell wall; the membranous material follows Hooke's law, and E ≈ 3 × 107 dyn/cm2 for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13°C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function Eσ = E(σ) approaches saturation. The corresponding stress-strain diagrams, σ = σ(εν), and the graphs, Eσ = E(σ) and Eσ = E(t) are given. The cyto-elastic phenomena at the membrane are discussed. PMID:5689191

Mechanical behavior of cells in microinjection: a minimum potential energy study.

PubMed

Liu, Fei; Wu, Dan; Chen, Ken

2013-08-01

Microinjection is a widely used technique to deliver foreign materials into biological cells. We propose a mathematical model to study the mechanical behavior of a cell in microinjection. Firstly, a cell is modeled by a hyperelastic membrane and interior cytoplasm. Then, based on the fact that the equilibrium configuration of a cell would minimize the potential energy, the energy function during microinjection is analyzed. With Lagrange multiplier and Rayleigh-Ritz technique, we successfully minimize the potential energy and obtain the equilibrium configuration. Upon this model, the injection force, the injection distance, the radius of the microinjector and the membrane stress are studied. The analysis demonstrates that the microinjector radius has a significant influence on the cell mechanical behavior: (1) the larger radius generates larger injection force and larger interior pressure at the same injection distance; (2) the radius determines the place where the membrane is most likely to rupture by governing the membrane stress distribution. For a fine microinjector with radius less than 20% of the cell radius, the most likely rupture point located at the edge of the contact area between the microinjector and the membrane; however, it may move to the middle of the equilibrium configuration as the radius increases. To verify our model, some experiments were conducted on zebrafish egg cells. The results show that the computational analysis agrees with the experimental data, which supports the findings from the theoretical model. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of egg weight on composition, embryonic growth, and expression of amino acid transporter genes in yolk sac membranes and small intestines of the domestic pigeon (Columba livia).

PubMed

Chen, M X; Li, X G; Yan, H C; Wang, X Q; Gao, C Q

2016-06-01

The objective of this study was to investigate the effect of egg weight on the composition of the egg, the growth of the embryo, and the expression of amino acid transporter genes in the yolk sac membranes and small intestines of the domestic pigeon (Columba livia). A total of 240 fertilized eggs were collected and divided into two groups based on the weight of the eggs, light (LE) and heavy (HE). The composition of 20 eggs from each group was measured, and the remaining eggs were weighed and placed in an incubator. On embryonic days (E) 9, 11, 13, and 15 and day of hatch (DOH), 15 embryos/hatchlings from each group were measured for embryonic growth, and samples were collected. The HE had heavier yolk and albumen weights than the LE (P < 0.01). Compared with the LE, the HE had heavier yolk-free embryonic body and yolk sac weights from E13 to DOH (P < 0.05). Additionally, the HE had larger yolk sac membrane weights from E13 to E15 (P < 0.05) and had more residual yolk sac content on DOH than those of the LE (P < 0.01). The yolk absorption was greater for the HE than for the LE from E11 to E13 (P < 0.05). Furthermore, the abundance of CAT2 and PepT1 mRNA in the yolk sac membranes was greater in the HE than in the LE on E13 (P < 0.05). Compared with the LE, the gene expression of EAAT2 in the intestine on E13 was greater in the HE, whereas the expression of EAAT3 was lower in the HE (P < 0.05). Taken together, our results suggest that egg weight influenced the composition of the eggs, embryonic development, and expression of amino acid transporter genes in the yolk sac membranes and small intestines of pigeon embryos. © 2016 Poultry Science Association Inc.

Developmental Regulation of a Plasma Membrane Arabinogalactan Protein Epitope in Oilseed Rape Flowers.

PubMed Central

Pennell, RI; Janniche, L; Kjellbom, P; Scofield, GN; Peart, JM; Roberts, K

1991-01-01

We have identified and characterized the temporal and spatial regulation of a plasma membrane arabinogalactan protein epitope during development of the aerial parts of oilseed rape using the monoclonal antibody JIM8. The JIM8 epitope is expressed by the first cells of the embryo and by certain cells in the sexual organs of flowers. During embryogenesis, the JIM8 epitope ceases to be expressed by the embryo proper but is still found in the suspensor. During differentiation of the stamens and carpels, expression of the JIM8 epitope progresses from one cell type to another, ultimately specifying the endothecium and sperm cells, the nucellar epidermis, synergid cells, and the egg cell. This complex temporal sequence demonstrates rapid turnover of the JIM8 epitope. There is no direct evidence for any cell-inductive process in plant development. However, if cell-cell interactions exist in plants and participate in flower development, the JIM8 epitope may be a marker for one set of them. PMID:12324592

Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

PubMed Central

Chen, M.S.; Almeida, E.A.C.; Huovila, A.-P.J.; Takahashi, Y.; Shaw, L.M.; Mercurio, A.M.; White, J.M.

1999-01-01

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2). PMID:9971748

Transovarial persistence of Babesia ovata DNA in a hard tick, Haemaphysalis longicornis, in a semi-artificial mouse skin membrane feeding system.

PubMed

Umemiya-Shirafuji, Rika; Hatta, Takeshi; Okubo, Kazuhiro; Sato, Moeko; Maeda, Hiroki; Kume, Aiko; Yokoyama, Naoaki; Igarashi, Ikuo; Tsuji, Naotoshi; Fujisaki, Kozo; Inoue, Noboru; Suzuki, Hiroshi

2017-12-20

Bovine piroplasmosis, a tick-borne protozoan disease, is a major concern for the cattle industry worldwide due to its negative effects on livestock productivity. Toward the development of novel therapeutic and vaccine approaches, tick-parasite experimental models have been established to clarify the development of parasites in the ticks and the transmission of the parasites by ticks. A novel tick-Babesia experimental infection model recently revealed the time course of Babesia ovata migration in its vector Haemaphysalis longicornis, which is a dominant tick species in Japan. However, there has been no research on the transovarial persistence of B. ovata DNA using this experimental infection model. Here we assessed the presence of B. ovata DNA in eggs derived from parthenogenetic H. longicornis female ticks that had engorged after semi-artificial mouse skin membrane feeding of B. ovata-infected bovine red blood cells. The oviposition period of the engorged female ticks was 21-24 days in the semi-artificial feeding. Total egg weight measured daily reached a peak by day 3 in all female ticks. Nested PCR revealed that 3 of 10 female ticks laid B. ovata DNA-positive eggs after the semi-artificial feeding. In addition, B. ovata DNA was detected at the peak of egg weight during oviposition, indicating that B. ovata exist in the eggs laid a few days after the onset of oviposition in the tick. These findings will contribute to the establishment of B. ovata-infected H. longicornis colonies under laboratory conditions.

Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy1

PubMed Central

Kryzak, Cassie A.; Moraine, Maia M.; Kyle, Diane D.; Lee, Hyo J.; Cubeñas-Potts, Caelin; Robinson, Douglas N.; Evans, Janice P.

2013-01-01

ABSTRACT Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm. PMID:23863404

Nanoliposomes for encapsulation and delivery of the potential antitumoral methyl 6-methoxy-3-(4-methoxyphenyl)-1 H-indole-2-carboxylate

NASA Astrophysics Data System (ADS)

Abreu, Ana S.; Castanheira, Elisabete Ms; Queiroz, Maria-João Rp; Ferreira, Paula Mt; Vale-Silva, Luís A.; Pinto, Eugénia

2011-08-01

A potential antitumoral fluorescent indole derivative, methyl 6-methoxy-3-(4-methoxyphenyl)-1 H-indole-2-carboxylate, was evaluated for the in vitro cell growth inhibition on three human tumor cell lines, MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), and NCI-H460 (non-small cell lung cancer), after a continuous exposure of 48 h, exhibiting very low GI50 values for all the cell lines tested (0.25 to 0.33 μM). This compound was encapsulated in different nanosized liposome formulations, containing egg lecithin (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG), DSPC, cholesterol, dihexadecyl phosphate, and DSPE-PEG. Dynamic light scattering measurements showed that nanoliposomes with the encapsulated compound are generally monodisperse and with hydrodynamic diameters lower than 120 nm, good stability and zeta potential values lower than -18 mV. Dialysis experiments allowed to monitor compound diffusion through the lipid membrane, from DPPC/DPPG donor liposomes to NBD-labelled lipid/DPPC/DPPG acceptor liposomes.

Subcellular Metabolite and Lipid Analysis of Xenopus laevis Eggs by LAESI Mass Spectrometry

PubMed Central

Reschke, Brent R.; Henderson, Holly D.; Powell, Matthew J.; Moody, Sally A.; Vertes, Akos

2014-01-01

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis. PMID:25506922

Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

PubMed

Shrestha, Bindesh; Sripadi, Prabhakar; Reschke, Brent R; Henderson, Holly D; Powell, Matthew J; Moody, Sally A; Vertes, Akos

2014-01-01

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

Comparative study on histological structures of the vitelline membrane of hen and duck egg observed by cryo-scanning electron microscopy.

PubMed

Chung, Wen-Hsin; Lai, Kung-Ming; Hsu, Kuo-chiang

2010-02-10

The histological structures of the vitelline membranes (VM) of hen and duck eggs were observed by cryo-scanning electron microscopy (cryo-SEM), and the chemical characteristics were also compared. The outer layer surface (OLS) of duck egg VM showed networks constructed by fibrils and sheets (0.1-5.2 microm in width), and that of hen egg presented networks formed only by sheets (2-6 microm in width). Thicker fibrils (0.5-1.5 microm in width) with different arrangement were observed on the inner layer surface (ILS) of duck egg VM as compared to those (0.3-0.7 microm in width) of hen egg VM. Upon separation, the outer surface of the outer layer (OSOL) and the inner surface of the inner layer (ISIL) of hen and duck egg VMs were quite similar to fresh VM except that the OSOL of duck egg VM showed networks constructed only by sheets. Thin fibrils interlaced above a bumpy or flat structure were observed at the exposed surface of the outer layer (ESOL) of hen and duck egg VMs. The exposed surfaces of inner layers (ESIL) of hen and duck egg VMs showed similar structures of fibrils, which joined, branched, and ran in straight lines for long distances up to 30 microm; however, the widths of the fibrils shown in ESOL and ESIL of duck egg VM were 0.1 and 0.7-1.4 microm, respectively, and were greater than those (<0.1 and 0.5-0.8 microm) of hen egg VM. The continuous membranes of both hen and duck egg VMs were still attached to the outer layers when separated. The content of protein, the major component of VM, was higher in duck egg VM (88.6%) than in hen egg VM (81.6%). Four and six major SDS-soluble protein patterns with distinct localization were observed in hen and duck egg VMs, respectively. Overall, the different histological structures of hen and duck egg VMs were suggested to be majorly attributable to the diverse protein components.

Progressive chorion morphology during egg development in Samia ricini (Donovan).

PubMed

Renthlei, Collin Z; Raghuvarman, Arumugam; Kharbuli, Besterwell; Dey, Sudip

2010-03-01

The egg of Samia ricini (Donovan), is oval or laterally flattened ellipsoid, freshly laid eggs are candid white while the chorion is colorless and semi-transparent. The surface of the chorion is covered with network patterns of polygons and their shapes are common in the whole surface region. The boundaries between polygons made ridges had distinct acropyles at three-cell junctions. The numbers of aeropyles are variable according to their structures both in the lateral flat and marginal regions. During the course of egg development, no significant structural changes were observed in either the polygonal structures or the overall morphology of the egg. However, the size of the aeropyles kept on changing as the egg matures. The aeropyle increases initially upto day-9 of egg development and then decreases as it approach hatching. Lines of weaknesses were not observed at time of hatching or close to it. Hatching process of the newly emerge larvae are through gnawing. The larva eats their way out through the chorion membrane mostly from the anterior region. Egg buster or spine which aid in hatching are not present in the newly emerge larvae.This article was published online on 25 September 2009. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 6 January 2010.

Microencapsulation of bovine spermatozoa for use in artificial insemination: a review.

PubMed

Nebel, R L; Vishwanath, R; McMillan, W H; Saacke, R G

1993-01-01

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus of potential use in artificial insemination. The polymers poly-l-lysine, polyvinylamine and protamine sulfate have proven best for membranes. Encapsulation has been successful with capsules ranging in size from 0.75 to 1.5 mm, and with sperm concentrations from 45 to 180 x 10(6) cells mL-1. Successful extenders include CUE, CAPROGEN, and egg yolk-citrate-glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under ageing and physical stress) is negatively related to membrane thickness which ranges from 1.92 to 5.32 microns (depending on the concentration of polymer used) and positively related to concentration of sperm encapsulated. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when cows are inseminated at conventional times. Uterine retention of inseminates is favoured by capsules having a 'sticky' membrane. Using current procedures, preliminary homospermic fertility studies indicate that sperm encapsulated with poly-l-lysine or protamine sulfate may achieve normal fertility.

Combination of super chilling and high carbon dioxide concentration techniques most effectively to preserve freshness of shell eggs during long-term storage.

PubMed

Yanagisawa, T; Ariizumi, M; Shigematsu, Y; Kobayashi, H; Hasegawa, M; Watanabe, K

2010-01-01

This study was made to examine the combined effects of stored temperature and carbon dioxide atmosphere on shell egg quality. The shell eggs were packed into polyethylene terephthalate/polyethylene (PET/PE) pouches and stored at 0 degrees C (super chilling), 10 degrees C, and 20 degrees C, respectively for 90 d. The atmospheric carbon dioxide concentration was controlled to obtain the 3 concentration levels of high (about 2.0%), medium (about 0.5%), and low (below 0.01%). Changes in Haugh unit (HU) values, weakening of vitelline membranes, and generation of volatiles were analyzed to evaluate the freshness of shell eggs. Results showed that, compared with the other combinations, the technique of super chilling and high carbon dioxide concentration enabled shell eggs to be most effectively stored for 90 d, based on estimations of the statistical significances of differences in HU values, and on maintaining the initial HU values during storage. In addition, the storage of shell eggs using this combination technique was found to significantly prevent the weakening of the vitelline membrane based on the estimations of numbers of eggs without vitelline membrane breakage when eggs broke, and significantly lowered the incidence of hexanal in the yolk from exposure to the gas chromatographic-mass spectrometric analyses of volatiles. Thus, these results confirmed that the combination of super chilling and high carbon dioxide concentration was the most effective technique for preserving shell eggs during a long term of 90 d compared with other combination techniques.

The post-thaw quality of ram sperm held for 0 to 48 h at 5 degrees C prior to cryopreservation.

PubMed

Purdy, P H

2006-06-01

The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from the 0 or 48 h holding time (281 sperm; P<0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P>0.05). These results indicate that ram sperm can be held at 5 degrees C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.

Evidence of possible vertical transmission of Tembusu virus in ducks.

PubMed

Zhang, Ying; Li, Xiuli; Chen, Hao; Ti, Jinfeng; Yang, Guoping; Zhang, Lu; Lu, Yunjian; Diao, Youxiang

2015-09-30

In 2013, Tembusu virus (TMUV) infection was successively observed on several breeding duck farms in Shandong province, China. Affected ducks showed consistently acute anorexia, diarrhea and egg production drop. 125 hatching eggs produced by TMUV infected breeding ducks from four duck farms were collected. Among them, 35 hatching eggs were selected randomly from all before incubation for vitelline membrane samples collection. The rest of 90 hatching eggs were incubated routinely. As a result, 16 hatching eggs were found non-embryonated, 28 duck embryos died during incubation and 46 newly hatched ducklings were obtained. Vitelline membranes of non-embryonated hatching eggs, vitelline membrane, brain or liver samples of dead embryos and brain samples of newly hatched ducklings were collected for virus detection. Samples collected from one egg, embryo or duckling were treated as one. Consequently, 18 of 35 (51.43%) hatching eggs, 2 of 16 (12.50%) non-embryonated duck eggs, 17 of 28 (60.71%) dead duck embryos and 5 of 46 (10.87%) newly hatched ducklings were detected positive for TMUV using NS3-based RT-PCR. Overall, 42 of 125 (33.6%) eggs were positive for TMUV. A virus strain, designated as TMUV-SDDE, was isolated from one of these dead duck embryos which were detected TMUV positive. The results of phylogenetic analysis showed that E gene of TMUV-SDDE virus was closely related to other TMUV strains isolated in China during 2010-2013. Pathogenicity studies showed that TMUV-SDDE strain was virulent to ducklings. This is the first report that TMUV is isolated from duck embryos. The findings provide evidence of possible vertical transmission of TMUV from breeding ducks to ducklings. Copyright © 2015 Elsevier B.V. All rights reserved.

[Expression and localization of transmembrane protein CMTM2 in human testis and sperm].

PubMed

Zhang, X W; Lan, K; Yang, W B; Li, Q; Zhao, Y P; Yin, H Q; Kite, B; Bai, W J; Xu, T

2017-08-18

To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system. The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction. CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction. Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. The expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis. And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca).

PubMed

Zivkovic, Danica; Créton, Robbert; Zwaan, Gideon; de Bruijn, Willem C; Dohmen, M René

1990-11-01

During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca 2+ /Mg 2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca 2+ /Mg 2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca 2+ -stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

PubMed

Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

2005-08-01

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

Effects of supplementation with fish oil and n-3 PUFAs enriched egg yolk phospholipids on anhedonic-like response and body weight in the rat chronic mild stress model of depression.

PubMed

Rutkowska, M; Trocha, M; Szandruk, M; Słupski, W; Rymaszewska, J

2013-08-01

Polyunsaturated fatty acids play an important role in the human organism. They guarantee a normal function of nervous cells, influence neurotransmission, and build some elements of cellular membranes. Several reports indicate an association between a deficiency of polyunsaturated fatty acids and depression. The aim of this study was to examine the effects of diet supplemented with fish oil, which is rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) and n-3 PUFAs enriched phospholipids ("super lecithin") obtained from designed eggs on anhedonic-like response and body weight in the rat chronic mild stress (CMS) model of depression. The results showed that neither fish oil nor n-3 PUFAs enriched egg yolk phospholipids supplementation reversed disturbances caused by CMS, such as anhedonic-like state or reduction of body weight gain.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, G.E.

When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase.more » The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.« less

Scanning electron microscope studies of sea urchin fertilization. I. Eggs with vitelline layers.

PubMed

Tegner, M J; Epel, D

1976-07-01

The surface coats of sea urchin eggs and the events of fertilization which take place on these surfaces were examined with the scanning electron microscope (SEM). Gametes of Stronglyocentrotus purpuratus and Lytechinus pictus were considered in detail; eggs of seven other echinoids were examined for comparative purposes. Jelly coats, preserved by varying the pH of fixation, were found to vary in morphology and solubility properties between species. The vitelline layers of the nine echinoids are characterized by arrays of projections which are impressions of cytoplasmic microvilli in the vitelline layer. After sperm bind to the egg surface via the acrosomal process, fine filaments, apparently an egg response to insemination, further connect some sperm heads and tails to the egg. The cortical reactions spread out as a wave from where the fertilizing sperm fused with the egg; separation of the vitelline layer proceeds as a smooth wave from S. purpuratus eggs and as a series of localized separations in L. pictus eggs. The fertilization membranes of S. purpuratus and Allocentrotus fragilis zygotes are expanded replicas of their respective vitelline layers, suggesting that fertilization membranes are formed by an unfolding of the vitelline layer.

Interactions of the baicalin and baicalein with bilayer lipid membranes investigated by cyclic voltammetry and UV-Vis spectroscopy.

PubMed

Zhang, Ying; Wang, Xuejing; Wang, Lei; Yu, Miao; Han, Xiaojun

2014-02-01

The baicalin and baicalein are the major flavonoids found in Radix Scutellariae, an essential herb in traditional Chinese medicine for thousands of years. The interactions of the baicalin and baicalein with lipid bilayer membranes were studied using cyclic voltammetry and UV-Vis spectroscopy. The thickness d of supported bilayer lipid membranes was calculated as d=4.59(±0.36) nm using AC impedance spectroscopy. The baicalein interacted with egg PC bilayer membranes in a dose-dependent manner. The responses of K3Fe(CN)6 on lipid bilayer membrane modified Pt electrode linearly increased in a concentration range of baicalein from 6.25μM to 25μM with a detection limit of 0.1μM and current-concentration sensitivity of 0.11(±0.01) μA/μM, and then reached a plateau from 25μM to 50μM. However the baicalin showed much weaker interactions with egg PC bilayer membranes. UV-Vis spectroscopy also confirmed that the baicalein could interact with egg PC membranes noticeably, but the interaction of baicalin with membranes was hard to be detected. The results provide useful information on understanding the mechanism of action of Radix Scutellariae in vivo. © 2013.

Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot.

PubMed

Zhang, Qian; Lin, Hong; Sui, Jianxin; Wang, Jingxue; Cao, Limin

2015-01-01

In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry.

Egg serpins: The chicken and/or the egg dilemma.

PubMed

Dombre, Clara; Guyot, Nicolas; Moreau, Thierry; Monget, Philippe; Da Silva, Mylène; Gautron, Joël; Réhault-Godbert, Sophie

2017-02-01

Twenty-seven serpins belonging to clade A, B, C, D, E, F, G, H and I serpins are currently referenced in chicken genome databases. Phylogenetic analysis of chicken serpins revealed that ovalbumin (Serpinb14) and its paralogs ovalbumin-related protein Y (Serpinb14b) and ovalbumin-related protein X (Serpinb14c) are found in bird species. These clade B serpins are specifically expressed in reproductive tissues and exported in the egg where they constitute major protein components. These data suggest that these three paralogs have probably appeared in birds to face new environments and ensure the extra-uterine development of an embryo in a shell egg. Twelve other serpins have been identified in the newly produced egg, some of them having a specific distribution in the respective egg structures (eggshell, egg white, vitelline membrane and egg yolk). The physiological role of these egg serpins remain largely unexplored, but there is increasing evidence in literature or by homologies with their mammalian counterparts, that some of them participate in cell proliferation, tissue remodeling and/or angiogenesis associated with folliculogenesis and development of extraembryonic structures, eggshell biomineralization, egg defense and nutrition of the embryo. A better knowledge of the phylogenetic evolution of these 15 serpins in other oviparous species, on their egg distribution, on their regulation during embryonic development (activation/degradation/transfer) and on their functional specificity, is needed to better appreciate their role and their bird-specificity. These review shed light on the multiple possibilities that offer the avian egg model to study the role of serpins in reproduction and developmental biology. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

ELECTRIC IMPEDANCE OF ARBACIA EGGS

PubMed Central

Cole, Kenneth S.; Cole, Robert H.

1936-01-01

The alternating current resistance and capacity of suspensions of unfertilized and fertilized eggs of Arbacia punctulata have been measured at frequencies from 103 to 1.64 x 107 cycles per second. The unfertilized egg has a static plasma membrane capacity of 0.73 µf./cm.2 which is practically independent of frequency. The fertilized egg has a static membrane capacity of 3.1 µf./cm.2 at low frequencies which decreases to a value of 0.55 µf./cm.2 at high frequencies. The decrease follows closely the relaxation dispersion of the dielectric constant if the dissipation of such a system is ignored. It is considered more probable that the effect is due to a fertilization membrane of 3.1 µf./cm.2 capacity lifted 1.5 µ. from the plasma membrane, the interspace having the conductivity of sea water. The suspensions show a frequency-dependent capacity at low frequencies which may be attributable to surface conductance. The equivalent low frequency internal specific resistance of both the unfertilized and fertilized egg is about 186 ohm cm. or about 6 times that of sea water, while the high frequency data extrapolate to a value of about 4 times sea water. There is evidence at the highest frequencies that the current is penetrating the nucleus and other materials in the cytoplasm. If this effect were entirely due to the nucleus it would lead to a very approximate value of 0.1 µf./cm.2 for the capacity of the nuclear membrane. The measurements do not indicate any change in this effect on fertilization. PMID:19872952

Membrane damage-induced vesicle–vesicle fusion of dysferlin-containing vesicles in muscle cells requires microtubules and kinesin

PubMed Central

McDade, Joel R.; Michele, Daniel E.

2014-01-01

Mutations in the dysferlin gene resulting in dysferlin-deficiency lead to limb-girdle muscular dystrophy 2B and Myoshi myopathy in humans. Dysferlin has been proposed as a critical regulator of vesicle-mediated membrane resealing in muscle fibers, and localizes to muscle fiber wounds following sarcolemma damage. Studies in fibroblasts and urchin eggs suggest that trafficking and fusion of intracellular vesicles with the plasma membrane during resealing requires the intracellular cytoskeleton. However, the contribution of dysferlin-containing vesicles to resealing in muscle and the role of the cytoskeleton in regulating dysferlin-containing vesicle biology is unclear. Here, we use live-cell imaging to examine the behavior of dysferlin-containing vesicles following cellular wounding in muscle cells and examine the role of microtubules and kinesin in dysferlin-containing vesicle behavior following wounding. Our data indicate that dysferlin-containing vesicles move along microtubules via the kinesin motor KIF5B in muscle cells. Membrane wounding induces dysferlin-containing vesicle–vesicle fusion and the formation of extremely large cytoplasmic vesicles, and this response depends on both microtubules and functional KIF5B. In non-muscle cell types, lysosomes are critical mediators of membrane resealing, and our data indicate that dysferlin-containing vesicles are capable of fusing with lysosomes following wounding which may contribute to formation of large wound sealing vesicles in muscle cells. Overall, our data provide mechanistic evidence that microtubule-based transport of dysferlin-containing vesicles may be critical for resealing, and highlight a critical role for dysferlin-containing vesicle–vesicle and vesicle–organelle fusion in response to wounding in muscle cells. PMID:24203699

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

PubMed Central

Fernandes, K.S.; Silva, A.H.M.; Mendanha, S.A.; Rezende, K.R.; Alonso, A.

2013-01-01

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation. PMID:24068194

Effects of supplementing n-3 fatty acid enriched eggs and walnuts on cardiovascular disease risk markers in healthy free-living lacto-ovo-vegetarians: a randomized, crossover, free-living intervention study

PubMed Central

2014-01-01

Background Plant and marine n-3 fatty acids (FA) may favorably modify select markers of cardiovascular disease risk. Whether supplementing the habitual diet of lacto-ovo-vegetarians (LOV) with walnuts (containing α-linolenic acid, ALA) and n-3 FA enriched eggs (containing primarily docosahexaenoic acid, DHA and ALA) would have equivalent effects on CVD risk factors is explored in this study. Methods In this study, 20 healthy free-living LOVs following their habitual diet were randomly assigned in a crossover design to receive one of three supplements: n-3 FA enriched egg (6/week), walnuts (28.4 g, 6/week) or a standard egg, 6/week (control) for 8 weeks each with 4-wk washout between treatments. Erythrocyte membrane fatty acids, serum lipids and inflammatory markers were measured at the end of each treatment. Results Dietary compliance was observed by an expected increase in erythrocyte membrane ALA following the walnut treatment and in DHA following the n-3 FA enriched egg treatment. Walnut treatment lowered serum triacylglycerol, total cholesterol and Apo B (p < 0.05) compared to the standard egg but not the n-3 FA enriched egg treatment. However, walnut treatment significantly reduced total: HDL cholesterol ratio compared to both egg treatments. There were no differences between treatments for any of the inflammatory markers. Conclusions For LOV, a direct source of DHA such as n-3 FA enriched eggs seems necessary to increase membrane levels of DHA. However for producing an overall favorable blood lipid profile, daily consumption of a handful of walnuts rich in ALA may be a preferred option for lacto-ovo vegetarian. PMID:24673793

Effects of phalloidin microinjection and localization of fluorescein-labeled phalloidin in living sand dollar eggs.

PubMed

Hamaguchi, Y; Mabuchi, I

1982-01-01

Effects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 microM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 microM, though the latter seemed to be more sensitive to phalloidin than the former. Fluorescein-labeled phalloidin (FL-phalloidin) was injected into eggs in order to investigate F-actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL-phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rod-like structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL-phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.

PRESSURE-VOLUME RELATIONSHIP OF THE FUNDULUS EGG IN SEA WATER AND IN SUCROSE

PubMed Central

Kao, C. Y.

1956-01-01

Upon activation, an internal hydrostatic pressure develops within the Fundulus egg, and compresses the egg proper to a reduced volume. When the perivitelline pressure is abolished by a highly hypertonic sucrose solution, the egg volume increases. As sucrose penetrates the chorion, the volume again decreases. The relation between P and V in these conditions is inverse, and approximates a rectangular hyperbola. The limiting factor causing most of the deviation is shown to be the incompressible fraction. It is concluded that the volume of the egg proper is controlled by the perivitelline pressure, and that the effect of hypertonic sucrose solution is exerted by lowering the pressure and thereby increasing membrane permeability non-specifically. It is also shown that some permanent alterations occur within the plasma membrane during activation that reduce the permeance, and thereby, increase the incompressible fraction. PMID:13357739

Factors affecting the toxicity of methylmercury injected into eggs

USGS Publications Warehouse

Heinz, G.H.; Hoffman, D.J.; Kondrad, S.L.; Erwin, C.A.

2006-01-01

We developed a standardized protocol for comparing the sensitivities of the embryos of different bird species to methylmercury when methylmercury was injected into their eggs. During the course of developing this protocol, we investigated the effects of various factors on the toxicity of the injected methylmercury. Most of our experiments were done with chicken (Gallus domesticus), mallard (Anas platyrhynchos), and ring-necked pheasant (Phasianus colchicus) eggs, all of which were purchased in large numbers from game farms. A smaller amount of work was done with double-crested cormorant (Phalacrocorax auritus) eggs collected from the wild. Several solvents were tested, and corn oil at a rate of 1 ??l/g egg contents was selected for the final standardized protocol because it had minimal toxicity to embryos and because methylmercury dissolved in corn oil yielded a dose-response curve in a range of egg concentrations that was similar to the range that causes reproductive impairment when the mother deposits methylmercury into her own eggs. The embryonic stage at which eggs were injected with corn oil altered mercury toxicity; at early stages, the corn oil itself was toxic. Therefore, in the final protocol we standardized the time of injection to occur when each species reached the morphologic equivalent of a 3-day-old chicken embryo. Although solvents can be injected directly into the albumen of an egg, high embryo mortality can occur in the solvent controls because of the formation of air bubbles in the albumen. Our final protocol used corn oil injections into the air cell, which are easier and safer than albumen injections. Most of the methylmercury, when dissolved in corn oil, injected into the air cell passes through the inner shell membrane and into the egg albumen. Most commercial incubators incubate eggs in trays with the air cell end of the egg pointing upward, but we discovered that mercury-induced mortality was too great when eggs were held in this orientation. In addition, some species of bird eggs require incubation on their sides with the eggs being rolled 180?? for them to develop normally. Therefore, we adopted a procedure of incubating the eggs of all species on their sides and rolling them 180?? every hour. Little has been published about the conditions of temperature, humidity, and the movements to which eggs of wild birds need to be subjected for them to hatch optimally under artificial incubation. Not unexpectedly, hatching success in an artificial incubator is generally less than what natural incubation by the parents can achieve. However, the survival of control embryos of most wild bird species was good (generally ??? 80%) up to within 1 or 2 days of hatching when we incubated the eggs at 37.5??C (or 37.6??C for gallinaceous species) at a relative humidity that resulted in an approximate 15% to 16% loss in egg weight by the end of incubation and by incubating the eggs on their sides and rolling them 180??/h. To improve statistical comparisons, we used survival through 90% of incubation as our measurement to compare survival of controls with survival of eggs injected with graded concentrations of mercury. ?? 2006 Springer Science+Business Media, Inc.

Factors affecting the toxicity of methylmercury injected into eggs

USGS Publications Warehouse

Heinz, G.H.; Hoffman, D.J.; Kondrad, S.L.; Erwin, C.A.

2006-01-01

We developed a standardized protocol for comparing the sensitivities of the embryos of different bird species to methylmercury when methylmercury was injected into their eggs. During the course of developing this protocol, we investigated the effects of various factors on the toxicity of the injected methylmercury. Most of our experiments were done with chicken (Gallus domesticus), mallard (Anas platyrhynchos), and ring-necked pheasant (Phasianus colchicus) eggs, all of which were purchased in large numbers from game farms. A smaller amount of work was done with double-crested cormorant (Phalacrocorax auritus) eggs collected from the wild. Several solvents were tested, and corn oil at a rate of 1 :l/g egg contents was selected for the final standardized protocol because it had minimal toxicity to embryos and because methylmercury dissolved in corn oil yielded a dose?response curve in a range of egg concentrations that was similar to the range that causes reproductive impairment when the mother deposits methylmercury into her own eggs. The embryonic stage at which eggs were injected with corn oil altered mercury toxicity; at early stages, the corn oil itself was toxic. Therefore, in the final protocol we standardized the time of injection to occur when each species reached the morphologic equivalent of a 3-day-old chicken embryo. Although solvents can be injected directly into the albumen of an egg, high embryo mortality can occur in the solvent controls because of the formation of air bubbles in the albumen. Our final protocol used corn oil injections into the air cell, which are easier and safer than albumen injections. Most of the methylmercury, when dissolved in corn oil, injected into the air cell passes through the inner shell membrane and into the egg albumen. Most commercial incubators incubate eggs in trays with the air cell end of the egg pointing upward, but we discovered that mercury-induced mortality was too great when eggs were held in this orientation. In addition, some species of bird eggs require incubation on their sides with the eggs being rolled 180? for them to develop normally. Therefore, we adopted a procedure of incubating the eggs of all species on their sides and rolling them 180? every hour. Little has been published about the conditions of temperature, humidity, and the movements to which eggs of wild birds need to be subjected for them to hatch optimally under artificial incubation. Not unexpectedly, hatching success in an artificial incubator is generally less than what natural incubation by the parents can achieve. However, the survival of control embryos of most wild bird species was good (generally > 80%) up to within 1 or 2 days of hatching when we incubated the eggs at 37.5?C (or 37.6?C for gallinaceous species) at a relative humidity that resulted in an approximate 15% to 16% loss in egg weight by the end of incubation and by incubating the eggs on their sides and rolling them 180?/h. To improve statistical comparisons, we used survival through 90% of incubation as our measurement to compare survival of controls with survival of eggs injected with graded concentrations of mercury.

Effect of storage duration on the rheological properties of goose liquid egg products and eggshell membranes.

PubMed

Kumbar, V; Nedomova, S; Trnka, J; Buchar, J; Pytel, R

2016-07-01

In practice, goose eggs are increasingly used and, therefore, the rheological properties have to be known for processing. The eggs of geese (Landes Goose, Anser anser f. domestica) were stored for one, 2, 3, 4, 6, and 8 wk at a constant temperature 4°C. First of all, the egg quality parameters were described in terms of egg weight, egg weight loss, egg shape index, yolk height, albumen height, yolk index, albumen index, and Haugh units. In the next step the rheological behavior of liquid egg products (egg yolk, albumen, and whole liquid egg) was studied using a concentric cylinder viscometer. Flow curves of all liquid egg products exhibited non-Newtonian shear thinning behavior. This behavior can be described using the Herschel-Bulkley model and for technical application using the Ostwald-de Waele model. The effect of the storage duration on the rheological behavior is different for the different liquid egg products. With the exception of very low shear rates, the viscosity of the egg yolk as well as of the whole liquid egg decreases with storage time. At lower shear rates there is a tendency toward increased albumen viscosity with storage duration. The storage duration also affects the mechanical properties of the eggshell membrane. This effect has been evaluated in terms of the ultimate tensile strength, fracture strain, and fracture toughness. All these parameters increased with the loading rate, but decreased during the egg storage. These mechanical phenomena should be respected, namely in the design of the egg model for the numerical simulation of the egg behavior under different kinds of the mechanical loading. © 2016 Poultry Science Association Inc.

Optimization of fluorimetric lipid membrane biosensor sensitivity through manipulation of membrane structure and nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine concentration

NASA Astrophysics Data System (ADS)

Shrive, Jason D. A.; Krull, Ulrich J.

1995-01-01

In the work reported here, surface concentrations of 0.027 and 0.073 molecules nm-2 of the fluorescent membrane probe molecule nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) were shown to yield optimum sensitivity for fluorimetric transduction of membrane structural perturbations for lipid membrane-based biosensor development. These optima were obtained through correlation of experimental data with theoretical predictions of optimum surface concentrations based on a model for NBD-PE self quenching previously published by our group. It was also determined that membrane structural heterogeneity improves the sensitivity of NBD-PE labeled membrane transducers. Together with fluorescence microscopy, observations of surface potential change upon compression or expansion of phosphatidylcholine (PC)/phosphatidic acid (PA) monolayers were used to qualitatively indicate the degree of structural heterogeneity in these membranes. It was determined that sub-microscopic domains must exist in microscopically homogeneous egg PC/egg PA membranes in order to facilitate the observed NBD-PE self-quenching responses upon alteration of bulk pH and therefore, membrane surface electrostatics and structure.

Metabolic similarities between fertilization and phagocytosis. Conservation of a peroxidatic mechanism

PubMed Central

1979-01-01

At the time of fertilization, sea urchin eggs release a peroxidase which, together with H2O2 generated by a respiratory burst, is responsible for hardening of the fertilization membrane. We demonstrate here that the ovoperoxidase of unfertilized eggs is located in cortical granules and, after fertilization, is concentrated in the fertilization membrane. Fertilization of sea urchin eggs or their parthenogenetic activation with the ionophor A23187 also results in (a) the conversion of iodide to a trichloroacetic acid-precipitable form (iodination), (b) the deiodination of eggs exogenously labeled with myeloperoxidase and H2O2, (c) the degradation of thyroxine as measured by the recovery of the released radioiodine at the origin and in the inorganic iodide spot on paper chromatography, and (d) the conversion of estradiol to an alcohol-precipitable form (estrogen binding). The iodination reaction and the binding of estradio occurs predominantly in the fertilization membrane where the ovoperoxidase is concentrated. From the estimation of the kinetics of incorporation of iodine, we determine that the peroxidative system is active for 30 min after fertilization, long after hardening of the fertilization membrane is complete. Most of the bound iodine is lost during the hatching process. Iodination of albumin is catalyzed by the material released from the egg during fertilization, when combined with H2O2 and iodide. Iodination, thyroxine degradation, and estradiol binding are inhibited by azide, cyanide, aminotriazole, methimazole, ascorbic acid and ergothioneine, all of which can inhibit peroxidase-catalyzed reactions. These responses of the sea urchin egg to fertilization are strikingly similar to the changes induced in polymorphonuclear leukocytes by phagocytosis and, in both instances, a peroxidative mechanism may be involved. PMID:372484

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise.

PubMed

Chiba, K; Alderton, J M; Hoshi, M; Steinhardt, R A

1999-05-01

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle. Copyright 1999 Academic Press.

Detection and molecular characterization of Methicillin Resistant Staphylococcus aureus from table eggs

USDA-ARS?s Scientific Manuscript database

Background: Table eggs are nutritionally important food consumed globally. Despite being protected inside the hard shell and a semi-permeable membrane, the egg contents may be contaminated with microbes and thus become a possible carrier of infectious agents to humans. A number of medically signific...

Detection and molecular characterization of methicillin resistant Staphylococcus aureus from table eggs

USDA-ARS?s Scientific Manuscript database

Table eggs are nutritionally important food consumed globally. Despite being protected inside the hard shell and a semi-permeable membrane, the egg contents may be contaminated with microbes and thus become a possible carrier of infectious agents to humans. A number of medically significant bacteria...

Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct.

PubMed

Orihuela, Pedro A; Zuñiga, Lidia M; Rios, Mariana; Parada-Bustamante, Alexis; Sierralta, Walter D; Velásquez, Luis A; Croxatto, Horacio B

2009-11-30

Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

rab3 mediates cortical granule exocytosis in the sea urchin egg.

PubMed

Conner, S; Wessel, G M

1998-11-15

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking. Copyright 1998 Academic Press.

The formation of zona radiata in Pseudosciaena crocea revealed by light and transmission electron microscopy.

PubMed

Ma, Xiao-Xin; Zhu, Jun-Quan; Zhou, Hong; Yang, Wan-Xi

2012-02-01

The egg envelope is an essential structure occurring during oogenesis. It plays an important role during the process of fertilization in the large yellow croaker Pseudosciaena crocea. Elucidation of egg envelope formation helps us to understand fertilization mechanisms in teleosts. In the present work, we studied the formation of egg envelope in P. crocea by light microscopy, as well as by transmission and scanning electron microscopy. Four layers exist outside the oocyte plasmalemma, i.e., theca cell layer, basal membrane, granulosa cell layer and zona radiata. According to our observation, zona radiata is a multilaminar structure just like the same structure reported in teleosts, but the origin of this structure is a little different. Before it is formed, a peripheral space filled with different density of vesicles is the place where zona radiata is formed. Zona radiata (Z1) is secreted only by oocyte itself, it belongs to the primary envelope; zona radiata 2 (Z2) and zona radiata 3 (Z3) belong to the secondary envelope, because the two layers are formed after granulosa cells appear, and microvilli participate this process. It is very interesting that Z2 and Z3 are situated between Z1 and the granulosa cell first, but they translocate to the other side of Z1. This microanatomy difference may due to the participation of microvilli. The new finding about egg envelope formation in P. crocea will help us to do further investigation on fertilization mechanisms and will make artificial breeding possible which may contribute to the resource recovery of this species. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effects of toxic compounds in Montipora capitata on exogenous and endogenous zooxanthellae performance and fertilization success.

PubMed

Hagedorn, Mary; Farrell, Ann; Carter, Virginia; Zuchowicz, Nikolas; Johnston, Erika; Padilla-Gamiño, Jacqueline; Gunasekera, Sarath; Paul, Valerie

2015-01-01

Studies have identified chemicals within the stony coral genus Montipora that have significant biological activities. For example, Montiporic acids A and B and other compounds have been isolated from the adult tissue and eggs of Montipora spp. and have displayed antimicrobial activity and cytotoxicity in cultured cells. The ecological role of these toxic compounds is currently unclear. This study examines the role these toxins play in reproduction. Toxins were found in the eggs and larvae of the coral Montipora capitata. Releasing these toxins by crushing both the eggs and larvae resulted in irreversible inhibition of photosynthesis in endogenous and exogenous zooxanthellae within minutes. Moreover, these toxins were stable, as frozen storage of eggs and larvae did not affect toxicity. Photosynthetic competency of Porites compressa zooxanthellae treated with either frozen or fresh, crushed eggs was inhibited similarly (P > 0.05, ANCOVA). Addition of toxic eggs plugs to live P. compressa fragments caused complete tissue necrosis under the exposed area on the fragments within 1 week. Small volumes of M. capitata crushed eggs added to sperm suspensions reduced in vitro fertilization success by killing the sperm. After 30 min, untreated sperm maintained 90 ± 1.9% SEM motility while those treated with crushed eggs were rendered immotile, 4 ± 1.4% SEM. Flow cytometry indicated membrane disruption of the immotile sperm. Fertilization success using untreated sperm was 79 ± 4% SEM, whereas the success rate dropped significantly after exposure to the crushed eggs, 1.3 ± 0% SEM. Unlike the eggs and the larvae, M. capitata sperm did not reduce the photosynthetic competency of P. compressa zooxanthellae, suggesting the sperm was nontoxic. The identity of the toxins, cellular mechanism of action, advantage of the toxins for M. capitata and their role on the reef are still unknown.

Filamentous actin organization in the unfertilized sea urchin egg cortex.

PubMed

Henson, J H; Begg, D A

1988-06-01

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.

Crocodilian perivitelline membrane-bound sperm detection.

PubMed

Augustine, Lauren

2017-05-01

Advanced reproductive technologies (ART's) are often employed with various taxa to enhance captive breeding programs and maintain genetic diversity. Perivitelline membrane-bound (PVM-bound) sperm detection has previously been demonstrated in avian and chelonian species as a useful technique for breeding management. In the absence of embryotic development within an egg, this technique can detect the presence of sperm trapped on the oocyte membrane confirming breeding, male reproductive status, and pair compatibility. PVM-bound sperm were successfully detected in three clutches of Cuban crocodile (Crocodylus rhombifer) eggs at the Smithsonian's National Zoological Park (NZP) for the first time in any crocodilian species. PVM-bound sperm were detected in fresh and incubated C. rhombifer eggs, as well as eggs that were developing (banded) and those that were not (not banded). The results of this study showed significant differences in average sperm densities per egg between clutches (p = 0.001). Additionally, there was not a significant difference within clutches between eggs that banded and those that did not band (Clutch A, p = 0.505; Clutch B, p = 0.665; Clutch C, p = 0.266). The results of this study demonstrate the necessity to microscopically examine eggs that do not develop (do not band), to determine if sperm is present, which can help animal managers problem solve reproductive shortcomings. PVM-bound sperm detection could be a useful technique in assessing crocodilian breeding programs, as well as have potential uses in studies assessing sperm storage, artificial insemination, and artificial incubation. This article is a U.S. Government work and is in the public domain in the USA.

Opto-injection into single living cells by femtosecond near-infrared laser

NASA Astrophysics Data System (ADS)

Peng, Cheng

This dissertation presents a novel technique to deliver membrane impermeable molecules into single living cells with the assistance of femtosecond (fs) near-infrared (NIR) laser pulses. This approach merges ultrafast laser technology with key biological, biomedical, and medical applications, such as gene transfection, gene therapy and drug delivery. This technique promises several major advantages, namely, very high transfection efficiency, high cell survival rate (≈100%) and fully preserved cell viabilities. It is also a promising method to deliver molecules into cells that are difficult or even completely resistant to established physical methods, such as microinjection by glass pipettes, electroporation, and biolistics. In this work, the system for fs NIR opto-injection was designed and built. Successful fs NIR opto-injection has been performed on several cell systems including single mammalian cells (bovine aortic endothelial cells), marine animal eggs (Spisula solidissima oocytes), and human cancer cells (fibrosarcoma HT1080) cultured in a tissue-like environment. The connections between laser parameters and cell responses were explored through further experiments and in-depth analyses, especially the relationship between dye uptake rate and incident laser intensity, and the relationship between pore size created on cell membranes and incident laser intensity. Dye uptake rate of the target cells was observed to depend on incident laser intensity. Pore size was found dependent on incident laser intensity. The conclusion was made that laser-induced breakdown and plasma-induced ablation in cell membrane are the physical principles that govern the process of fs NIR opto-injection.

Colonization of Lutzomyia shannoni (Diptera: Psychodidae) utilizing an artificial blood feeding technique.

PubMed

Mann, Rajinder S; Kaufman, Phillip E

2010-12-01

Laboratory colonization of hematophagous insects must include an efficient method of blood feeding, preferably by artificial means. Strict rules for obtaining animal use permits, extensive animal maintenance costs, and indirect anesthesia effects on animal health warrant the development of an artificial membrane feeding technique for sand fly colonization in laboratories. An attempt was made to colonize Lutzomyia shannoni using an artificial blood feeding membrane to replace the use of live animals commonly used for sand fly blood-feeding purposes. Lutzomyia shannoni readily fed through a pig intestine membrane exposed at an angle of 45°. However, it did not feed through a chicken skin membrane. Olfactory attractants were unable to improve blood-feeding efficiency. Plaster of Paris was the most suitable oviposition substrate. Female L. shannoni adults laid no eggs on moist sand substrate. Sand fly adults held in groups of ten or more laid higher numbers of eggs than did individually maintained sand flies. Inclusion of the L. longipalpis oviposition hormone dodecanoic acid or the presence of previously laid eggs did not stimulate L. shannoni oviposition. The average L. shannoni egg, larval, and pupal duration were 9.3, 36.7, and 17.8 days, respectively. The addition of a 20% sugar solution improved adult female longevity. Females survived longer (14.8 days) than males (11.9 days). Lutzomyia shannoni was successfully colonized in the laboratory for up to four generations using this artificial membrane technique. © 2010 The Society for Vector Ecology.

Microscopic analysis of Spodoptera frugiperda (Lepidoptera: Noctuidae) embryonic development before and after treatment with azadirachtin, lufenuron, and deltamethrin.

PubMed

Correia, Alicely A; Wanderley-Teixeira, Valéria; Teixeira, Alvaro A C; Oliveira, José V; Gonçalves, Gabriel G A; Cavalcanti, MaríIia G S; Brayner, Fábio A; Alves, Luiz C

2013-04-01

The botanical insecticides, growth regulators, and pyrethroids have an effect on the biology of Spodoptera frugiperda (Smith). However, no emphasis has been given to the effect of these insecticides on embryonic development of insects, in histological level. Thus, this research aimed to examine by light and scanning electron microscopy S. frugiperda eggs and to describe the embryonic development, before and after immersion treatment, using commercial concentrations and lower concentrations than commercial ones, of the compounds lufenuron (Match), azadirachtin (AzaMax), and deltamethrin (Decis-positive control). For light microscopy semithin sections of eggs were used, and for scanning electron microscopy, images of the surface of eggs, treated and untreated with insecticides. The morphological characteristics of S. frugiperda eggs, in general, were similar to those described in the literature for most of the insects in the order Lepidoptera. Spherical eggs slightly flattened at the poles, with chorion, yolk, vitelline membrane, and embryo formation. In both microscopic analysis, we observed that insecticides acted immediately and independent of concentration, resulting absence, or incomplete embryo, presented yolk granules widely dispersed, without vitellophage formation, chorion disintegration, disorganized blastoderm, presenting vacuoles, yolk region with amorphous cells, and formation of completely uncharacterized appendages. Thus, we conclude that the compounds lufenuron and azadirachtin interfere on S. frugiperda embryonic development.

Preparation of the egg membrane bandage contained the antibacterial Ag nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jin; Duan, Guangwen; Fu, Yunzhi, E-mail: yzhfu@hainu.edu.cn

Silver nanoparticles were synthesized using a rapid, single step, and completely green biosynthetic method employing aqueous aloe leaf extracts as both the reducing and capping agent. Transmission electron microscopy analysis revealed the average size of silver nanoparticles approximately 18.05 nm. Fourier transform infrared spectroscopy observation showed the estimation of two kinds of binding sites between aqueous aloe leaf and aqueous aloe leaf with silver nanoparticles. In addition, the critical roles of the concentration of silver nitrate, temperature, and reaction time in the formation of silver nanoparticles had been illustrated. Furthermore, silver nanoparticles were deposited on egg membrane bandage, forming amore » new egg membrane bandage that contained silver nanoparticles that exhibiting excellent antibacterial effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, which was 2.5 times stronger than the commercially available bandage. - Graphical Abstract: Display Omitted.« less

An Electron Microscope Study of the Rat Ovum

PubMed Central

Sotelo, J. Roberto; Porter, Keith R.

1959-01-01

This paper reports on the fine structure of rat oocytes at stages before ovulation, during maturation, fertilization, and early cleavage. The study includes parallel observations on light and electron microscope preparations with attempted correlations. The follicular cells of the ovarian egg are described as sending long processes through the zona pellucida to the egg surface where they mingle with thin projections from the egg itself. No open communication between follicle cell cytoplasm and egg cytoplasm was observed. During maturation and fertilization both types of processes are withdrawn from the zona. The germinal vesicle and later the pronuclei of the fertilized egg are characterized by numerous large nucleoli. These have the form of thick walled vesicles with diameters as great as 8 to 10 µ. The wall is dense in the EM image and appears to consist in part of small granules. The cytoplasm shows several inclusions including mitochondria of usual form and a Golgi component which has the typical fine structure and the distribution described by earlier light studies. Small dense particles, presumably RNP particles, are distributed throughout the cytoplasmic matrix and show no preference for membranes. The endoplasmic reticulum of the oocyte is represented by a scattering only of vesicles, but begins a more extensive and elaborate development with the onset of segmentation. One inclusion of the ooplasm, similar in size to mitochondria, receives special attention. It is a vesicular structure, containing a large number of small vesicles (10 to 50 mµ in diameter) and frequently a central density or nucleoid. They are referred to as multivesicular bodies. Such bodies are found in small number in the ovarian egg, but increase greatly in number during maturation and fertilization. It appears from the micrographs of eggs in these latter stages that these vesicular bodies break down and liberate their content of small vesicles to the surrounding ooplasm. Comments are provided on the apparent significance of the various observations. PMID:13654454

Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Winkle, L.J.; Campione, A.L.

1991-07-01

The results of histochemical and immunocytochemical studies have been used elsewhere to support the hypothesis that Na+/K(+)-ATPase expression is initiated or increases dramatically in preimplantation mouse conceptuses just before they begin to cavitate. Moreover, localization of the enzyme in the inner membrane of the mural trophoblast is thought to be involved directly in formation and maintenance of the blastocyst cavity. Presumably, Na+/K(+)-ATPase extrudes the cation, Na+, and therefore water into the cavity. The cation transporting activity of the enzyme can be determined by measuring ouabain-sensitive Rb+ uptake by cells. Therefore, we measured Rb+ uptake in mouse eggs and preimplantation conceptusesmore » at various stages of development. 86Rb+ uptake by conceptuses increased linearly with time for at least 60 min in medium containing 0.7 mM total Rb+ plus K+ in the absence or presence of 1.0 mM ouabain, and ouabain inhibited more than 70% of 86Rb+ uptake. The ouabain concentration at 1/2 of maximum inhibition of the ouabain-sensitive component of 86Rb+ uptake was about 10-20 microM in eggs and conceptuses at all stages of preimplantation development. Moreover, ouabain-sensitive Rb+ uptake had a twofold higher Vmax value in blastocysts than in eggs or conceptuses at earlier stages of development (i.e., approximately 173 vs 70-100 fmole.conceptus-1.min-1), although the total cell surface area also was probably about two times greater in blastocysts than in eggs or other conceptuses. Ouabain-sensitive Rb+ transport in eggs and conceptuses may have occurred via a single ouabain-sensitive Rb+ transporter with a Hill coefficient of 1.5-1.8 (Hill plots). When it was assumed that the Hill coefficient had a value of 2.0, however, eggs and conceptuses appeared to contain at least two forms of Na+/K(+)-ATPase activity.« less

Cortical Isolation from Xenopus laevis Oocytes and Eggs.

PubMed

Sive, Hazel L; Grainger, Robert M; Harland, Richard M

2007-06-01

INTRODUCTIONIn Xenopus laevis, the cortex is the layer of gelatinous cytoplasm that lies just below the plasma membrane of the egg. Rotation of the cortex relative to the deeper cytoplasm soon after fertilization is intimately linked to normal dorsal axis specification. The cortex can be dissected from the egg to analyze its composition and activity or to clone associated RNAs. This protocol describes a procedure for isolating the vegetal cortex of the fertilized egg.

Effect of Ca2+ on Vesicle Fusion on Solid Surface: An In vitro Model of Protein-Accelerated Vesicle Fusion

NASA Astrophysics Data System (ADS)

Shinozaki, Youichi; Siitonen, Ari M.; Sumitomo, Koji; Furukawa, Kazuaki; Torimitsu, Keiichi

2008-07-01

Lipid vesicle fusion is an important reaction in the cell. Calcium ions (Ca2+) participate in various important biological events including the fusion of vesicles with cell membranes in cells. We studied the effect of Ca2+ on the fusion of egg yolk phosphatidylcholine/brain phosphatidylserine (eggPC/brainPS) lipid vesicles on a mica substrate with fast scanning atomic force microscopy (AFM). When unattached and unfused lipid vesicles on mica were rinsed away, discrete patches of fused vesicles were observed under high Ca2+ concentrations. At 0 mM Ca2+, lipid vesicles were fused on mica and formed continuous supported lipid bilayers (SLBs) covering almost the entire mica surface. The effect of Ca2+ on SLB formation was offset by a Ca2+ chelating agent. When lipid vesicles were added during AFM observation, vesicles fused on mica and covered almost all areas even under high Ca2+ concentrations. These results indicate that force between AFM tip and vesicles overcomes the Ca2+-reduced fusion of lipid vesicles.

Multiplication of Salmonella Enteritidis in egg yolks after inoculation outside, on, and inside vitelline membranes and storage at different temperatures

USDA-ARS?s Scientific Manuscript database

Prompt refrigeration to restrict bacterial growth is a widely acknowledged practice for reducing the risk of egg-borne transmission of Salmonella Enteritidis to consumers. A recently published federal regulation for S. Enteritidis control requires eggs to be refrigerated within 36 after they are la...

Evaluation of disinfectants and antiseptics to eliminate bacteria from the surface of turkey eggs and hatch gnotobiotic poults

USDA-ARS?s Scientific Manuscript database

Bird eggs and are in contact with intestinal microbiota prior to oviposition, but are protected from bacterial translocation by a glycoprotein cuticle layer, the shell and internal membranes. In a preliminary study, turkey eggs were hatched in a germ-free environment. Firmicutes 16S rRNA gene was de...

Origin, differentiation and functional ultrastructure of egg envelopes in the cestode Echinococcus multilocularis Leuckart, 1863 (Cyclophyllidea: Taeniidae).

PubMed

Świderski, Zdzisław; Miquel, Jordi; Azzouz-Maache, Samira; Pétavy, Anne-Françoise

2017-07-01

The origin, differentiation and functional ultrastructure of oncospheral or egg envelopes in Echinococcus multilocularis Leuckart, 1863 were studied by transmission electron microscopy (TEM) and cytochemistry. The purpose of our study is to describe the formation of the four primary embryonic envelopes, namely vitelline capsule, outer envelope, inner envelope and oncospheral membrane, and their transformation into the oncospheral or egg envelopes surrounding the mature hexacanth. This transformation takes place in the preoncospheral phase of embryonic development. The vitelline capsule and oncospheral membrane are thin membranes, while the outer and inner envelopes are thick cytoplasmic layers formed by two specific types of blastomeres: the outer envelope by cytoplasmic fusion of two macromeres and the inner envelope by cytoplasmic fusion of three mesomeres. Both outer and inner envelopes are therefore cellular in origin and syncytial in nature. During the advanced phase of embryonic development, the outer and inner envelopes undergo great modifications. The outer envelope remains as a metabolically active layer involved in the storage of glycogen and lipids for the final stages of egg development and survival. The inner envelope is the most important protective layer because of its thick layer of embryophoric blocks that assures oncospheral protection and survival. This embryophore is the principal layer of mature eggs, affording physical and physiological protection for the differentiated embryo or oncosphere, since the outer envelope is stripped from the egg before it is liberated. The embryophore is very thick and impermeable, consisting of polygonal blocks of an inert keratin-like protein held together by a cementing substance. The embryophore therefore assures extreme resistance of eggs, enabling them to withstand a wide range of environmental temperatures and physicochemical conditions.

Modeling of Fluid-Membrane Interaction in Cellular Microinjection Process

NASA Astrophysics Data System (ADS)

Karzar-Jeddi, Mehdi; Diaz, Jhon; Olgac, Nejat; Fan, Tai-Hsi

2009-11-01

Cellular microinjection is a well-accepted method to deliver matters such as sperm, nucleus, or macromolecules into biological cells. To improve the success rate of in vitro fertilization and to establish the ideal operating conditions for a novel computer controlled rotationally oscillating intracytoplasmic sperm injection (ICSI) technology, we investigate the fluid-membrane interactions in the ICSI procedure. The procedure consists of anchoring the oocyte (a developing egg) using a holding pipette, penetrating oocyte's zona pellucida (the outer membrane) and the oolemma (the plasma or inner membrane) using an injection micropipette, and finally to deliver sperm into the oocyte for fertilization. To predict the large deformation of the oocyte membranes up to the piercing of the oolemma and the motion of fluids across both membranes, the dynamic fluid-pipette-membrane interactions are formulated by the coupled Stokes' equations and the continuum membrane model based on Helfrich's energy theory. A boundary integral model is developed to simulate the transient membrane deformation and the local membrane stress induced by the longitudinal motion of the injection pipette. The model captures the essential features of the membranes shown on optical images of ICSI experiments, and is capable of suggesting the optimal deformation level of the oolemma to start the rotational oscillations for piercing into the oolemma.

Two independent forms of endocytosis maintain embryonic cell surface homeostasis during early development

PubMed Central

Covian-Nares, J. Fernando; Smith, Robert M.; Vogel, Steven S.

2008-01-01

Eukaryotic cells have multiple forms of endocytosis which maintain cell surface homeostasis. One explanation for this apparent redundancy is to allow independent retrieval of surface membranes derived from different types of vesicles. Consistent with this hypothesis we find that sea urchin eggs have at least two types of compensatory endocytosis. One is associated with retrieving cortical vesicle membranes, and formed large endosomes by a mechanism that was inhibited by agatoxin, cadmium, staurosporine and FK506. The second type is thought to compensate for constitutive exocytosis, and formed small endosomes using a mechanism that was insensitive to the above mentioned reagents, but was inhibited by phenylarsine oxide (PAO), and by microinjection of mRNA encoding Src kinase. Both mechanisms could act concurrently, and account for all of the endocytosis occurring during early development. Inhibition of either form did not trigger compensation by the other form, and phorbol ester treatment rescued the endocytotic activity blocked by agatoxin, but not the retrieval blocked by PAO. PMID:18281031

A critical evaluation of the utility of eggshells for estimating mercury concentrations in avian eggs

USGS Publications Warehouse

Peterson, Sarah; Ackerman, Joshua T.; Eagles-Smith, Collin A.; Hartman, C. Alex; Herzog, Mark P.

2017-01-01

Eggshells are a potential tool for non-lethally sampling contaminant concentrations in bird eggs, yet few studies have examined their utility to represent mercury exposure. We assessed mercury concentrations in eggshell components for 23 bird species and determined whether they correlated with total mercury (THg) in egg contents. We designed a multi-experiment analysis to examine how THg is partitioned into eggshell components, specifically hardened eggshells, material adhered to the eggshell, and inner eggshell membranes. THg concentrations in eggshells were much lower than in egg contents, and almost all of the THg within the eggshell was contained within material adhered to eggshells and inner eggshell membranes, and specifically not within calcium-rich hardened eggshells. Despite having very little mercury, THg concentrations in hardened eggshells had the strongest correlation with egg contents among all eggshell components. However, species with the same THg concentrations in eggshells had different THg concentrations in egg contents, indicating that there is no global predictive equation among species for the relationship between eggshell and egg content THg concentrations. Further, for all species, THg concentrations in eggshells decreased with relative embryo age. Although the majority of mercury in eggshells was contained within other eggshell components and not within hardened eggshells, THg in hardened eggshells can be used to estimate THg concentrations in egg contents, if embryo age and species are addressed.

The research about microscopic structure of emulsion membrane in O/W emulsion by NMR and its influence to emulsion stability.

PubMed

Xie, Yiqiao; Chen, Jisheng; Zhang, Shu; Fan, Kaiyan; Chen, Gang; Zhuang, Zerong; Zeng, Mingying; Chen, De; Lu, Longgui; Yang, Linlin; Yang, Fan

2016-03-16

This paper discussed the influence of microstructure of emulsion membrane on O/W emulsion stability. O/W emulsions were emulsified with equal dosage of egg yolk lecithin and increasing dosage of co-emulsifier (oleic acid or HS15). The average particle size and centrifugal stability constant of emulsion, as well as interfacial tension between oil and water phase were determined. The microstructure of emulsion membrane had been studied by (1)H/(13)C NMR, meanwhile the emulsion droplets were visually presented with TEM and IFM. With increasing dosage of co-emulsifier, emulsions showed two stable states, under which the signal intensity of characteristic group (orient to lipophilic core) of egg yolk lecithin disappeared in NMR of emulsions, but that (orient to aqueous phase) of co-emulsifiers only had some reduction at the second stable state. At the two stable states, the emulsion membranes were neater in TEM and emulsion droplets were rounder in IFM. Furthermore, the average particle size of emulsions at the second stable state was bigger than that at the first stable state. Egg yolk lecithin and co-emulsifier respectively arranged into monolayer and bilayer emulsion membrane at the two stable states. The microstructure of emulsion membrane was related to the stability of emulsion. Copyright © 2016 Elsevier B.V. All rights reserved.

Eggshell ultrastructure and delivery of pharmacological inhibitors to the early embryo of R. prolixus by ethanol permeabilization of the extraembryonic layers.

PubMed

Bomfim, Larissa; Vieira, Priscila; Fonseca, Ariene; Ramos, Isabela

2017-01-01

Most vectors of arthropod-borne diseases produce large eggs with hard and opaque eggshells. In several species, it is still not possible to induce molecular perturbations to the embryo by delivery of molecules using microinjections or eggshell permeabilization without losing embryo viability, which impairs basic studies regarding development and population control. Here we tested the properties and permeability of the eggshell of R. prolixus, a Chagas disease vector, with the aim to deliver pharmacological inhibitors to the egg cytoplasm and allow controlled molecular changes to the embryo. Using field emission scanning and transmission electron microscopy we found that R. prolixus egg is coated by three main layers: exochorion, vitelline layer and the plasma membrane, and that the pores that allow gas exchange (aeropiles) have an average diameter of 10 μm and are found in the rim of the operculum at the anterior pole of the egg. We tested if different solvents could permeate through the aeropiles and reach the egg cytoplasm/embryo and found that immersions of the eggs in ethanol lead to its prompt penetration through the aeropiles. A single five minute-immersion of the eggs/embryos in pharmacological inhibitors, such as azide, cyanide and cycloheximide, solubilized in ethanol resulted in impairment of embryogenesis in a dose dependent manner and DAPI-ethanol solutions were also able to label the embryo cells, showing that ethanol penetration was able to deliver those molecules to the embryo cells. Multiple immersions of the embryo in the same solutions increased the effect and tests using bafilomycin A1 and Pepstatin A, known inhibitors of the yolk proteolysis, were also able to impair embryogenesis and the yolk protein degradation. Additionally, we found that ethanol pre-treatments of the egg make the aeropiles more permeable to aqueous solutions, so drugs diluted in water can be carried after the eggs are pre-treated with ethanol. Thus, we found that delivery of pharmacological inhibitors to the embryo of R. prolixus can be performed simply by submersing the fertilized eggs in ethanol with no need for additional methods such as microinjections or electroporation. We discuss the potential importance of this methodology to the study of this vector developmental biology and population control.

Organochlorine contaminants in double-crested cormorants from Green Bay, WI: I. Large-scale extraction and isolation from eggs using semi-permeable membrane dialysis

USGS Publications Warehouse

Meadows, J.C.; Tillitt, D.E.; Schwartz, T.R.; Schroeder, D.J.; Echols, K.R.; Gale, R.W.; Powell, D.C.; Bursian, S.J.

1996-01-01

A 41.3-kg sample of double-crested cormorant (Phalacrocorax auritus) egg contents was extracted, yielding over 2 L of egg lipid. The double-crested cormorant (DCC) egg extract, after clean-up and concentration, was intended for use in egg injection studies to determine the embryotoxicity of the organic contaminants found within the eggs. Large-scale dialysis was used as a preliminary treatment to separate the extracted contaminants from the co-extracted sample lipids. The lipid was dialyzed in 80×5 cm semi-permeable membrane devices (SPMDs) in 50-ml aliquants. After the removal of 87 g of cholesterol by freeze-fractionation, the remaining lipid carryover (56 g) was removed by 100 routine gel permeation chromatography (GPC) operations. A 41,293-g sample was thus extracted and purified to the extent that it could easily be placed at a volume of 5 ml, the volume calculated to be necessary for the egg injection study. Analyses were performed comparing contaminant concentrations in the final purified extract to those present in the original egg material, in the extract after dialysis and cholesterol removal, and in the excluded materials. Recoveries of organochlorine pesticides through dialysis and cholesterol ranged from 96% to 135%. Total polychlorinated biphenyls in the final extract were 96% of those measured in the original egg material. Analysis of excluded lipid and cholesterol indicated that 92% of the polychlorinated dibenzo-dioxins and-furans were separated into the final extract.

The Effects of Commercial Cool Water Washing of Shell Eggs on Haugh Unit, Vitelline Membrane Strength, Aerobic Microorganisms and Fungi

USDA-ARS?s Scientific Manuscript database

Current egg washing practices utilize wash water temperatures averaging 49°C, and have been found to increase internal egg temperature by 6.7 to 7.8°C. These high temperatures create a more optimal environment for bacterial growth, including Salmonella Enteritidis (SE), if it is present. SE is the...

Liposomes for Topical Use: A Physico-Chemical Comparison of Vesicles Prepared from Egg or Soy Lecithin

PubMed Central

Budai, Lívia; Kaszás, Nóra; Gróf, Pál; Lenti, Katalin; Maghami, Katayoon; Antal, István; Klebovich, Imre; Petrikovics, Ilona; Budai, Marianna

2013-01-01

Developments in nanotechnology and in the formulation of liposomal systems provide the opportunity for cosmetic dermatology to design novel delivery systems. Determination of their physico-chemical parameters has importance when developing a nano-delivery system. The present study highlights some technological aspects/characteristics of liposomes formulated from egg or soy lecithins for topical use. Alterations in the pH, viscosity, surface tension, and microscopic/macroscopic appearance of these vesicular systems were investigated. The chemical composition of the two types of lecithin was checked by mass spectrometry. Caffeine, as a model molecule, was encapsulated into multilamellar vesicles prepared from the two types of lecithin: then zeta potential, membrane fluidity, and encapsulation efficiency were compared. According to our observations, samples prepared from the two lecithins altered the pH in opposite directions: egg lecithin increased it while soy lecithin decreased it with increased lipid concentration. Our EPR spectroscopic results showed that the binding of caffeine did not change the membrane fluidity in the temperature range of possible topical use (measured between 2 and 50 °C). Combining our results on encapsulation efficiency for caffeine (about 30% for both lecithins) with those on membrane fluidity data, we concluded that the interaction of caffeine with the liposomal membrane does not change the rotational motion of the lipid molecules close to the head group region. In conclusion, topical use of egg lecithin for liposomal formulations can be preferred if there are no differences in the physico-chemical properties due to the encapsulated drugs, because the physiological effects of egg lecithin vesicles on skin are significantly better than that of soy lecithin liposomes. PMID:24482779

Histological study on maturation, fertilization and the state of gonadal region following spawning in the model sea anemone, Nematostella vectensis

PubMed Central

Moiseeva, Elizabeth; Rabinowitz, Claudette; Rinkevich, Baruch

2017-01-01

The starlet sea-anemone Nematostella vectensis has emerged as a model organism in developmental biology. Still, our understanding of various biological features, including reproductive biology of this model species are in its infancy. Consequently, through histological sections, we study here key stages of the oogenesis (oocyte maturation/fertilization), as the state of the gonad region immediately after natural spawning. Germ cells develop in a secluded mesenterial gastrodermal zone, where the developing oocytes are surrounded by mucoid glandular cells and trophocytes (accessory cells). During vitellogenesis, the germinal vesicle in oocytes migrates towards the animal pole and the large polarized oocytes begin to mature, characterized by karyosphere formation. Then, the karyosphere breaks down, the chromosomes form the metaphase plate I and the eggs are extruded from the animal enclosed in a sticky, jelly-like mucoid mass, along with numerous nematosomes. Fertilization occurs externally at metaphase II via swimming sperm extruded by males during natural spawning. The polar bodies are ejected from the eggs and are situated within a narrow space between the egg’s vitelline membrane and the adjacent edge of the jelly coat. The cortical reaction occurs only at the polar bodies’ ejection site. Several spermatozoa can penetrate the same egg. Fertilization is accompanied by a strong ooplasmatic segregation. Immediately after spawning, the gonad region holds many previtellogenic and vitellogenic oocytes, though no oocytes with karyosphere. Above are the first histological descriptions for egg maturation, meiotic chromosome’s status at fertilization, fertilization and the gonadal region’s state following spawning, also documenting for the first time the ejection of the polar body. PMID:28796817

Parthenogenesis in unfertilized eggs of Coturnix chinensis, the Chinese painted quail, and the effect of egg clutch position on embryonic development.

PubMed

Parker, H M; McDaniel, C D

2009-04-01

Parthenogenesis, embryonic development of an unfertilized egg, was studied for many years in turkeys. In fact, as many as 49% of unfertilized Beltsville Small White turkey eggs develop embryos. However, no research exists on parthenogenesis in quail. The Chinese painted quail is a close relative of the more common Japanese quail and, unlike turkeys or chickens, the small Chinese painted quail reaches sexual maturity rapidly, making it a great candidate for further research on parthenogenesis. Obviously, a better understanding of avian parthenogenesis should increase our knowledge of avian fertilization and early embryonic development. Therefore, we determined if unfertilized Chinese painted quail hens produce embryos. Second, we explored the possibility that position of the egg within the clutch influences parthenogenesis. When initial secondary sexual plumage was apparent at 4 wk of age, male chicks were separated from females to prevent fertilization. Hens were placed in individual cages near sexual maturity, at approximately 6 wk of age. Individual eggs were collected daily and labeled with hen number and date. Eggs were stored for 0 to 3 d at 20 degrees C before incubation at 37.5 degrees C. After 10 d of incubation, approximately 4,000 eggs from 300 laying hens were examined for embryonic development under a magnifying lamp. On average, 4.8% of the unfertilized eggs contained an abortive form of embryonic development consisting of undifferentiated cells and unorganized membranes. Approximately 27% of the laying hens produced at least 1 egg with parthenogenic development. However, about 10% (30) of these hens exhibited a predisposition for parthenogenesis by producing 2 or more unfertilized eggs with embryonic development. Twenty percent of the eggs from 2 hens produced embryonic development. Additionally, the first egg laid in a clutch was most likely to produce embryonic development, with a steady decline in the percentage of eggs with embryonic development as position in the clutch increased. In conclusion, the Chinese painted quail does exhibit parthenogenesis and clutch position influences the rate of naturally occurring parthenogenesis.

A family of cellular proteins related to snake venom disintegrins.

PubMed

Weskamp, G; Blobel, C P

1994-03-29

Disintegrins are short soluble integrin ligands that were initially identified in snake venom. A previously recognized cellular protein with a disintegrin domain was the guinea pig sperm protein PH-30, a protein implicated in sperm-egg membrane binding and fusion. Here we present peptide sequences that are characteristic for several cellular disintegrin-domain proteins. These peptide sequences were deduced from cDNA sequence tags that were generated by polymerase chain reaction from various mouse tissue and a mouse muscle cell line. Northern blot analysis with four sequence tags revealed distinct mRNA expression patterns. Evidently, cellular proteins containing a disintegrin domain define a superfamily of potential integrin ligands that are likely to function in important cell-cell and cell-matrix interactions.

Differing strategies of patterning of follicular cells in higher and lower brachycerans (Diptera: Brachycera).

PubMed

Tworzydlo, Waclaw; Jablonska, Anna; Kisiel, Elzbieta; Bilinski, Szczepan M

2005-10-01

In all higher dipterans (Brachycera), including the fruitfly, Drosophila melanogaster, each egg chamber (ovarian follicle) consists of a group (clone) of germ cells (one oocyte and 15 accompanying nurse cells) that is surrounded by a layer of somatic mesodermal follicular cells (FCs). As oogenesis progresses the initially uniform FCs diversify into several morphologically and functionally distinct subpopulations. In D. melanogaster some of these subpopulations, e.g., border, centripetal, and dorsolateral cells, undertake coordinated migration or rearrangement over the surface of the germ cells. During the final stages of oogenesis these subpopulations participate in the formation of a complex, regionally specialized eggshell. In representatives of lower brachycerans (Orthorrhapha), only FCs that undertake active, directed migration are the border cells. These cells originate at the anterior pole of the ovarian follicle and migrate between the nurse cells to the anterior pole of the oocyte. Reduced motility of FCs in lower brachycerans results in the absence of certain FC subpopulations in their egg chambers and subsequent simplicity of their eggshells. We found that the lack of some FC subpopulations coincided with the appearance of lamellipodium-like protrusions of the oocyte. These protrusions penetrated between the apposing membranes of nurse and FCs and partially enveloped the nurse cell compartment. Analysis of whole-mount preparations stained with rhodamine-conjugated phalloidin revealed that the protrusions contained microfilaments and that their tips were equipped with actin-rich filopodium-like processes. We also found that in some lower brachycerans (representatives of the family Rhagionidae), the FCs located at the posterior pole of the oocyte, became enlarged and morphologically similar to the anterior border cells. These findings indicate that in higher dipterans the processes leading to the formation of a functional egg are variable and often markedly different from those in the model organism, D. melanogaster. genesis 43:49-58, 2005. (c) 2005 Wiley-Liss, Inc.

Why does a cleavage plane develop parallel to the spindle axis in conical sand dollar eggs? A key question for clarifying the mechanism of contractile ring positioning.

PubMed

Yoshigaki, Tomoyoshi

2003-03-21

Three types of models have been proposed about how the mitotic apparatus determines the position of the cleavage furrow in animal cells. In the first and second types, the contractile ring appears in a cortical region that least and most astral microtubules reach, respectively. The third type is that the spindle midzone positions the contractile ring. In the previous study, a new model was proposed through analyses of cytokinesis in sand dollar and sea urchin eggs. Gradients of the surface density of microtubule plus ends are assumed to drive membrane proteins whose accumulation causes the formation of contractile-ring microfilaments. In the present study, the validity of each model is examined by simulating the furrow formation in conical sand dollar eggs with the mitotic apparatus oriented perpendicular to the cone axis. The new model predicts that unilateral furrows with cleavage planes roughly parallel to the spindle axis appear between the mitotic apparatus and the vertex besides the normally positioned furrow. The predictions are consistent with the observations by Rappaport & Rappaport (1994, Dev. Biol.164, 258-266). The other three types of models do not predict the formation of the ectopic furrows. Furthermore, it is pointed out that only the new model has the ability to explain the geometrical relationship between the mitotic apparatus and the contractile ring under various experimental conditions. These results strongly suggest the real existence of the membrane proteins postulated in the model.

Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

PubMed

Fernández-Bussy, Rodrigo; Mouguelar, Valeria; Banchio, Claudia; Coux, Gabriela

2015-04-01

In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.

Detection of microlesions induced by heavy ions using liposomes filled with fluorescent dye

NASA Technical Reports Server (NTRS)

Koniarek, J. P.; Thomas, J. L.; Vazquez, M.

2004-01-01

In cells irradiation by heavy ions has been hypothesized to produce microlesions, regions of local damage. In cell membranes this damage is thought to manifest itself in the form of holes. The primary evidence for microlesions comes from morphological studies of cell membranes, but this evidence is still controversial, especially since holes also have been observed in membranes of normal, nonirradiated, cells. However, it is possible that damage not associated with histologically discernable disruptions may still occur. In order to resolve this issue, we developed a system for detecting microlesions based on liposomes filled with fluorescent dye. We hypothesized that if microlesions form in these liposomes as the result of irradiation, then the entrapped dye will leak out into the surrounding medium in a measurable way. Polypropylene vials containing suspensions of vesicles composed of either dipalmitoyl phosphatidylcholine, or a combination of egg phosphatidylcholine and cholesterol were irradiated at the Brookhaven National Laboratory using 56Fe ions at 1 GeV/amu. In several cases we obtained a significant loss of the entrapped dye above the background level. Our results suggest that holes may form in liposomes as the result of heavy ion irradiation, and that these holes are large enough to allow leakage of cell internal contents that are at least as large as a 1 nm diameter calcein molecule. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct

PubMed Central

2009-01-01

Background Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Methods Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Results Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Conclusion Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct. PMID:19948032

Effects of Toxic Compounds in Montipora capitata on Exogenous and Endogenous Zooxanthellae Performance and Fertilization Success

PubMed Central

Hagedorn, Mary; Farrell, Ann; Carter, Virginia; Zuchowicz, Nikolas; Johnston, Erika; Padilla-Gamiño, Jacqueline; Gunasekera, Sarath; Paul, Valerie

2015-01-01

Studies have identified chemicals within the stony coral genus Montipora that have significant biological activities. For example, Montiporic acids A and B and other compounds have been isolated from the adult tissue and eggs of Montipora spp. and have displayed antimicrobial activity and cytotoxicity in cultured cells. The ecological role of these toxic compounds is currently unclear. This study examines the role these toxins play in reproduction. Toxins were found in the eggs and larvae of the coral Montipora capitata. Releasing these toxins by crushing both the eggs and larvae resulted in irreversible inhibition of photosynthesis in endogenous and exogenous zooxanthellae within minutes. Moreover, these toxins were stable, as frozen storage of eggs and larvae did not affect toxicity. Photosynthetic competency of Porites compressa zooxanthellae treated with either frozen or fresh, crushed eggs was inhibited similarly (P > 0.05, ANCOVA). Addition of toxic eggs plugs to live P. compressa fragments caused complete tissue necrosis under the exposed area on the fragments within 1 week. Small volumes of M. capitata crushed eggs added to sperm suspensions reduced in vitro fertilization success by killing the sperm. After 30 min, untreated sperm maintained 90 ± 1.9% SEM motility while those treated with crushed eggs were rendered immotile, 4 ± 1.4% SEM. Flow cytometry indicated membrane disruption of the immotile sperm. Fertilization success using untreated sperm was 79 ± 4% SEM, whereas the success rate dropped significantly after exposure to the crushed eggs, 1.3 ± 0% SEM. Unlike the eggs and the larvae, M. capitata sperm did not reduce the photosynthetic competency of P. compressa zooxanthellae, suggesting the sperm was nontoxic. The identity of the toxins, cellular mechanism of action, advantage of the toxins for M. capitata and their role on the reef are still unknown. PMID:25714606

Cell cycle in egg cell and its progression during zygotic development in rice.

PubMed

Sukawa, Yumiko; Okamoto, Takashi

2018-03-01

Rice egg is arrested at G1 phase probably by OsKRP2. After fusion with sperm, karyogamy, OsWEE1-mediated parental DNA integrity in zygote nucleus, zygote progresses cell cycle to produce two-celled embryo. In angiosperms, female and male gametes exist in gametophytes after the complementation of meiosis and the progression of nuclear/cell division of the haploid cell. Within the embryo sac, the egg cell is specially differentiated for fertilization and subsequent embryogenesis, and cellular programs for embryonic development, such as restarting the cell cycle and de novo gene expression, are halted. There is only limited knowledge about how the cell cycle in egg cells restarts toward zygotic division, although the conversion of the cell cycle from a quiescent and arrested state to an active state is the most evident transition of cell status from egg cell to zygote. This is partly due to the difficulty in direct access and analysis of egg cells, zygotes and early embryos, which are deeply embedded in ovaries. In this study, precise relative DNA amounts in the nuclei of egg cells, developing zygotes and cells of early embryos were measured, and the cell cycle of a rice egg cell was estimated as the G1 phase with a 1C DNA level. In addition, increases in DNA content in zygote nuclei via karyogamy and DNA replication were also detectable according to progression of the cell cycle. In addition, expression profiles for cell cycle-related genes in egg cells and zygotes were also addressed, and it was suggested that OsKRP2 and OsWEE1 function in the inhibition of cell cycle progression in egg cells and in checkpoint of parental DNA integrity in zygote nucleus, respectively.

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane.

PubMed

Derde, Melanie; Nau, Françoise; Guérin-Dubiard, Catherine; Lechevalier, Valérie; Paboeuf, Gilles; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique

2015-04-01

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts

PubMed Central

1993-01-01

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase- like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M- phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine- treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation. PMID:8253833

Brief hypo-osmotic shock causes test cell death, prevents neurula rotation, and disrupts left-right asymmetry in Ciona intestinalis.

PubMed

Katsumoto, Shimpei; Hatta, Kohei; Nakagawa, Masashi

2013-05-01

Ascidian Ciona intestinalis tadpole larvae exhibit left-right asymmetry. The photoreceptors are situated on the right side of the sensory vesicle, and the tail curls along the left side of the trunk within the chorion. In tailbud embryos, the Ci-pitx gene is expressed in the left-side epidermis. It was previously reported that embryos generated from naked eggs, which lack the chorionic membrane and accessory cells (follicle cells attached to the outside of the chorion and test cells covering the inner surface of the chorion), show bilateral expression of Ci-pitx. This suggested that the chorion or accessory cells are needed for generation of asymmetry. Here, we show that a brief treatment with 60% artificial seawater (ASW) before, but not after, the neurula stage results in bilateral expression of Ci-pitx in the chorion of tailbud embryos, loss of follicle cells, and randomization of both the direction of tail curling and the locations of photoreceptors in larvae. This treatment also impaired the transient counterclockwise rotation within the chorion at the neurula stage. Nearly all test cells in the chorion died following 60% ASW treatment. These results suggest that dead test cells blocked the neural rotation and impaired left-right asymmetry. We also showed that tailbud embryos and larvae generated from defolliculated eggs produced by 80% ASW treatment, in which the test cells were alive, showed normal left-right asymmetry, suggesting that the follicle cells were not essential for asymmetric morphogenesis.

Late steps of egg cell differentiation are accelerated by pollination in Zea mays L.

PubMed

Mól, R; Idzikowska, K; Dumas, C; Matthys-Rochon, E

2000-04-01

Egg cells were analysed cytologically during the female receptivity period in maize (Zea mays L., line A 188). Three classes of egg cell were distinguished: type A--small, non-vacuolated cells with a central nucleus; type B--larger cells with small vacuoles surrounding the perinuclear cytoplasm located in the middle of the cell; type C--big cells with a large apical vacuole and the mid-basal perinuclear cytoplasm. The less-dense cytoplasm of the vacuolated egg cells usually contained numerous cup- or bell-shaped mitochondria. The three egg types appear to correspond to three late stages of egg cell differentiation. The frequencies of each of the three egg types were monitored in developing maize ears before and after pollination. In young ears, with the silks just extending out of the husks, small A-type cells were found in about 86% of ovules. Their frequency decreased to about 58% at the optimum silk length, remained unchanged in non-pollinated ears, and fell to 16% at the end of the female receptivity period. However, after pollination and before fertilisation the frequency of these cells decreased to about 33%, and the larger vacuolated egg cells (types B and C) prevailed. At various stages of the receptivity period, pollination accelerated changes in the egg population, increasing the number of ovules bearing larger, vacuolated egg cells. Experiments with silk removal demonstrated that putative pollination signals act immediately after pollen deposition and are not species-specific.

Nutritional effects of egg shell membrane supplements on chicken performance and immunity

USDA-ARS?s Scientific Manuscript database

Eggshell membranes (ESM) contain a variety of proteins and peptides which help in the development of embryo and provide protection to it. Many of the peptides and proteins associated with ESM have antimicrobial, immune-modulatory, and adjuvant properties. We hypothesized that the membrane byproducts...

Avian egg shape: Form, function, and evolution.

PubMed

Stoddard, Mary Caswell; Yong, Ee Hou; Akkaynak, Derya; Sheard, Catherine; Tobias, Joseph A; Mahadevan, L

2017-06-23

Avian egg shape is generally explained as an adaptation to life history, yet we currently lack a global synthesis of how egg-shape differences arise and evolve. Here, we apply morphometric, mechanistic, and macroevolutionary analyses to the egg shapes of 1400 bird species. We characterize egg-shape diversity in terms of two biologically relevant variables, asymmetry and ellipticity, allowing us to quantify the observed morphologies in a two-dimensional morphospace. We then propose a simple mechanical model that explains the observed egg-shape diversity based on geometric and material properties of the egg membrane. Finally, using phylogenetic models, we show that egg shape correlates with flight ability on broad taxonomic scales, suggesting that adaptations for flight may have been critical drivers of egg-shape variation in birds. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigen-receptor-mediated functions.

PubMed Central

Eris, J M; Basten, A; Brink, R; Doherty, K; Kehry, M R; Hodgkin, P D

1994-01-01

B-cell tolerance to soluble protein self antigens such as hen egg lysozyme (HEL) is mediated by clonal anergy. Anergic B cells fail to mount antibody responses even in the presence of carrier-primed T cells, suggesting an inability to activate or respond to T helper cells. To investigate the nature of this defect, B cells from tolerant HEL/anti-HEL double-transgenic mice were incubated with a membrane preparation from activated T-cell clones expressing the CD40 ligand. These membranes, together with interleukin 4 and 5 deliver the downstream antigen-independent CD40-dependent B-cell-activating signals required for productive T-B collaboration. Anergic B cells responded to this stimulus by proliferating and secreting antibody at levels comparable to or better than control B cells. Furthermore, anergic B cells presented HEL acquired in vivo and could present the unrelated antigen, conalbumin, targeted for processing via surface IgD. In contrast, the low immunoglobulin receptor levels on anergic B cells were associated with reduced de novo presentation of HEL and a failure to upregulate costimulatory ligands for CD28. These defects in immunoglobulin-receptor-mediated functions could be overcome in vivo, suggesting a number of mechanisms for induction of autoantibody responses. Images PMID:7514304

Evidence for conjugated linoleic acid-induced embryonic mortality that is independent of egg storage conditions and changes in egg relative fatty acids.

PubMed

Leone, V A; Stransky, D L; Aydin, R; Cook, M E

2009-09-01

Three experiments were performed to determine the effect of conjugated linoleic acid (CLA) on embryonic development in the absence of vitelline membrane disruption. In experiment 1, when eggs from control and CLA (0.5%)-fed hens were stored at 21 or 15 degrees C for 48 h, mineral movement between the yolk and albumen was not observed (with the exception of Mg and Na). Also, it was found that CLA-induced changes in yolk fatty acid content (e.g., increased saturated fatty acids and CLA) had begun to change after 5 d of feeding hens CLA, and no differences were detected in fatty acid composition after 14 d. In experiment 2, the hatchability of eggs incubated directly after oviposition or stored 24 h at 21 or 15 degrees C was determined from hens fed control or 0.5% CLA diets. Regardless of storage conditions, CLA reduced hatchability. These data showed that CLA elicits negative effects on hatchability independent of vitelline membrane disruption or egg storage condition. In experiment 3, eggs were collected from hens fed 0 or 1% CLA daily for 3 wk, stored at 21 degrees C for 24 h, and incubated. Not only did CLA decrease hatchability, the data showed as the days of CLA feeding increased, the days of survival during incubation decreased. Average days of embryonic survival during incubation for the CLA group diminished to 18.0, 13.4, and 6.3 d for wk 1, 2, and 3 of CLA feeding, respectively, and control remained at 20.6, 20.8, and 19.8 for the 3 wk. These studies suggested that without the disruption of the vitelline membrane, hatchability and embryonic days of survival were significantly reduced by maternal CLA feeding in comparison to control-fed hens. Evidence that embryos die earlier the longer the hens are fed CLA, even though no additional changes in the fatty acid content of eggs were found, suggested that factors other than storage and egg yolk fatty acid composition played a role in CLA-induced embryonic mortality.

Vertical transmission of microcystins to Nile crocodile (Crocodylus niloticus) eggs.

PubMed

Singo, Alukhethi; Myburgh, Jan G; Laver, Peter N; Venter, Elizabeth A; Ferreira, Gezina C H; Rösemann, Gertruida M; Botha, Christo J

2017-08-01

Cyanobacteria or blue green algae are known for their extensive and highly visible blooms in eutrophic, stagnant freshwater bodies. Climate change and global warming have also contributed to a rise in toxic cyanobacterial blooms. One of the most important cyanobacteria is Microcystis aeruginosa, which can synthesize various microcystins that can affect the health of terrestrial and aquatic animals. Commercial Nile crocodile (Crocodylus niloticus) farming in South Africa is based on keeping breeders (adult males and females) in big dams on farms (captive-bred approach). Unfortunately, cyanobacterial blooms in the breeder dams are a concern to farm owners, managers and veterinarians. The main objectives of this research project were to determine if microcystins were present in the contents of crocodile eggs and the liver and yolk of dead hatchlings, and to determine if the reduced hatchability on commercial farms might be caused by these toxins. Furthermore, the concentration of microcystins in the breeder dam was monitored on a monthly basis spanning the ovulation and egg laying period. During the hatching season microcystin concentrations in unfertilised eggs, egg shell membranes and in the yolk and liver of dead hatchlings were determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Microcystins were detected in Nile crocodile egg and hatchling samples. Microcystin (MC-LR, MC-RR, MC-YR) concentrations in the crocodile egg and hatchling samples collected from clutches with a good hatching rate (≥90%) ranged between 0 and 1.76 ng g -1 , with the highest concentration in the egg shell membranes. Microcystin concentrations in samples collected from clutches with a bad hatching rate (≤10%) ranged from 0 - 1.63 ng g -1 with the highest concentration detected in the hatchling yolk. However, the concentrations were probably underestimated as the percentage recovery from spiked samples was very low with the extraction method employed. Bayesian analysis suggests that the liver, yolk and unfertilised egg all have similar microcystin concentrations, while the membranes have (with moderate to high certainty) higher microcystin concentrations. There appears to be no difference in microcystin concentrations among good and bad clutches across all tissue types or within a specific tissue type, but due to the small sample size, it was not possible to determine whether microcystin affected the hatchability of Nile crocodile eggs. However, vertical transmission of microcystin variants to the Nile crocodile egg does occur and the possible implications for the survival of wild Nile crocodile populations should be ascertained. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk.

PubMed

Axnér, E; Lagerson, E

2016-04-01

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal-protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris-based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5-ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (C-FDA) were evaluated 5 min post-thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin-based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin-based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin. © 2016 Blackwell Verlag GmbH.

Dual behavior of N-acetylcysteine during ethanol-induced oxidative stress in embryonic chick brains.

PubMed

Bauer, Alison K; Fitzgerald, Mary; Ladzinski, Adam T; Lenhart Sherman, Sydney; Maddock, Benjamin H; Norr, Zoe M; Miller, Robert R

2017-10-01

Ethanol (EtOH) causes oxidative stress in embryos. Because N-acetylcysteine (NAC) failures and successes in ameliorating EtOH-induced oxidative stress have been reported, the objective was to determine if exogenous NAC ameliorated EtOH-induced oxidative stress within embryonic chick brains. Control eggs were injected with approximately 25 µl of water on day 0, 1, and 2 of development (E 0-2 ). Experimental eggs were injected with dosages of either 3.0 mmol EtOH/kg egg; 747 µmol NAC/kg egg; 3.0 mmol EtOH and 747 µmol NAC/kg egg; 1000 µmol NAC/kg egg; or 3.0 mmol EtOH and 1000 µmol NAC/kg during the first 3 days of development (E 0-2 ). At 11 days of development (E 11 ; late embryogenesis), brains were harvested and subsequently assayed for oxidative stress markers including the loss of long-chain membrane polyunsaturated fatty acids (PUFAs); the accumulation of lipid hydroperoxides (LPO); decreased glutathione (GSH) and glutathione/glutathione disulfide (GSSG) levels; and decreased glutathione peroxidase (GPx) activities. EtOH (3 mmol/kg egg), medium NAC (747 µmol/kg egg), and EtOH and medium NAC promoted oxidative stress. These treatments caused decreased brain membrane long-chain PUFAs; increased LPO levels; decreased GSH levels and GSH/GSSG levels; and decreased Se-dependent GPx activities. High NAC dosages (1000 µmol/kg egg) attenuated EtOH-induced oxidative stress within EtOH and high NAC-treated chick brains. Exogenous EtOH and/or medium NAC propagated oxidative stress. Meanwhile, high NAC ameliorated EtOH-induced oxidative stress.

Molecular Characterization of Severin from Clonorchis sinensis Excretory/Secretory Products and Its Potential Anti-apoptotic Role in Hepatocarcinoma PLC Cells

PubMed Central

He, Lei; Wang, Xiaoyun; Liang, Pei; Chen, Wenjun; Bian, Meng; Ren, Mengyu; Lin, Jinsi; Liang, Chi; Xu, Jin; Wu, Zhongdao; Li, Xuerong; Huang, Yan; Yu, Xinbing

2013-01-01

Background Clonorchiasis, caused by the infection of Clonorchis sinensis (C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma and hepatocellular carcinoma (HCC). It has been well known that the excretory/secretory products of C. sinensis (CsESPs) play key roles in clonorchiasis associated carcinoma. From genome and transcriptome of C. sinensis, we identified one component of CsESPs, severin (Csseverin), which had three putative gelsolin domains. Its homologues are supposed to play a vital role in apoptosis resistance of tumour cell. Methodology/Principal Findings There was significant similarity in tertiary structures between human gelsolin and Csseverin by bioinformatics analysis. We identified that Csseverin expressed at life stage of adult worm, metacercaria and egg by the method of quantitative real-time PCR and western blotting. Csseverin distributed in vitellarium and intrauterine eggs of adult worm and tegument of metacercaria by immunofluorence assay. We obtained recombinant Csseverin (rCsseverin) and confirmed that rCsseverin could bind with calciumion in circular dichroism spectrum analysis. It was demonstrated that rCsseverin was of the capability of actin binding by gel overlay assay and immunocytochemistry. Both Annexin V/PI assay and mitochondrial membrane potential assay of human hepatocarcinoma cell line PLC showed apoptosis resistance after incubation with different concentrations of rCsseverin. Morphological analysis, apoptosis-associated changes of mitochondrial membrane potential and Annexin V/PI apoptosis assay showed that co-incubation of PLC cells with rCsseverin in vitro led to an inhibition of apoptosis induced by serum-starved for 24 h. Conclusions/Significance Collectively, the molecular properties of Csseverin, a molecule of CsESPs, were characterized in our study. rCsseverin could cause obvious apoptotic inhibition in human HCC cell line. Csseverin might exacerbate the process of HCC patients combined with C. sinensis infection. PMID:24367717

Development of Meteorus pulchricornis and regulation of its noctuid host, Pseudaletia separata.

PubMed

Suzuki, M; Tanaka, T

2007-10-01

The solitary endoparasitoid Meteorus pulchricornis can parasitize many lepidopteran host species successfully. In the case of parasitization of Pseudaletia separata, developmental duration of M. pulchricornis was 8-9 days from egg to larval emergence and 6 days from prepupa to adult emergence. Successful parasitism by M. pulchricornis decreased with host age. Following parasitization of day-0 4th host instar, the parasitoid embryo, whilst still enclosed in serosal cell membrane, hatched out of the egg chorion 2 days after oviposition. Subsequently, the 1st instar parasitoid emerged from the surrounding serosal cell membrane. Serosal cells dissociated and developed as teratocytes 3.5 days after oviposition. One embryo of M. pulchricornis gave rise to approximately 1200 teratocytes, a number that remained constant until 6 days after parasitization, but decreased drastically to 200 at 7 days post-oviposition. The teratocytes of M. pulchricornis were round- or oval-shaped and grew from 65 microm at 4 days to 200 microm in the long axis at 6 days post-parasitization. At 4 days post-parasitization, many cells or cell clusters with lipid particles were observed in the hemocoels of parasitized hosts. In addition, paraffin sections of parasitized hosts revealed that many teratocytes were attached to the host's fat body and contributed to disrupting the fat body tissue. Further, examination of the total hemocyte count (THC) during parasitization revealed that THC was maintained at low levels. Surprisingly, a temporal decrease followed by restoration of THC was observed in hosts injected with virus-like particles of M. pulchricornis (MpVLPs) plus venom, which contrasts with the constant THC suppression seen in parasitized hosts. This indicates that MpVLP function is temporal and is involved in regulation of the host during early parasitism. Therefore, teratocytes, a host regulation factor in late parasitism, could be involved in keeping THC at a low level.

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.

PubMed

Maxwell, W M; Johnson, L A

1999-12-01

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa.

PubMed

Akhter, S; Ansari, M S; Andrabi, S M H; Rakha, B A; Ullah, N; Khalid, M

2012-10-01

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen. © 2011 Blackwell Verlag GmbH.

Calibrating compound-specific stable nitrogen isotope analysis in avian eggs as bioarchives for paleoreconstruction

NASA Astrophysics Data System (ADS)

Preciado, C., Jr.

2016-12-01

Compound-specific stable isotope analysis (CSIA) has become a powerful tool for reconstructing consumer-resource relationships in modern and ancient systems. Stable nitrogen isotope analysis (δ15N) of "trophic" and "source" amino acids provides independent proxies of trophic position and the δ15N value at the base of the food web, respectively. When applied to avian egg tissues (e.g., shell protein, membrane), which can be preserved in the archaeological record, this approach can be used to address complex questions in food web architecture and biogeochemical cycling. In this study, we examined how sample-processing protocols affected the chromatography and reliability of δ15N values of individual amino acids in avian (chicken and penguin) egg components (shell protein, membrane, yolk, and albumen) via gas chromatography-combustion-isotope ratio mass spectrometry. "Unprocessed" egg tissues underwent standard acid hydrolysis protocols prior to derivatization, and resulted in poor chromatography with highly variable δ15N values across replicates. "Processed" samples were eluted through cation exchange columns (Dowex 50WX*400) prior to derivatization, followed by a P-buffer (KH2PO4 + Na2HPO4)-chloroform centrifugation extraction to remove confounding peaks and matrix impurities. These additional procedures greatly improved the chromatography of the "processed" samples, revealing better peak separation and baseline integration as well as lower δ15N variability. Additionally, a standard lipid extraction was necessary for yolk but not membrane, albumen, and shell. While the additional procedures applied to the "columned" samples did result in a significant reduction in sample yield ( 20%), it was non-fractionating and thus only affected the total sample sizes necessary for δ15N CSIA (shell protein 50mg and membrane, yolk, and albumen 0.5mg). The protocols developed here will streamline CSIA of egg tissues for future work in avian ecology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santella, Luigia, E-mail: santella@szn.it; Limatola, Nunzia; Chun, Jong T.

The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggsmore » at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm–egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent “excitable media” that quickly respond to the stimulus with the Ca{sup 2+} swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca{sup 2+} signals and in the control of monospermic fertilization. - Highlights: • Besides microtubules, microfilaments may anchor the nucleus to oocyte surface. • The cortical Ca{sup 2+} flash and wave at fertilization mirror electrical membrane change. • Artificial egg activation lacks microvilli extension in the perivitelline space. • Calcium is necessary but not sufficient for cortical granules exocytosis. • Actin cytoskeleton modulates Ca{sup 2+} release at oocyte maturation and fertilization.« less

A lectin of a non-invasive apple snail as an egg defense against predation alters the rat gut morphophysiology.

PubMed

Ituarte, Santiago; Brola, Tabata Romina; Fernández, Patricia Elena; Mu, Huawei; Qiu, Jian-Wen; Heras, Horacio; Dreon, Marcos Sebastián

2018-01-01

The eggs of the freshwater Pomacea apple snails develop above the water level, exposed to varied physical and biological stressors. Their high hatching success seems to be linked to their proteins or perivitellins, which surround the developing embryo providing nutrients, sunscreens and varied defenses. The defensive mechanism has been unveiled in P. canaliculata and P. maculata eggs, where their major perivitellins are pigmented, non-digestible and provide a warning coloration while another perivitellin acts as a toxin. In P. scalaris, a species sympatric to the former, the defense strategy seems different, since no toxin was found and the major perivitellin, PsSC, while also colored and non-digestible, is a carbohydrate-binding protein. In this study we examine the structure and function of PsSC by sequencing its subunits, characterizing its carbohydrate binding profile and evaluating its effect on gut cells. Whereas cDNA sequencing and database search showed no lectin domain, glycan array carbohydrate binding profile revealed a strong specificity for glycosphingolipids and ABO group antigens. Moreover, PsSC agglutinated bacteria in a dose-dependent manner. Inspired on the defensive properties of seed lectins we evaluated the effects of PsSC on intestinal cells both in vitro (Caco-2 and IEC-6 cells) and in the gastrointestinal tract of rats. PsSC binds to Caco-2 cell membranes without reducing its viability, while a PsSC-containing diet temporarily induces large epithelium alterations and an increased absorptive surface. Based on these results, we propose that PsSC is involved in embryo defenses by altering the gut morphophysiology of potential predators, a convergent role to plant defensive lectins.

Isolation of Individual Egg Cells and Zygotes in Alstroemeria Followed by Manual Selection with a Microcapillary-connected Micropump

PubMed Central

HOSHINO, YOICHIRO; MURATA, NAHO; SHINODA, KOICHI

2006-01-01

• Aims To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. • Scope An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining. • Conclusions The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary. PMID:16621859

Effect of feed grinding methods with and without expansion on prececal and total tract mineral digestibility as well as on interior and exterior egg quality in laying hens.

PubMed

Hafeez, A; Mader, A; Ruhnke, I; Männer, K; Zentek, J

2016-01-01

The grinding of cereals by various milling methods as well as thermal treatment of feed may influence mineral digestibility and egg quality. The present study investigated the effect of feed produced by disc mill (D) and wedge-shaped disc mill (WSD), as mash (M) or expandate (E) on apparent ileal absorption (AIA) and apparent total digestibility (ATD) of calcium, phosphorus, magnesium, zinc, manganese, copper and iron, as well as on egg quality in laying hens. A total of 192 hens (Lohmann Brown) aged 19 wk, were assigned using a randomized design with a 2 × 2 factorial arrangement. Four experimental diets were offered ad libitum. Eggs were analyzed for weight, shape index, area, shell weight per unit surface area, yolk color, air cell, blood spot, Haugh unit, albumen and yolk measures (index, weight, height, width and length), shell measures (surface area, stability, density, thickness and membrane weight), as well as percent contents of albumen, yolk, shell, and shell membrane. The ATD for phosphorus, manganese, and copper was higher in WSD compared with D treatment (P = 0.028, P = 0.028 and P = 0.016, respectively). The interaction between milling methods and thermal treatment influenced ATD of copper (P = 0.033), which was higher in WSD+M group (41.0 ± 20.2) compared with D+E group (-3.21 ± 25.1), whereas no differences were observed for D+M (1.90 ± 37.8) and WSD+E (8.02 ± 36.2) groups. Egg stability tended to be higher in E compared with M treatment (P = 0.055). Albumen weight, percentage albumen weight, and albumen: yolk were higher and percentage yolk weight was lower in D compared with WSD treatment (P = 0.043, P = 0.027, P = 0.024, and P = 0.041, respectively). Number of blood spots was higher in E than M treatment (P = 0.053). In conclusion, use of a wedge-shaped disc mill resulted in higher ATD for phosphorus, manganese, and copper than use of a disc mill; however, digestibility for majority of minerals as well as egg quality parameters was comparable. Therefore, feed produced by either disc mill or wedge-shaped disc mill as mash or expandate may be used for laying hens without negative effects on egg quality. © 2015 Poultry Science Association Inc.

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

PubMed Central

Zhou, Jian; McDowell, William; Park, Jungeun; Haug, Jeff; Staehling, Karen; Tang, Hong; Xie, Ting

2011-01-01

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms. PMID:22216012

Egg drop syndrome virus enters duck embryonic fibroblast cells via clathrin-mediated endocytosis.

PubMed

Huang, Jingjing; Tan, Dan; Wang, Yang; Liu, Caihong; Xu, Jiamin; Wang, Jingyu

2015-12-02

Previous studies of egg drop syndrome virus (EDSV) is restricted to serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV are poorly understood. Therefore, we aimed to explore the entry pathway of EDSV into duck embryonic fibroblast (DEF) cells as well as the infection characteristics and proliferation of EDSV in primary DEF and primary chicken embryo liver (CEL) cells. Transmission electron microscopy revealed that the virus triggered DEF cell membrane invagination as early as 10 min post-infection and that integrated endocytic vesicles formed at 20 min post-infection. The virus yield in EDSV-infected DEF cells treated with chlorpromazine (CPZ), sucrose, methyl-β-cyclodextrin (MβCD), or NH4Cl was measured by quantitative real-time PCR. Compared with the mock treatment, CPZ and sucrose greatly inhibited the production of viral progeny in a dose-dependent manner, while MβCD treatment did not result in a significant difference. Furthermore, NH4Cl had a strong inhibitory effect on the production of EDSV progeny. In addition, indirect immunofluorescence demonstrated that virus particles clustered on the surface of DEF cells treated with CPZ or sucrose. These results indicate that EDSV enters DEF cells through clathrin-mediated endocytosis followed by a pH-dependent step, which is similar to the mechanism of entry of human adenovirus types 2 and 5. Copyright © 2015 Elsevier B.V. All rights reserved.

A method to assess the migration properties of cell-derived microparticles within a living tissue.

PubMed

Hoang, Thang Q; Rampon, Christine; Freyssinet, Jean-Marie; Vriz, Sophie; Kerbiriou-Nabias, Danièle

2011-09-01

Cells undergoing activation or apoptosis exhibit plasma membrane changes, leading to the formation of shed vesicles (microparticles, MP). Although their effects on recipient cells in vitro, and their ability to support inflammatory or thrombotic events in the circulation have been studied, the spreading of such vesicles in tissues is still elusive. Our aim was to set up a method to examine the behavior of these vesicles in vivo. We examined the persistence of green-fluorescent microparticles (fMP), prepared after Ca2+ ionophore activation (iono-fMP) or apoptogenic treatment (eto-fMP) of human Jurkat T lymphoblastic or non-hematopoietic embryonic kidney (HEK) cell lines, following injection in zebrafish embryos 2h after egg fertilization. One hour post-injection, iono-fMP issued from both cell types formed a fluorescent dispersal in the intercellular space of embryos. In contrast, eto-fMP or MP deprived of sialic acid at their membrane, gathered together at the site of injection. We propose a method characterizing the abilities of MP to spread in the intercellular space. We showed that MP produced by apoptosis of T cells and those deprived of sialic acid at their membrane do not diffuse within the living cells. On the contrary, MP shed upon calcium induced activation of T and HEK cells, diffuse at a distance and spread in the intercellular space. The fate of injected MP relies on the type of induction rather than the cell species and results provide a model to test the ability of vesicles to interact locally or to spread outside of the site of production. Copyright © 2011 Elsevier B.V. All rights reserved.

Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

PubMed Central

Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G.

2013-01-01

In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+pe) of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/+pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos. PMID:24027530

Osmosis and the Marvelous Membrane.

ERIC Educational Resources Information Center

Cocanour, Barbara; Bruce, Alease S.

1985-01-01

Shows how the natural membrane of a decalcified chicken egg can demonstrate the principle of osmosis within a single class period. Various glucose and saline solutions used, periods of time, physiological effects experiments, and correction for differences in initial weights are noted. (DH)

Fertilization selection on egg and jelly-coat size in the sand dollar Dendraster excentricus.

PubMed

Levitan, D R; Irvine, S D

2001-12-01

Organisms with external fertilization are often sperm limited, and in echinoids, larger eggs have a higher probability of fertilization than smaller eggs. This difference is thought to be a result of the more frequent sperm-egg collisions experienced by larger targets. Here we report how two components of egg target size, the egg cell and jelly coat, contributed to fertilization success in a selection experiment. We used a cross-sectional analysis of correlated characters to estimate the selection gradients on egg and jelly-coat size in five replicate male pairs of the sand dollar Dendraster excentricus. Results indicated that eggs with larger cells and jelly coats were preferentially fertilized under sperm limitation in the laboratory. The selection gradients were an average of 922% steeper for egg than for jelly-coat size. The standardized selection gradients for egg and jelly-coat size were similar. Our results suggest that fertilization selection can act on both egg-cell and jelly-coat size but that an increase in egg-cell volume is much more likely to increase fertilization success than an equal change in jelly-coat volume. The strengths of the selection gradients were inversely related to the correlation of egg traits across replicate egg clutches. This result suggests the importance of replication in studies of selection of correlated characters.

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer

2005-07-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requiresmore » permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.« less

Laser microbeam irradiation and renucleation of mouse eggs. Final progress report, July 1, 1979-December 31, 1983

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, T.P.

1983-01-01

We have combined laser microbeam irradiation of mouse egg nuclei with fusion to donor cell nuclei in order to develop a new procedure for transferring nuclei into mammalian eggs. We have been using virus-treated cells injected into the perivitelline for fusion with egg cells. Binucleate cells inside the zona pellucida were often produced indicating nuclear transfer between cells had occurred. To prevent the formation of such abortive polyploidy, host nuclei were inactivated with a laser microbeam. The subsequent cleavage of the microirradiated eggs has been studied.

Microfiltration of enzyme treated egg whites for accelerated detection of viable Salmonella

USDA-ARS?s Scientific Manuscript database

We report detection of <13 CFU of Salmonella per 25 g egg white within 7 h by concentrating the bacteria using microfiltration through 0.2-lm cutoff polyethersulfone hollow fiber membranes. A combination of enzyme treatment, controlled cross-flow on both sides of the hollow fibers, and media selecti...

Extended longevity of queen honey bees compared to workers is associated with peroxidation-resistant membranes.

PubMed

Haddad, Laura Saade; Kelbert, Louie; Hulbert, A J

2007-07-01

In the honey bee (Apis mellifera), depending on what they are fed, female eggs become either workers or queens. Although queens and workers share a common genome, the maximum lifespan of queens is an order-of-magnitude longer than workers. The mechanistic basis of this longevity difference is unknown. In order to test if differences in membrane composition could be involved we have compared the fatty acid composition of phospholipids of queen and worker honey bees. The cell membranes of both young and old honey bee queens are highly monounsaturated with very low content of polyunsaturates. Newly emerged workers have a similar membrane fatty acid composition to queens but within the first week of hive life, they increase the polyunsaturate content and decrease the monounsaturate content of their membranes, probably as a result of pollen consumption. This means their membranes likely become more susceptible to lipid peroxidation in this first week of hive life. The results support the suggestion that membrane composition might be an important factor in the determination of maximum lifespan. Assuming the same slope of the relationship between membrane peroxidation index and maximum lifespan as previously observed for mammal and bird species, we propose that the 3-fold difference in peroxidation index of phospholipids of queens and workers is large enough to account for the order-of-magnitude difference in their longevity.

Impact of commercial housing systems and nutrient and energy intake on laying hen performance and egg quality parameters1

PubMed Central

Karcher, D. M.; Jones, D. R.; Abdo, Z.; Zhao, Y.; Shepherd, T. A.; Xin, H.

2015-01-01

The US egg industry is exploring alternative housing systems for laying hens. However, limited published research related to cage-free aviary systems and enriched colony cages exists related to production, egg quality, and hen nutrition. The laying hen's nutritional requirements and resulting productivity are well established with the conventional cage system, but diminutive research is available in regards to alternative housing systems. The restrictions exist with limited availability of alternative housing systems in research settings and the considerable expense for increased bird numbers in a replicate due to alternative housing system design. Therefore, the objective of the current study was to evaluate the impact of nutrient and energy intake on production and egg quality parameters from laying hens housed at a commercial facility. Lohmann LSL laying hens were housed in three systems: enriched colony cage, cage-free aviary, and conventional cage at a single commercial facility. Daily production records were collected along with dietary changes during 15 production periods (28-d each). Eggs were analyzed for shell strength, shell thickness, Haugh unit, vitelline membrane properties, and egg solids each period. An analysis of covariance (ANCOVA) coupled with a principal components analysis (PCA) approach was utilized to assess the impact of nutritional changes on production parameters and monitored egg quality factors. The traits of hen-day production and mortality had a response only in the PCA 2 direction. This finds that as house temperature and Met intake increases, there is an inflection point at which hen-day egg production is negatively effected. Dietary changes more directly influenced shell parameters, vitelline membrane parameters, and egg total solids as opposed to laying hen housing system. Therefore, further research needs to be conducted in controlled research settings on laying hen nutrient and energy intake in the alternative housing systems and resulting impact on egg quality measures. PMID:25630672

Impact of commercial housing systems and nutrient and energy intake on laying hen performance and egg quality parameters.

PubMed

Karcher, D M; Jones, D R; Abdo, Z; Zhao, Y; Shepherd, T A; Xin, H

2015-03-01

The US egg industry is exploring alternative housing systems for laying hens. However, limited published research related to cage-free aviary systems and enriched colony cages exists related to production, egg quality, and hen nutrition. The laying hen's nutritional requirements and resulting productivity are well established with the conventional cage system, but diminutive research is available in regards to alternative housing systems. The restrictions exist with limited availability of alternative housing systems in research settings and the considerable expense for increased bird numbers in a replicate due to alternative housing system design. Therefore, the objective of the current study was to evaluate the impact of nutrient and energy intake on production and egg quality parameters from laying hens housed at a commercial facility. Lohmann LSL laying hens were housed in three systems: enriched colony cage, cage-free aviary, and conventional cage at a single commercial facility. Daily production records were collected along with dietary changes during 15 production periods (28-d each). Eggs were analyzed for shell strength, shell thickness, Haugh unit, vitelline membrane properties, and egg solids each period. An analysis of covariance (ANCOVA) coupled with a principal components analysis (PCA) approach was utilized to assess the impact of nutritional changes on production parameters and monitored egg quality factors. The traits of hen-day production and mortality had a response only in the PCA 2 direction. This finds that as house temperature and Met intake increases, there is an inflection point at which hen-day egg production is negatively effected. Dietary changes more directly influenced shell parameters, vitelline membrane parameters, and egg total solids as opposed to laying hen housing system. Therefore, further research needs to be conducted in controlled research settings on laying hen nutrient and energy intake in the alternative housing systems and resulting impact on egg quality measures. © The Author 2015. Published by Oxford University Press on behalf of Poultry Science Association.

Adhesion and growth inhibitory effect of chicken egg yolk antibody (IgY) on Salmonella enterica serovars Enteritidis and Typhimurium in vitro.

PubMed

Chalghoumi, Raja; Théwis, André; Beckers, Yves; Marcq, Christopher; Portetelle, Daniel; Schneider, Yves-Jacques

2009-06-01

The protective effects of powder preparation of egg yolk immunoglobulin Y (IgY), specific to Salmonella Enteritidis and Salmonella Typhimurium outer membrane proteins (OMP), against these two Salmonella sp. serovars were investigated in vitro in two different assays: adhesion-prevention and growth-inhibition. The adhesion-prevention assay was conducted using polarized monolayers of the human intestinal epithelial Caco-2 cell line. First, the conditions of Salmonella adherence to Caco-2 cells were optimized, and interferences of bacteria with the transepithelial electrical resistance (TER) of fully differentiated Caco-2 cell monolayers and the lactate dehydrogenase release upon exposure of the cells to Salmonella were evaluated. Both Salmonella sp. serovars were able to adhere to Caco-2 cells and decreased TER. Results from the adhesion-prevention assay demonstrated that specific IgY reduced the decrease in TER of the infected Caco-2 cell monolayers and blocked the Salmonella sp. adhesion in a concentration-dependent manner (p < 0.05). Nonspecific IgY also exhibited an inhibitory effect on these two parameters, but to a lesser extent than that of the specific IgY (p < 0.05). The protective effect of nonspecific IgY could be attributed to the low-density lipoprotein component of the water-soluble fraction of egg yolks that may not have been eliminated during ultrafiltration. The growth-inhibition assay revealed that specific IgY had an inhibitory effect on the bacterial growth, markedly during the late exponential phase, whereas nonspecific IgY failed to do so. Taken together, these results suggest that the in vitro growth inhibitory effect of specific IgY on Salmonella spp. resulted from the specific binding activity of these IgY to Salmonella sp. OMP. Passive immunization with Salmonella sp. OMP-specific IgY could thus be useful to prevent Salmonella colonization in broiler chickens and the subsequent carcass contamination during processing.

Effects of phosphonium-based ionic liquids on phospholipid membranes studied by small-angle X-ray scattering.

PubMed

Kontro, Inkeri; Svedström, Kirsi; Duša, Filip; Ahvenainen, Patrik; Ruokonen, Suvi-Katriina; Witos, Joanna; Wiedmer, Susanne K

2016-12-01

The effects of ionic liquids on model phospholipid membranes were studied by small-angle X-ray scattering, dynamic light scattering (DLS) and zeta potential measurements. Multilamellar 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes and large unilamellar vesicles composed of l-α-phosphatidylcholine (eggPC) and l-α-phosphatidylglycerol (eggPG) (80:20mol%) or eggPC, eggPG, and cholesterol (60:20:20mol%) were used as biomimicking membrane models. The effects of the phosphonium-based ionic liquids: tributylmethylphosphonium acetate, trioctylmethylphosphonium acetate, tributyl(tetradecyl)-phosphonium acetate, and tributyl(tetradecyl)-phosphonium chloride, were compared to those of 1-ethyl-3-methyl-imidazolium acetate. With multilamellar vesicles, the ionic liquids that did not disrupt liposomes decreased the lamellar spacing as a function of concentration. The magnitude of the effect depended on concentration for all studied ionic liquids. Using large unilamellar vesicles, first a slight decrease in the vesicle size, then aggregation of vesicles was observed by DLS for increasing ionic liquid concentrations. At concentrations just below those that caused aggregation of liposomes, large unilamellar vesicles were coated by ionic liquid cations, evidenced by a change in their zeta potential. The ability of phosphonium-based ionic liquids to affect liposomes is related to the length of the hydrocarbon chains in the cation. Generally, the ability of ionic liquids to disrupt liposomes goes hand in hand with inducing disorder in the phospholipid membrane. However, trioctylmethylphosphonium acetate selectively extracted and induced a well-ordered lamellar structure in phospholipids from disrupted cholesterol-containing large unilamellar vesicles. This kind of effect was not seen with any other combination of ionic liquids and liposomes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs.

PubMed

Snow, P; Yim, D L; Leibow, J D; Saini, S; Nuccitelli, R

1996-11-25

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.

Upright Imaging of Drosophila Egg Chambers

PubMed Central

Manning, Lathiena; Starz-Gaiano, Michelle

2015-01-01

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective. PMID:25867882

The dynamics of plasma membrane PtdIns(4,5)P(2) at fertilization of mouse eggs.

PubMed

Halet, Guillaume; Tunwell, Richard; Balla, Tamas; Swann, Karl; Carroll, John

2002-05-15

A series of intracellular Ca2+ oscillations are responsible for triggering egg activation and cortical granule exocytosis at fertilization in mammals. These Ca2+ oscillations are generated by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Using confocal imaging to simultaneously monitor Ca2+ and plasma membrane PtdIns(4,5)P(2) in single living mouse eggs we have sought to establish the relationship between the kinetics of PtdIns(4,5)P(2) metabolism and the Ca2+ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane PtdIns(4,5)P(2) either during the latent period or during the subsequent Ca2+ oscillations. When phosphatidylinositol 4-kinase is inhibited with micromolar wortmannin a limited decrease in plasma membrane PtdIns(4,5)P(2) is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P(2), we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P(2) that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P(2) follows the increase in Ca2+, is blocked by Ca2+ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P(3). Inhibition of Ca2+-dependent exocytosis of cortical granules, without interfering with Ca2+ transients, inhibits the PtdIns(4,5)P(2) increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally, there is no increase in PtdIns(4,5)P(2) in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane PtdIns(4,5)P(2) and that one of the pathways for increasing PtdIns(4,5)P(2) at fertilization is invoked by exocytosis of cortical granules.

Nucleus positioning within Drosophila egg chamber.

PubMed

Bernard, Fred; Lepesant, Jean-Antoine; Guichet, Antoine

2017-10-19

Both types of Drosophila egg chamber germ cells, i.e. oocyte and nurse cells, have to control their nucleus positions in order to produce a viable gamete. Interestingly, while actin microfilaments are crucial to position the nuclei in nurse cells, these are the microtubules that are important for oocyte nucleus to migrate and adopt the correct position. In this review, we discuss the mechanisms underlying these positioning processes in the two cell types with respect to the organization and dynamics of the actin and microtubule skeleton. In the nurse cells it is essential to keep firmly the nuclei in a central position to prevent them from obstructing the ring canals when the cytoplasmic content of the cells is dumped into the oocyte cells toward the end of oogenesis. This is achieved by the assembly of thick filopodia-like actin cables anchored to the plasma membrane, which grow inwardly and eventually encase tightly the nuclei in a cage-like structure. In the oocyte, the migration at an early stage of oogenesis of the nucleus from a posterior location to an anchorage site at an asymmetric anterior position, is an essential step in the setting up of the dorsoventral polarity axis of the future embryo. This process is controlled by an interplay between MT networks that just start to be untangled. Although both mechanisms have evolved to fulfill cell-type specific cell processes in the context of fly oogenesis, interesting parallels can be drawn with other nuclear positioning mechanisms in the mouse oocyte and the developing muscle respectively. Copyright © 2017. Published by Elsevier Ltd.

Oocyte membrane localization of vitellogenin receptor coincides with queen flying age, and receptor silencing by RNAi disrupts egg formation in fire ant virgin queens.

PubMed

Lu, Hsiao-Ling; Vinson, S B; Pietrantonio, Patricia V

2009-06-01

In ant species in which mating flights are a strategic life-history trait for dispersal and reproduction, maturation of virgin queens occurs. However, the specific molecular mechanisms that mark this transition and the effectors that control premating ovarian growth are unknown. The vitellogenin receptor (VgR) is responsible for vitellogenin uptake during egg formation in insects. In the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), virgin queens have more abundant VgR transcripts than newly mated queens, but limited egg formation. To elucidate whether the transition to egg production involved changes in VgR expression, we investigated both virgin and mated queens. In both queens, western blot analysis showed an ovary-specific VgR band (approximately 202 kDa), and immunofluorescence analysis of ovaries detected differential VgR localization in early- and late-stage oocytes. However, the VgR signal was much lower in virgin queens ready to fly than in mated queens 8 h post mating flight. In virgin queens, the receptor signal was first observed at the oocyte membrane beginning at day 12 post emergence, coinciding with the 2 weeks of maturation required before a mating flight. Thus, the membrane localization of VgR appears to be a potential marker for queen mating readiness. Silencing of the receptor in virgin queens through RNA interference abolished egg formation, demonstrating that VgR is involved in fire ant ovary development pre mating. To our knowledge, this is the first report of RNA interference in any ant species and the first report of silencing of a hymenopteran VgR.

Sperm characteristics following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins in the collared peccary (Pecari tajacu).

PubMed

Souza, Ana Liza Paz; Lima, Gabriela Liberalino; Peixoto, Gislayne Christianne Xavier; de Souza Castelo, Thibério; Oliveira, Maria Glaucia Carlos; de Paula, Valéria Veras; Silva, Alexandre Rodrigues

2015-12-01

The aim of the current study was to compare sperm quality characteristics of the collared peccary (Pecari tajacu) following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins (LDL). Semen from 11 adult males was obtained by electroejaculation and evaluated for sperm motility, vigor, morphology as well as membrane integrity analyzed by the hypo-osmotic swelling (HOS) test and a fluorescent staining. Moreover, the semen was diluted in a Tris-based extender containing 20% egg yolk (control group) or 5, 10 or 20% LDL (treatment groups). The semen samples were frozen in liquid nitrogen and thawed in a water bath for 60s at 37°C. The treatments did not affect (p>0.05) sperm vigor, morphology or membrane integrity analyzed by the HOS test. However, post-thaw sperm motility was significantly higher (p<0.05) in the extender supplemented with 20% LDL (36.4 ± 5.3%) compared with the egg yolk extender and extender supplemented with 10% LDL. Furthermore, the percentage of membrane-intact frozen-thawed spermatozoa analyzed by the fluorescent staining was significantly higher (p<0.05) in the extender supplemented with 20% LDL (27.4 ± 6.5%) than in the other groups. In conclusion, 20% LDL can be used to substitute the whole egg yolk as a cryoprotective additive for freezing semen of the collared peccary. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Effects of posture on the respiratory mechanics of the chick embryo.

PubMed

Menna, Tara M; Mortola, Jacopo P

2002-10-01

In the chicken embryo, pulmonary ventilation and pulmonary gas exchange begin approximately one day before the completion of hatching. We asked to what extent the posture inside the egg, and the presence of the eggshell and membranes, may alter the mechanical behaviour of the respiratory system. The passive mechanical properties of the respiratory system were studied in chicken embryos during the internal pipping phase (rupture of the air cell) or the external pipping phase (hole in the eggshell). Tracheal pressure and changes in lung volume were recorded during mechanical ventilation, first, with the embryo curled up inside the egg, then again after exteriorization from the eggshell. In the internal pippers, respiratory system compliance increased and expiratory resistance decreased after exteriorization, whereas the mean inspiratory impedance did not change. In the external pippers, exteriorization had no significant effects on respiratory compliance, resistance, or impedance, and the values were similar to those of newly hatched chicks. We conclude that, in the chicken embryo, at a time when pulmonary ventilation becomes an important mechanism for gas exchange, the curled up posture inside the egg does not provide any significant mechanical constraint to breathing.

Transmission and Scanning Electron Microscopy of the Accessory Cells and Chorion During Development of Ciona intestinalis Type B Embryos and the Impact of Their Removal on Cell Morphology.

PubMed

Thompson, Helen; Shimeld, Sebastian M

2015-06-01

Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

PubMed

Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

2007-08-15

Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

SALT EFFECTS ON EGGS AND NAUPLII OF ARTEMIA SALINA L

PubMed Central

Boone, Eleanor; Baas-Becking, L. G. M.

1931-01-01

Eggs of Artemia salina L., the brine shrimp, are easily obtainable in large quantities. Ecdysis takes place in two stages: (a) extrusion of the inner membrane, and (b) ecdysis of the nauplius from that membrane. The conditions which allow for the former are much more varied than those for the latter. Nauplii form in only solutions of a few sodium salts; and, in Mg, Ca, and Sr salts, potassium is very toxic. The possible environment for the nauplii (1 M total molarity) has been ascertained for chlorides of Na, K, Mg, and Ca. The facts observed account for the peculiar distribution of the organism. PMID:19872620

Exercise and neuromodulators: choline and acetylcholine in marathon runners

NASA Technical Reports Server (NTRS)

Conlay, L. A.; Sabounjian, L. A.; Wurtman, R. J.

1992-01-01

Certain neurotransmitters (i.e., acetylcholine, catecholamines, and serotonin) are formed from dietary constituents (i.e., choline, tyrosine and tryptophan). Changing the consumption of these precursors alters release of their respective neurotransmitter products. The neurotransmitter acetylcholine is released from the neuromuscular junction and from brain. It is formed from choline, a common constituent in fish, liver, and eggs. Choline is also incorporated into cell membranes; membranes may likewise serve as an alternative choline source for acetylcholine synthesis. In trained athletes, running a 26 km marathon reduced plasma choline by approximately 40%, from 14.1 to 8.4 uM. Changes of similar magnitude have been shown to reduce acetylcholine release from the neuromuscular junction in vivo. Thus, the reductions in plasma choline associated with strenuous exercise may reduce acetylcholine release, and could thereby affect endurance or performance.

The origin of the vertebrate skeleton

NASA Astrophysics Data System (ADS)

Pivar, Stuart

2011-01-01

The anatomy of the human and other vertebrates has been well described since the days of Leonardo da Vinci and Vesalius. The causative origin of the configuration of the bones and of their shapes and forms has been addressed over the ensuing centuries by such outstanding investigators as Goethe, Von Baer, Gegenbauer, Wilhelm His and D'Arcy Thompson, who sought to apply mechanical principles to morphogenesis. However, no coherent causative model of morphogenesis has ever been presented. This paper presents a causative model for the origin of the vertebrate skeleton, based on the premise that the body is a mosaic enlargement of self-organized patterns engrained in the membrane of the egg cell. Drawings illustrate the proposed hypothetical origin of membrane patterning and the changes in the hydrostatic equilibrium of the cytoplasm that cause topographical deformations resulting in the vertebrate body form.

Unravelling proteome changes of chicken egg whites under carbon dioxide modified atmosphere packaging.

PubMed

Xu, Lei; Jia, Fei; Luo, Changyao; Yu, Qianqian; Dai, Ruitong; Li, Xingmin

2018-01-15

Unfertilized chicken eggs within 24h of laying were chosen and stored at 25°C and 45% humidity for 0, 20, and 40days. The experimental group (EG) was the carbon dioxide-modified atmosphere packaging (CDMAP) group, whereas the control group (CG) contained eggs without special handling. Egg freshness indexes were measured. The proteome of the egg whites was determined by LC-MS/MS using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 87 proteins were detected. The results indicated that CDMAP can control the change in protein abundance. Using a correlation analysis between the protein abundance and freshness indexes of the EG, Beta-hexosaminidase, Trypsin inhibitor ClTI-1 and Apolipoprotein D were determined to be potential predictors of egg freshness. In comparing the proteomes of the EG and CG, it was concluded that CDMAP could affect the proteins related to egg vitelline membranes, eggshell matrix and metabolic intensity to maintain egg freshness. Copyright © 2017. Published by Elsevier Ltd.

Cryopreservation of canine semen - new challenges.

PubMed

Farstad, W

2009-07-01

Egg yolk (EY) protects cell membranes against cold shock, and it prevents or restores the loss of phospholipids from the membrane. EY has been widely used in semen extenders. It has been added to Tris-Glucose buffer and has been widely used for cooling and cryopreservation of canine semen. EY is not a defined entity, but a complex biological compound containing proteins, vitamins, phospholipids, glucose and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it also is a biologically hazardous compound. Hence, whole EY needs to be replaced by other chemically defined components for semen processing in dogs. Freezing poor semen does not improve its quality, so attention must be focused on how to cope with dogs whose semen does not freeze well, and on designing individual freezing extenders for semen from such males. Furthermore, differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. If the presence of long-chained polyunsaturated fatty acids contributes to increased membrane fluidity, this relationship may be biphasic, i.e. either too much membrane fluidity, or too little, could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen thawed dog spermatozoa. The protective effect of exogenous lipids may lie in close association with the membrane rather than in modification or rearrangement of the membrane. This also points at lipids as an important, if not entirely new group of substances, which may substitute standard EY-based diluents in preserving sperm survival during freezing. EY-derived phospholipids or lecithin could be used to replace whole EY. Vegetable lecithin is currently investigated to avoid using substances of animal origin. EY also contains antioxidants which prevent cells from oxidative damage due to the generation of reactive oxygen species. An increasing number of publications now recognize the significance of protecting sperm from this damage during processing by using dietary or diluent supplemented antioxidants. This paper aims at looking at some of the new challenges in freezing of dog semen.

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

NASA Astrophysics Data System (ADS)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Egg collagen content is increased by a diet supplemented with wood charcoal powder containing wood vinegar liquid.

PubMed

Yamauchi, K; Matsumoto, Y; Yamauchi, K

2016-10-01

The aims of the present study were to examine whether collagen exists in egg, particularly in egg yolk; to establish a Fourier transform-near infrared (FT-NIR) measurement method for collagen in egg and to assess the possibility of increasing the collagen content by feeding hens a diet containing wood charcoal powder containing wood vinegar liquid (WCV). The collagen in eggs from 67-week-old hens fed on the dietary 0 and 9.9 g/kg WCV diets was investigated using a combination of histochemical, matrix-assisted laser desorption ionisation with time-of-flight mass spectrometry (MALDI-TOF MS), Fourier transform infrared (FT-IR) and FT-NIR approaches. All approaches used to identify collagen in egg yolk yielded positive results. The collagen in egg yolk measured using colorimetry, collagen in egg yolk, egg white and eggshell membrane using FT-NIR and collagen in egg yolk determined by treating the egg yolk with collagenase were abundant after feeding a dietary WCV (p<0.05). These results suggest that egg yolk contains collagen, that the collagen in egg can be measured using FT-NIR, and that the collagen content of egg yolk can be increased by feeding dietary WCV diets.

Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells.

PubMed

Hussain, Althaf I; Cordeiro, Melissa; Sevilla, Elizabeth; Liu, Jonathan

2010-05-14

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable. Copyright 2010 Elsevier Ltd. All rights reserved.

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

PubMed Central

Prapaiwan, N.; Tharasanit, T.; Punjachaipornpol, S.; Yamtang, D.; Roongsitthichai, A.; Moonarmart, W.; Kaeoket, K.; Manee-in, S.

2016-01-01

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa. PMID:26954170

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.

PubMed

Prapaiwan, N; Tharasanit, T; Punjachaipornpol, S; Yamtang, D; Roongsitthichai, A; Moonarmart, W; Kaeoket, K; Manee-In, S

2016-05-01

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

Reduction of facial wrinkles by hydrolyzed water-soluble egg membrane associated with reduction of free radical stress and support of matrix production by dermal fibroblasts

PubMed Central

Jensen, Gitte S; Shah, Bijal; Holtz, Robert; Patel, Ashok; Lo, Donald C

2016-01-01

Objective The aim of this study was to evaluate the effects of water-soluble egg membrane (WSEM) on wrinkle reduction in a clinical pilot study and to elucidate specific mechanisms of action using primary human immune and dermal cell-based bioassays. Methods To evaluate the effects of topical application of WSEM (8%) on human skin, an open-label 8-week study was performed involving 20 healthy females between the age of 45 years and 65 years. High-resolution photography and digital analysis were used to evaluate the wrinkle depth in the facial skin areas beside the eye (crow’s feet). WSEM was tested for total antioxidant capacity and effects on the formation of reactive oxygen species by human polymorphonuclear cells. Human keratinocytes (HaCaT cells) were used for quantitative polymerase chain reaction analysis of the antioxidant response element genes Nqo1, Gclm, Gclc, and Hmox1. Evaluation of effects on human primary dermal fibroblasts in vitro included cellular viability and production of the matrix components collagen and elastin. Results Topical use of a WSEM-containing facial cream for 8 weeks resulted in a significant reduction of wrinkle depth (P<0.05). WSEM contained antioxidants and reduced the formation of reactive oxygen species by inflammatory cells in vitro. Despite lack of a quantifiable effect on Nrf2, WSEM induced the gene expression of downstream Nqo1, Gclm, Gclc, and Hmox1 in human keratinocytes. Human dermal fibroblasts treated with WSEM produced more collagen and elastin than untreated cells or cells treated with dbcAMP control. The increase in collagen production was statistically significant (P<0.05). Conclusion The topical use of WSEM on facial skin significantly reduced the wrinkle depth. The underlying mechanisms of this effect may be related to protection from free radical damage at the cellular level and induction of several antioxidant response elements, combined with stimulation of human dermal fibroblasts to secrete high levels of matrix components. PMID:27789968

Testing an egg yolk supplemented diet on boars to aid in sperm adaptation at 5°C.

PubMed

Casas, Isabel; Miller-Lux, Yvonne; Osborne, Betty; Bonet, Sergi; Althouse, Gary C

2015-01-01

In many species, extended semen can be stored at low temperatures to slow bacterial growth. However, boar semen performs poorly at temperatures below 15 °C and this poses unique challenges, as it is not easy to maintain a constant 15-19 °C during shipment. Some extenders have been formulated with egg yolk for storage at 5 °C but the addition of egg yolk is not applicable in the majority of commercial operations. The purpose of this study was to evaluate if boar dietary supplementation with powdered egg yolk imparts any protective effects on sperm quality when stored at 15 °C and 5 °C for up to 11 days in a conventional extender. Ten boars were fed a commercial diet with the addition of 0.11 Kg of powdered egg yolk for 10 weeks. Ejaculates collected on weeks 4, 6, 8, and 10 were processed for storage at both 15 °C and 5 °C and compared with ejaculates from boars fed a standard diet. Throughout an 11-day storage period, sperm quality was assessed including several motility and morphologic parameters and select plasma membrane properties (fluidity, integrity, and triacylglycerol content). Linear regression models were used to describe effects of treatment, storage day, week and temperature on all sperm parameters. Overall, there were minimal beneficial effects of egg yolk treatment on sperm quality parameters. Sperm from egg yolk supplemented boars did have a slower decline in viability and plasma membrane fluidity than that observed in the control sperm when stored at 5 °C (p < 0.001). Additionally, there was an increase in total morphologic abnormalities in sperm from egg yolk fed boars compared to controls at week 10 (p <  .001). In conclusion, the results of this study do not support a significant benefit to sperm quality or resistance to cold storage when feeding a 10-week dietary supplementation of 0.11 Kg powdered egg yolk to crossbred boars.

Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex.

PubMed

Sardet, Christian; Nishida, Hiroki; Prodon, Francois; Sawada, Kaichiro

2003-12-01

Localization of maternal mRNAs in the egg cortex is an essential feature of polarity in embryos of Drosophila, Xenopus and ascidians. In ascidians, maternal mRNAs such as macho 1, a determinant of primary muscle-cell fate, belong to a class of postplasmic RNAs that are located along the animal-vegetal gradient in the egg cortex. Between fertilization and cleavage, these postplasmic RNAs relocate in two main phases. They further concentrate and segregate in small posterior blastomeres into a cortical structure, the centrosome-attracting body (CAB), which is responsible for unequal cleavages. By using high-resolution, fluorescent, in situ hybridization in eggs, zygotes and embryos of Halocynthia roretzi, we showed that macho 1 and HrPEM are localized on a reticulated structure situated within 2 mum of the surface of the unfertilized egg, and within 8 mum of the surface the vegetal region and then posterior region of the zygote. By isolating cortices from eggs and zygotes we demonstrated that this reticulated structure is a network of cortical rough endoplasmic reticulum (cER) that is tethered to the plasma membrane. The postplasmic RNAs macho 1 and HrPEM were located on the cER network and could be detached from it. We also show that macho 1 and HrPEM accumulated in the CAB and the cER network. We propose that these postplasmic RNAs relocalized after fertilization by following the microfilament- and microtubule-driven translocations of the cER network to the poles of the zygote. We also suggest that the RNAs segregate and concentrate in posterior blastomeres through compaction of the cER to form the CAB. A multimedia BioClip 'Polarity inside the egg cortex' tells the story and can be downloaded at www.bioclips.com/bioclip.html

The stem cell debate continues: the buying and selling of eggs for research.

PubMed

Baylis, F; McLeod, C

2007-12-01

Now that stem cell scientists are clamouring for human eggs for cloning-based stem cell research, there is vigorous debate about the ethics of paying women for their eggs. Generally speaking, some claim that women should be paid a fair wage for their reproductive labour or tissues, while others argue against the further commodification of reproductive labour or tissues and worry about voluntariness among potential egg providers. Siding mainly with those who believe that women should be financially compensated for providing eggs for research, the new stem cell guidelines of the International Society for Stem Cell Research (ISSCR) legitimise both reimbursement of direct expenses and financial compensation for many women who supply eggs for research. In this paper, the authors do not attempt to resolve the thorny issue of whether payment for eggs used in human embryonic stem cell research is ethically legitimate. Rather, they want to show specifically that the ISSCR recommended payment practices are deeply flawed and, more generally, that all payment schemes that aim to avoid undue inducement of women risk the global exploitation of economically disadvantaged women.

Xenopus egg cytoplasm with intact actin.

PubMed

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

Exosomes Derived from Dendritic Cells Treated with Schistosoma japonicum Soluble Egg Antigen Attenuate DSS-Induced Colitis

PubMed Central

Wang, Lifu; Yu, Zilong; Wan, Shuo; Wu, Feng; Chen, Wei; Zhang, Beibei; Lin, Datao; Liu, Jiahua; Xie, Hui; Sun, Xi; Wu, Zhongdao

2017-01-01

Exosomes are 30–150 nm small membrane vesicles that are released into the extracellular medium via cells that function as a mode of intercellular communication. Dendritic cell (DC)-derived exosomes modulate immune responses and prevent the development of autoimmune diseases. Moreover, Schistosoma japonicum eggs show modulatory effects in a mouse model of colitis. Therefore, we hypothesized that exosomes derived from DCs treated with S. japonicum soluble eggs antigen (SEA; SEA-treated DC exosomes) would be useful for treating inflammatory bowel disease (IBD). Exosomes were purified from the supernatant of DCs treated or untreated with SEA and identified via transmission electron microscopy, western blotting and NanoSight. Acute colitis was induced via the administration of dextran sulfate sodium (DSS) in drinking water (5.0%, wt/vol). Treatment with exosomes was conducted via intraperitoneal injection (i.p.; 50 μg per mouse) from day 0 to day 6. Clinical scores were calculated based on weight loss, stool type, and bleeding. Colon length was measured as an indirect marker of inflammation, and colon macroscopic characteristics were determined. Body weight loss and the disease activity index of DSS-induced colitis mice decreased significantly following treatment with SEA-treated DC exosomes. Moreover, the colon lengths of SEA-treated DC exosomes treated colitis mice improved, and their mean colon macroscopic scores decreased. In addition, histologic examinations and histological scores showed that SEA-treated DC exosomes prevented colon damage in acute DSS-induced colitis mice. These results indicate that SEA-treated DC exosomes attenuate the severity of acute DSS-induced colitis mice more effectively than DC exosomes. The current work suggests that SEA-treated DC exosomes may be useful as a new approach to treat IBD. PMID:28959207

Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary.

PubMed

Chen, Bo; Gui, Jianfang

2004-07-01

Potential roles of C1q/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of C1q family with a C1q domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific C1q-like factor, CaOC1q-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOC1q-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOC1q-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization.

Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens.

PubMed

Pors, Susanne E; Pedersen, Ida J; Skjerning, Ragnhild Bager; Thøfner, Ida C N; Persson, Gry; Bojesen, Anders M

2016-11-15

Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have previously shown promising results in protection against infections and we hypothesized that OMVs could serve as an immunogen to protect egg-laying hens against G. anatis. To investigate the immunogenic potential of G. anatis OMVs, two in vivo studies in egg-laying hens were made. The trials assessedthe degree of protection provided by immunization with G. anatis OMV against challenge and the IgY responses in serum after immunization and challenge, respectively. A total of 64 egg-laying hens were included in the trials. OMVs for immunization were produced and purified from a high-producing G. anatis ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re-isolation remained unchanged. Furthermore, a high OMV-specific IgY response was induced by immunization and subsequent challenge of the hens. The results strongly indicate that immunization with G. anatis OMVs provides significant protection against G. anatis challenge and induces specific antibody responses with high titers of OMV-specific IgY in serum. The results therefore show great promise for OMV based vaccines aiming at providing protecting against G. anatis in egg-laying hens. Copyright © 2016 Elsevier B.V. All rights reserved.

Vitellogenin transcytosis in follicular cells of the honeybee Apis mellifera and the wasp Polistes simillimus.

PubMed

Dohanik, Virgínia Teles; Gonçalves, Wagner Gonzaga; Oliveira, Leandro Licursi; Zanuncio, José Cola; Serrão, José Eduardo

2018-05-13

Vitellogenin receptor (VgR) is a low-density lipoprotein receptor responsible for the mediated endocytosis of vitellogenin (Vg) during egg formation in insects. The maturing oocyte is enveloped by a follicular epithelium, which has large intercellular spaces during Vg accumulation (patency). However, Vg has been reported in the cytoplasm of follicular cells, indicating that there may be a transcellular route for its transport. This study verified the presence of VgR in the follicular cells of the ovaries of the honeybee Apis mellifera and the wasp Polistes simillimus in order to evaluate if Vg is transported via transcytosis in these insects. Antibodies specific for vitellogenin receptor (anti-VgR), vitellogenin (anti-Vg), and clathrin (anti-Clt) were used for immunolocalization. The results showed the presence of VgR on the apical and basal plasma membranes of follicular cells of the vitellogenic follicles in both species, indicating that VgR may have been transported from the basal to the apical cell domain, followed by its release into the perivitelline space, evidenced by the presence of apical plasma membrane projections containing VgR. Co-localization proved that Vg bind to VgR and that the transport of this protein is mediated by clathrin. These data suggest that, in these social insects, Vg is transported via clathrin-mediated VgR transcytosis in follicular cells.

Effect of Trimethylamine N-Oxide on Interfacial Electrostatics at Phospholipid Monolayer-Water Interfaces and Its Relevance to Cardiovascular Disease.

PubMed

Mondal, Jahur A

2016-05-05

Trimethylamine N-oxide (TMAO), a metabolite of choline containing dietary nutrients which are abundant in red meat, egg, and other animal foods, increases the risk of cardiovascular disease (e.g., atherosclerosis) by boosted accumulation of fatty deposits on artery wall. Hence, for the molecular level elucidation of the pathogenesis of atherosclerosis, it is important to understand the effect of TMAO at the endothelial cell membrane-blood interface (artery wall). Heterodyne-detected vibrational sum frequency generation (HD-VSFG) study of a zwitterionic phosphatidylcholine (PC) lipid monolayer-water interface (mimic of endothelial membrane-blood interface) shows that the interfacial water becomes increasingly H-up oriented in the presence of TMAO in the aqueous phase, revealing a dramatic change in the interfacial electrostatics. Examinations of charged lipid interfaces show that TMAO screens anionic phosphate less effectively than cationic choline, which confirms that TMAO increases the relative influence of the anionic phosphate by preferential screening of the cationic choline at the zwitterionic PC lipid interface where the phosphate and choline groups are simultaneously present. Together, it is conceivable that at an elevated TMAO level in serum would modify the electrostatics at the endothelial cell membrane-blood interface (artery wall), which may affect the influx/efflux of fatty deposits on artery wall, setting the stage for atherosclerosis.

Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

PubMed Central

Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

2014-01-01

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

Antagonistic activity of the fungus Pochonia chlamydosporia on mature and immature Toxocara canis eggs.

PubMed

Maciel, A S; Freitas, L G; Figueiredo, L D; Campos, A K; Mello, I N K

2012-07-01

In vitro tests were performed to evaluate the ability of 6 isolates of the nematophagous fungus Pochonia chlamydosporia to infect immature and mature Toxocara canis eggs on cellulose dialysis membrane. There was a direct relationship between the number of eggs colonized and the increase in the days of interaction, as well as between the number of eggs colonized and the increase in the concentration of chlamydospores (P<0.05). Immature eggs were more susceptible to infection than mature eggs. The isolate Pc-04 was the most efficient egg parasite until the 7th day, and showed no difference in capacity to infect mature and immature eggs in comparison to Pc-07 at 14 and 21 days of interaction, respectively. Isolate Pc-04 was the most infective on the two evolutionary phases of the eggs at most concentrations, but its ability to infect immature eggs did not differ from that presented by the isolates Pc-07 and Pc-10 at the inoculum level of 5000 chlamydospores. Colonization of infective larvae inside or outside the egg was observed in treatments with the isolates Pc-03, Pc-04, Pc-07 and Pc-10. The isolate Pc-04 of P. chlamydosporia has great biological capacity to destroy immature and mature T. canis eggs in laboratory conditions.

Higher Storage Temperature Causes Greater Salmonella enterica Serovar Typhimurium Internal Penetration of Artificially Contaminated, Commercially Available, Washed Free Range Eggs.

PubMed

Whiley, Alice; Fallowfield, Howard; Ross, Kirstin; McEvoy, Vanessa; Whiley, Harriet

2016-07-01

Foodborne salmonellosis is a major public health concern, with contaminated eggs identified as a significant source of infection. In Australia, the most prevalent cause of salmonellosis from eggs is Salmonella enterica subsp. enterica serovar Typhimurium. This study explored the effect of temperature after 1, 7, 14, 21, and 28 days of storage on commercially available washed free range eggs, artificially contaminated with Salmonella Typhimurium on the external surface. At each time point, the external surface of the egg, the crushed eggshell, and the internal egg yolk and albumen were analyzed for Salmonella. After 28 days of storage, 25% of eggs stored at 4°C, 50% of eggs stored at 14°C, and 100% of eggs stored at 23 and 35°C were internally contaminated with Salmonella. After 1 day of storage, more than 50% of all eggs had Salmonella present in the crushed shell after the external surface had been disinfected with ethanol. This is the first study to demonstrate that refrigeration reduced the potential for Salmonella Typhimurium to penetrate the eggshell membrane and internally contaminate table eggs commercially available in Australia. It also suggests that the processes of cracking eggs may be a source of cross-contamination within the kitchen.

Reproductive toxicity and gender differences induced by cadmium telluride quantum dots in an invertebrate model organism

NASA Astrophysics Data System (ADS)

Yan, Si-Qi; Xing, Rui; Zhou, Yan-Feng; Li, Kai-Le; Su, Yuan-Yuan; Qiu, Jian-Feng; Zhang, Yun-Hu; Zhang, Ke-Qin; He, Yao; Lu, Xiao-Ping; Xu, Shi-Qing

2016-09-01

Sexual glands are key sites affected by nanotoxicity, but there is no sensitive assay for measuring reproductive toxicity in animals. The aim of this study was to investigate the toxic effects of cadmium telluride quantum dots (CdTe-QDs) on gonads in a model organism, Bombyx mori. After dorsal vein injection of 0.32 nmol of CdTe-QDs per individual, the QDs passed through the outer membranes of gonads via the generation of ROS in the membranes of spermatocysts and ovarioles, as well as internal germ cells, thereby inducing early germ cell death or malformations via complex mechanisms related to apoptosis and autophagy through mitochondrial and lysosomal pathways. Histological observations of the gonads and quantitative analyses of germ cell development showed that the reproductive toxicity was characterized by obvious male sensitivity. Exposure to QDs in the early stage of males had severe adverse effects on the quantity and quality of sperm, which was the main reason for the occurrence of unfertilized eggs. Ala- or Gly-conjugated QDs could reduce the nanotoxicity of CdTe-QDs during germ cell development and fertilization of their offspring. The results demonstrate that males are preferable models for evaluating the reproductive toxicity of QDs in combined in vivo/in vitro investigations.

Teratogenic effects of external egg applications of methyl mercury in the mallard, Anas platyrhynchos

USGS Publications Warehouse

Hoffman, D.J.; Moore, Johnnie N.

1979-01-01

The embryotoxic potential of external applications of methyl mercury on mallard eggs was investigated to assess the possible impact of mercury transferred from the plumage of effluent-contaminated aquatic birds to their eggs. Eggs were treated on day 3 of development with microliter applications of methyl mercury that was dissolved with ethyl acetate into an aliphatic hydrocarbon vehicle. Mercury analysis by atomic absorption indicated that almost half of the mercury applied entered the eggs past the shell membranes within several days of treatment. Most mortality occurred within this period at doses of 9 microgram of mercury per egg or higher. Decreased embryonic growth resulted with similar doses. A significant incidence of malformations occurred at a dose of 1 microgram per egg. These malformations were mainly minor skeletal aberrations and incomplete ossification. With higher doses of mercury, defects included gross external ones such as micromella, gastroschisis, and eye and brain defects. Application of the aliphatic hydrocarbon vehicle did not result in any of these defects.

Presence of encircling granulosa cells protects against oxidative stress-induced apoptosis in rat eggs cultured in vitro.

PubMed

Tiwari, Meenakshi; Tripathi, Anima; Chaube, Shail K

2017-01-01

Increased oxidative stress (OS) due to in vitro culture conditions can affect the quality of denuded eggs during various assisted reproductive technologies (ARTs). Presence of intact granulosa cells may protect eggs from OS damage under in vitro culture conditions. The present study was aimed to investigate whether encircling granulosa cells could protect against hydrogen peroxide (H 2 O 2 )-induced egg apoptosis in ovulated cumulus oocyte complexes (COCs) cultured in vitro. The OS was induced by exposing COCs as well as denuded eggs with various concentrations of H 2 O 2 for 3 h in vitro. The morphological changes, total reactive oxygen species (ROS) as well as catalase expression, Bax/Bcl-2, cytochrome c levels and DNA fragmentation were analysed in COCs as well as denuded eggs. Our results suggest that H 2 O 2 treatment induced morphological apoptotic features in a concentration-dependent manner in denuded eggs cultured in vitro. The 20 µM of H 2 O 2 treatment induced OS by elevating total ROS level, reduced catalase and Bcl-2 expression levels with overexpression of Bax and cytochrome c and induced DNA fragmentation in denuded eggs cultured in vitro. The presence of encircling granulosa cells protected H 2 O 2 -induced morphological apoptotic features by preventing the increase of Bax, cytochrome c expression levels and DNA fragmentation in associated egg. However, 20 µM of H 2 O 2 was sufficient to induce peripheral granulosa cell apoptosis in COCs and degeneration in few denuded eggs cultured in vitro. Taken together our data suggest that the presence of encircling granulosa cells could be beneficial to protect ovulated eggs from OS damage under in vitro culture conditions during various ART programs.

Polyelectrolyte-Mediated Transport of Doxorubicin Through the Bilayer Lipid Membrane

NASA Astrophysics Data System (ADS)

Yaroslavov, Alexander A.; Kitaeva, Marina V.; Melik-Nubarov, Nikolay S.; Menger, Frederic M.

A model is developed for the effect of ionic polymers on the transport of doxorubicin, an antitumor drug, through a bilayer membrane. Accordingly, a protonated (cationic) form of doxorubicin binds to an anionic polymer, poly(acrylic acid), the resulting complex being several hundred nanometers in size. Nevertheless, large complex species associate with neutral egg lecithin liposomes by means of hydrophobic attraction between the doxorubicin and the liposome bilayer. Then, the doxorubicin enters the liposome interior which has been imparted with an acidic buffer to protonate the doxorubicin. The rate of transmembrane Dox permeation decreases when elevating the polyacid-to-doxorubicin ratio. A cationic polymer, polylysine, being coupled with liposomes containing the negative lipid cardiolipin, accelerates membrane transport of doxorubicin with the maximum rate at a complete neutralization of the membrane charge by an interacting polycation. The effect of a polycation on doxorubicin transport becomes more pronounced as small negative liposomes (60-80 nm in diameter) are changed to larger ones (approx. 600 nm in diameter). An opportunity thus opens up for the manipulation of the kinetics of drug uptake by cells and, ultimately, the control of the pharmaceutical action of drugs.

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

PubMed

Hess, Kenneth C; Jones, Brian H; Marquez, Becky; Chen, Yanqiu; Ord, Teri S; Kamenetsky, Margarita; Miyamoto, Catarina; Zippin, Jonathan H; Kopf, Gregory S; Suarez, Susan S; Levin, Lonny R; Williams, Carmen J; Buck, Jochen; Moss, Stuart B

2005-08-01

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

PubMed Central

Levasseur, Mark; Dumollard, Remi; Chambon, Jean-Philippe; Hebras, Celine; Sinclair, Maureen; Whitaker, Michael; McDougall, Alex

2013-01-01

The fertilising sperm triggers a transient Ca2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/Ccdc20 inhibitor, links release from metaphase II arrest with the Ca2+ transient and its degradation is triggered by the Ca2+-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca2+-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates. PMID:24194472

The action of two ethyl carbamates on acetylcholinesterase and reproductive organs of Rhipicephalus microplus.

PubMed

Prado-Ochoa, M G; Ramírez-Noguera, P; Díaz-Torres, R; Garrido-Fariña, G I; Vázquez-Valadez, V H; Velázquez-Sánchez, A M; Muñoz-Guzmán, M A; Angeles, E; Alba-Hurtado, F

2014-01-31

The effects produced by the new synthetic carbamates ethyl-(4-bromophenyl) carbamate and ethyl-(4-chlorophenyl) carbamate on the acetylcholinesterase (AChE) activity, egg structure and reproductive organs of two Rhipicephalus microplus strains were evaluated. Inhibition kinetic parameters showed that the studied carbamates are weak inhibitors and have a low affinity for R. microplus AChE. Histologically, in oocytes from carbamate-treated engorged female ticks, a loss of shape, cytoplasmic vacuoles, decreased chorion deposition, alterations in cytoplasmic granularity and irregular membranes were observed. In oocyte germinal vesicles, a loss of shape, nucleolar fragmentation and membrane alterations with degenerative signs were observed. The ovarian epithelium was vacuolated, flattened, eroded and contained pyknotic nuclei. These alterations were observed from the first day and persisted and increased in severity until day 7 post-treatment. The ovaries from carbamate-treated ticks had fewer stage IV-V oocytes and more stage I-II oocytes. Additionally, eggs produced by the treated ticks had a modified appearance, decreased size, a reduced superficial waxy layer and a loss of viability. The results of this study show that the effects of carbamates on R. microplus were independent of AChE inhibition and show that the morphological alterations in the reproductive organs were due to carbamate actions on the vitellogenesis and viability of the ovarian cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Deuterated fatty acids as Raman spectroscopic probes of membrane structure.

PubMed

Mendelsohn, R; Sunder, S; Bernstein, H J

1976-09-07

Raman spectra are reported for the C-D stretching region of stearic acid-d35 bound in egg lecithin multilayers. The temperature dependence of the spectra shows that the linewidth of the C-D stretching bands is a sensitive and non-perturbative probe of membrane hydrocarbon chain conformation. The utility of this approach for studying lipid conformation in membranes containing a significant fraction of non-lipid component is discussed.

Morphology and ultrastructure of the freshwater rotifer Brachionus bidentatus (Monogononta: Brachionidae) using scanning and transmission electron microscopy.

PubMed

Guerrero-Jiménez, Gerardo; Zavala-Padilla, Guadalupe; Silva-Briano, Marcelo; Rico-Martínez, Roberto

2013-12-01

The study of sexual reproductive behavior supported by ultrastructural evidence is important in rotifers to describe differences among potential cryptic species. In this research, the morphology of the rotifer Brachionus bidentatus is described at the ultrastructural level, using electronic microscopy, together with a brief description and discussion of its sexual reproductive behavior. The characteristics of the (a) male, (b) the female, (c) the sexual egg or cyst, (d) the partenogenic egg, (e) the no-fecundated sexual egg (male egg), and (f) the trophi, were described. Another part of this research is dedicated to the ultrastructure of the sex cells of the male rotifer B. bidentatus. Samples were obtained from La Punta pond in Cosio, Aguascalientes, Mexico (22 degrees 08' N - 102 degrees 24' W), and a culture was maintained in the laboratory. Fifty organisms, from different stages of the rotifer Brachionus bidentatus, were fixed in Formol at 4% and then prepared; besides, for the trophi, 25 female rotifer Brachionus bidentatus were prepared for observation in a JEOL 5900 LV scanning electronic microscope. In addition, for the observation of male sex cells, 500 males of Brachionus bidentatus were isolated, fixed and observed in a JEOL 1010 transmission microscope. Females of B. bidentatus in laboratory cultures had a lifespan of five days (mean+one SD = 4.69 +/- 0.48; N=13), and produced 4.5 +/- 3.67 (N=6) parthenogenetic eggs during such lifespan. In the case of non-fertilized sexual eggs, they produced up to 18 eggs (mean+one SD = 13 +/- 4.93; N=7). Sexual females produced a single cyst on average (mean +/- one SD = I +/- 0; N=20). For the sexual cycle, the time of copulation between male and female ranged from 10 to 40 seconds (mean +/- one SD = 17.33 +/- 10.55, N=7). The spermatozoa are composed of a celular body and a flagellum, the size of the body is of 300 nm while the flagellum measures 1 700nm. The rods have a double membrane. Their mean length is almost 2.45 microm +/- 0.74, N=6; and their mean wide is 0.773 microm +/- 0.241, N=11. The evidence on the specific ultrastructural characteristics of the rotifer B. bidentatus is notorious, even more in the male and in the cyst cell. Regarding the ultrastructure of the spermatozoa and the rods, compared to other species they only differ in size, despite their structural resemblance. Our study of the ultraestructure of this species adds useful information that along with molecular data will help clarify the taxonomy of brachionid rotifers.

A direct approach to the study of the effect of gravity on axis formation in birds.

PubMed

Eyal-Giladi, H; Goldberg, M; Refael, H; Avner, O

1994-01-01

A system has been developed to enable the normal development of aborted very early uterine avian embryos, outside the female's uterus. The shell-less aborted egg was put into a foster shell of a sister egg, previously laid by the same female. The empty space between the shell and aborted egg was filled with artificial uterine fluid. The reconstructed eggs were incubated at 42 degrees C for 30 hours in a vertical position. The atmosphere contained a high concentration of CO2 (8-10%). At the termination of the 30 h the eggs were transferred to incubation at 37 degrees C in normal atmospheric conditions. Normal development has been recorded for a certain percentage of eggs incubated up to 12 days. In other cases abnormalities, arrested development or development of extraembryonic membranes only, without a sign of an embryonic axis, have been observed. The three important conclusions from the above experiments were: 1. It is possible to develop a closed, self-contained system, disconnected from the female's body, that would support the development of early uterine embryos. 2. The incidence of embryo-less extraembryonic membranes in such a system, is correlated with the degree of detachment of the "yolk" from the outer envelopes. 3. Such a system can be further developed into an experiment suited for microgravity conditions which will be an alternative to an experiment with live birds. The experiment will be aimed at testing the importance of gravity in changing the radially symmetrical avian blastoderm into a bilaterally symmetrical blastoderm.

Do pregnant lizards resorb or abort inviable eggs and embryos? Morphological evidence from an Australian skink, Pseudemoia pagenstecheri.

PubMed

Blackburn, Daniel G; Weaber, Kera K; Stewart, James R; Thompson, Michael B

2003-05-01

Although pregnant viviparous squamates are sometimes claimed to be able to resorb inviable eggs and embryos from the uterus, definitive evidence for such resorption is not available. After placing pregnant female Pseudemoia pagenstecheri into conditions under which embryonic development is terminated, we periodically harvested the gravid oviducts and examined them histologically. Females contained abnormal and degenerating eggs and embryos that had died in various stages of development. Dead embryos had undergone extensive cytolysis, dissolution, and aseptic necrosis and vitelline masses showed signs of deterioration and passage down the oviduct. The uterine mucosa lay in direct contact with the vitelline material, with no intact shell membrane intervening between them. Yolk was sometimes displaced into the exocoelom and allantoic cavity due to rupture of the extraembryonic membranes. Histological examination revealed no evidence of the uptake of yolk by the uterine epithelium or its accumulation in the subepithelial connective tissue. In many specimens, the uterine epithelium showed minuscule, apical granules. The position, appearance, and staining properties of the granules suggests them to be secretory, a manifestation of placentotrophy. Our observations indicate that P. pagenstecheri females retain dead eggs and embryos for several weeks or longer, yet do not resorb them during that period. This lizard is the second placentotrophic skink species in which resorption has been suspected, but in which abortive eggs appear to be retained or extruded instead of being resorbed by the oviducts. Researchers should not assume that squamates can digest and resorb oviductal eggs without definitive morphological evidence. Copyright 2003 Wiley-Liss, Inc.

Egg incubation position affects toxicity of air cell administered PCB 126 (3,3?4,4?,5- pentachlorobiphenyl) in chicken (Gallus domesticus) embryos

USGS Publications Warehouse

McKernan, M.A.; Rattner, B.A.; Hale, R.C.; Ottinger, M.A.

2007-01-01

The avian egg is used extensively for chemical screening and determining the relative sensitivity of species to environmental contaminants (e.g., metals, pesticides, polyhalogenated compounds). The effect of egg incubation position on embryonic survival, pipping, and hatching success was examined following air cell administration of polychlorinated biphenyl (PCB) congener 126 (3,3',4,4',5-pentachlorobiphenyl [PCB 126]; 500?2,000 pg/g egg) on day 4 of development in fertile chicken (Gallus gallus) eggs. Depending on dose, toxicity was found to be up to nine times greater in vertically versus horizontally incubated eggs. This may be due to enhanced embryonic exposure to the injection bolus in vertically incubated eggs compared to more gradual uptake in horizontally incubated eggs. Following air cell administration of PCB 126, horizontal incubation of eggs may more closely approximate uptake and toxicity that has been observed with naturally incorporated contaminants. These data have implications for chemical screening and use of laboratory data for ecological risk assessments.

Nup155 regulates nuclear envelope and nuclear pore complex formation in nematodes and vertebrates

PubMed Central

Franz, Cerstin; Askjaer, Peter; Antonin, Wolfram; Iglesias, Carmen López; Haselmann, Uta; Schelder, Malgorzata; de Marco, Ario; Wilm, Matthias; Antony, Claude; Mattaj, Iain W

2005-01-01

Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear lamina formation and defects in chromosome segregation at anaphase. Nup155 depletion inhibited accumulation of nucleoporins at the nuclear periphery, including those recruited to chromatin early in NE formation. Electron microscopy analysis revealed that Nup155 is also required for the formation of a continuous nuclear membrane in vivo and in vitro. Time-course experiments indicated that Nup155 is recruited to chromatin at the time of NE sealing, suggesting that nuclear pore complex assembly has to progress to a relatively late stage before NE membrane assembly occurs. PMID:16193066

Early intrauterine embryonic development in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae), an invasive tapeworm of carp (Cyprinus carpio): an ultrastructural study.

PubMed

Bruňanská, Magdaléna; Mackiewicz, John S; Młocicki, Daniel; Swiderski, Zdzisław; Nebesářová, Jana

2012-02-01

Intrauterine embryonic development in the caryophyllidean tapeworm Khawia sinensis has been investigated using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Contrary to previous light microscopy findings that reported the release of non-embryonated eggs of K. sinenesis to the external environment, the present study documents various stages of embryonation (ovoviviparity) within the intrauterine eggs of this cestode. At the initial stage of embryonic development, each fertilised oocyte is accompanied by several vitellocytes that become enclosed within the operculate, electrondense shell. Cleavage divisions result in formation of blastomeres (up to about 24 cells) of various sizes. Mitotic divisions and apparent rosette arrangment of the blastomeres, the latter atypical within the Eucestoda, are observed for the first time in the intrauterine eggs of K. sinenesis. The early embryo enclosed within the electrondense shell is surrounded by a thin membraneous layer which in some enlarged regions shows presence of nuclei. Simultaneously to multiplication and differentiation, some of the blastomeres undergo deterioration. A progressive degeneration of the vitellocytes within eggs provides nutritive reserves, including lipids, for the developing embryo. The possible significance of this atypical timing of the intrauterine embryonic development to (1) the ecology of K. sinensis and that of a recent introduction of another invasive tapeworm, the caryophyllidean Atractolytocestus huronensis Anthony, 1958 to Europe; and (2) the affiliation of caryophyllideans with other lower cestodes, are discussed.

Inactivation of Salmonella in Shell Eggs by Hot Water Immersion and Its Effect on Quality.

PubMed

Geveke, David J; Gurtler, Joshua B; Jones, Deana R; Bigley, Andrew B W

2016-03-01

Thermal inactivation kinetics of heat resistant strains of Salmonella Enteritidis in shell eggs processed by hot water immersion were determined and the effects of the processing on egg quality were evaluated. Shell eggs were inoculated with a composite of heat resistant Salmonella Enteritidis (SE) strains PT8 C405, 2 (FSIS #OB030832), and 6 (FSIS #OB040159). Eggs were immersed in a circulating hot water bath for various times and temperatures. Come-up time of the coldest location within the egg was 21 min. SE was reduced by 4.5 log at both hot water immersion treatments of 56.7 C for 60 min and 55.6 °C for 100 min. Decimal reduction times (D-values) at 54.4, 55.6, and 56.7 °C were 51.8, 14.6, and 9.33 min, respectively. The z-value was 3.07 °C. Following treatments that resulted in a 4.5 log reduction (56.7 °C/60 min and 55.6 °C/100 min), the surviving population of SE remained static during 4 wk of refrigerated storage. After processing under conditions resulting in 4.5 log reductions, the Haugh unit and albumen height significantly increased (P < 0.01) and yolk index significantly decreased (P < 0.05). The shell dynamic stiffness significantly increased (P < 0.05), while static compression shell strength showed no significant difference (P < 0.05). Vitelline membrane strength significantly increased (P < 0.05); although, no significant difference (P < 0.05) was observed in vitelline membrane elasticity. In summary, the hot water immersion process inactivated heat resistant SE in shell eggs by 4.5 log, but also significantly affected several egg quality characteristics. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands

PubMed Central

Villarreal, Ashley M.; Adamson, Steven W.; Browning, Rebecca E.; Khem Raj, B.C.; Sajid, Muhammad Sohail; Karim, Shahid

2013-01-01

Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and A. americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases. PMID:23499931

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review. Copyright 2009 Wiley-Liss, Inc.

Embryonic Developmental Stages of African Giant Catfish Heterobranchus longifilis (Valenciennes, 1840) (Teleostei, Clariidae)

NASA Astrophysics Data System (ADS)

Wilfred-Ekprikpo, P. C.

2016-02-01

One of the major challenges confronting the globe is the issue of food insecurity. This problem results from inadequate protein intake by humans especially those people from the third world countries. In order to arrest this ugly situation, there is the need to increase protein production by intensifying aquaculture. In sub-saharan Africa, particularly Nigeria, the major aquaculture species is African mud catfish (Clarias gariepinus) and its production has increase protein output but the protein deficit is still wide. Thus, necessitating the need to develop other aquaculture species endemic to the region. One of these species is Heterobranchus and there successful breeding depending on a good understanding of their biology. The embryonic developmental stages of Heterobranchus longifilis in freshwater tanks were determined. The first cleavage cell division occurred 30 minutes after fertilization of eggs while, the morula stage was observed within 2 hours. The blastula stage occurred between 2 and 8 hours, while the gastrula stage occurred between 12 and 18 hours. Thereafter, neurulation period, and embryonic body formation appeared. The optical vesicle and auditory vesicle formed. Finally muscular contraction, tail formation, heartbeat and hatching occurred. The embryonic developmental stage of H. longifilis started immediately the oocyte (egg) was fertilized and terminated when the embryo hatched from the chorion membranous wall. The young larva emerged from the embryonic membrane at 24.46 hrs with vigorous lashing of the caudal region against the chorion membrane. The average weight and length of the yolk larvae were 0.005g and 0.43 cm respectively. The percentage fertilization and hatchability rates were 82.50 and 65.10% respectively. The experiment revealed that Heterobranchus longifilis could be a good aquaculture species.

Intracellular Immunohistochemical Detection of Tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria)

PubMed Central

Salvitti, Lauren R.; Wood, Susanna A.; Winsor, Leigh; Cary, Stephen Craig

2015-01-01

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring. PMID:25636158

A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band.

PubMed

Sardet, C; Chang, P

1985-09-01

We have examined the subequatorial accumulation of pigment granules (the so-called 'pigment band') in the egg of the sea urchin Paracentrotus lividus, which constitutes an unambiguous marker of animal-vegetal polarity. Most of the reddish pigment granules are situated at the periphery of the egg. They exhibit occasional saltatory movements and can aggregate into large patches. Pigment granules are retained as a band in the isolated cortex when the egg surface complex is isolated by shearing eggs attached to polylysine-coated surfaces with calcium-free isotonic solutions. Pigment granules remain as the main vesicular component of fertilized egg cortices or of unfertilized egg cortices perfused with calcium to provoke cortical granule exocytosis. They may be anchored to the isolated cortex through associations with the plasma membrane and with an extensive subsurface network of rough endoplasmic reticulum (rough ER). Pigment granules contain antimonate-precipitable calcium and, in this respect and many others, resemble acidic vesicles recently identified in the cortex of unpigmented sea urchin eggs. We discuss the similarities observed between granules and acidic vesicles in various urchin egg species and their possible functions.

Patterns of globalized reproduction: Egg cells regulation in Israel and Austria

PubMed Central

2012-01-01

Since the successful introduction of in vitro fertilization in 1978, medically assisted reproduction (MAR) has proliferated in multiple clinical innovations. Consequently, egg cells have become an object of demand for both infertility treatment and stem cell research, and this raises complex legal, ethical, social and economic issues. In this paper we compare how the procurement and use of human egg cells is regulated in two countries: Israel and Austria. Israel is known for its scientific leadership, generous public funding, high utilization and liberal regulation of assisted reproductive technology (ART). Austria lies at the other extreme of the regulatory spectrum in terms of restrictions on reproductive interventions. In both countries, however, there is a constant increase in the use of the technology, and recent legal developments make egg cells more accessible. Also, in both countries the scarcity of egg cells in concert with the rising demand for donations has led to the emergence of cross-border markets and global 'reproductive tourism' practices. In Israel, in particular, a scandal known as the 'eggs affair' was followed by regulation that allowed egg cell donations from outside the country under certain conditions. Cross-border markets are developed by medical entrepreneurs, driven by global economic gaps, made possible by trans-national regulatory lacunae and find expression as consumer demand. The transnational practice of egg cell donations indicates the emergence of a global public health issue, but there is a general lack of medical and epidemiological data on its efficacy and safety. We conclude that there is need for harmonisation of domestic laws and formulation of new instruments for international governance. PMID:22913734

Patterns of globalized reproduction: Egg cells regulation in Israel and Austria.

PubMed

Shalev, Carmel; Werner-Felmayer, Gabriele

2012-04-18

Since the successful introduction of in vitro fertilization in 1978, medically assisted reproduction (MAR) has proliferated in multiple clinical innovations. Consequently, egg cells have become an object of demand for both infertility treatment and stem cell research, and this raises complex legal, ethical, social and economic issues.In this paper we compare how the procurement and use of human egg cells is regulated in two countries: Israel and Austria. Israel is known for its scientific leadership, generous public funding, high utilization and liberal regulation of assisted reproductive technology (ART). Austria lies at the other extreme of the regulatory spectrum in terms of restrictions on reproductive interventions.In both countries, however, there is a constant increase in the use of the technology, and recent legal developments make egg cells more accessible. Also, in both countries the scarcity of egg cells in concert with the rising demand for donations has led to the emergence of cross-border markets and global 'reproductive tourism' practices. In Israel, in particular, a scandal known as the 'eggs affair' was followed by regulation that allowed egg cell donations from outside the country under certain conditions.Cross-border markets are developed by medical entrepreneurs, driven by global economic gaps, made possible by trans-national regulatory lacunae and find expression as consumer demand. The transnational practice of egg cell donations indicates the emergence of a global public health issue, but there is a general lack of medical and epidemiological data on its efficacy and safety. We conclude that there is need for harmonisation of domestic laws and formulation of new instruments for international governance.

Possible role of the 38 kDa protein, lacking in the gastrula-arrested Xenopus mutant, in gastrulation.

PubMed

Tanaka, Tetsuya S; Ikenishi, Kohji

2002-02-01

An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.

Fine structure of uterus and non-functioning paruterine organ in Orthoskrjabinia junlanae (Cestoda, Cyclophyllidea).

PubMed

Korneva, Janetta V; Kornienko, Svetlana A; Jones, Malcolm K

2016-06-01

Some cyclophyllidean cestodes provide protection for their eggs in the external environment by providing them with additional protective layers around the egg membranes. In attempting to examine such adaptations, the microanatomy and fine structure of the uterus of pregravid and gravid proglottids of the cyclophyllidean cestode Orthoskrjabinia junlanae, a parasite of mammals that inhabit a terrestrial but moist environment, were studied. In the initial stages of uterine development, developing embryos locate freely in the lumen of a saccate uterus that later partitions into chambers. Each chamber that forms encloses several embryos. The chambers are surrounded by muscle cells that synthesize extracellular matrix actively. The paruterine organs consist of stacks of flattened long outgrowths of muscular cells, interspersed with small lipid droplets. In the gravid proglottids, the size of paruterine organ increases and consists of flattened basal and small rounded apical parts separated by constrictions. The fine structure of the organ wall remains the same: sparse nuclei and stacks of flattened cytoplasmic outgrowths but internal invaginations or lumen in the paruterine organ are absent. Completely developed eggs remain localized in the uterus. Based on the comparative morpho-functional analysis of uterine and paruterine organs and uterine capsules in cestodes, we conclude that these non-functioning paruterine organ in O. junlanae is an example of an atavism. We postulate that the life cycle of the parasite, which infects mammals living in wet habitats, where threats of desiccation of parasite ova is reduced, has favoured a reversion to a more ancestral form of uterine development.

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

PubMed Central

Gans, Madeleine; Audit, Claudie; Masson, Michele

1975-01-01

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed. PMID:814037

AN INDIRECT METHOD TO ASSAY FOR MITOTIC CENTERS IN SAND DOLLAR (DENDRASTER EXCENTRICUS) EGGS

PubMed Central

Went, Hans A.

1966-01-01

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C14-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication. PMID:6008198

An indirect method to assay for mitotic centers in sand dollar (Dendraster excentricus) eggs.

PubMed

Went, H A

1966-09-01

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C(14)-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.

Parthenogenesis and somatic cell nuclear transfer in sheep oocytes using Polscope.

PubMed

Nandedkar, Pandit; Chohan, Parul; Patwardhan, Archana; Gaikwad, Santosh; Bhartiya, Deepa

2009-07-01

Parthenogenesis and Somatic cell nuclear transfer (SCNT) techniques, offer a unique approach to manipulate the genetic composition of derived human embryonic stem cells - an essential step if the full opportunities for disease modeling, drug discovery or individualized stem cell therapy are to be realized. The present study describes the use of sheep oocytes to acquire expertise and establish methods to reconstruct embryos for obtaining blastocysts before venturing into human SCNT where the oocytes are a very precious starting material. Maturation of sheep eggs in vitro for 20-24 hr resulted in 65% metaphase II (MII) eggs which were either parthenogenetically activated using calcium ionomycin or ethanol or subjected to SCNT using cumulus cell as somatic cell. Sixteen blastocysts were produced by parthenogenetic activation of 350 eggs whereas reconstructed embryos, after SCNT carried out in 139 eggs, progressed only up to morula stage. The procedure of parthenogenesis and SCNT will be useful to generate autologous ES cells using human eggs.

Production of human monoclonal antibody in eggs of chimeric chickens.

PubMed

Zhu, Lei; van de Lavoir, Marie-Cecile; Albanese, Jenny; Beenhouwer, David O; Cardarelli, Pina M; Cuison, Severino; Deng, David F; Deshpande, Shrikant; Diamond, Jennifer H; Green, Lynae; Halk, Edward L; Heyer, Babette S; Kay, Robert M; Kerchner, Allyn; Leighton, Philip A; Mather, Christine M; Morrison, Sherie L; Nikolov, Zivko L; Passmore, David B; Pradas-Monne, Alicia; Preston, Benjamin T; Rangan, Vangipuram S; Shi, Mingxia; Srinivasan, Mohan; White, Steven G; Winters-Digiacinto, Peggy; Wong, Susan; Zhou, Wen; Etches, Robert J

2005-09-01

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

Effect of in ovo supplementation of nano forms of zinc, copper, and selenium on post-hatch performance of broiler chicken.

PubMed

Joshua, P Patric; Valli, C; Balakrishnan, V

2016-03-01

Nanoparticles can bypass conventional physiological ways of nutrient distribution and transport across tissue and cell membranes, as well as protect compounds against destruction prior to reaching their targets. In ovo administration of nanoparticles, may be seen as a new method of nano-nutrition, providing embryos with an additional quantity of nutrients. The aim of the study is to examine the effect of in ovo supplementation of nano forms of zinc, copper and selenium on the hatchability and post hatch performance of broiler chicken. Nano form of zinc at 20, 40, 60 and 80 µg/egg, nano form of copper at 4, 8, 12 and 16 µg/egg and nano form of selenium at 0.075, 0.15, 0.225 and 0.3 µg/egg were in ovo supplemented (18(th) day incubation, amniotic route) in fertile broiler eggs. Control group in ovo fed with normal saline alone was also maintained. Each treatment had thirty replicates. Parameters such as hatchability, hatch weight and post hatch performance were studied. In ovo feeding of nano minerals were not harmful to the developing embryo and did not influence the hatchability. Significantly (p<0.05) best feed efficiency for nano forms of zinc (2.16), copper (2.46) and selenium (2.51) were observed, when 40, 4 and 0.225 µg/egg respectively were in ovo supplemented. Except in nano form of copper at 12 µg per egg which had significantly (p<0.05) highest breast muscle percentage there was no distinct trend to indicate that dressing percentage or breast muscle yield was influenced in other treatments. Nano forms of zinc, copper and selenium can be prepared at laboratory conditions. In ovo feeding of nano forms of zinc, copper and selenium at 18(th) day of incubation through amniotic route does not harm the developing embryo, does not affect hatchability.

Effect of in ovo supplementation of nano forms of zinc, copper, and selenium on post-hatch performance of broiler chicken

PubMed Central

Joshua, P. Patric; Valli, C.; Balakrishnan, V.

2016-01-01

Background and Aim: Nanoparticles can bypass conventional physiological ways of nutrient distribution and transport across tissue and cell membranes, as well as protect compounds against destruction prior to reaching their targets. In ovo administration of nanoparticles, may be seen as a new method of nano-nutrition, providing embryos with an additional quantity of nutrients. The aim of the study is to examine the effect of in ovo supplementation of nano forms of zinc, copper and selenium on the hatchability and post hatch performance of broiler chicken. Materials and Methods: Nano form of zinc at 20, 40, 60 and 80 µg/egg, nano form of copper at 4, 8, 12 and 16 µg/egg and nano form of selenium at 0.075, 0.15, 0.225 and 0.3 µg/egg were in ovo supplemented (18th day incubation, amniotic route) in fertile broiler eggs. Control group in ovo fed with normal saline alone was also maintained. Each treatment had thirty replicates. Parameters such as hatchability, hatch weight and post hatch performance were studied. Results: In ovo feeding of nano minerals were not harmful to the developing embryo and did not influence the hatchability. Significantly (p<0.05) best feed efficiency for nano forms of zinc (2.16), copper (2.46) and selenium (2.51) were observed, when 40, 4 and 0.225 µg/egg respectively were in ovo supplemented. Except in nano form of copper at 12 µg per egg which had significantly (p<0.05) highest breast muscle percentage there was no distinct trend to indicate that dressing percentage or breast muscle yield was influenced in other treatments. Conclusion: Nano forms of zinc, copper and selenium can be prepared at laboratory conditions. In ovo feeding of nano forms of zinc, copper and selenium at 18th day of incubation through amniotic route does not harm the developing embryo, does not affect hatchability. PMID:27057113

The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro.

PubMed

Byrne, Richard D; Larijani, Banafshé; Poccia, Dominic L

2016-01-01

FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.

In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen.

PubMed

Salmani, Hossein; Towhidi, Armin; Zhandi, Mahdi; Bahreini, Majid; Sharafi, Mohsen

2014-04-01

Soybean lecithin is a suitable plant-based cryoprotectant for freezing ruminant sperm. Optimum level of lecithin was not clear for goat semen cryopreservation. The objective of this study was to investigate the effects of different levels of soybean lecithin in semen extender on post-thaw sperm quality including CASA-motion parameters, viability, plasma membrane integrity and lipid peroxidation. Semen samples were collected from 4 Mahabadi bucks using an artificial vagina. Different concentrations of soy lecithin (SL, 0.5%, 1%, 1.5%, 2% and 2.5% w/v) were compared to 15% (v/v) egg yolk-based extender (TR-EY). No significant difference was observed for sperm progressive motility, viability or plasma membrane integrity in 1.5% SL media (33.8%, 66%, and 62.7%, respectively) and TR-EY medium (35.4%, 67.2%, and 64.9%, respectively). Sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) and rapid spermatozoa were improved with extender containing 1% and 1.5% SL, compared to TR-EY extender. Furthermore, egg yolk produced significantly higher malondialdehyde (4.02±0.21) than other groups. Results suggest that the optimal lecithin concentration in the semen extender was 1.5% and also soy lecithin can substitute for egg yolk during cryopreservation for caprine sperm. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon

Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin duringmore » nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.« less

Effects of sediment burial on grass carp, Ctenopharyngodon idella (Valenciennes,1844), eggs

USGS Publications Warehouse

George, Amy E.; Chapman, Duane C.; Deters, Joseph E.; Erwin, Susannah O.; Hayer, Cari-Ann

2015-01-01

It is thought that grass carp (Ctenopharyngodon idella) eggs must remain suspended in the water column in order to hatch successfully. Using sand, the effects of varying sediment levels on grass carp eggs were tested at different developmental states and temperatures. Survival was high (15–35%, depending on temperature and trial) in the unburied treatment where eggs rested on a sand bed but were not covered by sediment. Survival was lower in the partial burial (5–10%) and very low (0–4%) in the full burial treatment. In all treatments, delayed hatching (organisms remaining in membranes past the stage of hatching competence) was noted. Deformities such as missing heads and pericardial edema occurred at high rates in the partial and full burials. Eggs that come in contact with the benthos and are resuspended in the water column should be considered in embryonic drift models.

Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol.

PubMed

Silva, M A; Peixoto, G C X; Lima, G L; Bezerra, J A B; Campos, L B; Paiva, A L C; Paula, V V; Silva, A R

2012-08-01

The objective was to determine the effectiveness of a powdered coconut water-based extender (ACP-116c), plus various concentrations of egg-yolk and glycerol, as an alternative for cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were apportioned into aliquots that were diluted in Tris plus 10% egg yolk and 3% glycerol, or in ACP-116c plus 10 or 20% egg yolk and 1.5 or 3% glycerol. Samples were frozen in liquid nitrogen and, after 1 mo, thawed at 37 °C for 1 min. After thawing, samples were evaluated as reported for fresh semen, and also for sperm membrane integrity (fluorescent probes) and kinematic parameters (computerized analysis). Results were presented as means ± SEM. Freezing and thawing decreased sperm characteristics relative to fresh semen. Overall, ACP-116c plus 20% egg yolk and 3% glycerol provided better (P < 0.05) sperm motility and kinetic rating (48 ± 6.1% and 2.8 ± 0.2, respectively) after thawing than Tris extender (30.4 ± 5.7% and 2.4 ± 0.2). However, there were no differences (P > 0.05) among treatments with regard to the other sperm characteristics. Based on computerized motion analysis, total (26.5 ± 5.9%) and progressive (8.1 ± 2.2%) motility were best preserved (P < 0.05) with the above-mentioned treatment. In conclusion, a coconut water-based extender, ACP-116c, plus 20% egg yolk and 3% glycerol, was effective for cryopreservation of semen from collared peccaries. Copyright © 2012 Elsevier Inc. All rights reserved.

Is the idea of a fast block to polyspermy based on artifact?

PubMed

Dale, Brian

2014-08-01

This purpose of this review is to look at the experimental evidence, both kinetic and electrophysiological, that led to the hypothesis of a fast electrical block to polyspermy in sea urchin eggs. The idea of a fast partial block, forwarded in the 1950's, that would reduce the receptivity of the egg surface by 1/20th following its interaction with the fertilizing spermatozoon, was based on experiments that treated fertilization as a first order chemical reaction. Here, I outline the criticisms of the Rothschild theory and demonstrate that the hypothesis of a fast partial block to polyspermy is unfounded. Notwithstanding, it was suggested in the 1970's that the membrane depolarization, induced by the fertilizing spermatozoon, prevented the interaction of supernumerary spermatozoa, the fast electrical block to polyspermy. While trans-membrane voltage recording has permitted a better understanding of the sequence of events occurring at fertilization, there is no evidence that depolarization prevents the interaction of supernumerary spermatozoa. Sperm entry is prevented at positive and negative potentials, in the voltage clamp configuration, however this is an artifact caused by the currents injected into the egg employed to hold the voltage constant in a non-physiological range. At permissive voltages, around -20 mV, where the current required to hold the voltage is minimal, only one spermatozoon normally enters the egg. Thus, irrespective of the egg voltage, the fertilizing spermatozoon is, in any case, attached to a privileged interaction site that permits entry and distinguishes it from supernumerary spermatozoa. Competence for monospermy is acquired during oocyte maturation and data on cortical organization in echinoderm eggs points to the actin filament system for regulating sperm entry. Copyright © 2014 Elsevier Inc. All rights reserved.

Irregularly calcified eggs and eggshells of Caiman latirostris (Alligatoridae: Crocodylia).

PubMed

Fernández, Mariela Soledad; Simoncini, Melina Soledad; Dyke, Gareth

2013-05-01

We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9% (range 0-38%) across our sample compared to a mean normal success of 75%. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration.

Irregularly calcified eggs and eggshells of Caiman latirostris (Alligatoridae: Crocodylia)

NASA Astrophysics Data System (ADS)

Fernández, Mariela Soledad; Simoncini, Melina Soledad; Dyke, Gareth

2013-05-01

We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9 % (range 0-38 %) across our sample compared to a mean normal success of 75 %. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration.

High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

PubMed Central

Sot, Jesús; Manni, Marco M.; Viguera, Ana R.; Castañeda, Verónica; Cano, Ainara; Alonso, Cristina; Gil, David; Valle, Mikel; Alonso, Alicia; Goñi, Félix M.

2014-01-01

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition. PMID:25517149

Probing the tumor microenvironment: collection and induction

NASA Astrophysics Data System (ADS)

Williams, James K.; Padgen, Michael R.; Wang, Yarong; Entenberg, David; Gertler, Frank; Condeelis, John S.; Castracane, James

2012-03-01

The Nano Intravital Device, or NANIVID, is under development as an optically transparent, implantable tool to study the tumor microenvironment. Two etched glass substrates are sealed using a thin polymer membrane to create a reservoir with a single outlet. This reservoir is loaded with a hydrogel blend that contains growth factors or other chemicals to be delivered to the tumor microenvironment. When the device is implanted in the tumor, the hydrogel will swell and release these entrapped molecules, forming a gradient. Validation of the device has been performed in vitro using epidermal growth factor (EGF) and MenaINV, a highly invasive, rat mammary adenocarcinoma cell line. In both 2-D and 3-D environments, cells migrated toward the gradient of EGF released from the device. The chorioallantoic membrane (CAM) of White Leghorn chicken eggs is being utilized to grow xenograft tumors that will be used for ex vivo cell collection. Device optimization is being performed for in vivo use as a tool to collect the invasive cell population. Preliminary cell collection experiments in vivo were performed using a mouse model of breast cancer. As a second application, the device is being explored as a delivery vehicle for chemicals that induce controlled changes in the tumor microenvironment. H2O2 was loaded in the device and generated intracellular reactive oxygen species (ROS) in cells near the device outlet. In the future, other induction targets will be explored, including hypoglycemia and the manipulation of extracellular matrix stiffness.

[Morphology of gametes in sea urchins from Peter the Great Bay, Sea of Japan].

PubMed

Drozdov, A L; Vinnikova, V V

2010-01-01

The fine structure of the gametes in six sea urchin species of the Sea of Japan was studied. The spermatozoons in Strongylocentrotus nudus, S. intermedius, Echinocardium cordatum, Scaphechinus mirabilis, Sc. grizeus and Echinarachnius parma are species-specific. The conical head and symmetrically disposed ring-shape mitochondrion are common to regular sea urchin sperm cells. S. nudus is characterized by the bulb-shaped head of the zoosperm; S. intermedius, by a bullet-shaped one. The zoosperm spearhead and small amount of postacrosome material are common to irregular sea urchins; the sperm width: length ratio varies for different species, with the highest for Sc. mirabilis. The zoosperm of Sc. griseus is characterized by two lipid drops in the cell center. Asymmetrical mitochondrion disposal is usual for E. parma. Actin filaments are found in the postacrosome material in the zoosperm of cordiform sea urchins. The differences in the fine structure of zoosperm in eurybiont species Ech. cordatum inhabiting the Sea of Japan and coastal areas of the Northeast Atlantic may bear record to the complex existence of species Ech. cordatum. The fine structure of zoosperm is unique for each of the studied families, Strongylocentrotidae, Scutellidae, and Loveniidae. The eggs of all the species are characterized by vitelline and tremelloid membranes. The vitelline membrane is formed by cytoplasm protrusions; the area between them is filled with fubrillary material. The tremelloid membrane is formed by fubrillary material associated with apical parts of microvilli of the vitelline membrane. The irregular sea urchins Sc. griseus, Sc. mirabilis and E. parma are characterized by chromatophores situated in the tremelloid membrane, with the highest abundance in Sc. mirabilis.

Immunologic effects of low levels of ochratoxin A in ovo: utilization of a chicken embryo model.

PubMed

Harvey, R B; Kubena, L F; Naqi, S A; Gyimah, J E; Corrier, D E; Panigrahy, B; Phillips, T D

1987-01-01

Ochratoxin A (OA) was administered to 13-day-old chicken embryos via the chorioallantoic membrane. The 7-day LD50 value (day 20 incubation) of OA was calculated at 7.9 micrograms of OA. Ochratoxin-treated embryos (2.5 micrograms) had slight but significant changes in numbers of immunoglobulin-bearing cells in the bursa but not in the spleen. Chicks hatched from in ovo-treated eggs were challenged with 9 X 10(4) colony-forming units (CFU) of beta-hemolytic Escherichia coli (O1:K1) at 7 days of age via the thoracic air sac. Lesion scores of OA-treated chicks were equal to or less severe than those of controls. Hatchmates of the above chicks were vaccinated with a homologous killed E. coli bacterin (O1:K1) at both 2 and 4 weeks of age and challenged with 10(4) CFU of E. coli at 7 weeks. Post-challenge lesions were present in three vaccinated untreated controls and no OA-treated chicks. We conclude that although in ovo exposure to OA may marginally suppress immunoglobulin-bearing cells of bursa, chicks hatched from OA-treated eggs respond as well as controls to an antigen and resist infection by a virulent organism.

Animal cells dependent on exogenous phosphatidylcholine for membrane biogenesis.

PubMed Central

Esko, J D; Nishijima, M; Raetz, C R

1982-01-01

A Chinese hamster ovary cell (CHO) mutant (strain 58), defective in CDP-choline synthetase (cholinephosphate cytidylyltransferase; CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15), is temperature sensitive for growth and contains less than half of the normal amount of phosphatidylcholine under nonpermissive conditions [Esko, J. D. & Raetz, C. R. H. (1980) Proc. Natl. Acad. Sci. USA 77, 5192-5196]. We now report that the addition of 40 microM egg phosphatidylcholine or lysophosphatidylcholine to the medium suppresses the temperature sensitivity of mutant 58 and permits the growth of colonies at the restrictive temperature. Phospholipids with different polar headgroups, lipoprotein-bound phospholipids, sphingomyelin, and glycerophosphocholine do not support prolonged growth at 40 degrees C, whereas phosphatidylcholine analogs such as phosphatidyldimethylethanolamine, D-phosphatidylcholine, and beta-phosphatidylcholine are quite effective. A broad range of saturated phosphatidylcholines, especially those with fatty acids 12-18 carbons in length, suppresses the phenotype. Phospholipids containing ether-linked hydrocarbons are ineffective, whereas polyunsaturated phosphatidylcholines are toxic. Residual endogenous synthesis of phosphatidylcholine by the mutant is not stimulated under conditions of phenotypic bypass, but the uptake of exogenous lipid is enhanced considerably compared to the wild type. Our findings demonstrate that exogenous phospholipid can provide at least 50% of the phosphatidylcholine required for membrane biogenesis in animal cells and that uptake of exogenous phospholipids may be regulated. PMID:6281780

For love or money? The saga of Korean women who provided eggs for embryonic stem cell research.

PubMed

Baylis, Françoise

2009-01-01

In 2004 and 2005, Woo-Suk Hwang achieved international stardom with publications in Science reporting on successful research involving the creation of stem cells from cloned human embryos. The wonder and success all began to unravel, however, when serious ethical concerns were raised about the source of the eggs for this research. When the egg scandal had completely unfolded, it turned out that many of the women who provided eggs for stem cell research had not provided valid consents and that nearly 75% of the women egg providers had received cash or in-kind payments. Among those who did not receive direct benefits, some cited patriotism as their reason for participating in embryonic stem cell research, hence the question "for love or money?"--namely, patriotism versus payment. This paper summarizes the Hwang debacle with particular attention to the egg scandal and ends with some preliminary thoughts on patriotism as a motive for research participation.

Nest materials as a source of genetic data for avian ecological studies

USGS Publications Warehouse

Pearce, J.M.; Fields, R.L.; Scribner, K.T.

1997-01-01

We examined the utility of feathers and egg shell membranes, deposited in the nests of Spectacled Eiders (Somateria fischeri), as a source of DNA for genetic studies at both the population and individual level. The potential for feather DNA contamination as a result of female behavioral interactions (e.g. nest parasitism), reuse of nest sites from previous years, or other unknown occurrences was acknowledged and specifically tested. DNA was successfully extracted from both feathers and egg shell membranes and waterfowl microsatellite loci were used to construct individual genotypes. We found no difference in the genotypes obtained from nest feathers or blood of the incubating female. Detection of nest feather contamination was possible with as little as one feather when samples from multiple females were intentionally mixed. Triplicate DNA extractions from 33 nests provided a means of detecting contamination in 3 nests. Egg membranes proved a viable source of offspring DNA and can contribute valuable data to investigations of parentage when assayed jointly with maternal feather DNA. Nest materials provide an efficient, non-invasive method of genetic sampling that can be readily incorporated into field research. However, the natural history traits and mating strategies of a species must be considered during sample collection to identify the possible sources of nest materials (e.g., paternal, maternal, parasite, etc.). Specific experiments should also be designed to test sampling assumptions.

Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation.

PubMed

Baghshahi, H; Riasi, A; Mahdavi, A H; Shirazi, A

2014-12-01

Clove bud (Syzygium aromaticum) extract was added at concentrations of 0, 35, 75, and 115 μg/ml to ovine semen extenders in order to investigate the antioxidant activities of clove bud extract and its effects on semen quality parameters after cryopreservation of ram spermatozoa. The basic extender was composed of Tris, egg yolk, and glycerol. Two other extenders were prepared by substitution of egg yolk with either LDL or egg yolk+SDS. The DPPH inhibition test was employed to assess the antioxidant activity of clove bud extract. Results showed that, compared to vitamin E, clove bud extract had a higher antioxidant activity. Better sperm motility and movement characteristics (P<0.05) were observed in the semen diluted with medium containing egg yolk+SDS than in that containing egg yolk and LDL. Progressive motility and movement characteristics of the sperm were significantly improved (P<0.05) by adding 35 and/or 75 μg/ml of clove bud extract to semen extenders. Sperm viability and plasma membrane integrity were also higher (P<0.05) in the semen exposed to medium containing egg yolk+SDS and 75 μg of clove buds extract after cryopreservation processes. Higher levels of clove bud extract, however, had adverse effects on all the sperm quality parameters and significantly reduced (P<0.05) the motility, movement parameters, viability, and plasma membrane integrity of ovine sperm. It was concluded that the clove bud extract had an antioxidant potential that makes it useful for addition to semen extenders and that the best results are obtained with a maximum clove bud extract of 75 μg/ml. Moreover, the combination of egg yolk and a detergent was found to improve sperm quality after the cooling and freeze-thawing processes. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of a commercial housing system on egg quality during extended storage.

PubMed

Jones, D R; Karcher, D M; Abdo, Z

2014-05-01

Egg producers in the United States are utilizing a variety of commercial egg production systems to provide consumer choice and meet legislative requirements. Consumer egg grades in the United States were developed for conventional cage production, and it is unclear what effect alternative production systems might have on egg quality during retail and consumer home storage. The current study was undertaken to determine what changes in egg quality characteristics occur during extended cold storage for commercially produced conventional cage, enriched colony cage, and cage-free aviary eggs. During 12 wk of cold storage, egg weight, albumen height, Haugh unit, static compression shell strength, vitelline membrane strength and deformation, yolk index, shell dynamic stiffness, and whole egg total solids were monitored. Overall, aviary and enriched eggs were significantly (P < 0.05) heavier than conventional cage. Albumen height and Haugh unit (P < 0.05) were significantly greater for conventional cage than enriched eggs. Static compression shell strength was greatest (P < 0.05) for enriched eggs compared with aviary. No overall housing system effects for yolk measurements, shell dynamic stiffness, or whole egg total solids were observed. Albumen height, Haugh unit, and yolk quality measurements were all greatest at 0 and lowest at 12 wk of storage (P < 0.05). The rate of quality change among the housing systems for each measured attribute at 4, 6, and 12 wk was determined. Other than differences in the change of egg weight at 4 wk, no significant differences in the rate of quality decline were found among the housing systems. The results of the current study indicate that current US egg quality standards should effectively define quality for commercially produced conventional cage, enriched colony cage, and cage-free aviary eggs.

Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Saitou, Mitinori

2013-08-01

Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

Morphology, development, and evolution of fetal membranes and placentation in squamate reptiles.

PubMed

Blackburn, Daniel G; Flemming, Alexander F

2009-09-15

Current studies on fetal membranes of reptiles are providing insight into three major historical transformations: evolution of the amniote egg, evolution of viviparity, and evolution of placentotrophy. Squamates (lizards and snakes) are ideal for such studies because their fetal membranes sustain embryos in oviparous species and contribute to placentas in viviparous species. Ultrastructure of the fetal membranes in oviparous corn snakes (Pituophis guttatus) shows that the chorioallantois is specialized for gas exchange and the omphalopleure, for water absorption. Transmission and scanning electron microscopic studies of viviparous thamnophine snakes (Thamnophis, Storeria) have revealed morphological specializations for gas exchange and absorption in the intra-uterine environment that represent modifications of features found in oviparous species. Thus, fetal membranes in oviparous species show morphological differentiation for distinct functions that have been recruited and enhanced under viviparous conditions. The ultimate in specialization of fetal membranes is found in viviparous skinks of South America (Mabuya) and Africa (Trachylepis, Eumecia), in which placentotrophy accounts for nearly all of the nutrients for development. Ongoing research on these lizards has revealed morphological specializations of the chorioallantoic placenta through which nutrient transfer is accomplished. In addition, African Trachylepis show an invasive form of implantation, in which uterine epithelium is replaced by invading chorionic cells. Ongoing analysis of these lizards shows how integration of multiple lines of evidence can provide insight into the evolution of developmental and reproductive specializations once thought to be confined to eutherian mammals.

Fertilization Mechanisms in Flowering Plants

PubMed Central

Dresselhaus, Thomas; Sprunck, Stefanie; Wessel, Gary M.

2016-01-01

Compared to the animal kingdom, fertilization is particularly complex in flowering plants (angiosperms). Sperm cells of angiosperms have lost their motility and require transportation as a passive cargo by the pollen tube cell to the egg apparatus (egg cell and accessory synergid cells). Sperm cell release from the pollen tube occurs after intensive communication between the pollen tube cell and the receptive synergid, culminating in the lysis of both interaction partners. Following release of the two sperm cells they interact and fuse with two dimorphic female gametes (egg and central cell) forming the major seed components embryo and endosperm, respectively. This process is known as double fertilization. Here we review the current understanding of the processes of sperm cell reception, gamete interaction, their pre-fertilization activation and fusion as well as the mechanisms plants use to prevent the fusion of egg cells with multiple sperm cells. The role of Ca2+ is highlighted in these various processes and comparisons are drawn between fertilization mechanisms in flowering plants and other eukaryotes including mammals. PMID:26859271

EGG-4 and EGG-5 link events of the oocyte-to-embryo transition with meiotic cell cycle progression in Caenorhabditis elegans

PubMed Central

Parry, Jean M.; Velarde, Nathalie V.; Lefkovith, Ariel J.; Zegarek, Matthew H.; Hang, Julie S.; Ohm, Jonathan; Klancer, Richard; Maruyama, Rika; Druzhinina, Marina K.; Grant, Barth D.; Piano, Fabio; Singson, Andrew

2009-01-01

Summary The molecular underpinnings of the oocyte-to-embryo transition are poorly understood. Here we show that two protein tyrosine phosphatase-like (PTPL) family proteins, EGG-4 and EGG-5, are required for key events of the oocyte-to-embryo transition in Caenorhabditis elegans. The predicted EGG-4 and EGG-5 amino acid sequences are 99% identical and their functions are redundant. In embryos lacking EGG-4 and EGG-5 we observe defects in meiosis, polar body formation, the block to polyspermy, F-actin dynamics, and eggshell deposition. During oogenesis, EGG-4 and EGG-5 assemble at the oocyte cortex with the previously identified regulators or effectors of the oocyte-to-embryo transition EGG-3, CHS-1 and MBK-2 [1, 2]. All of these molecules share a complex interdependence with regards to their dynamics and subcellular localization. Shortly after fertilization, EGG-4 and EGG-5 are required to properly coordinate a redistribution of CHS-1 and EGG-3 away from the cortex during meiotic anaphase I. Therefore EGG-4 and EGG-5 are not only required for critical events of the oocyte-to-embryo transition but also link the dynamics of the regulatory machinery with the advancing cell cycle. PMID:19879147

Stem Cell Information: Glossary

MedlinePlus

... germ cells (those that would become sperm and eggs). Embryonic germ cells are thought to have properties ... the male gamete (sperm) and the female gamete (egg). Fetus - In humans, the developing human from approximately ...

The hen's egg chorioallantoic membrane (HET-CAM) test to predict the ophthalmic irritation potential of a cysteamine-containing gel: Quantification using Photoshop® and ImageJ.

PubMed

McKenzie, Barbara; Kay, Graeme; Matthews, Kerr H; Knott, Rachel M; Cairns, Donald

2015-07-25

A modified hen's egg chorioallantoic membrane (HET-CAM) test has been developed, combining ImageJ analysis with Adobe(®) Photoshop(®). The irritation potential of an ophthalmic medicine can be quantified using this method, by monitoring damage to blood vessels. The evaluation of cysteamine containing hyaluronate gel is reported. The results demonstrated that the novel gel formulation is non-irritant to the ocular tissues, in line with saline solution (negative control). In conclusion, the modification of the established HET-CAM test can quantify the damage to minute blood vessels. These results offer the possibility to formulate cysteamine in an ocular applicable gel formulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of egg shell quality and washing on Salmonella Infantis penetration.

PubMed

Samiullah; Chousalkar, K K; Roberts, J R; Sexton, M; May, D; Kiermeier, A

2013-07-15

The vast majority of eggs in Australia are washed prior to packing to remove dirt and fecal material and to reduce the microbial contamination of the egg shell. The egg contents can be an ideal growth medium for microorganisms which can result in human illness if eggs are stored improperly and eaten raw or undercooked, and it is estimated that egg-related salmonellosis is costing Australia $44 million per year. Egg shell characteristics such as shell thickness, amount of cuticle present, and thickness of individual egg shell layers can affect the ease with which bacteria can penetrate the egg shell and washing could partially or completely remove the cuticle layer. The current study was conducted to investigate the effects of egg washing on cuticle cover and effects of egg shell quality and cuticle cover on Salmonella Infantis penetration of the egg shell. A higher incidence of unfavorable ultrastructural variables of the mammillary layer such as late fusion, type B bodies, type A bodies, poor cap quality, alignment, depression, erosion and cubics were recorded in Salmonella penetrated areas of egg shells. The influence of egg washing on the ability of Salmonella Infantis on the egg shell surface to enter the egg internal contents was also investigated using culture-based agar egg penetration and real-time qPCR based experiments. The results from the current study indicate that washing affected cuticle cover. There were no significant differences in Salmonella Infantis penetration of washed or unwashed eggs. Egg shell translucency may have effects on Salmonella Infantis penetration of the egg shell. The qPCR assay was more sensitive for detection of Salmonella Infantis from egg shell wash and internal contents than traditional microbiological methods. The agar egg and whole egg inoculation experiments indicated that Salmonella Infantis penetrated the egg shells. Egg washing not only can be highly effective at removing Salmonella Infantis from the egg shell surface, but also allows subsequent trans-shell and trans-membrane penetration into the egg. Consequently, it is important to prevent recontamination of the egg after washing. Copyright © 2013 Elsevier B.V. All rights reserved.

Sulfation of Eggshell Proteins by Pipe Defines Dorsal-Ventral Polarity in the Drosophila embryo

PubMed Central

Zhang, Zhenyu; Stevens, Leslie M.; Stein, David

2009-01-01

Summary Drosophila embryonic dorsal-ventral (DV) polarity is controlled by a group of sequentially acting serine proteases located in the fluid-filled perivitelline space between the embryonic membrane and the eggshell, which generate the ligand for the Toll receptor on the ventral side of the embryo [1, 2, 3]. Spatial control of the protease cascade relies on the Pipe sulfotransferase, a fly homologue of vertebrate glycosaminoglycan modifying enzymes [4, 5, 6], which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. The identification of the Pipe enzymatic target has remained a major gap in our understanding of the mechanism controlling the perivitelline protease cascade, and hence embryonic DV patterning. Here we show that the protein Vitelline Membrane-Like (VML) [7] undergoes Pipe-dependent sulfation and, consistent with a role in conveying positional information from the egg chamber to the embryo, becomes incorporated into the eggshell at a position corresponding to the location of the follicle cells from which it was secreted. Although VML influences embryonic DV pattern in a sensitized genetic background, VML is not essential for DV axis formation, suggesting that there is redundancy in the composition of the Pipe enzymatic target. Correspondingly, we find that additional structural components of the vitelline membrane undergo Pipe-dependent sulfation. In identifying the elusive targets of Pipe, this ork points to the vitelline membrane as the source of signals that generate the Drosophila DV axis and provides a framework for understanding the mechanism controlling spatially-specific activation of serine protease activity during embryonic pattern formation. PMID:19540119

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction*

PubMed Central

De La Vega-Beltran, Jose Luis; Sánchez-Cárdenas, Claudia; Krapf, Darío; Hernandez-González, Enrique O.; Wertheimer, Eva; Treviño, Claudia L.; Visconti, Pablo E.; Darszon, Alberto

2012-01-01

Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction. PMID:23095755

Combined inhibition of autophagy and caspases fails to prevent developmental nurse cell death in the Drosophila melanogaster ovary.

PubMed

Peterson, Jeanne S; McCall, Kimberly

2013-01-01

During the final stages of Drosophila melanogaster oogenesis fifteen nurse cells, sister cells to the oocyte, degenerate as part of normal development. This process involves at least two cell death mechanisms, caspase-dependent cell death and autophagy, as indicated by apoptosis and autophagy markers. In addition, mutations affecting either caspases or autophagy partially reduce nurse cell removal, leaving behind end-stage egg chambers with persisting nurse cell nuclei. To determine whether apoptosis and autophagy work in parallel to degrade and remove these cells as is the case with salivary glands during pupariation, we generated mutants doubly affecting caspases and autophagy. We found no significant increase in either the number of late stage egg chambers containing persisting nuclei or in the number of persisting nuclei per egg chamber in the double mutants compared to single mutants. These findings suggest that there is another cell death mechanism functioning in the ovary to remove all nurse cell remnants from late stage egg chambers.

Calcium-responsive contractility during fertilization in sea urchin eggs.

PubMed

Stack, Christianna; Lucero, Amy J; Shuster, Charles B

2006-04-01

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins, there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both before and after fertilization and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed by and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. (c) 2006 Wiley-Liss, Inc.

Calcium-Responsive Contractility During Fertilization in Sea Urchin Eggs

PubMed Central

Stack, Christianna; Lucero, Amy J.; Shuster, Charles B.

2008-01-01

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both prior to- and following fertilization, and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed- and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs, but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. PMID:16470603

Antiproliferative Activity of Egg Yolk Peptides in Human Colon Cancer Cells.

PubMed

Yousr, Marwa N; Aloqbi, Akram A; Omar, Ulfat M; Howell, Nazlin K

2017-01-01

Egg yolk peptides were successfully prepared from egg yolk protein by-products after lecithin extraction. Defatted egg yolk protein was hydrolyzed with pepsin and pancreatin and purified by gel filtration to produce egg yolk gel filtration fraction (EYGF-33) with antiproliferative activity. The highlight of this study was that the peptide EYGF-33 (1.0 mg/ml) significantly inhibits cell viability of colon cancer cells (Caco-2) with no inhibitory effects on the viability of human colon epithelial normal cells (HCEC) after 48 h. Reduced cell viability can be explained by cell cycle arrest in the S-phase in which DNA replication normally takes place. EYGF-33 significantly enhanced the production of superoxide anions in the mitochondria of Caco-2 cells; this could activate a mitochondrial apoptotic pathway leading to typical Poly Adenosine diphosphate-ribose polymerase (PARP) cleavage as observed in the Western blot result. The induction of apoptotic cell death by EYGF-33 was supported by the externalization of phosphatidylserine (PS). However, further elucidation of the mechanism of EYGF-33-mediated apoptosis would provide further support for its use as a potential therapeutic and chemopreventive agent.

Novel CD44 receptor targeting multifunctional "nano-eggs" based on double pH-sensitive nanoparticles for co-delivery of curcumin and paclitaxel to cancer cells and cancer stem cells

NASA Astrophysics Data System (ADS)

Chen, Daquan; Wang, Guohua; Song, Weiguo; Zhang, Qiang

2015-10-01

Most anticancer drugs cannot kill cancer stem cells (CSCs) effectively, which lead to the failure of anticancer chemotherapy, such as relapse and metastasis. In this study, we prepared a multifunctional oligosaccharides of hyaluronan (oHA) conjugates, oHA-histidine-menthone 1,2-glycerol ketal (oHM). The oHM conjugates possess pH-sensitive menthone 1,2-glycerol ketal (MGK) as hydrophobic moieties and oHA as the target of CD44 receptor. Anticancer drugs, curcumin(Cur) and paclitaxel(PTX), were loaded into oHM micelles via self-assembly. Then, oHM micelles were mineralized through controlled deposition of inorganic calcium and phosphate ions on the nanoparticular shell via a sequential addition method to fabricate the "nano-eggs." The formed nano-eggs had a smaller size (120.6 ± 4.5 nm) than oHM micelles (158.6 ± 6.4 nm), indicating that mineralization made the appearance of compact nanoparticles. Interestingly, when the nano-eggs were put into the acidic conditions (pH 6.5), their outer shell(inorganic minerals) will be destroyed with the larger size, while the "nano-eggs" were stable under pH 7.4. For both nano-eggs and oHM micelles, the Cur and PTX were released in a sustained manner depending on the pH of the solution. However, the nano-eggs showed much lower released than the oHM micelles due to the dissolution of the inorganic minerals and pH-sensitive ketal at mildly acidic environments (pH 6.5). In vivo study, the nano-eggs could get to the tumor site more effectively than oHM micelles. CSCs were sorted by a side population assay from MDA-MB-231 breast cancer cell lines over-expressing CD44 receptors. Antitumor activity was also evaluated on MDA-MB-231 xenografts in nude mice. The antitumor efficacy indicated that nano-eggs with co-delivery of Cur and PTX produced the strongest antitumor efficacy, and nano-eggs showed strong activity against cancer stem cells. These double pH-sensitive nano-eggs may provide a promising strategy for drug delivery to both cancer cells and cancer stem cells.

Calcium and actin in the saga of awakening oocytes.

PubMed

Santella, Luigia; Limatola, Nunzia; Chun, Jong T

2015-04-24

The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggs at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm-egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent "excitable media" that quickly respond to the stimulus with the Ca(2+) swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca(2+) signals and in the control of monospermic fertilization. Copyright © 2015 Elsevier Inc. All rights reserved.

Isotonicity of liver and of kidney tissue in solutions of electrolytes.

PubMed

OPIE, E L

1959-07-01

Solutions of a wide variety of electrolytes, isotonic with liver or with kidney tissue, have approximately the same osmotic pressure as solutions of sodium chloride isotonic with tissues of the two organs respectively; that is, with solutions approximately twice as concentrated as the sodium chloride of mammalian blood plasma. The molar concentration of various electrolytes isotonic with liver or with kidney tissue immediately after its removal from the body is determined by the molecular weight, valency, and ion-dissociation of these electrolytes in accordance with the well known conditions of osmosis. The plasma membranes of liver and of kidney cells are imperfectly semipermeable to electrolytes, and those that enter the cell, though retarded in so doing, bring about injury which increases permeability to water. The osmotic activity of cells of mammalian liver and kidney immediately after their removal from the body resembles that of plant cells, egg cells of marine invertebrates, and mammalian red blood corpuscles and presumably represents a basic property of living cells by which osmotic pressure may be adjusted to functional need.

ISOTONICITY OF LIVER AND OF KIDNEY TISSUE IN SOLUTIONS OF ELECTROLYTES

PubMed Central

Opie, Eugene L.

1959-01-01

Solutions of a wide variety of electrolytes, isotonic with liver or with kidney tissue, have approximately the same osmotic pressure as solutions of sodium chloride isotonic with tissues of the two organs respectively; that is, with solutions approximately twice as concentrated as the sodium chloride of mammalian blood plasma. The molar concentration of various electrolytes isotonic with liver or with kidney tissue immediately after its removal from the body is determined by the molecular weight, valency, and ion-dissociation of these electrolytes in accordance with the well known conditions of osmosis. The plasma membranes of liver and of kidney cells are imperfectly semipermeable to electrolytes, and those that enter the cell, though retarded in so doing, bring about injury which increases permeability to water. The osmotic activity of cells of mammalian liver and kidney immediately after their removal from the body resembles that of plant cells, egg cells of marine invertebrates, and mammalian red blood corpuscles and presumably represents a basic property of living cells by which osmotic pressure may be adjusted to functional need. PMID:13664872

Emetine inhibits replication of RNA and DNA viruses without generating drug-resistant virus variants.

PubMed

Khandelwal, Nitin; Chander, Yogesh; Rawat, Krishan Dutt; Riyesh, Thachamvally; Nishanth, Chikkahonnaiah; Sharma, Shalini; Jindal, Naresh; Tripathi, Bhupendra N; Barua, Sanjay; Kumar, Naveen

2017-08-01

At a noncytotoxic concentration, emetine was found to inhibit replication of DNA viruses [buffalopoxvirus (BPXV) and bovine herpesvirus 1 (BHV-1)] as well as RNA viruses [peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)]. Using the time-of-addition and virus step-specific assays, we showed that emetine treatment resulted in reduced synthesis of viral RNA (PPRV and NDV) and DNA (BPXV and BHV-1) as well as inhibiting viral entry (NDV and BHV-1). In addition, emetine treatment also resulted in decreased synthesis of viral proteins. In a cell free endogenous viral polymerase assay, emetine was found to significantly inhibit replication of NDV, but not BPXV genome, suggesting that besides directly inhibiting specific viral polymerases, emetine may also target other factors essentially required for efficient replication of the viral genome. Moreover, emetine was found to significantly inhibit BPXV-induced pock lesions on chorioallantoic membrane (CAM) along with associated mortality of embryonated chicken eggs. At a lethal dose 50 (LD 50 ) of 126.49 ng/egg and at an effective concentration 50 (EC 50 ) of 3.03 ng/egg, the therapeutic index of the emetine against BPXV was determined to be 41.74. Emetine was also found to significantly delay NDV-induced mortality in chicken embryos associated with reduced viral titers. Further, emetine-resistant mutants were not observed upon long-term (P = 25) sequential passage of BPXV and NDV in cell culture. Collectively, we have extended the effective antiviral activity of emetine against diverse groups of DNA and RNA viruses and propose that emetine could provide significant therapeutic value against some of these viruses without inducing an antiviral drug-resistant phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

The discrimination of fish egg quality and viability by using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Ishigaki, Mika; Sato, Hidetoshi

2014-03-01

Sexual reproductive body can be produced from a fertilized ovum. Once the ovum is fertilized with sperm, it runs through the cell division, differentiates to all kinds of cells, and goes to make a complete body. However, not all of them are viable and some of them stop to ontogenesis showing the developmental abnormality. In order to discriminate the egg quality, we apply Raman spectroscopy for fish egg. After the measurement, these Raman data are checked up with the information about the eggs can survive or not, and we examine what factors are important in egg components to distinguish between "good quality" and "not good quality". We present the results of assessment of egg quality, and investigate whether Raman spectroscopy can be used to a discriminate of egg quality.

Cell lineages of the embryo of the nematode Caenorhabditis elegans.

PubMed

Deppe, U; Schierenberg, E; Cole, T; Krieg, C; Schmitt, D; Yoder, B; von Ehrenstein, G

1978-01-01

Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock--i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution video recording.

Characterization of interactions of eggPC lipid structures with different biomolecules.

PubMed

Corrales Chahar, F; Díaz, S B; Ben Altabef, A; Gervasi, C; Alvarez, P E

2018-01-01

In this paper we study the interactions of two biomolecules (ascorbic acid and Annonacin) with a bilayer lipid membrane. Egg yolk phosphatidylcholine (eggPC) liposomes (in crystalline liquid state) were prepared in solutions of ascorbic acid (AA) at different concentration levels. On the other hand, liposomes were doped with Annonacin (Ann), a mono-tetrahydrofuran acetogenin (ACG), which is an effective citotoxic substance. While AA pharmacologic effect and action mechanisms are widely known, those of Ann's are only very recently being studied. Both Fourier Transformed Infrared (FTIR) and Raman spectroscopic techniques were used to study the participation of the main functional groups of the lipid bilayer involved in the membrane-solution interaction. The obtained spectra were comparatively analyzed, studying the spectral bands corresponding to both the hydrophobic and the hydrophilic regions in the lipid bilayer. Electrochemical experiments namely; impedance spectroscopy (EIS) and cyclic voltamperometry (CV) were used as the main characterization techniques to analyse stability and structural changes of a model system of supported EggPC bilayer in connection with its interactions with AA and Ann. At high molar ratios of AA, there is dehydration in both populations of the carbonyl group of the polar head of the lipid. On the other hand, Ann promotes the formation of hydrogen bonds with the carbonyl groups. No interaction between AA and phosphate groups is observed at low and intermediate molar ratios. Ann is expected to be able to induce the dehydration of the phosphate groups without the subsequent formation of H bonds with them. According to the electrochemical analysis, the interaction of AA with the supported lipid membrane does not alter its dielectric properties. This fact can be related to the conservation of structured water of the phosphate groups in the polar heads of the lipid. On the other hand, the incorporation of Ann into the lipid membrane generates an increase in the number of defects while changes the dielectric constant. This, in turn, can be associated with the induced dehydration of the phosphate groups. Copyright © 2017 Elsevier B.V. All rights reserved.

Distinct roles for multiple Src family kinases at fertilization.

PubMed

O'Neill, Forest J; Gillett, Jessica; Foltz, Kathy R

2004-12-01

Egg activation at fertilization requires the release of Ca2+ from the endoplasmic reticulum of the egg. Recent evidence indicates that Src family kinases (SFKs) function in the signaling pathway that initiates this Ca2+ release in the eggs of many deuterostomes. We have identified three SFKs expressed in starfish (Asterina miniata) eggs, designated AmSFK1, AmSFK2 and AmSFK3. Antibodies made against the unique domains of each AmSFK protein revealed that all three are expressed in eggs and localized primarily to the membrane fraction. Both AmSFK1 and AmSFK3 (but not AmSFK2) are necessary for egg activation, as determined by injection of starfish oocytes with dominant-interfering Src homology 2 (SH2) domains, which specifically delay and reduce the initial release of Ca2+ at fertilization. AmSFK3 exhibits a very rapid and transient kinase activity in response to fertilization, peaking at 30 seconds post sperm addition. AmSFK1 kinase activity also increases transiently at fertilization, but peaks later, at 2 minutes. These results indicate that there are multiple SFKs present in starfish eggs with distinct, perhaps sequential, signaling roles.

Salmonella enterica Serovar Enteritidis Antimicrobial Peptide Resistance Genes Aid in Defense against Chicken Innate Immunity, Fecal Shedding, and Egg Deposition

PubMed Central

McKelvey, Jessica A.; Yang, Ming; Jiang, Yanhua

2014-01-01

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major etiologic agent of nontyphoid salmonellosis in the United States. S. Enteritidis persistently and silently colonizes the intestinal and reproductive tract of laying hens, resulting in contaminated poultry products. The consumption of contaminated poultry products has been identified as a significant risk factor for human salmonellosis. To understand the mechanisms S. Enteritidis utilizes to colonize and persist in laying hens, we used selective capture of transcribed sequences to identify genes overexpressed in the HD11 chicken macrophage cell line and in primary chicken oviduct epithelial cells. From the 15 genes found to be overexpressed in both cell types, we characterized the antimicrobial peptide resistance (AMPR) genes, virK and ybjX, in vitro and in vivo. In vitro, AMPR genes were required for natural morphology, motility, secretion, defense against detergents such as EDTA and bile salts, and resistance to antimicrobial peptides polymyxin B and avian β-defensins. From this, we inferred the AMPR genes play a role in outer membrane stability and/or modulation. In the intestinal tract, AMPR genes were involved in early intestinal colonization and fecal shedding. In the reproductive tract, virK was required in early colonization whereas a deletion of ybjX caused prolonged ovary colonization and egg deposition. Data from the present study indicate that AMPR genes are differentially utilized in various host environments, which may ultimately assist S. Enteritidis in persistent and silent colonization of chickens. PMID:25267840

Lipid composition of positively buoyant eggs of reef building corals

NASA Astrophysics Data System (ADS)

Arai, Iakayuki; Kato, Misako; Heyward, Andrew; Ikeda, Yutaka; Iizuka, Tokio; Maruyama, Tadashi

1993-07-01

Lipid composition of the eggs of three reef building corals, Acropora millepora, A. tenuis and Montipora digitata, were determined. Sixty to 70% of the egg dry weight was lipid, which consisted of wax esters (69.5 81.8%), triacylglycerols (1.1 8.4%) and polar lipids c/mainly phospholipids (11.9 13.2%). Montipora digitata also contained some polar lipids typical of the thylakoid membrane in chloroplasts, probably due to the presence of symbiotic zooxanthellae in the eggs. The wax esters appeared to be the major contributor to positive buoyancy of the eggs, and specific gravity of wax esters in A. millepora was estimated to be 0.92. Among the fatty acids of the wax esters, 34.9 51.3% was hexadecanoic acid (16:0) while the major fatty acids in polar lipids were octadecenoic acid (18:1), hexadecanoic acid (16:0), eicosapentaenoic acid (20:5) and eicosatetraenoic acid (20:4). The wax ester appears to be the main component of the 4.5 6.0 μm diameter lipid droplets which fill most of the central mass of the coral eggs.

Rearing and colonization of Lutzomyia evansi (Diptera: Psychodidae), a vector of visceral leishmaniasis in Colombia.

PubMed

Montoya-Lerma, J; Cadena-Peña, H; Jaramillo-Salazar, C

1998-01-01

The sandfly Lutzomyia evansi from a focus of visceral leishmaniasis in northern Columbia was reared and maintained under laboratory conditions for five generations. The average time for total development was 41.9 days (range = 35.1-49.6) at 25 degrees C and 89-95% of relative humidity. The mean number of eggs laid was lower in laboratory bred females either in pots (13.2 eggs/female) or vials (29.9 eggs/female) than in wild caught females (33.4 eggs/female). Immature mortality, mainly due to fungal and mite contamination, was higher during the first two instars than in the remaining immature stages. Adults were robust and healthy although difficult to feed on hamster or chick skin membrane. In summary, Lu. evansi is a colonizable species but requires specific conditions.

Effects of water hardness on size and hatching success of silver carp eggs

USGS Publications Warehouse

Rach, Jeff J.; Sass, Greg G.; Luoma, James A.; Gaikowski, Mark P.

2010-01-01

Eggs of silver carp Hypophthalmichthys molitrix absorb water after release from the female, causing them to become turgid and to increase substantially in size. The volume of water that diffuses within an egg is most likely determined by (1) the difference in ionic concentration between the egg and the water that surrounds it and (2) the elasticity of the egg membrane. Prior observations suggest that silver carp eggs may swell and burst in soft waters. If water hardness affects silver carp reproductive success in nonnative ecosystems, this abiotic factor could limit silver carp distribution or abundance. In this study, we tested the effect of water hardness on silver carp egg enlargement and hatching success. Groups of newly fertilized silver carp eggs were placed in water at one of five nominal water hardness levels (50, 100, 150, 200, or 250 mg/L as CaCO3) for 1 h to harden (absorb water after fertilization). Egg groups were then placed in separate incubation vessels housed in two recirculation systems that were supplied with either soft (50 mg/L as CaCO3) or hard (250 mg/L as CaCO3) water to evaluate hatching success. Tests were terminated within 24 h after viable eggs had hatched. Eggs that were initially placed in 50-mg/L water to harden were larger (i.e., swelled more) and had a greater probability of hatch than eggs hardened in other water hardness levels. Unlike the effect of water hardness during egg hardening, the water hardness during incubation appeared to have no effect on egg hatching success. Our research suggests that water hardness may not be a limiting factor in the reproduction, recruitment, and range expansion of silver carp in North America.

Detection, identification, and differentiation of sheep pox virus and goat pox virus from clinical cases in Giza Governorate, Egypt

PubMed Central

Mahmoud, M. A.; Khafagi, M. H.

2016-01-01

Aim: To isolate, identify, and differentiate Capripoxviruses (CaPV) (sheep pox virus and goat pox virus) infections by egg inoculation, transmission electron microscopy (TEM), and 30 kDa RNA polymerase subunit gene-based polymerase chain reaction (PCR) (RPO30) in clinically affected animals in Hawamdia township of Giza Governorate, Egypt. Materials and Methods: A total of 37 scab samples were collected from clinically suspected field cases of sheep pox and goat pox. These samples were collected during (2014-2015) during different outbreaks of sheep pox and goat pox from Hawamdia township of Giza Governorate, Egypt. The samples were subjected to egg inoculation, TEM, and (RPO30) gene-based PCR. By using the egg inoculation: Previously prepared 37 scab samples (n=23 sheep and n=14 goats) were inoculated on the chorioallantoic membrane of specific pathogen free (SPF) embryonated chicken eggs (12 days old age). In the presence of the suitable percentage of humidity and candling, the inoculated eggs were incubated at 37°C. By using the TEM: Samples showed positive pock lesions on the chorioallantoic membranes, were fixed in glutaraldehyde, then processed and sectioned for TEM. Using the (RPO30) gene-based PCR assay, 30 of positive samples after egg inoculation (n=19 sheep and n=11 goats) were screened. Results: Using the egg inoculation, a characteristic pock lesions for poxviruses were seen in 30/37 (n=19 sheep and n=11 goats) (81.08%). Using the TEM, examination of the positive samples after egg inoculation revealed positive result in 23/30 (n=15 sheep and n=8 goats) (76.66%). The positive results represented by the presence of negatively stained oval-shape virus particles. Using the (RPO30) gene-based PCR assay, out of 30 total of positive samples after egg inoculation (n=19 sheep and n=11 goats) were screened, 27 (90%) samples (n=17 sheep and n=10 goats) were positive. The given band sizes of sheep and goats were 172 and 152 bp, respectively. Conclusion: PCR assay depended on RPO30 gene can be used lonely for the detection, identification, and differentiation of CaPVs. RPO30 gene-based PCR assay in combination with gene sequencing helps in molecular epidemiological studies of CaPV infection. PMID:28096619

Detection, identification, and differentiation of sheep pox virus and goat pox virus from clinical cases in Giza Governorate, Egypt.

PubMed

Mahmoud, M A; Khafagi, M H

2016-12-01

To isolate, identify, and differentiate Capripoxviruses (CaPV) (sheep pox virus and goat pox virus) infections by egg inoculation, transmission electron microscopy (TEM), and 30 kDa RNA polymerase subunit gene-based polymerase chain reaction (PCR) (RPO30) in clinically affected animals in Hawamdia township of Giza Governorate, Egypt. A total of 37 scab samples were collected from clinically suspected field cases of sheep pox and goat pox. These samples were collected during (2014-2015) during different outbreaks of sheep pox and goat pox from Hawamdia township of Giza Governorate, Egypt. The samples were subjected to egg inoculation, TEM, and (RPO30) gene-based PCR. By using the egg inoculation: Previously prepared 37 scab samples (n=23 sheep and n=14 goats) were inoculated on the chorioallantoic membrane of specific pathogen free (SPF) embryonated chicken eggs (12 days old age). In the presence of the suitable percentage of humidity and candling, the inoculated eggs were incubated at 37°C. By using the TEM: Samples showed positive pock lesions on the chorioallantoic membranes, were fixed in glutaraldehyde, then processed and sectioned for TEM. Using the (RPO30) gene-based PCR assay, 30 of positive samples after egg inoculation (n=19 sheep and n=11 goats) were screened. Using the egg inoculation, a characteristic pock lesions for poxviruses were seen in 30/37 (n=19 sheep and n=11 goats) (81.08%). Using the TEM, examination of the positive samples after egg inoculation revealed positive result in 23/30 (n=15 sheep and n=8 goats) (76.66%). The positive results represented by the presence of negatively stained oval-shape virus particles. Using the (RPO30) gene-based PCR assay, out of 30 total of positive samples after egg inoculation (n=19 sheep and n=11 goats) were screened, 27 (90%) samples (n=17 sheep and n=10 goats) were positive. The given band sizes of sheep and goats were 172 and 152 bp, respectively. PCR assay depended on RPO30 gene can be used lonely for the detection, identification, and differentiation of CaPVs. RPO30 gene-based PCR assay in combination with gene sequencing helps in molecular epidemiological studies of CaPV infection.

Methods for rearing Mesostoma ehrenbergii in the laboratory for cell biology experiments, including identification of factors that influence production of different egg types.

PubMed

Hoang, Carina; Ferraro-Gideon, Jessica; Gauthier, Kimberley; Forer, Arthur

2013-10-01

Mesostoma ehrenbergii spermatocytes are uniquely useful to study various aspects of cell division. Their chromosomes are large in size and few in number, with only three bivalent and four univalent chromosomes. During prometaphase, bipolar bivalents oscillate regularly to and from the poles for 1-2 hours. The univalents remain at the poles but occasionally move from one pole to the other. In addition, a precocious cleavage furrow forms during prometaphase and remains partially constricted until anaphase. Attempts to rear these animals indefinitely in laboratory conditions, however, have been mostly unsuccessful because of their reproductive strategy. M. ehrenbergii are hermaphroditic flatworms that can produce viviparous offspring (termed S eggs) and/or diapausing eggs (termed D eggs) and they follow either one of two reproductive patterns: (1) they first form S eggs and following the delivery of these eggs produce D eggs, or (2) they only produce D eggs. When only D eggs are formed, which is common under laboratory conditions, the stocks die out until the D eggs hatch, which is irregular and creates unpredictable wait times. Consequently, in order to maintain M. ehrenbergii stocks to study their spermatocytes, we examined various factors that might influence egg-type production. Feeding them daily and keeping them at 25°C favours S egg production. Currently, our cultures have reached the 53rd generation. We herein describe our rearing and dissection methods, and some experiments which led to our present rearing methods. © 2013 International Federation for Cell Biology.

A simplified method for correcting contaminant concentrations in eggs for moisture loss.

USGS Publications Warehouse

Heinz, Gary H.; Stebbins, Katherine R.; Klimstra, Jon D.; Hoffman, David J.

2009-01-01

We developed a simplified and highly accurate method for correcting contaminant concentrations in eggs for the moisture that is lost from an egg during incubation. To make the correction, one injects water into the air cell of the egg until overflowing. The amount of water injected corrects almost perfectly for the amount of water lost during incubation or when an egg is left in the nest and dehydrates and deteriorates over time. To validate the new method we weighed freshly laid chicken (Gallus gallus) eggs and then incubated sets of fertile and dead eggs for either 12 or 19 d. We then injected water into the air cells of these eggs and verified that the weights after water injection were almost identical to the weights of the eggs when they were fresh. The advantages of the new method are its speed, accuracy, and simplicity: It does not require the calculation of a correction factor that has to be applied to each contaminant residue.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandler, D.P.; Welt, M.; Leung, F.C.

An efficient one-step injection technique for gene insertion into fertilized rainbow trout (Oncorhynchus mykiss) eggs is described, and basic parameters affecting egg survival are reported. Freshly fertilized rainbow trout eggs were injected in the perivitelline space with a recombinant mouse metallothionein-genomic bovine growth hormone (bGH) DNA construct using a 30-gauge hypodermic needle and a standard microinjection system. Relative to control, site of injection and DNA concentration did not affect the egg survival, but injections later than 3--4 hours post fertilization were detrimental. The injection technique permitted treatment of 100 eggs/hr with survivals up to 100%, resulting in a 4% DNAmore » uptake rate as indicated by DNA dot blot analysis. Positive dot blot results also indicated that the injected DNA is able to cross the vitelline membrane and persist for 50--60 days post hatching, obviating the need for direct injection into the germinal disk. Results are consistent with previous transgenic fish work, underscoring the usefulness of the technique for generating transgenic trout and salmonids. 24 refs., 6 figs., 3 tabs.« less

[Application of asymmetrical flow field-flow fractionation for size characterization of low density lipoprotein in egg yolk plasma].

PubMed

Zhang, Wenhui; Cai, Chunxue; Wang, Jing; Mao, Zhen; Li, Yueqiu; Ding, Liang; Shen, Shigang; Dou, Haiyang

2017-08-08

Home-made asymmetrical flow field-flow fractionation (AF4) system, online coupled with ultraviolet/visible (UV/Vis) detector was employed for the separation and size characterization of low density lipoprotein (LDL) in egg yolk plasma. At close to natural condition of egg yolk, the effects of cross flow rate, sample loading, and type of membrane on the size distribution of LDL were investigated. Under the optimal operation conditions, AF4-UV/Vis provides the size distribution of LDL. Moreover, the precision of AF4-UV/Vis method proposed in this work for the analysis of LDL in egg yolk plasma was evaluated. The intra-day precisions were 1.3% and 1.9% ( n =7) and the inter-day precisions were 2.4% and 2.3% ( n =7) for the elution peak height and elution peak area of LDL, respectively. Results reveal that AF4-UV/Vis is a useful tool for the separation and size characterization of LDL in egg yolk plasma.

First paleoparasitological record of acanthocephalan eggs from Northwestern Patagonia (Late Holocene, Argentina).

PubMed

Beltrame, María Ornela; Fernández, Fernando Julián; Sardella, Norma Haydeé

2015-06-01

Eggs representative of an acanthocephalan were found in an ancient fragment of raptor pellet, probably belonged to the barn owl, Tyto alba, from the archeological site named "Epullán Chica cave." This site is a cave located at the southern of Neuquén Province, Patagonia, Argentina. The fragment of pellet was found in a layer with charcoals dated at 1980±80 years B.P. A total of 56 eggs were found. Eggs were brown colored and thick-shelled, and presented four membranes, the outer lightly sculpted. The embryos presented hooks in one extremity. Measurements ranged from 87.5 to 107.5μm long and 50 to 57.5μm wide. Eggs were very well-preserved, and were identified as belonged to Class Archiacanthocephala, Order Oligacanthorhynchida, Family Oligacanthorhynchidae, probably Macracanthorhynchus Travassos, 1917, or an unidentified species. This is the first report of small mammal acanthocephalans from ancient material worldwide. Copyright © 2015 Elsevier B.V. All rights reserved.

ELECTRIC IMPEDANCE OF ASTERIAS EGGS

PubMed Central

Cole, Kenneth S.; Cole, Robert H.

1936-01-01

The alternating current resistance and capacity of suspensions of unfertilized eggs of Asterias forbesi have been measured at frequencies from one thousand to sixteen million cycles per second. The plasma membrane of the egg has a static capacity of 1.10µf/cm.2 which is practically independent of frequency. The suspensions show a capacity dependent on frequency at low frequencies which may be attributable to surface conductance. The specific resistance of the cytoplasm is between 136 and 225 ohm cm. (4 to 7 times sea water), indicating a relatively high concentration of non-electrolytes. At frequencies above one million cycles there is definite evidence of another element of which the nucleus is presumably a part. PMID:19872951

Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol.

PubMed

Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

2012-03-01

The present study determined the effect of different types of sugars (lactose, fructose, glucose and sorbitol) used in egg yolk-based extender on the post-thawed boar semen quality. Twenty-two ejaculates from 6 fertility-proven Yorkshire boars were cryopreserved by liquid nitrogen vapor method. Sperm motility, viability, acrosome integrity and intact functional plasma membrane were determined at 0, 2 and 4 hr after thawing. It was found that the lactose-based extender resulted in a higher percentage of post-thawed sperm motility, viability, intact acrosome and functional plasma membrane than sorbitol-based extender (P<0.05) and fructose-based extender yielded a higher post-thawed sperm motility and viability than sorbitol-based extender (P<0.05). It could be concluded that sorbitol was not an effective sugar for the cryopreservation in boar semen.

The reaction time of organ-forming substance in goldfish EGG and its relationship with mesodermal formation

NASA Astrophysics Data System (ADS)

Zhang, Shicui; Wu, Shangqin

1988-12-01

Fertilized goldfish eggs were dechorionated with a pair of forceps and were cut off along or a little above the equator into animal and vegetative parts at desired stages with a glass needle or ligated into two connected fragments before cleavage with baby hair loop. Some of the ligated eggs were detached by further fastening soon after ligation, and some released later at different stages (2-cell, 16-cell, 128-cell, 512-cell, mid-blastula) to let the organ-forming substance (OFS) enter the blastoderm. The cholinesterase (ChE) in the resulting embryos was assayed. The results are as follows. 1. All the 142 embryos developed from the animal hemispheres cut off or ligated off before cleavage gave rise to hyperblastula in which no ChE activity was observed. 2. All 50 embryos obtained from animal halves isolated at the 8-cell stage produced ChE. 3. Embryos developed from the eggs released before the 512-cell stage formed ChE, but the later the releasing of the hair knots, the smaller the number of ChE-producing embryos. 4. After the 512-cell stage (excluding this stage), neither ChE nor tissue differentiation occurred in the embryos developed from the unfastened eggs though their OFS flow was set free. Since ChE is thought to be a muscle-specific enzyme in the early developmental stage, it is concluded that the OFS in goldfish egg appears to be indispensable for the establishment of the mesoderm.

Cellular biophysics during freezing of rat and mouse sperm predicts post-thaw motility.

PubMed

Hagiwara, Mie; Choi, Jeung Hwan; Devireddy, Ramachandra V; Roberts, Kenneth P; Wolkers, Willem F; Makhlouf, Antoine; Bischof, John C

2009-10-01

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5 degrees C/min and 20 degrees C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters L(pg) and E(Lp). A "combined fit" (to 5 degrees C/min and 20 degrees C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded L(pg) = 0.007 microm min(-1) atm(-1) and E(Lp) = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded L(pg) = 0.005 microm min(-1) atm(-1) and E(Lp) = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%-15% normalized cell water at -30 degrees C during freezing in cryopreservation media. These predicted rates range from 53 degrees C/min to 70 degrees C/min and from 28 degrees C/min to 36 degrees C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50 degrees C/min and 80 degrees C/min for rat and at 20 degrees C/min with a sharp drop at 50 degrees C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.

Launch Conditions Might Affect the Formation of Blood Vessel in the Quail Chorioallantoic Membrane

NASA Technical Reports Server (NTRS)

Henry, M. K.; Unsworth, B. R.; Sychev, B. R.; Guryeva, T. S.; Dadasheva, O. A.; Piert, S. J.; Lagel, K. E.; Dubrovin, L. C.; Jahns, G. C.; Boda, K.;

Effectiveness of cross-flow microfiltration for removal of microorganisms associated with unpasteurized liquid egg white from process plant.

PubMed

Mukhopadhyay, S; Tomasula, P M; Van Hekken, D; Luchansky, J B; Call, J E; Porto-Fett, A

2009-08-01

Thermal preservation is used by the egg industry to ensure the microbiological safety of liquid egg white (LEW); however, it does not eliminate all microorganisms and impairs some of the delicate functional properties of LEW. In this study, a pilot-scale cross-flow microfiltration (MF) process was designed to remove the natural microflora present in commercial LEW, obtained from a local egg-breaking plant, while maintaining the nutritional and functional properties of the LEW. LEW, containing approximately 10(6 +/- 1.7) colony forming units (CFU) per milliliter of total aerobic bacteria, was microfiltered using a ceramic membrane with a nominal pore size of 1.4 microm, at a cross-flow velocity of 6 m/s. To facilitate MF, LEW was screened, homogenized, and then diluted (1 : 2, w/w) with distilled water containing 0.5% sodium chloride. Homogenized LEW was found to have a threefold lower viscosity than unhomogenized LEW. Influence of MF temperature (25 and 40 degrees C) and pH (6 and 9) on permeate flux, transmission of egg white nutrients across the membrane, and microbial removal efficiency were evaluated. The pH had a significantly greater influence on permeate flux than temperature. Permeate flux increased by almost 148% when pH of LEW was adjusted from pH 9 to pH 6 at 40 degrees C. Influence of temperature on permeate flux, at a constant pH, however, was found to be inconclusive. Microbial removal efficiency was at least 5 log(10) CFU/mL. Total protein and SDS-PAGE analysis indicated that this MF process did not alter the protein composition of the permeate, compared to that of the feed LEW, and that the foaming properties of LEW were retained in the postfiltered samples.

Effect of rainbow trout (Oncorhynchus mykiss) seminal plasma on the post-thaw quality of ram semen cryopreserved in a soybean lecithin-based or egg yolk-based extender.

PubMed

Ustuner, Burcu; Alcay, Selim; Toker, M Berk; Nur, Zekariya; Gokce, Elif; Sonat, Fusun Ak; Gul, Zulfiye; Duman, Muhammed; Ceniz, Cafer; Uslu, Aydın; Sagirkaya, Hakan; Soylu, M Kemal

2016-01-01

The aim of the current study was to evaluate the effects of different concentrations of rainbow trout seminal plasma (RTSP) (0.1%, 1% and 10%) in extenders containing either egg yolk or lecithin for use in Awassi ram semen cryopreservation. Pooled sperm were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in egg yolk or lecithin extender containing no RTSP, 0.1%, 1% or 10% RTSP (v/v). Semen samples were assessed for sperm motility, plasma membrane integrity [hypoosmotic swelling test (HOST) and Hoechst 33258] and defective acrosomes [FITC-conjugated Pisum sativum agglutinin (PSA-FITC)] at the following five time points: after dilution with extender A; after equilibration; and post-thaw at 0h, 3h and 5h. Malondialdehyde (MDA) was examined only after thawing. Freezing and thawing procedures (dilution, equilibration and post-thaw incubation at 0h, 3h and 5h) negatively affected the motility (P<0.001) and acrosome integrity (P<0.001). Additionally, freezing and thawing negatively affected the plasma membrane integrity, as determined by the HOST and Hoechst 33258 (P<0.001). The extender group affected the motility (P<0.001) and the HOST results (P<0.001). Levels of MDA in the egg yolk extender with 1% RTSP group were significantly lower than in the lecithin control group (P<0.05). In conclusion, the egg yolk extender groups that were supplemented with 10% and 1% RTSP provided greater cryoprotective effects for semen survivability during 5h incubation than the other extender groups. Copyright © 2015 Elsevier B.V. All rights reserved.

Incubation temperature and hemoglobin dielectric of chicken embryos incubated under the influence of electric field.

PubMed

Shafey, T M; Al-Batshan, H A; Shalaby, M I; Ghannam, M M

2006-01-01

Eggs from a layer-type breeder flock (Baladi, King Saud University) between 61 and 63 weeks of age were used in 3 trials to study the effects of electric field (EF) during incubation on the internal temperature of incubation, and eggs and hemoglobin (Hb) dielectric of chicken embryos at 18 days of age. Dielectric relative permittivity (epsilon') and conductivity (sigma) of Hb were examined in the range of frequency from 20 to 100 kHz. The values of dielectric increment (Deltaepsilon') and the relaxation times (tau) of Hb molecules were calculated. The internal temperature of eggs was measured in empty (following the removal of egg contents) and fertilized eggs in trials 1 and 2, respectively. The level of the EF was 30 kV/m, 60 Hz. EF incubation of embryos influenced the temperature of incubation and electrical properties of Hb molecules and did not influence the temperature of incubation and internal environment of eggs when empty eggs were incubated. EF incubation of fertilized eggs significantly raised the temperature of incubation, egg air cell, and at the surface of the egg yolk by approximately 0.09, 0.60, and 0.61 degrees F, respectively and Hb epsilon', sigma, Deltaepsilon', and tau as a function of the range of frequency of 20 to 100 kHz when compared with their counterparts of the control group. It was concluded that the exposure of fertilized chicken eggs to EF of 30 kV/m, 60 Hz, during incubation altered dielectric properties of Hb and that probably affected cell to cell communication and created the right environment for enhancing the growing process and heat production of embryos consequently increasing the temperature of the internal environment of the egg, and incubation.

The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes.

PubMed

Mouguelar, Valeria S; Cabada, Marcelo O; Coux, Gabriela

2011-05-01

Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and β1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-β1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. β1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.

Intracytoplasmic sperm injection

MedlinePlus Videos and Cool Tools

... occurs outside of the body. First, egg cells are harvested and transferred to a special media in ... be checked to ensure that various genetic disorders are not present. Typically, several egg cells are harvested ...

Automatic stage identification of Drosophila egg chamber based on DAPI images

PubMed Central

Jia, Dongyu; Xu, Qiuping; Xie, Qian; Mio, Washington; Deng, Wu-Min

2016-01-01

The Drosophila egg chamber, whose development is divided into 14 stages, is a well-established model for developmental biology. However, visual stage determination can be a tedious, subjective and time-consuming task prone to errors. Our study presents an objective, reliable and repeatable automated method for quantifying cell features and classifying egg chamber stages based on DAPI images. The proposed approach is composed of two steps: 1) a feature extraction step and 2) a statistical modeling step. The egg chamber features used are egg chamber size, oocyte size, egg chamber ratio and distribution of follicle cells. Methods for determining the on-site of the polytene stage and centripetal migration are also discussed. The statistical model uses linear and ordinal regression to explore the stage-feature relationships and classify egg chamber stages. Combined with machine learning, our method has great potential to enable discovery of hidden developmental mechanisms. PMID:26732176

Twin plants from supernumerary egg cells in Arabidopsis.

PubMed

Kong, Jixiang; Lau, Steffen; Jürgens, Gerd

2015-01-19

Sexual reproduction of flowering plants is distinguished by double fertilization—the two sperm cells delivered by a pollen tube fuse with the two gametic cells of the female gametophyte, the egg and the central cell—inside the ovule to give rise to the embryo and the nutritive endosperm, respectively. The pollen tube is attracted by nongametic synergid cells, and how these two cells of the female gametophyte are specified is currently unclear. Here, we show that ALTERED MERISTEM PROGRAM 1 (AMP1), encoding a protein associated with the endoplasmic reticulum, is required for synergid cell fate during Arabidopsis female gametophyte development. Loss of AMP1 function leads to supernumerary egg cells at the expense of synergids, enabling the generation of dizygotic twins. However, if twin embryos are formed, endosperm formation is prevented, eventually resulting in ovule abortion. The latter can be overcome by the delivery of supernumerary sperm cells in tetraspore (tes) pollen, enabling the formation of twin plants. Thus, both primary and supernumerary egg cells are fully functional in amp1 mutant plants. Sporophytic AMP1 expression is sufficient to prevent cell-fate change of synergids, indicating that one or more AMP1-dependent mobile signals from outside the female gametophyte can contribute to its patterning, in addition to the previously reported lateral inhibition between gametophytic cells. Our results provide insight into the mechanism of synergid fate specification and emphasize the importance of specifying only one egg cell within the female gametophyte to ensure central-cell fertilization by the second sperm cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

The survival of salmonellas in shell eggs cooked under simulated domestic conditions.

PubMed Central

Humphrey, T. J.; Greenwood, M.; Gilbert, R. J.; Rowe, B.; Chapman, P. A.

1989-01-01

Strains of Salmonella enteritidis, S. typhimurium and S. senftenberg inoculated into the yolks of shell eggs were found to survive forms of cooking where some of the yolk remained liquid. Survival was largely independent of the size of the initial inoculum. The organisms also grew rapidly in eggs stored at room temperature and after 2 days the number of cells per gram of yolk exceeded log10 8.0. With this level of contamination viable cells could be recovered from eggs cooked in any manner. PMID:2673824

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing

PubMed Central

Donis, Ruben O.; Chen, i-Mei; Davis, C Todd; Foust, Angie; Hossain, M. Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, odewijk; Neumeier, Elisabeth; Ziegler, Thedi

2018-01-01

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production. PMID:24975811

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing.

PubMed

Donis, Ruben O; Davis, C Todd; Foust, Angie; Hossain, M Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, Odewijk; Neumeier, Elisabeth; Ziegler, Thedi

2014-11-12

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production. Published by Elsevier Ltd.

Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification.

PubMed

Figueroa, E; Merino, O; Risopatrón, J; Isachenko, V; Sánchez, R; Effer, B; Isachenko, E; Farias, J G; Valdebenito, I

2015-01-15

This study was designed to test a vitrification method in Atlantic salmon spermatozoa and determine the capacity of seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium consisted of a standard buffer for fish spermatozoa (Cortland medium) + 10% DMSO + 2% BSA + 0.13-M sucrose + SP at concentrations of 30% (G30), 40% (G40), or 50% (G50). Fresh sperm was used as a control. To freeze the samples, 30-μL suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. The resulting spheres were placed in cryotubes for storage in liquid nitrogen. The cryotubes with the vitrified spermatozoa were thawed by placing them in a water bath at 37 °C for 45 seconds. After thawing, the following sperm quality parameters were determined by flow cytometry: DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling), plasma membrane integrity (SYBR-14/PI, staining technique), and mitochondrial membrane potential (JC-1 staining). An optical microscope was used to assess subjectively sperm motility, whereas fertility was determined by the presence of neurulation using five replicates per treatment in a sample of 30 eggs. Spermatozoa quality variables were preserved best when the highest concentration of SP (50%) was used (DNA fragmentation, 9.2%; plasma membrane integrity, 98.6%; mitochondrial membrane integrity, 47.2%; motility, 44.1%; and fertility, 46.2%). Copyright © 2015 Elsevier Inc. All rights reserved.

Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine.

PubMed

Proulx, P; Aubry, H; Brglez, I; Williamson, D G

1985-04-01

Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid - bile salt micelles, such that the lecithin - bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid - bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of space-fertilized eggs and formation of primordial germ cells in the embryos of medaka fish

NASA Astrophysics Data System (ADS)

Ijiri, K.

In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly `space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.

Characterization of S100A11, a suppressive factor of fertilization, in the mouse female reproductive tract.

PubMed

Hanaue, Mayu; Miwa, Naofumi; Uebi, Tatsuya; Fukuda, Yusuke; Katagiri, Yukiko; Takamatsu, Ken

2011-02-01

We recently found that Xenopus dicalcin, present in the extracellular egg-coating envelope, suppresses the efficiency of fertilization in vitro through binding to envelope-constituent glycoproteins. In the present study, we explored the mouse counterpart of Xenopus dicalcin, specifically its localization in the female reproductive tract and its action on mouse fertilization. Our homology and phylogenetic analyses using known S100 proteins showed that S100A11 is most closely related to Xenopus dicalcin. S100A11 was localized in the cytosol of luteal cells, but not in the follicle, in the mouse ovary, and also in the cytosol of the oviductal epithelial cells. In addition, our quantitative analyses revealed preferential expression of S100A11 in the ampullary region of the oviduct and at the estrus stage during the mouse estrous cycle. In the cumulus cell-oocyte complex dissected from the oviduct following ovulation, S100A11 was present in the plasma membrane of cumulus cells, but not in the zona pellucida, which is comparable with Ca(2+) -dependent binding of exogenously applied S100A11 to the plasma membrane of cumulus cells. Pretreatment of the cumulus cell-oocyte complex with recombinant S100A11 substantially reduced the efficiency of in vitro fertilization, but S100A10, the next closest S100 protein to Xenopus dicalcin, had no effect. These results suggested that S100A11 is the mouse counterpart of Xenopus dicalcin, suppresses the fertilization process through its action on cumulus cells, and thereby plays a key role in fertilization success in the mouse. Copyright © 2010 Wiley-Liss, Inc.

Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen

PubMed Central

Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

2009-01-01

The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L−1 of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L−1 of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50°C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P < 0.05), and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk (group IV) improved both progressive motility (P < 0.05) and acrosome integrity (P < 0.01). The use of DHA-enriched hen egg yolk alone (group II) did not enhance any of the post-thawed semen qualities (P > 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity. PMID:19633681

Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen.

PubMed

Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

2009-09-01

The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50 degrees C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P < 0.05), and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk (group IV) improved both progressive motility (P < 0.05) and acrosome integrity (P < 0.01). The use of DHA-enriched hen egg yolk alone (group II) did not enhance any of the post-thawed semen qualities (P > 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders.

PubMed

Layek, S S; Mohanty, T K; Kumaresan, A; Parks, J E

2016-09-01

Since the inception of bovine semen cryopreservation, egg yolk and milk based extenders have been used to protect sperm from the detrimental effects of cooling and freezing. In recent years, demand for alternatives to conventional commercial extenders has arisen as the risk of introducing exotic diseases through transporting egg yolk based products has been recognized. Egg yolk can also interfere with sperm evaluation and the presence of particulate material in the extender may reduce fertility. Soybeans contain lecithin, a phospholipid fraction that can substitute for high molecular weight lipoprotein and phospholipids from egg yolk and prevent or ameliorate damage to the sperm plasma membrane that occurs during extension, cooling, and cryopreservation. Soy lecithin based extenders have been evaluated for processing and freezing bovine semen, although extender from soybean milk has not been studied as extensively. Commercially available soy lecithin based extenders are used increasingly but remain under scrutiny and are not universally accepted. With these observations in mind, this review is intended to examine effects of conventional cryopreservation procedures, methods of assessment, and potential for developing soybean extract as an acceptable alternative to traditional egg yolk and milk based extenders for bull sperm cryopreservation. Copyright © 2016. Published by Elsevier B.V.

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

PubMed Central

Gillespie, Peter J.; Gambus, Agnieszka; Blow, J. Julian

2012-01-01

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication. PMID:22521908

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

PubMed

Nishio, Shunsuke; Kohno, Yoshinori; Iwata, Yuki; Arai, Mayumi; Okumura, Hiroki; Oshima, Kenzi; Nadano, Daita; Matsuda, Tsukasa

2014-11-01

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs. © 2014 by the Society for the Study of Reproduction, Inc.

The utilization of Habrobracon and artemia as experimental materials in bioastronautic studies

NASA Technical Reports Server (NTRS)

Grosch, D. S.

1972-01-01

In the reproductive performance of female braconids striking contrasts were revealed between the results from the actual biosatellite flight and those from experiments when the recovered vehicle was subjected to the forces of simulated launching and recovery. Second week decreases in egg production due to the radiation damage of cells in mitosis were minimized for the females irradiated during space flight. It was demonstrated that females irradiated for two days during orbital flight laid as many eggs during the second week as the unirradiated ground-based controls. After the 10th day their oviposition records exceeded control values. The hatchability of eggs deposited by Biosatellite II females was excellent. Explanations were sought for the space flight's cancellation of the characteristic radiation-induced decrease in egg production, and for the exceptionally good hatchability of eggs derived from most of the cell types in the irradiated ovarioles. Eggs from only two classes of cells showed enhanced embryonic lethality: those poised in meiotic metaphase during their mother's orbital flight, and those from oocytes beginning vitellogenesis.

Modified egg as a nutritional supplement during peak brain development: a new target for fortification.

PubMed

Shapira, Niva

2009-01-01

Though eggs have the unique capacity, like breastmilk, to concentrate essential nutrients required for early growth and brain development of offspring - i.e. n-3 PUFA, increasingly deficient and sources contaminated - cholesterol and allergy concerns often exclude them from perinatal recommendations. Egg's potential contribution of key nutrients required for peak brain development are re-evaluated vis-à-vis fortification, accessibility, and risks. Contributions of standard (USDA) and fortified (selected market-available) egg compositions to perinatal requirements for critical brain-supporting nutrients were compared to human and cow milks, and risks and recommendations evaluated. Standard egg has already higher concentrations/kcal of iron, selenium, zinc, choline, vitamins B12 and E, and essential amino acids (plus taurine) than human milk. Fortified egg could further yield significant n-3 PUFA % recommendations for pregnancy-lactation (total n-3 69.6-75.0% [DRI=1400-1300 mg/day]), including DHA (120.1-129.3%, mostly approximately 80% [calculated DRI=140-130 mg/day]), plus antioxidant vitamins A (9.0-15.2%) and E (51.6-65.3%), and minerals iodine (33.6-44.5%) and selenium (33.7-39.3%); % recommendations for children (1-3 y) even more. Cholesterol, important for nerve membranes and learning, may not be generally contraindicated in childbearing-aged women (approximately 10.5% hypercholesterolemia), and early-life egg exposure may increase tolerance. Egg-inclusive perinatal nutrition programs have shown significant contributions. Eggs, especially target-fortified, may provide a unique nutritional supplement for peak brain development continously during pregnancy, nursing, and infancy (from 6 months), especially vs. insufficiencies. Missing nutritional opportunities by egg exclusion vs. concerns of hypercholesterolemia or allergy could be addressed individually, rather than as general recommendations, warranting further research and targeted egg design.

In vitro effect of Chrysosporium indicum and Chrysosporium keratinophylum on Toxocara canis eggs.

PubMed

Bojanich, María V; Basualdo, Juan A; Giusiano, Gustavo

2017-12-05

The degree of antagonism exercised by fungi on geohelminth development varies according to the morphological alterations caused by different fungal species. Saprophytic fungi may exert ovicidal or ovistatic effects. The aim of this study was to apply scanning electron microscopy (SEM) to observe the action of two soil saprophytic species of Chrysosporium (C. indicum and C. keratinophylum) on Toxocara canis eggs. The fungal strains to be tested were incubated for 28 days at 28°C in 2% water agar with a suspension of unembryonated T. canis eggs. A suspension of T. canis eggs in 2% water agar was used as control group. The assay was done in triplicate for each fungus and the control group. SEM observations were performed on the 4th, 7th, 14th, 21st, and 28th day after inoculation. The effect of the fungi on eggs was evaluated in accordance with the alterations observed on the surface and the changes in the normal characteristics of the eggs. Hyphae around the eggs, appresoria penetrating the shell and changes in the typical egg membrane were observed in this assay. Type 3 effect (alterations that occur both in the embryo and the shell, and hyphal penetration of the eggs) was the prevalent effect. SEM allowed us to observe clearly the morphological alterations in T. canis eggs due to the effect of C. indicum and C. keratinophylum. Both saprophytic species of Chrysosporium alter the egg structure and alterations increase as exposure increases. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Isolation of poxvirus from debilitating cutaneous lesions on four immature grackles (Quiscalus sp.)

USGS Publications Warehouse

Docherty, D.E.; Long, R.I.; Flickinger, Edward L.; Locke, L.N.

1991-01-01

Poxvirus was isolated from nodules on four immature grackles (Quiscalus sp.) collected in two residential areas of Victoria, Texas. All of the birds were emaciated and had nodules on the eyelids, bill, legs, toes, and areas of the skin on the wings, neck, and ventral abdomen. These pox nodules were extensive and probably interfered with both sight and flight. The preliminary diagnosis was confirmed by virus isolation, histopathology, and electron microscopy. Poxvirus was isolated on the chorioallantoic membrane of embryonated hen's eggs and in Muscovy duck embryo fibroblast cell culture. Phaenicia calliphoridae (blowfly) larvae were found in one of the pox nodules, raising the possibility of mechanical transmission of the virus by contaminated adult blowflies.

Oleic acid transfer from microsomes to egg lecithin liposomes: participation of fatty acid binding protein.

PubMed

Catalá, A; Avanzati, B

1983-11-01

Oleic acid transfer from microsomes or mitochondria to egg lecithin liposomes was stimulated by fatty acid binding protein. By gel filtration, it could be demonstrated that this protein incorporates oleic acid into liposomes. Fatty acid binding protein transfer activity was higher using microsomes rather than mitochondria, which suggests a selective interaction with different kinds of membranes. Transfer of oleic acid by this soluble protein is greater than that of stearic acid. The results indicate that fatty acid binding protein may participate in the intracellular transport of fatty acids.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

PubMed

Jadi, R S; Sudeep, A B; Kumar, Satyendra; Arankalle, V A; Mishra, A C

2010-08-01

Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID(50)) and indirect immunofluorescence assay (IFA). All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Differentiation of Aedes aegypti and Aedes notoscriptus (Diptera: Culicidae) eggs using scanning electron microscopy.

PubMed

Faull, Katherine J; Williams, Craig R

2016-05-01

Aedes notoscriptus and Aedes aegypti are both peri-domestic, invasive container-breeding mosquitoes. While the two potential arboviral vectors are bionomically similar, their sympatric distribution in Australia is limited. In this study, analyses of Ae. aegypti and Ae. notoscriptus eggs were enabled using scanning electron microscopy. Significant variations in egg length to width ratio and outer chorionic cell field morphology between Ae. aegypti and Ae. notoscriptus enabled distinction of the two species. Intraspecific variations in cell field morphology also enabled differentiation of the separate populations of both species, highlighting regional and global variation. Our study provides a comprehensive comparative analysis of inter- and intraspecific egg morphological and morphometric variation between two invasive container-breeding mosquitoes. The results indicate a high degree of intraspecific variation in Ae. notoscriptus egg morphology when compared to the eggs of Ae. aegypti. Comparative morphological analyses of Ae. aegypti and Ae. notoscriptus egg attributes using SEM allows differentiation of the species and may be helpful in understanding egg biology in relation to biotope of origin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunochemical and biological characterization of outer membrane proteins of Porphyromonas endodontalis.

PubMed Central

Ogawa, T; Kuribayashi, S; Shimauchi, H; Toda, T; Hamada, S

1992-01-01

Outer membrane proteins (OMP) of Porphyromonas endodontalis HG 370 (ATCC 35406) were prepared from the cell envelope fraction of the organisms. The cell envelope that had been obtained by sonication of the whole cells was extracted in 2% lithium dodecyl sulfate and then successively chromatographed with Sephacryl S-200 HR and DEAE-Sepharose Fast Flow. Two OMP fractions, OMP-I and OMP-II, were obtained, and their immunochemical properties and induction of specific antibodies were examined. The OMP-I preparation consisted of a major protein with an apparent molecular mass of 31 kDa and other moderate to minor proteins of 40.3, 51.4, 67, and 71.6 kDa, while the OMP-II preparation contained 14-, 15.5-, 27-, and 44-kDa proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. OMP-I was found to form hydrophilic diffusion pores by incorporation into artificial liposomes composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that OMP-I exhibited significant porin activity. However, the liposomes containing heat-denatured OMP-I were scarcely active. Spontaneous and antigen-specific immunoglobulin M (IgM)-, IgG-, and IgA-secreting spot-forming cells (SFC) enzymatically dissociated into single-cell suspensions from chronically inflamed periapical tissues and were enumerated by enzyme-linked immunospot assay. In patients with radicular cysts or dental granulomas, the major isotype of spontaneous SFC was IgG. In radicular cysts, the OMP-II-specific IgG SFC represented 0.13% of the total IgG SFC, while the antigen-specific IgA or IgM SFC was not observed. It was also found that none of these mononuclear cells produced antibodies specific for OMP-I or lipopolysaccharide of P. endodontalis. Images PMID:1328059

Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

PubMed Central

Huang, Wenjing; Arai, Fumihito; Kawahara, Tomohiro

2015-01-01

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research. PMID:25768929

Marchantia MpRKD Regulates the Gametophyte-Sporophyte Transition by Keeping Egg Cells Quiescent in the Absence of Fertilization.

PubMed

Rövekamp, Moritz; Bowman, John L; Grossniklaus, Ueli

2016-07-11

Unlike in animals, the life cycle of land plants alternates between two multicellular generations, the haploid gametophyte and the diploid sporophyte [1]. Gamete differentiation initiates the transition from the gametophyte to the sporophyte generation and, upon maturation, the egg cell establishes a quiescent state that is maintained until fertilization. This quiescence represents a hallmark of the gametophyte-sporophyte transition. The underlying molecular mechanisms are complex and best characterized in the flowering plant Arabidopsis thaliana [2-4]. However, only few genes with egg cell-specific expression or defects have been identified [5-10]. Intriguingly, ectopic expression of members of a clade of RWP-RK domain (RKD)-containing transcription factors, which are absent from animal genomes [11-13], can induce an egg cell-like transcriptome in sporophytic cells of A. thaliana. Yet, to date, loss-of-function experiments have not produced phenotypes affecting the egg cell, likely due to genetic redundancy and/or cross-regulation among the five RKD genes of A. thaliana [10]. To reduce genetic complexity, we explored the genome of Marchantia polymorpha, a liverwort belonging to the basal lineage of extant land plants [14-17]. Based on sequence homology, we identified a single M. polymorpha RKD gene, MpRKD, which is orthologous to all five A. thaliana RKD genes. Analysis of the MpRKD expression pattern and characterization of lines with reduced MpRKD activity indicate that it functions as a regulator of gametophyte development and the gametophyte-sporophyte transition. In particular, MpRKD is required to establish and/or maintain the quiescent state of the egg cell in the absence of fertilization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of Vascular Growth in the Chorioallantoic Membrane of Japanese Quail Eggs

NASA Technical Reports Server (NTRS)

Montague, Idoreyin P.

2004-01-01

The Microgravity Research Program is part of NASA's Office of Biological and Physical Research (OBPR). The mission of the Microgravity Fluid Physics research program is to facilitate and conduct the best possible fluid physics research using the space environment and make this knowledge available to the scientific community and the public at large. During the summer of 2004, I worked in this division with Dr. Patricia Parsons-Wingerter. Dr. Parsons was working on several projects that used the chorioallantoic membrane (CAM) of Japanese quail eggs. The CAM develops in the eggs of birds and reptiles and is a very vascular fetal membrane composed of the fused chorion and adjacent wall of the allantois. The CAM is formed on day 4 of incubation and its primary job is to mediate gas exchanges with the extra embryonic environment. The CAM of our Japanese quail eggs is easily identifiable to us because it is transparent and it sits on top of the yolk with the embryo in the center. The CAM is of interest because of its many applications in the field of medicine as it relates to vascular remodeling and angiogenesis. Angiogenesis is simply the growth or formation of new blood vessels and anti-angiogenesis is the inhibition of said vessels. Angiogenesis occurs naturally in a healthy body for healing wounds and for restoring blood flow to tissues after injury and in females during the monthly reproductive cycle. In many serious diseases, like several types of cancer and those that affect the heart and cardiovascular system, the body loses control over angiogenesis. These diseases, which are dependent on angiogenesis, result when new blood vessels either grow excessively or insufficiently. The chorioallantoic membrane of our Japanese quail eggs gives a good model of angiogenesis. We used angiogenic regulators to inhibit or stimulate vascular growth in the CAM in a healthy manner and they induced distinct vascular patterns in vivo. Certain dominant regulators can be recognized by their unique vascular patterns and from these patterns; we can deduce specific alterations in vascular remodeling and angiogenesis. This will aid us in early-stage diagnosis and customized therapies for patients with angiogenic-dependent diseases. This particular research is important to NASA because cardiovascular health issues are the second highest of ten categories that have been defined as risk factors in human space exploration. Also, cardiovascular-related diseases have been the leading cause of death in America since 1981. Therefore, this kind of research in the field of cardiovascular health is of great importance to humans on earth and in space. This particular research is important to NASA because cardiovascular health is of great importance to humans on earth and in space.

Effect of Fatty Acyl Group and Sterol Composition on Sensitivity of Lecithin Liposomes to Imidazole Antimycotics

PubMed Central

Yamaguchi, Hideyo; Iwata, Kazuo

1979-01-01

The specific affinity for membrane lipids and the membrane selectivity of three imidazole derivatives, clotrimazole, miconazole, and econazole, were studied using various types of liposomes with respect to the lecithin fatty acyl group composition and the liposome content and composition of sterol as membrane models. The sensitivity of liposomes to these drugs was primarily dependent upon the lecithin fatty acyl group composition. With sterol-free liposome systems, each imidazole induced maximum release of trapped glucose as a marker from the unsaturated dioleoyl lecithin liposomes, minimum release from the saturated dipalmitoyl lecithin liposomes, and intermediate release from egg lecithin liposomes. The sensitivity of the dipalmitoyl lecithin liposomes to any imidazole drug was not influenced by the incorporation of cholesterol or ergosterol. On the other hand, clotrimazole-induced permeability changes of liposomes prepared from unsaturated dioleoyl lecithin or egg lecithin were greatly enhanced by the incorporation of ergosterol, whereas they were suppressed by cholesterol incorporation. The sensitivity of liposomes prepared from these unsaturated lecithins to miconazole and econazole was also augmented by ergosterol incorporation, although it was scarcely altered by cholesterol incorporation. Negatively charged liposomes were more sensitive to the three imidazole drugs than positively charged liposomes. PMID:525988

Effect of fatty acyl group and sterol composition on sensitivity of lecithin liposomes to imidazole antimycotics.

PubMed

Yamaguchi, H; Iwata, K

1979-05-01

The specific affinity for membrane lipids and the membrane selectivity of three imidazole derivatives, clotrimazole, miconazole, and econazole, were studied using various types of liposomes with respect to the lecithin fatty acyl group composition and the liposome content and composition of sterol as membrane models. The sensitivity of liposomes to these drugs was primarily dependent upon the lecithin fatty acyl group composition. With sterol-free liposome systems, each imidazole induced maximum release of trapped glucose as a marker from the unsaturated dioleoyl lecithin liposomes, minimum release from the saturated dipalmitoyl lecithin liposomes, and intermediate release from egg lecithin liposomes. The sensitivity of the dipalmitoyl lecithin liposomes to any imidazole drug was not influenced by the incorporation of cholesterol or ergosterol. On the other hand, clotrimazole-induced permeability changes of liposomes prepared from unsaturated dioleoyl lecithin or egg lecithin were greatly enhanced by the incorporation of ergosterol, whereas they were suppressed by cholesterol incorporation. The sensitivity of liposomes prepared from these unsaturated lecithins to miconazole and econazole was also augmented by ergosterol incorporation, although it was scarcely altered by cholesterol incorporation. Negatively charged liposomes were more sensitive to the three imidazole drugs than positively charged liposomes.

Anti-tumour activity of two novel compounds in cisplatin-resistant testicular germ cell cancer.

PubMed

Nitzsche, B; Gloesenkamp, C; Schrader, M; Hoffmann, B; Zengerling, F; Balabanov, S; Honecker, F; Höpfner, M

2012-11-20

Resistance to cisplatin-based chemotherapy is associated with poor prognosis in testicular germ cell cancer, emphasising the need for new therapeutic approaches. In this respect, the therapeutic concept of anti-angiogenesis is of particular interest. In a previous study, we presented two novel anti-angiogenic compounds, HP-2 and HP-14, blocking the tyrosine kinase activity of angiogenic growth factor receptors, such as vascular endothelial growth factor receptor-2 (VEGFR-2), and related signalling pathways in testicular cancer. In this study, we investigated the efficacy of these new compounds in platinum-resistant testicular germ cell tumours (TGCTs), in vitro and in vivo. Drug-induced changes in cell proliferation of the cisplatin-sensitive TGCT cell line 2102EP and its cisplatin-resistant counterpart 2102EP-R, both expressing the VEGFR-2, were evaluated by crystal violet staining. Both compounds inhibited the growth of cisplatin-resistant TGCT cells in a dose-dependent manner. In combination experiments with cisplatin, HP-14 revealed additive growth-inhibitory effects in TGCT cells, irrespective of the level of cisplatin resistance. Anti-angiogenic effects of HP compounds were confirmed by tube formation assays with freshly isolated human umbilical vein endothelial cells. Using TGCT cells inoculated onto the chorioallantoic membrane of fertilised chicken eggs (chicken chorioallantoic membrane assay), the anti-angiogenic and anti-proliferative potency of the novel compounds was also demonstrated in vivo. Gene expression profiling revealed changes in the expression pattern of genes related to DNA damage detection and repair, as well as in chaperone function after treatment with both cisplatin and HP-14, alone or in combination. This suggests that HP-14 can revert the lost effectiveness of cisplatin in the resistant cells by altering the expression of critical genes. The novel compound HP-14 effectively inhibits the growth of cisplatin-resistant TGCT cells and suppresses tumour angiogenesis. Thus, HP-14 may be an interesting new agent that should be further explored for TGCT treatment, especially in TGCTs that are resistant to cisplatin.

Fate of egg proteins during the development of Columba livia domestica embryo.

PubMed

Shbailat, Seba Jamal; Aslan, Ibtisam Omar

2018-01-01

The transfer of egg white into the yolk and consumption of yolk proteins by the embryo are largely unexplored in the pigeon Columba livia domestica. Here, we investigated the route of egg white transfer as well as the degradation and uptake of yolk proteins by the pigeon embryo. Initially, we tested the electrophoretic patterns of proteins in different egg compartments throughout development. Then, we used lysozyme as a reference protein to follow the egg white transfer, and we measured its activity using Micrococcus lysodeikticus as a substrate. Moreover, we determined the general protease activity during different developmental stages in the yolk using casein. Finally, we examined the expression of aminopeptidase-N (APN) and oligopeptide transporter PepT1 genes in the yolk sac membrane (YSM) from incubation day 8 until day 17. Several electrophoretic bands of presumptive egg white proteins appeared in different egg compartments. Also, lysozyme activity was detected chronologically in the egg compartments. It appeared on day 12 in the amniotic and intestinal fluids and on day 14 in the yolk. Moreover, protease activity in the yolk increased significantly on day 14 and thereafter. APN expression was largest on day 8 and reduced generally afterward, whereas PepT1 expression peaked between days 13 and 15 but then reduced substantially. Our results suggest that the egg white proteins move through the amnion and intestine into the yolk where they undergo degradation by the activated proteases. Furthermore, the YSM appears to have a role in protein consumption, and this role decreases toward hatch. © 2018 Wiley Periodicals, Inc.

New Forces Yet Undetermined: The Challenge of Biodefense

DTIC Science & Technology

2009-09-01

processed. Smallpox virus was grown on the allantoic membrane of a fertile chicken egg. After examination of the membrane, it was a simple matter to place...Virologists have observed that it was common to put such vials in the back of a freezer and for old-fashioned-style labels to sometimes fall-off. Small- pox ...concern are naturally occurring. These include, for example, anthrax (in the Western United States), hanta virus (in New Mexico ), and tularemia (in Texas

Proceedings of the Symposium on Current Concepts and Approaches on Animal Test Alternatives

DTIC Science & Technology

1993-09-01

vertebrate model, more specifically, the chorloallantoic membrane (CAN) of a developing chicken embryo, has been proposed for use as an alternative...and Drug Administration in Washington, D.C. He has a B.S. in pharmacy (University of New Mexico ) and a Ph.D. in pharmacology (University of Colorado...model is represented by the choriallantoic membrane system (CAM).20,21 This system uses fertilized chicken eggs, whose vascular network (or CAM) is

31P and 1H NMR Studies of the Molecular Organization of Lipids in the Parallel Artificial Membrane Permeability Assay.

PubMed

Assmus, Frauke; Ross, Alfred; Fischer, Holger; Seelig, Joachim; Seelig, Anna

2017-01-03

The parallel artificial membrane permeability assay (PAMPA) has emerged as a widely used primary in vitro screen for passive permeability of potential drug candidates. However, the molecular structure of the permeation barrier (consisting of a filter-supported dodecane-egg lecithin mixture) has never been characterized. Here, we investigated the long-range order of phospholipids in the PAMPA barrier by means of 31 P static solid-state NMR. Diffusion constants of PAMPA membrane components were derived from liquid state NMR and, in addition, drug distribution between the PAMPA lipid phase and buffer (log D PAMPA at pH 7.4) was systematically investigated. Increasing concentration of n-dodecane to the system egg lecithin-water (lamellar phase, L α ) induces formation of inverted hexagonal (H ii ) and isotropic phases. At n-dodecane concentrations matching those used in PAMPA (9%, w/v) a purely "isotropic" phase was observed corresponding to lipid aggregates with a diameter in the range 4-7 nm. Drug distribution studies indicate that these reverse micelles facilitate the binding to, and in turn the permeation across, the PAMPA dodecane barrier, in particular for amphiphilic solutes. The proposed model for the molecular architecture and function of the PAMPA barrier provides a fundamental, hitherto missing framework to evaluate the scope but also limitations of PAMPA for the prediction of in vivo membrane permeability.

Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples.

PubMed

Macuhova, K; Kumagai, T; Akao, N; Ohta, N

2010-12-01

We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.

Caffeine alleviates the deterioration of Ca2+ release mechanisms and fragmentation of in vitro aged mouse eggs

PubMed Central

Zhang, Nan; Wakai, Takuya; Fissore, Rafael. A.

2011-01-01

The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others found that in these eggs the intracellular calcium ([Ca2+]i) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca2+ release is not well known. Here, we investigated if the function of IP3R1, the major Ca2+ release channel at fertilization, was undermined in in vitro aged mouse eggs. We found that in aged eggs IP3R1 displayed reduced function, as many of the changes acquired during maturation that enhance IP3R1 Ca2+ conductivity such as phosphorylation, receptor reorganization and increased Ca2+ store content ([Ca2+]ER) were lost with increasing postovulatory time. IP3R1 fragmentation, possibly associated with the activation of caspase-3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP3R1 function and maintained [Ca2+]ER content. Caffeine also maintained mitochondrial membrane potential as measured by JC-1 fluorescence. We therefore conclude that [Ca2+]i responses in aged eggs are undermined by reduced IP3R1 sensitivity, decreased [Ca2+]ER and compromised mitochondrial function, and that addition of caffeine ameliorates most of these aging-associated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs. PMID:22095868

Embryonated chicken eggs as an alternative model for mixed Clostridium perfringens and Eimeria tenella infection in chickens.

PubMed

Alnassan, Alaa Aldin; Shehata, Awad Ali; Kotsch, Marianne; Lendner, Matthias; Daugschies, Arwid; Bangoura, Berit

2013-06-01

The chorioallantoic membrane (CAM) of chicken embryo eggs is a suitable model for viral and bacterial infections. In the present study, a new approach for testing the pathogenesis and virulence of Clostridium perfringens and Eimeria tenella dual infections as a model using the CAM of embryonated chicken eggs was developed. For this purpose, 24 specific pathogen-free (SPF) embryonated chicken eggs were divided into four groups (n = 6) and designated group E, group CP, group CPE, and NC. Sporozoites of E. tenella (20,000 sporozoites) were inoculated into 10-day-old embryonated SPF chicken eggs (groups E and CPE) via allantoic sac route. At 15-day-old, eggs of groups CP and CPE were infected with 10 (4)  cfu C. perfringens via the same route. Assessment of pathogenicity was assessed using gross and histopathological lesions. Embryo mortality reached 17 % after mono-infection with C. perfringens and/or E. tenella and 50 % in the mixed-infected group. Lesions in the CAMs were most numerous and most severe in co-infected eggs (group CPE), reaching the maximum score of 3 in 50 % of the inoculated eggs (P < 0.01). In Eimeria spp.-infected eggs (group E), lesions of score were between 1 and 2. Mono-infection with C. perfringens did not lead to a significant occurrence of lesions. Histopathological investigations of the CAM revealed clusters of Gram-positive bacteria, infiltration with leukocytes, lymphocytes, and developmental stages of E. tenella in the co-infected group. These data suggest that embryonated eggs could be an in ovo model for studying the pathogenesis of mixed infection with Eimeria and C. perfringens.

Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shirakashi, Ryo, E-mail: aa21150@iis.u-tokyo.ac.jp; Mischke, Miriam; Fischer, Peter

2012-11-09

Highlights: Black-Right-Pointing-Pointer Electrorotation offers a non-invasive tool for dielectric analysis of fish embryos. Black-Right-Pointing-Pointer The three-shell dielectric model matches the rotation spectra of medaka eggs. Black-Right-Pointing-Pointer The capacitance value suggests a double-membrane structure of yolk envelope. -- Abstract: The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to earlymore » somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.« less

Maternal control of the Drosophila dorsal–ventral body axis

PubMed Central

Stein, David S.; Stevens, Leslie M.

2016-01-01

The pathway that generates the dorsal–ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)-α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral-to-dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity. PMID:25124754

Effects of the tumor-vasculature-disrupting agent verubulin and two heteroaryl analogues on cancer cells, endothelial cells, and blood vessels.

PubMed

Mahal, Katharina; Resch, Marcus; Ficner, Ralf; Schobert, Rainer; Biersack, Bernhard; Mueller, Thomas

2014-04-01

Two analogues of the discontinued tumor vascular-disrupting agent verubulin (Azixa®, MPC-6827, 1) featuring benzo-1,4-dioxan-6-yl (compound 5 a) and N-methylindol-5-yl (compound 10) residues instead of the para-anisyl group on the 4-(methylamino)-2-methylquinazoline pharmacophore, were prepared and found to exceed the antitumor efficacy of the lead compound. They were antiproliferative with single-digit nanomolar IC50 values against a panel of nine tumor cell lines, while not affecting nonmalignant fibroblasts. Indole 10 surpassed verubulin in seven tumor cell lines including colon, breast, ovarian, and germ cell cancer cell lines. In line with docking studies indicating that compound 10 may bind the colchicine binding site of tubulin more tightly (Ebind =-9.8 kcal mol(-1) ) than verubulin (Ebind =-8.3 kcal mol(-1) ), 10 suppressed the formation of vessel-like tubes in endothelial cells and destroyed the blood vessels in the chorioallantoic membrane of fertilized chicken eggs at nanomolar concentrations. When applied to nude mice bearing a highly vascularized 1411HP germ cell xenograft tumor, compound 10 displayed pronounced vascular-disrupting effects that led to hemorrhages and extensive central necrosis in the tumor. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microgravity effects during fertilization, cell division, development, and calcium metabolism in sea urchins

NASA Technical Reports Server (NTRS)

Schatten, Heide

1996-01-01

The overall objectives of this project are to explore the role of microgravity during fertilization, early development, cytoskeletal organization, and skeletal calcium deposition in a model development system: the sea urchin eggs and embryos. While pursuing these objectives, we have also helped to develop, test, and fly the Aquatic Research Facility (ARF) system. Cells were fixed at preselected time points to preserve the structures and organelles of interest with regards to cell biology events during development. The protocols used for the analysis of the results had been developed during the earlier part of this research and were applied for post-flight analysis using light and (immuno)fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy. The structures of interest are: microtubules during fertilization, cell division, and cilia movement; microfilaments during cell surface restructuring and cell division; centrosomes and centrioles during cell division, cell differentiation, and cilia formation and movement; membranes, Golgi, endoplasmic reticulum, mitochondria, and chromosomes at all stages of development; and calcium deposits during spicule formation in late-stage embryos. In addition to further explore aspects important or living in space, several aspects of this research are also aimed at understanding diseases that affect humans on Earth which may be accelerated in space.

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashem, Anwar M.; Department of Microbiology, Faculty of Medicine, King Abdulaziz University, Jeddah; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON

Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it couldmore » be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.« less

Survival of Salmonella enterica serovar infantis on and within stored table eggs.

PubMed

Lublin, Avishai; Maler, Ilana; Mechani, Sara; Pinto, Riky; Sela-Saldinger, Shlomo

2015-02-01

Contaminated table eggs are considered a primary source of foodborne salmonellosis globally. Recently, a single clone of Salmonella enterica serovar Infantis emerged in Israel and became the predominant serovar isolated in poultry. This clone is currently the most prevalent strain in poultry and is the leading cause of salmonellosis in humans. Because little is known regarding the potential transmission of this strain from contaminated eggs to humans, the objective of this study was to evaluate the ability of Salmonella Infantis to survive on the eggshell or within the egg during cold storage or at room temperature. Salmonella cells (5.7 log CFU per egg) were inoculated on the surface of 120 intact eggs or injected into the egg yolk (3.7 log CFU per egg) of another 120 eggs. Half of the eggs were stored at 5.5 ± 0.3°C and half at room temperature (25.5 ± 0.1°C) for up to 10 weeks. At both temperatures, the number of Salmonella cells on the shell declined by 2 log up to 4 weeks and remained constant thereafter. Yolk-inoculated Salmonella counts at cold storage declined by 1 log up to 4 weeks and remained constant, while room-temperature storage supported the growth of the pathogen to a level of 8 log CFU/ml of total egg content, as early as 4 weeks postinoculation. Examination of egg content following surface inoculation revealed the presence of Salmonella in a portion of the eggs at both temperatures up to 10 weeks, suggesting that this strain can also penetrate through the shell and survive within the egg. These findings imply that Salmonella enterica serovar Infantis is capable of survival both on the exterior and interior of table eggs and even multiply inside the egg at room temperature. Our findings support the need for prompt refrigeration to prevent Salmonella multiplication during storage of eggs at room temperature.

The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells.

PubMed

Williams, Michael J

2009-03-25

When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes) to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg) is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1) 1. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface. The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1.

The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells

PubMed Central

Williams, Michael J

2009-01-01

Background When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes) to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. Results Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg) is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1) [1]. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface. Conclusion The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1. PMID:19320973

Egg Component-Composited Inverse Opal Particles for Synergistic Drug Delivery.

PubMed

Liu, Yuxiao; Shao, Changmin; Bian, Feika; Yu, Yunru; Wang, Huan; Zhao, Yuanjin

2018-05-23

Microparticles have a demonstrated value in drug delivery systems. The attempts to develop this technology focus on the generation of functional microparticles by using innovative but accessible materials. Here, we present egg component-composited microparticles with a hybrid inverse opal structure for synergistic drug delivery. The egg component inverse opal particles were produced by using egg yolk to negatively replicate colloid crystal bead templates. Because of their huge specific surface areas, abundant nanopores, and complex nanochannels of the inverse opal structure, the resultant egg yolk particles could be loaded with different kinds of drugs, such as hydrophobic camptothecin (CPT), by simply immersing them into the corresponding drug solutions. Attractively, additional drugs, such as the hydrophilic doxorubicin (DOX), could also be encapsulated into the particles through the secondary filling of the drug-doped egg white hydrogel into the egg yolk inverse opal scaffolds, which realized the synergistic drug delivery for the particles. It was demonstrated that the egg-derived inverse opal particles were with large quantity and lasting releasing for the CPT and DOX codelivery, and thus could significantly reduce cell viability, and enhance therapeutic efficacy in treating cancer cells. These features of the egg component-composited inverse opal microparticles indicated that they are ideal microcarriers for drug delivery.

Development of Urinary Bladder Pre-Neoplasia by Schistosoma haematobium Eggs and Chemical Carcinogen in Mice

PubMed Central

Chala, Bayissa; Choi, Min-Ho; Moon, Kyung Chul; Kim, Hyung Suk; Kwak, Cheol; Hong, Sung-Tae

2017-01-01

Schistosoma haematobium is a biocarcinogen of human urinary bladder (UB). The present study investigated developing UB cancer mouse model by injecting S. haematobium eggs into the bladder wall and introduction of chemical carcinogens. Histopathological findings showed mild hyperplasia to epithelial vacuolar change, and high grade dysplasia. Squamous metaplasia was observed in the S. haematobium eggs+NDMA group at week 12 but not in other groups. Immunohistochemistry revealed significantly high expression of Ki-67 in urothelial epithelial cells of the S. haematobium eggs+BBN group at week 20. The qRT-PCR showed high expression of p53 gene in S. haematobium eggs group at week 4 and S. haematobium eggs+BBN group at week 20. E-cadherin and vimentin showed contrasting expression in S. haematobium eggs+BBN group. Such inverse expression of E-cadherin and vimentin may indicate epithelial mesenchymal transition in the UB tissue. In conclusion, S. haematobium eggs and nitrosamines may transform UB cells into squamous metaplasia and dysplasia in correlation with increased expression of Ki-67. Marked decrease in E-cadherin and increase in p53 and vimentin expressions may support the transformation. The present study introduces a promising modified animal model for UB cancer study using S. haematobium eggs. PMID:28285503

The avian chorioallantoic membrane in ovo--a useful model for bacterial invasion assays.

PubMed

Adam, Rüdiger; Mussa, Shueb; Lindemann, Dirk; Oelschlaeger, Tobias A; Deadman, Mary; Ferguson, David J P; Moxon, Richard; Schroten, Horst

2002-09-01

The aim of this study was to evaluate the practicability of the chick embryo chorioallantoic membrane (CAM) with special regard to the 'natural air sac' technique (NAST) of preparation for in-vivo research on the invasive potential of bacterial strains of various enterobacterial species. It was sought to establish an experimental system more closely resembling in-vivo conditions than cell lines on one hand, and cheaper and easier to handle than established animal models on the other. Fertilized eggs of the domestic fowl were incubated. The CAM was prepared atraumatically at the natural air space of the egg, and a cannula was inserted for subsequent extraction of allantoic fluid (AF) below the CAM. The CAM was then inoculated with either one out of five strains of Klebsiella pneumoniae, an Escherichia coli K-12 strain or a Salmonella typhimurium strain, either alone or in combinations, respectively. AF samples were extracted at certain time points, and the presence of bacteria was determined by cultivation. Penetration and mortality ratios of the infected embryos were calculated. In addition, the mode of crossing the epithelial barrier was examined by electron microscopy. Differing rates of invasion through the CAM and rates of mortality of the chicken embryos demonstrated a clear dependency on the inoculated bacterial strain. Low invading bacteria could be distinguished from intermediate strains, and from strains exerting a strong capability of invasion and killing of the embryos. Simultaneous monotopical inoculation of Klebsiella and E. coli showed a permissive effect of co-incubated Klebsiella on the invasiveness of E. coli. The chick embryo CAM prepared by NAST has shown to be a useful model for in vivo studies on invasion capabilities, pathogenicity and interactions of inoculated bacteria.

Insertion and self-diffusion of a monotopic protein, the Aquifex aeolicus sulfide quinone reductase, in supported lipid bilayers.

PubMed

Harb, Frédéric; Prunetti, Laurence; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne; Tinland, Bernard

2015-10-01

Monotopic proteins constitute a class of membrane proteins that bind tightly to cell membranes, but do not span them. We present a FRAPP (Fluorescence Recovery After Patterned Photobleaching) study of the dynamics of a bacterial monotopic protein, SQR (sulfide quinone oxidoreductase) from the thermophilic bacteria Aquifex aeolicus, inserted into two different types of lipid bilayers (EggPC: L-α-phosphatidylcholine (Egg, Chicken) and DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine) supported on two different types of support (mica or glass). It sheds light on the behavior of a monotopic protein inside the bilayer. The insertion of SQR is more efficient when the bilayer is in the fluid phase than in the gel phase. We observed diffusion of the protein, with no immobile fraction, and deduced from the diffusion coefficient measurements that the resulting inserted object is the same whatever the incubation conditions, i.e. homogeneous in terms of oligomerization state. As expected, the diffusion coefficient of the SQR is smaller in the gel phase than in the fluid phase. In the supported lipid bilayer, the diffusion coefficient of the SQR is smaller than the diffusion coefficient of phospholipids in both gel and fluid phase. SQR shows a diffusion behavior different from the transmembrane protein α-hemolysin, and consistent with its monotopic character. Preliminary experiments in the presence of the substrate of SQR, DecylUbiquinone, an analogue of quinone, component of transmembrane electrons transport systems of eukaryotic and prokaryotic organisms, have been carried out. Finally, we studied the behavior of SQR, in terms of insertion and diffusion, in bilayers formed with lipids from Aquifex aeolicus. All the conclusions that we have found in the biomimetic systems applied to the biological system.

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

PubMed

Oka, M T; Arai, T; Hamaguchi, Y

1990-12-01

Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.

Design and Synthesis of Archaea-Inspired Tetraether Lipids

NASA Astrophysics Data System (ADS)

Koyanagi, Takaoki

Maintaining the correct ion homeostasis across membranes is a major challenge in both nature and artificial systems. Archaea, have evolved to solve membrane permeability problems to survive in extreme environments by incorporating unique structural features found in their lipid. Specifically, inclusion of phytanyl side chains, ether glycerol linkages, tethering of lipids, cycloalkanes, and different polar lipid headgroups into their lipid membrane are believed to contribute to membrane stability. We sought to gain a better understanding of the functional benefits attributed to these structural features to membrane stability to design a new class of synthetic Archaea inspired lipid membranes that can be used to overcome limitations (i.e. unstable in serum environment, high background leakage, and prone to hydrolysis) found in current lipid based technologies. Leakage experiments revealed liposomes made from GMGTPC (glycerol monoalkyl glycerol tetraether lipid with phosphatidylcholine headgroup) demonstrated a two order magnitude reduction in membrane leakage to small ions when compared with liposomes made from EggPC. Additionally, liposomes composed of GMGTPC-CH (cyclohexane integrated) lipid displayed an additional 40% decrease in membrane leakage to small ions when compared with liposomes made from GMGTPC lipids. Furthermore, leakage experiments revealed a higher degree of tolerance to headgroup modifications to membrane leakage for liposomes made from GMGT lipid analogs when compared with liposomes made from POPC. After designing an optimal tetraether lipid scaffold that incorporates key Archaeal structural features for membrane leakage, we explored to integrate strategies employed by eukaryotes to improve membrane properties (i.e. addition of cholesterol). Liposomes made from the hybrid lipid, GcGTPC-CH, displayed a five-fold decrease in membrane leakage when compared with liposomes made from GMGTPC-CH, while maintaining functional membrane properties similar to membranes made from diacyl lipids. Lastly, we engineered a thiol responsive hybrid lipid, GcGT(S-S)PC-CH, that demonstrated similar membrane stability in serum as GcGTPC-CH. Gratifyingly, doxorubicin loaded liposomes composed of GcGT(S-S)PC-CH liposomes displayed a 4 or 20-fold increase in toxicity to HeLa cells when compared with liposomes made from GcGTPC-CH or Doxil, respectively. This work represents a first step towards development of stimuli-responsive tetraether lipids that may offer advantages in membrane properties to be used in cancer therapy.

External control of the Drosophila melanogaster egg to imago development period by specific combinations of 3D low-frequency electric and magnetic fields.

PubMed

Makarov, Vladimir I; Khmelinskii, Igor

2016-01-01

We report that the duration of the egg-to-imago development period of the Drosophila melanogaster, and the imago longevity, are both controllable by combinations of external 3-dimensional (3D) low-frequency electric and magnetic fields (LFEMFs). Both these periods may be reduced or increased by applying an appropriate configuration of external 3D LFEMFs. We report that the longevity of D. melanogaster imagoes correlates with the duration of the egg-to-imago development period of the respective eggs. We infer that metabolic processes in both eggs and imago are either accelerated (resulting in reduced time periods) or slowed down (resulting in increased time periods). We propose that external 3D LFEMFs induce electric currents in live systems as well as mechanical vibrations on sub-cell, whole-cell and cell-group levels. These external fields induce media polarization due to ionic motion and orientation of electric dipoles that could moderate the observed effects. We found that the longevity of D. melanogaster imagoes is affected by action of 3D LFEMFs on the respective eggs in the embryonic development period (EDP). We interpret this effect as resulting from changes in the regulation mechanism of metabolic processes in D. melanogaster eggs, inherited by the resulting imagoes. We also tested separate effects of either 3D electric or 3D magnetic fields, which were significantly weaker.

Challenges in Radiofrequency Pasteurization of Shell Eggs: Coagulation Rings.

PubMed

Lau, Soon Kiat; Thippareddi, Harshavardhan; Jones, David; Negahban, Mehrdad; Subbiah, Jeyamkondan

2016-10-01

A total of 50 different configurations of simple radiofrequency (RF) heating at 27.12 MHz of a shell egg were simulated using a finite element model with the purpose of pasteurizing the egg. Temperature-dependent thermal and dielectric properties of the yolk, albumen, and shell were measured, fitted, and introduced into the model. A regression equation that relates the top electrode voltage to the gap between the electrodes and vertical position of the egg was developed. Simulation and experimental results had good agreement in terms of temperature deviation (root mean squared error ranged from 0.35 °C to 0.48 °C) and both results demonstrated the development of a "coagulation ring" around the air cell. The focused heating near the air cell of the egg prevented pasteurization of the egg due to its impact on quality (coagulation). Analysis of the electric field patterns offered a perspective on how nonuniform RF heating could occur in heterogeneous food products. The results can be used to guide development of RF heating for heterogeneous food products and further development of RF pasteurization of eggs. © 2016 Institute of Food Technologists®.

Development and standardization of a short-term assay for evaluating polluted estuarine and coastal environments: The Medaka Embryo-Larval Assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Helmstetter, M.F.

1992-01-01

The eggs of the Japanese medaka (Oryzias latipes) were employed to develop a routine, standardized assay which can assess the acute and sublethal impacts of individual toxicants and complex mixtures. The eggs of this Cyprinodont minnow were topically treated with each toxicant or mixture dissolved in membrane permeable dimethylsulfoxide (DMSO) solvent. Nine chemicals were initially evaluated with two, pentachlorophenol (PCP) and tributyltin chloride (TBTCl), subsequently selected for extensive evaluations of salinity tolerance and the accuracy of the topical exposure method for representing a typical immersion exposure. The amount of chemical actually penetrating the eggs was determined, as were the optimummore » rearing conditions for the eggs. To determine the usefulness of this assay in field studies, surveys of lower Chesapeake Bay tributary water and sediment samples were performed using the MELA approach. The results indicate that the proposed assay is very useful for the evaluation of the developmental effects of individual toxicants as well as complex mixtures. The salinity tolerance studies indicated that salinity (up to 20ppt for PCP and 35ppt for TBTCl) elicited no significant effect on the toxicity of the two test substances on developing embryos. In addition, if the bioconcentration factor (BCF) is well established for a chemical, it may be possible to extrapolate to an equivalent medium concentration for a given topical dose. The permeability data indicate that the toxicants penetrate into the eggs at levels well correlated to their octanol-water partition coefficients, suggesting passive transport, even in the presence of a membrane permeable carrier. Finally, the field studies demonstrate that the MELA approach is very useful for evaluating the relative toxic/teratogenic potential of numerous site, simultaneously. The data from the MELA treatments often correlated well with other biological assays and with chemical data on a site specific basis.« less

Schistosoma mansoni Phosphoenolpyruvate Carboxykinase, a Novel Egg Antigen: Immunological Properties of the Recombinant Protein and Identification of a T-Cell Epitope

PubMed Central

Asahi, Hiroko; Osman, Ahmed; Cook, Rosemary M.; LoVerde, Philip T.; Stadecker, Miguel J.

2000-01-01

In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4+ T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4+ Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4+ Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4+ Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4+ Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease. PMID:10816489

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila.

PubMed

Mukai, Masanori; Kato, Hirotaka; Hira, Seiji; Nakamura, Katsuhiro; Kita, Hiroaki; Kobayashi, Satoru

2011-01-01

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Inference of genetic network of Xenopus frog egg: improved genetic algorithm.

PubMed

Wu, Shinq-Jen; Chou, Chia-Hsien; Wu, Cheng-Tao; Lee, Tsu-Tian

2006-01-01

An improved genetic algorithm (IGA) is proposed to achieve S-system gene network modeling of Xenopus frog egg. Via the time-courses training datasets from Michaelis-Menten model, the optimal parameters are learned. The S-system can clearly describe activative and inhibitory interaction between genes as generating and consuming process. We concern the mitotic control in cell-cycle of Xenopus frog egg to realize cyclin-Cdc2 and Cdc25 for MPF activity. The proposed IGA can achieve global search with migration and keep the best chromosome with elitism operation. The generated gene regulatory networks can provide biological researchers for further experiments in Xenopus frog egg cell cycle control.

GEMINI-TITAN (GT)-3 - WEIGHTLESSNESS EXPERIMENT - AMES RESEARCH CENTER (ARC), CA

NASA Image and Video Library

1965-03-01

S65-18762 (March 1965) --- Effects of the weightless environment on cell division, the basic growth process for living tissue, will be studied during the Gemini-Titan 3 flight scheduled for March 23, 1965. A spiny black sea urchin (upper left) is stimulated by mild electric shock or potassium chloride. As a result it sheds many thousands of eggs. When fertilized, these eggs become actively dividing cells very similar in basic processes to cells of other animals, including humans. These pictures show stages of cell division. At upper right is a single cell; at lower right cell divisions have produced many cells. Cell photos are magnified about 700 times, and all cells shown are too small to be seen by the naked eye. (Photos at upper right and lower left are of sea urchin eggs. Group of cells at lower right are from a sand dollar, which like the sea urchin, is an Echinoderm. Its eggs are virtually identical and are used interchangeably with those of the sea urchin in NASA Ames Center weightlessness experiments.) The Gemini experiment will involve cell division like that shown here. This will take place during several hours of weightlessness aboard the Gemini spacecraft. The experiment will be flown back to laboratories at Cape Kennedy after spacecraft recovery. It has been designed so that any abnormal cell division found by postflight analysis should suggest that the weightless environment has effects on individual cells. This might mean hazards for prolonged periods of manned spaceflight.

Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter?

PubMed

Wang, Rui

2002-11-01

Bearing the public image of a deadly "gas of rotten eggs," hydrogen sulfide (H2S) can be generated in many types of mammalian cells. Functionally, H2S has been implicated in the induction of hippocampal long-term potentiation, brain development, and blood pressure regulation. By acting specifically on KATP channels, H2S can hyperpolarize cell membranes, relax smooth muscle cells, or decrease neuronal excitability. The endogenous metabolism and physiological functions of H2S position this gas well in the novel family of endogenous gaseous transmitters, termed "gasotransmitters." It is hypothesized that H2S is the third endogenous signaling gasotransmitter, besides nitric oxide and carbon monoxide. This positioning of H2S will open an exciting field-H2S physiology-encompassing realization of the interaction of H2S and other gasotransmitters, sulfurating modification of proteins, and the functional role of H2S in multiple systems. It may shed light on the pathogenesis of many diseases related to the abnormal metabolism of H2S.

Reduced egg shell permeability affects embryonic development and hatchling traits in Lycodon rufozonatum and Pelodiscus sinensis.

PubMed

Tang, Wenqi; Zhao, Bo; Chen, Ye; DU, Weiguo

2018-01-01

The response of embryos to unpredictable hypoxia is critical for successful embryonic development, yet there remain significant gaps in our understanding of such responses in reptiles with different types of egg shell. We experimentally generated external regional hypoxia by sealing either the upper half or bottom half of the surface area of eggs in 2 species of reptiles (snake [Lycodon rufozonatum] with parchment egg shell and Chinese soft-shelled turtle [Pelodiscus sinensis] with rigid egg shell), then monitored the growth pattern of the opaque white patch in turtle eggs (a membrane that attaches the embryo to the egg shell and plays an important role in gas exchange), the embryonic heart rate, the developmental rate and the hatchling traits in turtle and snake eggs in response to external regional hypoxia. The snake embryos from the hypoxia treatments facultatively increased their heart rate during incubation, and turtle embryos from the upper-half hypoxia treatment enhanced their growth of the opaque white patch. Furthermore, the incubation period and hatching success of embryos were not affected by the hypoxia treatment in these 2 species. External regional hypoxia significantly affected embryonic yolk utilization and offspring size in the snake and turtle. Compared to sham controls, embryos from the upper-half hypoxia treatment used less energy from yolk and, therefore, developed into smaller hatchlings, but embryos from the bottom-half hypoxia treatment did not. © 2017 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

Sexually dimorphic tridimensionally preserved pterosaurs and their eggs from China.

PubMed

Wang, Xiaolin; Kellner, Alexander W A; Jiang, Shunxing; Wang, Qiang; Ma, Yingxia; Paidoula, Yahefujiang; Cheng, Xin; Rodrigues, Taissa; Meng, Xi; Zhang, Jialiang; Li, Ning; Zhou, Zhonghe

2014-06-16

The pterosaur record is generally poor, with little information about their populations, and pterosaur eggs are even rarer, with only four isolated and flattened eggs found to date. We report here a population of a new sexually dimorphic pterosaur species (Hamipterus tianshanensis gen. et sp. nov.), with five exceptionally well-preserved three-dimensional eggs, from the Early Cretaceous deposit in northwestern China. About 40 male and female individuals in total were recovered, but the actual number associated might be in the hundreds. All of the discovered skulls have crests, which exhibit two different morphologies in size, shape, and robustness. The eggs show pliable depressions with cracking and crazing on the outer surface. The eggshell, observed by scanning electron microscopy and energy-dispersive spectroscopy, comprises a thin calcareous external hard shell followed by a soft membrane. These fossils shed new light on the reproductive strategy, ontogeny, and behavior of pterosaurs. The cranial crests show sexually dimorphic morphologies, with presumed males and females differing in crest size, shape, and robustness. Ontogenetic variation is reflected mainly in the expansion of the rostrum. The eggs have some external rigidity of the general pliable eggshell, and the microstructure of the eggshell is similar to that of some modern "soft" snake eggs. We suggest that this new pterosaur nested in colonies and thus exhibited gregarious behavior, a possible general trend for at least derived pterodactyloid pterosaurs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila.

PubMed

Andersen, Darcy; Horne-Badovinac, Sally

2016-04-15

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers consist of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a 'molecular corset' mechanism, whereby structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. Here, we provide evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ. © 2016. Published by The Company of Biologists Ltd.

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila

PubMed Central

Andersen, Darcy; Horne-Badovinac, Sally

2016-01-01

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers consist of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a ‘molecular corset’ mechanism, whereby structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. Here, we provide evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ. PMID:26952985

Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms

PubMed Central

Steiner, Jessica Kathryne; Tasaki, Junichi; Rouhana, Labib

2016-01-01

Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova. PMID:27149082

Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms.

PubMed

Steiner, Jessica Kathryne; Tasaki, Junichi; Rouhana, Labib

2016-05-01

Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova.

Local neuropeptide signaling modulates serotonergic transmission to shape the temporal organization of C. elegans egg-laying behavior

PubMed Central

Banerjee, Navonil; Bhattacharya, Raja; Francis, Michael M.

2017-01-01

Animal behaviors are often composed of distinct alternating behavioral states. Neuromodulatory signals are thought to be critical for establishing stable behavioral states and for orchestrating transitions between them. However, we have only a limited understanding of how neuromodulatory systems act in vivo to alter circuit performance and shape behavior. To address these questions, we have investigated neuromodulatory signaling in the context of Caenorhabditis elegans egg-laying. Egg-laying activity cycles between discrete states–short bursts of egg deposition (active phases) that alternate with prolonged quiescent periods (inactive phases). Here using genetic, pharmacological and optogenetic approaches for cell-specific activation and inhibition, we show that a group of neurosecretory cells (uv1) located in close spatial proximity to the egg-laying neuromusculature direct the temporal organization of egg-laying by prolonging the duration of inactive phases. We demonstrate that the modulatory effects of the uv1 cells are mediated by peptides encoded by the nlp-7 and flp-11 genes that act locally to inhibit circuit activity, primarily by inhibiting vesicular release of serotonin from HSN motor neurons. This peptidergic inhibition is achieved, at least in part, by reducing synaptic vesicle abundance in the HSN motor neurons. By linking the in vivo actions of specific neuropeptide signaling systems with the generation of stable behavioral outcomes, our study reveals how cycles of neuromodulation emanating from non-neuronal cells can fundamentally shape the organization of a behavioral program. PMID:28384151

Assessment of bovine herpesvirus 4 based vector in chicken.

PubMed

Donofrio, Gaetano; Manarolla, Giovanni; Ravanetti, Lara; Sironi, Giuseppe; Cavirani, Sandro; Cabassi, Clotilde Silvia; Flammini, Cesidio Filippo; Rampin, Tiziana

2008-03-01

The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the ability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, monkeys, hamsters, rats, mice, and chickens. In this report, the feasibility to use BoHV-4 based vector in chicken was investigated. Although BoHV-4 was able to replicate, leading to a cytopathic effect in a chicken cell line and infect the chorion allantoic membrane of embryonated eggs, however it was not pathogenic even when a large dose of virus was injected into the chicken. An immune response could be produced against heterologous antigen delivered by a recombinant BoHV-4. These data suggest the feasibility of using BoHV-4 based vector for vaccination purposes in chickens.

GAMMA IRRADIATION OF SALMONELLA SPECIES IN FROZEN WHOLE EGG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Comer, A.G.; Anderson, G.W.; Garrard, E.H.

1963-06-01

A study was undertaken to establish the survival pattern of 18 Salmonella species in frozen whole egg exposed to gamma radiation from a Co/sup 60/ source as part of a program to investigate the possibilities of using radiation for the destruction of micro-organisms in foods. A definite variation in sensitivity to radiation was found among these 18 species commonly occurring in frozen egg. The dose levels, applied at a dose rate of 0.491 Mrad/hr, required for a 10/sup 7/-fold reduction of these species ranged from 0.36 to 0.54 Mrad. Variation in sensitivity to radiation was also detected with three culturesmore » of S. pullorum that appeared similar except in origin. Variation in sensitivity within all species of Salmonella studied apparently indicates that no one species can be considered most resistant. These studies reveal that a level of 0.54 Mrad of gamma radiation would reduce the salmonellae to a safe level in frozen egg. However, this is based on the supposition that 10/sup 5/ cells is a reasonable number to consider necessary for the initiation of salmonellosis. The most heavily contaminated frozen egg melange examined contained approximates 2 x 10/sup 5/ cells of salmonellae/ml. Of these, any process giving a 10/sup 7/-fold reduction would leave only 2 x 10/sup -2/ cells/ml, i.e., 2 cells/100 ml or about 10 cells per lb. Apparently it would be necessary to consume several tons of uncooked melange to obtain an infective dose and therefore the 10/sup 7/-fold reduction is adequate from the viewpoint of the public health hazard. Although off-flavors develop in frozen whole egg irradiated with 0.5 Mrad, they disappear on storage and the irradiated product was found acceptable for commercial use. Thus irradiation of frozen whole egg melange with 0.54 Mrad would not be expected to markedly increase the unfavorable alterations in the product, indicating that gamma radiation possesses a distinct potential for removing Salmonella from frozen whole egg melange. (BBB)« less

[PKA-regulated phosphorylation status of S149 and S321 sites of CDC25B inhibits mitosis of fertilized mouse eggs].

PubMed

Xiao, Jian-Ying; Liu, Chao; Sun, Xiao-Han; Yu, Bing-Zhi

2012-02-25

To further test whether protein kinase A (PKA) can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase (22 h after hCG injection) were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 (0-50 μmol/L), the G(2) phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 μmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B (CDC25B) on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor (MPF) activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 μmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development of mouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G(2)/M transition in the mitotic cell cycle of fertilized mouse eggs.

Using a novel micro-sampling technique to monitor the effects of methylmercury on the eggs of wild birds

USGS Publications Warehouse

Klimstra, J.D.; Stebbins, K.R.; Heinz, G.H.

2007-01-01

Methylmercury is the predominant chemical form of mercury reported in the eggs of wild birds. The embryo is the life stage at which birds are most sensitive to methylmercury. Protective guidelines have been based largely on captive-breeding studies done with chickens (Gallus domesticus), mallards (Anas platyrhynchos), and ring-necked pheasants (Phasianus colchicus). Typically these studies are cost and time prohibitive. In the past, researchers have used either egg injections or the ?sample egg? technique to determine contaminant effects on bird eggs. Both techniques have their limitations. As an alternative to the above methods and because most of the methylmercury is found in the albumen we have developed a novel, less invasive technique, to micro-sample the albumen of eggs in the field. An albumen sample would be analyzed and then compared to the hatching success of that egg. Using the micro-sampling procedure, the egg is oriented with the blunt end up and the pointed end down. A vent hole is drilled at the top to relieve pressure. Approximately one third up from the bottom, a withdrawal site is drilled just until the inner shell membrane is exposed. A syringe with a 21 or 18 gauge needle is gently inserted just into the egg and approximately 200?300?l of albumen is removed. Almost concurrently this site and then the vent are sealed. Thus far we have experimented with both chicken and mallard eggs in the laboratory. We sampled chicken eggs at days 0 and 3 of incubation with a hatching success of 76% and 70%, respectively. Neither group was significantly different from control eggs (P=0.52, 0.54). Field studies are in progress using this technique in which birds are allowed to incubate their own eggs. We envision micro-sampling to be a tool that researchers and managers could use in the field to determine the effects of mercury or other contaminants in bird populations. Micro-sampling would reduce the impact on the sampled population and could be used to monitor sensitive species without impacting reproduction and recruitment.

Effects of Egg Shell Membrane Hydrolysates on Anti-Inflammatory, Anti-Wrinkle, Anti-Microbial Activity and Moisture-Protection.

PubMed

Yoo, Jinhee; Park, Kimoon; Yoo, Youngji; Kim, Jongkeun; Yang, Heejin; Shin, Youngjae

2014-01-01

This study was conducted to examine the effects of eggshell membrane hydrolysates (ESMH) on the anti-inflammatory, anti-wrinkle, anti-microbial activity, and moisture-protection for cosmetic use. Whole ESMH (before fractionation), and fraction I (>10 kDa), fraction II (3-10 kDa), and fraction III (<3 kDa) of the hydrolysates were assessed in this experiment. As lipopolysaccharide (LPS) and IFN-γ caused the inflammation on Raw264.7 cell, whole ESMH and fraction I showed to be effective in inhibiting the induction of cell inflammation depending on the concentration, and also showed outstanding effect to suppress the skin inflammation. Fraction I inhibited collagenase and elastase activities to a greater extent than the other fractions, while all fractions had antibiotic effects at concentrations of 10 mg/disc and 20 mg/disc. In addition, it showed the moisture protection effects of skin on the holding amount and losing amount of moisture in upper-inner arm of the human body with a relatively low loss rate in skin, which confirmed that the hydrolyzed fractions of ESM helps to form the superior protective layer of moisture. It was concluded that ESMH fractions with different molecular weights, especially the 10 kDa fraction, have anti-lipopolysaccharide, anti-IFN-γ-induced inflammation, anti- collagenase and elastase activities, and thus can be used as a cosmetic agent to protect skin.

Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses.

PubMed

Chiapponi, Chiara; Zanni, Irene; Garbarino, Chiara; Barigazzi, Giuseppe; Foni, Emanuela

2010-01-01

Influenza A virus isolation is undertaken routinely in embryonated chicken eggs, but to improve virus detection various cell lines can be used. The CACO-2 cell line was compared to the MDCK cell line and embryonated chicken eggs for the isolation of H1N1, H1N2, H3N2 swine influenza A virus subtypes from clinical specimens. From 2006 to 2008, 104 influenza A samples found positive by PCR from 42 respiratory outbreaks in Italian swine farms were examined by virus isolation. Sixty swine influenza A viruses were isolated (16 H1N1, 28 H1N2 and 16 H3N2) and their growth behaviour on the different substrates was examined. 16/16 H1N1, 28/28 H1N2 and 8/16 of H3N2 viruses were isolated from the CACO-2 cell line, while 7/16 H1N1, 3/28 H1N2 and 16/16 H3N2 viruses were isolated using embryonated chicken eggs. Only 9/16 H1N1, 1/28 H1N2 and 6/16 H3N2 viruses replicated in MDCK cells. A link was found between viral hemagglutinin and the isolation rate on the various substrates. The CACO-2 line was statistically more sensitive (Fisher's exact test, p<0.01) compared to the MDCK cells and embryonated chicken eggs for the isolation of H1N1 and H1N2 subtypes. In contrast influenza A H3N2 virus was isolated more readily in embryonated chicken eggs than in cultured cells (Fisher's exact test, p<0.01).

Delayed fertilization of anuran amphibian (Xenopus) eggs leads to reduced numbers of primordial germ cells

NASA Technical Reports Server (NTRS)

Wakahara, M.; Neff, A. W.; Malacinski, G. M.

1984-01-01

Several media were tested for the extent to which they promoted high fertilization efficiencies in ovulated, stripped Xenopus eggs. One medium was selected for maintaining eggs in a 'delayed fertilization' (DelF) condition. DelF eggs displayed several unusual characteristics, including shift of the center of gravity, prominent sperm entrance site, and occasional polyspermy. The frequency of normal pattern formation varied according to the length of time eggs were maintained in the DelF condition. Various developmental abnormalities were observed during gastrulation, neurulation, and organogenesis. Most abnormalities appeared, however, to be related to morphogenesis of the endoderm. Primordial germ cell (PGC) development was examined in DelF eggs which displayed normal external morphological features at the swimming tadpole stage. PGC counts were usually normal in short-duration (eg, 5 hr) DelF eggs, but frequently substantially reduced or completely diminished in longer-duration (eg, 25h) tadpoles. Six spawnings were compared and shown to exhibit considerable variability in fertility, morphogenesis, and PGC development. Yolk platelet shifts and developmental parameters were examined in two additional spawnings. The subcortical cytoplasm in which the germ plasm is normally localized appeared to be disrupted in longer duration DelF eggs. That observation may account for low PGC counts in DelF tadpoles.

Factors affecting thermal resistance of Salmonella enterica serovar enteritidis ODA 99-30581-13 in shell egg contents and use of heat-ozone combinations for egg pasteurization.

PubMed

Perry, Jennifer J; Yousef, Ahmed E

2013-02-01

Infection of laying hens with Salmonella enterica serovar Enteritidis leads to deposition of the pathogen into the albumen or yolk of forming eggs. Heat treatment can inactivate internalized Salmonella Enteritidis in shell eggs, but factors such as the nature and location of contamination may influence the efficacy of thermal treatments. In the current research, natural contamination was mimicked by introducing small inocula of Salmonella Enteritidis into different locations of shell eggs and incubating inoculated eggs. These pathogen-containing eggs were heated at 57°C for 40 min, and temperature within eggs was monitored at the locations of inocula. Comparison of inactivation at equivalent internal temperatures revealed similar levels of lethality regardless of inoculum location. Refrigeration between incubation and heat treatment did not increase thermal resistance of cells in albumen but decreased cell inactivation in yolk. Sequential application of heat and gaseous ozone allows for the development of a process capable of decontaminating shell eggs with minimal thermal treatment and impact on egg quality. Inoculated eggs were subjected to (i) an immersion heating process similar to that used in commercial pasteurization or (ii) immersion heating, at reduced duration, followed by vacuum (50.8 kPa) and treatment with ozone gas (maximum 160 g/m(3)) under pressure (∼187.5 kPa). All treatments tested produced greater than 5-log inactivation, which is required for "pasteurization" processes. Differences were observed in the visual quality of eggs depending on treatment parameters. Application of ozone subsequent to heating allows for a significant reduction in heating time without decreasing process lethality.

Sperm fertility and viability following 48h of refrigeration: evaluation of different extenders for the preservation of bull semen in liquid state.

PubMed

Crespilho, A M; Nichi, M; Guasti, P N; Freitas-Dell'Aqua, C P; Sá Filho, M F; Maziero, R R; Dell'aqua, J A; Papa, F O

2014-05-01

Two experiments were conducted to compare the effectiveness of different extenders conventionally used for semen cryopreservation to maintain the viability and fertility of cooled bull semen. In Experiment 1, sperm samples obtained from 20 Nellore bulls were preserved at 5°C for 48h using two extenders containing 20% of egg yolk [Tris (TRIS-R) and Botu-Bov(®) (BB)] and another composed of 1% soy lecithin [Botu-Bov(®)-Lecithin (BB-L)] as substitutes for animal origin products. The samples were evaluated at 6, 24 and 48h for plasma and acrosomal membrane integrity, quantification of thiobarbituric acid reactive substances (ng of TBARS/10(8) cells) and sperm motility parameters by computer-assisted semen analysis (CASA). In Experiment 2, pregnancy rate (P/AI) of 973 fixed-time artificially inseminated Nellore cows were compared when cows were inseminated with conventionally cryopreserved semen in TRIS-egg yolk glycerol (TRIS-C Control, n=253) or semen cooled for 48h in TRIS-R (n=233), BB (n=247) or BB-L (n=240). Although none of the extenders used was effective on maintaining total progressive motility and cellular integrity throughout the 48-h of the refrigeration period (P<0.01), BB-L conferred greater protection against oxidative stress (P<0.05) than egg yolk-based medias. The P/AI for semen samples preserved in TRIS-C, TRIS-R, BB and BB-L were 39.92(a), 25.32(b), 26.32(b) and 33.33(ab), respectively. These results demonstrate that the three conventional extenders used for semen cryopreservation do not provide the protection required to maintain bull semen fertility under refrigeration for a 48-h period, resulting in reduced pregnancy rates. However, the use of lecithin-based medium instead of egg yolk results in greater protection against lipid peroxidation, producing P/AI results comparable to those obtained using frozen semen. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrochemical immunosensor based on ensemble of nanoelectrodes for immunoglobulin IgY detection: application to identify hen's egg yolk in tempera paintings.

PubMed

Bottari, Fabio; Oliveri, Paolo; Ugo, Paolo

2014-02-15

A nanostructured electrochemical biosensor for detecting proteins of interest in work of art, in particular in tempera paintings, is presented. To determine egg yolk we focus here on the determination of immunoglobulin IgY. The transducers are nanoelectrode ensembles (NEEs), prepared via membrane templated electroless deposition of gold. Because of their geometrical and diffusion characteristics, NEEs are characterized by significantly low detection limits, moreover they display the capability of capturing proteins by interaction with the polycarbonate membrane of the NEE. At first, the proteic component of the paint is extracted by ultrasonication in an aqueous buffer, then IgY is captured by incubation on the NEE. The immunoglobulin is detected by treatment with anti-IgY labeled with horse radish peroxidase (Anti-IgY-HRP). The binding of the Anti-IgY-HRP is detected by recording the electrocatalytic signal caused by addition of H2O2 and methylene blue. The sensor detection capabilities are tested by analyzing both paint models, prepared in the lab, and real samples, from paintings of the XVIII-XX century. Multivariate exploratory analysis is applied to classify the voltammetric patterns, confirming the capability to differentiate egg-yolk tempera from other kind of tempera binders as well as from acrylic or oil paints. © 2013 Elsevier B.V. All rights reserved.

Dimethyleacetamide improves the cryosurvivability of Indian red jungle fowl (Gallus gallus murghi) sperm.

PubMed

Rakha, B A; Ansari, M S; Akhter, S; Zafar, Z; Naseer, A; Hussain, I; Santiago-Moreno, J; Blesbois, E

2017-11-01

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

PubMed

Brito, Maíra M; Lúcio, Cristina F; Angrimani, Daniel S R; Losano, João Diego A; Dalmazzo, Andressa; Nichi, Marcílio; Vannucchi, Camila I

2017-01-02

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Binding of glycated ovocystatin to rat renal brush border membranes.

PubMed

Golab, Krzysztof; Gburek, Jakub; Konopska, Bogusława; Krotkiewski, Hubert; Warwas, Maria

2013-10-01

Glycated proteins are considered as one of the factors involved in the pathogenesis of diabetic complications, including nephropathy. These proteins are formed endogenously under conditions of hyperglycemia, as well as being provided with food containing sugars, which was subjected to high temperature. Examples are egg products. One of the proteins found in eggs in a relatively high concentration is chicken cystatin (ovocystatin). It is now believed that some proteins can passage the intestinal epithelium by transcytosis directly into the bloodstream. Thus, glycated protein present in food can be an additional source of glycotoxins. The aim of this study was to compare the affinity of native and glycated cystatin to the brush border membranes of rat kidney. Kinetic analysis was performed with surface plasmon resonance technique using sensor chip L1. Dissociation constants for native and glycated cystatin (Kd ) were 2.76 μmol/L and 3.82 μmol/L, respectively. The results of our study indicate that glycation only slightly affects binding of cystatin to brush border membranes. This suggests that glycated cystatin and other glycated proteins may also be efficiently taken up in the kidney proximal tubule. The observation may be important for understanding the mechanisms involved in the development of diabetic nephropathy. © 2013 Japanese Society of Animal Science.

Stabilized filter-supported bilayer lipid membranes (BLMs) for automated flow monitoring of compounds of clinical, pharmaceutical, environmental and industrial interest

PubMed Central

Siontorou, Christina G.

1997-01-01

This paper describes the results of analytical applications of electrochemical biosensors based on bilayer lipid membranes (BLMs) for the automated rapid and sensitive flow monitoring of substrates of hydrolytic enzymes, antigens and triazine herbicides. BLMs, composed of mixtures of egg phosphatidylcholine (egg PC) and dipalmitoylphosphatidic acid (DPPA), were supported on ultrafiltration membranes (glass microfibre or polycarbonate filters) which were found to enhance their stability for flow experiments. The proteins (enzymes, antibodies) were incorporated into a floating lipid matrix at an air-electrolyte interface, and then a casting procedure was used to deliver the lipid onto the filter supports for BLM formation. Injections of the analyte were made into flowing streams of the carrier electrolyte solution and a current transient signal was obtained with a magnitude related to the analyte concentration. Substrates of hydrolytic enzyme reactions (acetylcholine, urea and penicillin) could be determined at the micromolar level with a maximum rate of 220 samples/h, whereas antigens (thyroxin) and triazine herbicides (simazine, atrazine and propazine) could be monitored at the nanomolar level in less than 2 min. The time of appearance of the transient response obtained for herbicides was increased to the order of simazine, atrazine and propazine which has permitted analysis of these triazines in mixtures. PMID:18924789

Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis

PubMed Central

Nawaratna, Sujeevi S. K.; Gobert, Geoffrey N.; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P.; Jones, Malcolm K.

2015-01-01

Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

Antioxidant, Antiproliferative, and Antiangiogenesis Effects of Polyphenol-Rich Seaweed (Sargassum muticum)

PubMed Central

Namvar, Farideh; Mohamad, Rosfarizan; Baharara, Javad; Zafar-Balanejad, Saeedeh; Fargahi, Fahimeh; Rahman, Heshu Sulaiman

2013-01-01

In the present study, we evaluated the effect of brown seaweeds Sargassum muticum methanolic extract (SMME), against MCF-7 and MDA-MB-231 breast cancer cell lines proliferation. This algae extract was also evaluated for reducing activity and total polyphenol content. The MTT assay results indicated that the extracts were cytotoxic against breast cancer cell lines in a dose-dependent manner, with IC50 of 22 μg/ml for MCF-7 and 55 μg/ml for MDA-MB-231 cell lines. The percentages of apoptotic MCF-7-treated cells increased from 13% to 67% by increasing the concentration of the SMME. The antiproliferative efficacy of this algal extract was positively correlated with the total polyphenol contents, suggesting a causal link related to extract content of phenolic acids. Cell cycle analysis showed a significant increase in the accumulation of SMME-treated cells at sub-G1 phase, indicating the induction of apoptosis by SMME. Further apoptosis induction was confirmed by Hoechst 33342 and AO/PI staining. Also SMME implanted in vivo into fertilized chicken eggs induced dose-related antiangiogenic activity in the chorioallantoic membrane (CAM). Our results imply a new insight on the novel function of Sargassum muticum polyphenol-rich seaweed in cancer research by induction of apoptosis, antioxidant, and antiangiogenesis effects. PMID:24078922

Mechanisms underlying differential food allergy response to heated egg.

PubMed

Martos, Gustavo; Lopez-Exposito, Ivan; Bencharitiwong, Ramon; Berin, M Cecilia; Nowak-Węgrzyn, Anna

2011-04-01

Egg white proteins are usually subjected to heating, making them edible for the majority of children with egg allergy. We sought to investigate the underlying mechanisms responsible for the reduced allergenicity displayed by heat-treated egg white allergens. C3H/HeJ mice were orally sensitized with ovalbumin (OVA) or ovomucoid and challenged with native or heated proteins to evaluate their allergenicity. Immunoreactivity was assessed by immunoblotting using sera from children with egg allergy. In vitro gastrointestinal digestion of native and heated OVA and ovomucoid was studied by SDS-PAGE and liquid chromatography. Intestinal uptake of intact native and heated OVA and ovomucoid by human intestinal epithelial (Caco-2) cells was investigated. Rat basophil leukemia cells passively sensitized with mouse serum and human basophils passively sensitized with serum from children with egg allergy were used to assess the effector cell activation by heated, digested, and transported OVA and ovomucoid. Heated OVA and ovomucoid did not induce symptoms of anaphylaxis in sensitized mice when administered orally. Heating did not completely destroy IgE-binding capacity of OVA or ovomucoid but enhanced in vitro digestibility of OVA. Digestion of both OVA and ovomucoid diminished mediator release in rat basophil leukemia assay and basophil activation. Heating of allergens prevented transport across human intestinal epithelial cells in a form capable of triggering basophil activation or T-cell activation. Heat treatment reduces allergenicity of OVA and ovomucoid. This is partially a result of the enhanced gastrointestinal digestibility of heated OVA and the inability of heated OVA or ovomucoid to be absorbed in a form capable of triggering basophils. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

PubMed Central

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca2+-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca2+-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach. PMID:26112481

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface.

PubMed

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

Development of a membrane adsorber based capture step for the purification of yellow fever virus.

PubMed

Pato, Tânia P; Souza, Marta Cristina O; Silva, Andréa N M R; Pereira, Renata C; Silva, Marlon V; Caride, Elena; Gaspar, Luciane P; Freire, Marcos S; Castilho, Leda R

2014-05-19

Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2±30.3%) and efficient DNA clearance (0.9±0.3 ng/dose). Copyright © 2014 Elsevier Ltd. All rights reserved.

Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

PubMed

Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

2010-01-01

Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

The impact of humic and fulvic acids on the dynamic properties of liposome membranes: the ESR method.

PubMed

Man, Dariusz; Pisarek, Izabella; Braczkowski, Michał; Pytel, Barbara; Olchawa, Ryszard

2014-06-01

This paper presents the results of research on the influence of two fractions of humic substances (HS): fulvic acids (FA) and humic acids (HA), as a function of concentration, on the liposome membranes formed from egg yolk lecithin (EYL). The concentration of HS in relation to EYL changed from 0% to 10% by weight. The influence of HS on various areas of membranes: interphase water-lipid, in the lipid layer just below the polar part of the membrane and in the middle of the lipid bilayer, was investigated by different spin labels (TEMPO, DOXYL 5, DOXYL 16). The study showed that HA slightly decreased the fluidity of the analyzed membranes on the surface layer, while FA significantly liquidated the center of the lipid bilayer. The strong effect of both fractions of HS on the concentration of free radicals as a function of time was also described.

Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig.

PubMed

Liu, Ying; Ostrup, Olga; Li, Rong; Li, Juan; Vajta, Gábor; Kragh, Peter M; Schmidt, Mette; Purup, Stig; Hyttel, Poul; Klærke, Dan; Callesen, Henrik

2014-08-01

In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

GEMINI-TITAN (GT)-III - WEIGHTLESSNESS EXPERIMENT - AMES RESEARCH CENTER (ARC), CA

NASA Image and Video Library

1965-03-19

S65-18766 (March 1965) --- Diagram of experiment planned for the Gemini-Titan 3 mission scheduled on March 23, 1965, to find out if there are effects of weightlessness on individual living cells. The round canister (top) shows the experiment package. It will contain eight identical chambers, each with sections of sperm, eggs and fixative. Cells are eggs of the spiny, black sea animal, the sea urchin. Bottom panel shows the three stages of each chamber. From left in the first stage, sperm, eggs and fixative are separated. By turning the handle, astronauts will fertilize a certain portion of the eggs, which will begin to divide. At 20 minutes after launch, further turns of the handle will force fixative into two chambers and stop cell division. At 70 minutes after launch, cell division in four more chambers will be stopped, and just prior to re-entry, growth of the remaining two chambers will be terminated by a turn of the handle. This system will allow study after the flight of how cells divided after various time periods in weightlessness. Abnormalities would suggest weightlessness effects on living tissue and possible hazard to prolonged manned spaceflight.

DECOUPLING OF PROTEIN AND RNA SYNTHESIS DURING DEUTERIUM PARTHENOGENESIS IN SEA URCHIN EGGS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gross, P.R.; Spindel, W.; Cousineau, G.H.

1963-10-29

The parthenogenetic activation of cell division and suppression of nucleic acid synthesis by deuterium in eggs of sea urchins was investigated. D/ sub 2/O treatment was found to evoke a high rate of protein synthesis in the eggs that was maintained for several hours. However, eggs whose protein synthesis was activated and that were making labeled cytasters showed no increment in RNA synthesis over controls. (P.C.H.)

Egg shell membrane improves immunity of post hatch poultry: A aparadigm for nutritional immunomodulation

USDA-ARS?s Scientific Manuscript database

Improving natural immunity can reduce susceptibility to infection, improving animal health and eliminating prophylactic use of antibiotics. However, there are very few affective means to enhance natural immunity to a wide range of infections. Considering the natural diets of most birds and mammals,...

Effect of high pressure treatment on liquid whole egg

NASA Astrophysics Data System (ADS)

Németh, Csaba; Dalmadi, István; Mráz, Balázs; Friedrich, László; Zeke, Ildikó; Juhász, Réka; Suhajda, Ágnes; Balla, Csaba

2012-06-01

In our tests, we artificially infected liquid whole egg samples with Salmonella enteritidis, Listeria monocytogenes, and Staphylococcus aureus bacteria, and then treated the samples in "Food Lab900" high hydrostatic pressure (HHP) instrument for 3-17 min at 200-400 MPa. Subsequently, the change of the viable cell count of the specific bacteria has been tested. In addition to the samples infected with various bacteria, non-infected samples were also treated in each test and the change in viable cell count, colour and viscosity of the samples upon the effect of the treatment. In summary, it can be concluded that in each test of our investigations, the viable cell count of S. enteritidis critical for egg products is reduced significantly, while the reduction of the total viable cell count was around two magnitudes. Additionally, based on our results, microbial destruction, reduction of enthalpy (denaturation of egg white) caused by the treatment at HPP, and colour change are primarily affected by the pressure level, while the changes in rheological properties are also significantly affected by the duration of high pressure treatment (p<0.05).

Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

PubMed

McCormack, M; Brecher, P

1987-06-15

Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes.

Cell cycle in ascidian eggs and embryos.

PubMed

McDougall, Alex; Chenevert, Janet; Lee, Karen W; Hebras, Celine; Dumollard, Remi

2011-01-01

In ascidians the cell cycle machinery has been studied mainly in oocytes while ascidian embryos have been used to dissect the mechanism that controls asymmetric cell division (ACD). Here we overview the most specific and often exceptional points and events in cell cycle control in ascidian oocytes and early embryos. Mature stage IV eggs are arrested at metaphase I due to cytostatic factor (CSF). In vertebrates, unfertilized eggs are arrested at metaphase II by CSF. Meta II-CSF is mediated by the Mos/MEK/MAPK/Erp1 pathway, which inhibits the ubiquitin ligase APC/C(cdc20) preventing cyclin B destruction thus stabilizing MPF activity. CSF is inactivated by the fertilization Ca(2+) transient that stimulates the destruction of Erp1 thus releasing APC/C(cdc20) from inhibition. Although many of the components of CSF are conserved between the ascidian and the vertebrates, the lack of Erp1 in the ascidians (and indeed other invertebrates) is notable since the Mos/MAPK pathway nonetheless mediates Meta I-CSF. Moreover, since the fertilization Ca(2+) transient targets Erp1, it is not clear how the sperm-triggered Ca(2+) transient in ascidians (and again other invertebrates) stimulates cyclin B destruction in the absence of Erp1. Nonetheless, like mammalian eggs, sperm trigger a series of Ca(2+) oscillations that increases the rate of cyclin B destruction and the subsequent loss of MAPK activity leading to meiotic exit in ascidians. Positive feedback from MPF maintains the Ca(2+) oscillations in fertilized ascidian eggs ensuring the eventual loss of MPF stimulating the egg-to-embryo transition. Embryonic cell cycles in the ascidian are highly stereotyped where both the rate of cell division and the orientation of cell division planes are precisely controlled. Three successive rounds of ACD generate two small posterior germ cell precursors at the 64 cell stage. The centrosome-attracting body (CAB) is a macroscopic cortical structure visible by light microscopy that causes these three rounds of ACD. Entry into mitosis activates the CAB causing the whole mitotic spindle to rotate and migrate toward the cortical CAB leading to a highly ACD whereby one small cell is formed that inherits the CAB and approximately 40 maternal postplasmic/PEM RNAs including the germ cell marker vasa.

Chick ex ovo culture and ex ovo CAM assay: how it really works.

PubMed

Dohle, Daniel S; Pasa, Susanne D; Gustmann, Sebastian; Laub, Markus; Wissler, Josef H; Jennissen, Herbert P; Dünker, Nicole

2009-11-30

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci. For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used(1-4), no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay(5,6), (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye(7-9), (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM(10-12), (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice(13) as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM(14). In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.

Separation of macromolecular proteins and rejection of toxic heavy metal ions by PEI/cSMM blend UF membranes.

PubMed

Kanagaraj, P; Nagendran, A; Rana, D; Matsuura, T; Neelakandan, S

2015-01-01

The charged surface modifying macromolecule (cSMM) was blended into the casting solution of poly(ether imide) (PEI) to prepare surface modified ultrafiltration membranes by phase inversion technique. The separation of proteins including bovine serum albumin, egg albumin, pepsin and trypsin was investigated by the fabricated membranes. On increasing cSMM content, solute rejection decreases whereas membrane flux increases. The pore size and surface porosity of the 5 wt% cSMM blend PEI membranes increases to 41.4 Å and 14.8%, respectively. Similarly, the molecular weight cut-off of the membranes ranged from 20 to 45 kDa, depending on the various compositions of the prepared membranes. The toxic heavy metal ions Cu(II), Cr(III), Zn(II) and Pb(II) from aqueous solutions were subjected to rejection by the prepared blended membrane with various concentration of polyethyleneimine (PETIM) as water soluble polymeric ligand. It was found that the rejection behavior of metal ion depends on the PETIM concentration and the stability complexation of metal ion with ligand. Copyright © 2014 Elsevier B.V. All rights reserved.

Human Cloning

DTIC Science & Technology

2006-07-20

parthenogenesis , to produce human embryos. ACT researchers obtained eggs from seven women, ages 24 to 32, who were paid $3,000 to $5,000. In the SCNT...cumulus cell nucleus began to divide but division stopped at the four-to-six-cell stage. In parthenogenesis , an egg cell is treated with chemicals...Human Subject Protection regulations] as of the date of enactment of this Act, that is derived by fertilization, parthenogenesis , cloning, or any

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm "Ming" lethal egg mutant.

PubMed

Chen, Anli; Gao, Peng; Zhao, Qiaoling; Tang, Shunming; Shen, Xingjia; Zhang, Guozheng; Qiu, Zhiyong; Xia, Dingguo; Huang, Yongping; Xu, Yunmin; He, Ningjia

2013-02-25

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation. Copyright © 2012 Elsevier B.V. All rights reserved.

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ostrup, Olga, E-mail: osvarcova@gmail.com; Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo; Norwegian Center for Stem Cell Research, Oslo

Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression.more » This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.« less

An evolutionary insight into the hatching strategies of pipefish and seahorse embryos.

PubMed

Kawaguchi, Mari; Nakano, Yuko; Kawahara-Miki, Ryouka; Inokuchi, Mayu; Yorifuji, Makiko; Okubo, Ryohei; Nagasawa, Tatsuki; Hiroi, Junya; Kono, Tomohiro; Kaneko, Toyoji

2016-03-01

Syngnathiform fishes carry their eggs in a brood structure found in males. The brood structure differs from species to species: seahorses carry eggs within enclosed brood pouch, messmate pipefish carry eggs in the semi-brood pouch, and alligator pipefish carry eggs in the egg compartment on abdomen. These egg protection strategies were established during syngnathiform evolution. In the present study, we compared the hatching mode of protected embryos of three species. Electron microscopic observations revealed that alligator pipefish and messmate pipefish egg envelopes were thicker than those of seahorses, suggesting that the seahorse produces a weaker envelope. Furthermore, molecular genetic analysis revealed that these two pipefishes possessed the egg envelope-digesting enzymes, high choriolytic enzyme (HCE), and low choriolytic enzyme (LCE), as do many euteleosts. In seahorses, however, only HCE gene expression was detected. When searching the entire seahorse genome by high-throughput DNA sequencing, we did not find a functional LCE gene and only a trace of the LCE gene exon was found, confirming that the seahorse LCE gene was pseudogenized during evolution. Finally, we estimated the size and number of hatching gland cells expressing hatching enzyme genes by whole-mount in situ hybridization. The seahorse cells were the smallest of the three species, while they had the greatest number. These results suggest that the isolation of eggs from the external environment by paternal bearing might bring the egg envelope thin, and then, the hatching enzyme genes became pseudogenized. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:XX-XX, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

PubMed

Yap, Jin Yan; Wirasinha, Rushika C; Chan, Anna; Howard, Debbie R; Goodnow, Christopher C; Daley, Stephen R

2018-02-07

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3 + regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes. © 2018 John Wiley & Sons Ltd.

A rapid, sensitive, and cost-efficient assay to estimate viability of potato cyst nematodes.

PubMed

van den Elsen, Sven; Ave, Maaike; Schoenmakers, Niels; Landeweert, Renske; Bakker, Jaap; Helder, Johannes

2012-02-01

Potato cyst nematodes (PCNs) are quarantine organisms, and they belong to the economically most relevant pathogens of potato worldwide. Methodologies to assess the viability of their cysts, which can contain 200 to 500 eggs protected by the hardened cuticle of a dead female, are either time and labor intensive or lack robustness. We present a robust and cost-efficient viability assay based on loss of membrane integrity upon death. This assay uses trehalose, a disaccharide present at a high concentration in the perivitelline fluid of PCN eggs, as a viability marker. Although this assay can detect a single viable egg, the limit of detection for regular field samples was higher, ≈10 viable eggs, due to background signals produced by other soil components. On the basis of 30 nonviable PCN samples from The Netherlands, a threshold level was defined (ΔA(trehalose) = 0.0094) below which the presence of >10 viable eggs is highly unlikely (true for ≈99.7% of the observations). This assay can easily be combined with a subsequent DNA-based species determination. The presence of trehalose is a general phenomenon among cyst nematodes; therefore, this method can probably be used for (for example) soybean, sugar beet, and cereal cyst nematodes as well.

Detection of ricin by using gold nanoclusters functionalized with chicken egg white proteins as sensing probes.

PubMed

Selvaprakash, Karuppuchamy; Chen, Yu-Chie

2017-06-15

Ricin produced from the castor oil plant, Ricinus communis, is a well-known toxin. The toxin comprises A and B chains. Ricin A chain can cause toxicity by inhibiting protein synthesis, and ricin B can bind to the galactose ligand on the cell membrane of host cells. Inhalation or ingestion of ricin may even lead to death. Therefore, rapid and convenient sensing methods for detecting ricin in suspicious samples must be developed. In this study, we generated protein encapsulated gold nanoclusters (AuNCs@ew) with bright photoluminescence by using chicken egg white proteins as starting materials to react with aqueous tetrachloroaurate. The generated nanoclusters, which were mainly composed of chicken ovalbumin-encapsulated AuNCs, can recognize ricin B because of the presence of Galβ(1→4)GlcNAc ligands on chicken ovalbumin. The generated conjugates of AuNCs@ew and ricin B were heavy and readily settled down under centrifugation (13,000rpm, 60min). Thus, bright spots resulting from the conjugates at the bottom of the sample vials were easily visualized by the naked eye under ultraviolet light illumination. The limit of detection (LOD) was ~4.6µM. The LOD was reduced to ~400nM when fluorescence spectroscopy was used as the detection tool, while the LOD can be further improved to ~7.8nM when using matrix-assisted laser desorption/ionization mass spectrometry as the detection method. We also demonstrated the feasibility of using the proposed approach to selectively detect ricin B chain in complex samples. Copyright © 2016 Elsevier B.V. All rights reserved.

N-hexane alters the maturation of oocytes and induces apoptosis in mice.

PubMed

Liu, Jin; Huang, Lei; Sun, Yan; Li, Yu Chen; Zhu, Jian Lin; Wang, Wen Xiang; Zhang, Wen Chang

2013-09-01

This study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes. Cell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis. Germinal vesicle breakdown (GVBD) and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane. After fertilization, the number of eggs in the mouse was significantly reduced by n-hexane. Mitochondrial membrane potentials (ΔΨm) were altered in mouse oocytes that were leading to apoptosis of the oocytes. N-hexane might have affected the maturation of oocytes, causing alteration of ΔΨm and leading to apoptosis which maybe one of the most important mechanisms. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Suppression of tumor-induced angiogenesis by taspine isolated from Radix et Rhizoma Leonticis and its mechanism of action in vitro.

PubMed

Zhang, Yanmin; He, Langchong; Meng, Liang; Luo, Wenjuan; Xu, Xuemei

2008-04-08

The present study was to demonstrate the effect of taspine isolated from Radix et Rhizoma Leonticis on tumor angiogenesis and its mechanism of action. The anti-angiogenic effect in vivo was evaluated on chicken chorioallantoic membrane (CAM) neovascularisation model and CAM transplantation tumor model. Taspine exerted inhibitory influence on CAM angiogenesis and the growth and microvessel density (MVD) of CAM transplantation tumor at concentrations of 0.5-2μg/egg. The mechanism was demonstrated through detecting vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) protein secretion by enzyme-linked immunosorbent assay (ELISA), as well as mRNA expression of VEGF, Flt-1 and Flk-1/KDR by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that taspine down-regulated the VEGF and bFGF secretion in human non-small cell lung cancer cell (A549 cell) and human umbilical vein endothelial cell (HUVEC), and the VEGF and Flk-1/KDR mRNA expression in HUVEC. Additionally, the effect of taspine on HUVEC migration was detected with the method of cell scrape. The result indicated that taspine inhibited HUVEC migration in a dose-dependent manner. These findings suggest that taspine might be a promising candidate as angiogenesis inhibitors.

DELAY OF CLEAVAGE OF THE ARBACIA EGG BY ULTRAVIOLET RADIATION

PubMed Central

Blum, Harold F.; Price, Judith P.

1950-01-01

While our data do not permit us to state the exact locus or mode of action of ultraviolet radiation in the Arbacia egg, certain general conclusions may be reached. The amount of delay of cleavage of these eggs is determined by two principal factors: (1) The extent of an effect, resulting from photochemical action induced by ultraviolet radiation, which is reversible in a biological sense, the reversibility not being directly dependent upon the process of cell division. (2) The sensitivity of the cell division process to the effects of the ultraviolet-induced photochemical reaction. This factor varies with the stage of cell division, the cell being insensitive during a period corresponding to most of mitosis. It seems likely that these findings may apply to cell division in general, but, since the quantitative relationships observed must, in this case, reflect the integration of two semi-independent factors, the over-all picture may appear quite different for different kinds of cells. PMID:15410486

Phospholipids and products of their hydrolysis as dietary preventive factors for civilization diseases.

PubMed

Parchem, Karol; Bartoszek, Agnieszka

2016-12-31

The results of numerous epidemiological studies indicate that phospholipids play an important role in the prevention of chronic diseases faced by contemporary society. Firstly, these compounds are responsible for the proper functioning of cell membranes, by ensuring liquidity and permeability, which is pivotal for normal activity of membrane proteins, including receptors. These mechanisms are at the core of prevention of cancer, autoimmune or neurological disorders. Secondly, structure and properties of phospholipids cause that they are highly available source of biologically active fatty acids. Thirdly, also products of endogenous hydrolysis of phospholipids exhibit biological activity. These include lysophospholipids formed as a result of disconnecting free fatty acid from glycerophospholipids in the reaction catalyzed by phospholipase A, phosphatidic acid and hydrophilic subunits released by the activity of phospholipase D. The bioactive products of hydrolysis also include ceramides liberated from phosphosphingolipids after removal of a hydrophilic unit catalyzed by sphingomyelinase. Phospholipids are supplied to the human body with food. A high content of phospholipids is characteristic for egg yolk, liver, pork and poultry, as well as some soy products. Particularly beneficial are phospholipids derived from seafood because they are a rich source of essential fatty acids of the n-3 family.

Highly Resolved Sub-Terahertz Vibrational Spectroscopy of Biological Macromolecules and Bacteria Cells

DTIC Science & Technology

2016-07-01

between average background spectrum and chicken egg - white lysozyme protein spectrum...spectroscopic signatures were conducted using human insulin protein and chicken egg -white lysozyme protein. Proteins with different structures...the comparison between the average background THz spectrum (black line in Figure 13) and the chicken egg -white lysozyme THz spectrum (blue line

In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens.

PubMed

Shanmugasundaram, Revathi; Selvaraj, Ramesh K

2013-01-01

The CD4(+)CD25(+) cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4(+)CD25(+) cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4(+)CD25(-) cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4(+)CD25(+) cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4(+)CD25(+) cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4(+)CD25(+) cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. At 16 d posthatch, the anti-chicken CD25 antibody injection decreased CD4(+)CD25(+) cell percentages in the thymus, spleen, and cecal tonsils. Chickens hatched from anti-CD25-antibody-injected eggs had approximately 25% of CD4(+)CD25(+) cells in the cecal tonsils and thymus compared with those in the cecal tonsils and thymus of the control group. The CD4(+)CD25(-) cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of interferon-γ and interleukin-2 mRNA than CD4(+)CD25(-) cells from the control group. It could be concluded that injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted the CD4(+)CD25(+) cells until 25 d posthatch.

Somatic insulin signaling regulates a germline starvation response in Drosophila egg chambers

PubMed Central

Burn, K. Mahala; Shimada, Yuko; Ayers, Kathleen; Lu, Feiyue; Hudson, Andrew M.; Cooley, Lynn

2014-01-01

Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers. PMID:25481758

The zinc spark is an inorganic signature of human egg activation.

PubMed

Duncan, Francesca E; Que, Emily L; Zhang, Nan; Feinberg, Eve C; O'Halloran, Thomas V; Woodruff, Teresa K

2016-04-26

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent "zinc spark." The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.

The zinc spark is an inorganic signature of human egg activation

PubMed Central

Duncan, Francesca E.; Que, Emily L.; Zhang, Nan; Feinberg, Eve C.; O’Halloran, Thomas V.; Woodruff, Teresa K.

2016-01-01

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent “zinc spark.” The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development. PMID:27113677

Chicks produced in the Magellanic penguin (Spheniscus magellanicus) after cloacal insemination of frozen-thawed semen.

PubMed

O'Brien, Justine Kellie; Steinman, Karen J; Montano, Gisele A; Dubach, Jean M; Robeck, Todd R

2016-07-01

The in vitro and in vivo functionality of cryopreserved spermatozoa was examined over two breeding seasons in a zoological colony of Magellanic penguins (Spheniscus magellanicus). Frozen-thawed semen was inseminated into five anesthetized females, over a total of eight egg production cycles, with a different male used for each artificial insemination (AI) within each season. Females were maintained within the colony in cordoned nest sites to prevent copulation with their paired male, and were inseminated every 3-10 days until the first oviposition. Semen frozen from seven males using a straw method retained 39.8%, 25.7%, 74.0%, and 52.1% of its initial total motility, progressive motility, average path velocity, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (P < 0.05) during freeze-thawing from 76.7% immediately prior to freezing to 65.3% post-thawing. Conceptive females received 1.6 ± 0.2 inseminations before the first oviposition, with 19.2 ± 1.6 × 10(6) motile, morphologically normal spermatozoa per insemination. Overall fertility was 53.3% (8/15 eggs), hatchability was 50.0% (4/8), and genetic analyses confirmed that all embryos and hatchlings were sired by the AI male. Fertile eggs were laid at 4.0-12.1 days after AI, indicating that frozen-thawed spermatozoa resided in the female reproductive tract for up to ∼7.2 days prior to fertilization. Results demonstrate that frozen-thawed Magellanic penguin spermatozoa are fully functional in vivo and support the use of genome banking and AI as tools for managing the sustainability of zoological penguin populations. Zoo Biol. 35:326-338, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

From material science to avant-garde cuisine. The art of shaping liquids into spheres.

PubMed

Fu, Haohao; Liu, Yingzhe; Adrià, Ferran; Shao, Xueguang; Cai, Wensheng; Chipot, Christophe

2014-10-09

Employing avant-garde cuisine techniques, in particular sodium alginates, liquid food can be shaped into spheres, thereby conferring to the former original and sometimes unexpected forms and textures. To achieve this result, rational understanding of the science that underlies food physical chemistry is of paramount importance. In this contribution, the process of spherification is dissected for the first time at the atomic level by means of classical molecular dynamics simulations. Our results show that a thin membrane consisting of intertwined alginate chains forms in an aqueous solution containing calcium ions, thereby encapsulating in a sphere the aliment in its liquid state. They also show why the polysaccharide chains will not cohere into such a membrane in a solution of sodium ions. Analysis of the trajectories reveals the emergence of so-called egg-box spatial arrangements, which connect the alginate chains by means of repeated chelation of one calcium ion by two carboxylate groups. Free-energy calculations delineating the formation of these egg-box structures further illuminate the remarkable stability of such tridimensional organizations, which ensures at room temperature the spontaneous growth of the polysaccharide membrane. Spherification has been also examined for liquid aliments of different nature, modeled by charged, hydrophilic and hydrophobic compounds. The membrane-encapsulated food is shaped into robust and durable spheres, irrespective of the liquid core material. By reconciling the views of spherification at small and large scales, the present study lays the groundwork for the rational design of innovative cooking techniques relevant to avant-garde cuisine.

Numbers of Salmonella enteritidis in the contents of naturally contaminated hens' eggs.

PubMed Central

Humphrey, T. J.; Whitehead, A.; Gawler, A. H.; Henley, A.; Rowe, B.

1991-01-01

Over 5700 hens eggs from 15 flocks naturally infected with Salmonella enteritidis were examined individually for the presence of the organism in either egg contents or on shells. Thirty-two eggs (0.6%) were positive in the contents. In the majority, levels of contamination were low. Three eggs, however, were found to contain many thousands of cells. In eggs where it was possible to identify the site of contamination, the albumen was more frequently positive than the yolk. Storage at room temperature had no significant effect on the prevalence of salmonella-positive eggs but those held for more than 21 days were more likely (P less than 0.01) to be heavily contaminated. In batches of eggs where both shells and contents were examined, 1.1% were positive on the former site and 0.9% in the latter. PMID:2050203

Amblyomma imitator Ticks as Vectors of Rickettsia rickettsii, Mexico

PubMed Central

Oliveira, Karla A.; Pinter, Adriano; Medina-Sanchez, Aaron; Boppana, Venkata D.; Wikel, Stephen K.; Saito, Tais B.; Shelite, Thomas; Blanton, Lucas; Popov, Vsevolod; Teel, Pete D.; Walker, David H.; Galvao, Marcio A.M.; Mafra, Claudio

2010-01-01

Real-time PCR of Amblyomma imitator tick egg masses obtained in Nuevo Leon State, Mexico, identified a Rickettsia species. Sequence analyses of 17-kD common antigen and outer membrane protein A and B gene fragments showed to it to be R. rickettsii, which suggested a potential new vector for this bacterium. PMID:20678325

Effects of the phosphatase inhibitors, okadaic acid, ATPgammaS, and calyculin A on the dividing sand dollar egg.

PubMed

Hamaguchi, Yukihisa; Kuriyama, Ryoko

2002-06-01

The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.

Why do proteins aggregate? “Intrinsically insoluble proteins” and “dark mediators” revealed by studies on “insoluble proteins” solubilized in pure water

PubMed Central

Song, Jianxing

2013-01-01

In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the β-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded with integral membrane proteins, if proteins originally emerged in unsalted prebiotic media. PMID:24555050

Different routes lead to apoptosis in unfertilized sea urchin eggs.

PubMed

Philippe, Laetitia; Tosca, Lucie; Zhang, Wen Ling; Piquemal, Marion; Ciapa, Brigitte

2014-03-01

Results obtained in various species, from mammals to invertebrates, show that arrest in the cell cycle of mature oocytes is due to a high ERK activity. Apoptosis is stimulated in these oocytes if fertilization does not occur. Our previous data suggest that apoptosis of unfertilized sea urchin eggs is the consequence of an aberrant short attempt of development that occurs if ERK is inactivated. They contradict those obtained in starfish, another echinoderm, where inactivation of ERK delays apoptosis of aging mature oocytes that are nevertheless arrested at G1 of the cell cycle as in the sea urchin. This suggests that the cell death pathway that can be activated in unfertilized eggs is not the same in sea urchin and in starfish. In the present study, we find that protein synthesis is necessary for the survival of unfertilized sea urchin eggs, contrary to starfish. We also compare the effects induced by Emetine, an inhibitor of protein synthesis, with those triggered by Staurosporine, a non specific inhibitor of protein kinase that is widely used to induce apoptosis in many types of cells. Our results indicate that the unfertilized sea urchin egg contain different mechanisms capable of leading to apoptosis and that rely or not on changes in ERK activity, acidity of intracellular organelles or intracellular Ca and pH. We discuss the validity of some methods to investigate cell death such as measurements of caspase activation with the fluorescent caspase indicator FITC-VAD-fmk or acidification of intracellular organelles, methods that may lead to erroneous conclusions at least in the sea urchin model.

Bacteriological and pathological studies of egg peritonitis in commercial layer chicken in Namakkal area.

PubMed

Srinivasan, Palani; Balasubramaniam, Gurusamypalayam Amirthalingam; Murthy, Thippichettipalayam Ramasamy Gopala Krishna; Balachandran, Perumal

2013-12-01

To detect the various bacteriological agents and pathological changes in commercial layer chicken affected with egg yolk peritonitis in Namakkal region of India. A total of 6 572 layer chicken from 85 commercial farms were subjected for the study, out of which 1 715 showed various types of oviduct abnormalities. Among the 1 715, 264 birds from six farms were identified as egg peritonitis on the basis of postmortem examination. Trachea, lung, heart blood, liver, peritoneal exudate, oviduct (infundibulum, magnum, uterus) and cloacal swabs were collected from the 264 birds with egg peritonitis lesion for screening of bacterial agents. Signalment, clinical signs and pathological changes were recorded in the affected flocks. The results of the present investigation indicated that the E. coli associated egg peritonitis was responsible for 15.39% of the reproductive tract abnormalities in commercial layers between 21 and 80 week of age. In the affected flocks egg production drop and mortality varied from 3% to 20% and 0.5% to 7.0% respectively. It was noticed during peak egg production (21 to 60 week) and southwest monsoon season (58%). Statistical analysis of age, season and egg production by Chi square test of independence revealed highly significant difference. E. coli was isolated as a pure culture and concurrent with other bacterial agents in 226 and 38 birds respectively. Among the fifteen E. coli serotypes identified serotype O166, O64 and O111 were predominant. Necropsy examination of affected birds revealed the presence of amorphous or insipissiated yolk material in the abdominal cavity with inflammatory changes in the ovary, oviduct and intestine. Microscopically the oviduct surface epithelium showed degeneration and desquamation, moderate to marked infiltration of inflammatory cells especially heterophils and lymphocytes in various regions and lumen contained serofibrinous exudate, inflammatory and desquamated epithelial cells with bacterial microcolonies. Ovarian follicles revealed hyperemia, degeneration of granulosa cells and infiltration of inflammatory cells. Intestine showed degenerative, necrotic and inflammatory lesion. The findings of this study showed that the egg peritonitis might be caused by either the translocation of intestinal E. coli into the peritoneal cavity or by the movement of cloacal E. coli into the oviduct followed by ascension of these bacteria up the oviduct, through the infundibulum, and into the peritoneal cavity. To control the egg peritonitis faecal contamination with E. coli should be minimized. Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.

Mutations of novel influenza A(H10N8) virus in chicken eggs and MDCK cells.

PubMed

Yang, Jian; Zhang, Ting; Guo, Li; Hu, Yongfeng; Li, Jinlin; Su, Haoxiang; Xiao, Yan; Ren, Xianwen; Dong, Jie; Sun, Lilian; Xiao, Yan; Li, Li; Yang, Fan; Wang, Jianwei; Yuan, Hui; Jin, Qi

2014-09-01

The recent emergence of human infection with influenza A(H10N8) virus is an urgent public health concern. Genomic analysis showed that the virus was conserved in chicken eggs but presented substantial adaptive mutations in MDCK cells. Our results provide additional evidence for the avian origin of this influenza virus.

Hen Egg as an Antioxidant Food Commodity: A Review

PubMed Central

Nimalaratne, Chamila; Wu, Jianping

2015-01-01

Intake of antioxidants through diet is known to be important in reducing oxidative damage in cells and improving human health. Although eggs are known for their exceptional, nutritional quality, they are not generally considered as antioxidant foods. This review aims to establish the importance of eggs as an antioxidant food by summarizing the current knowledge on egg-derived antioxidants. Eggs have various natural occurring compounds including the proteins ovalbumin, ovotransferrin and lysozyme in egg white, as well as phosvitin, carotenoids and free aromatic amino acids in egg yolk. Some lipophilic antioxidants such as vitamin E, carotenoids, selenium, iodine and others can be transferred from feed into egg yolk to produce antioxidant-enriched eggs. The bioactivity of egg antioxidants can be affected by food processing, storage and gastrointestinal digestion. Generally thermal processing methods can promote loss of antioxidant properties in eggs due to oxidation and degradation, whereas gastrointestinal digestion enhances the antioxidant properties, due to the formation of new antioxidants (free amino acids and peptides). In summary, in addition to its well-known nutritional contribution to our diet, this review emphasizes the role of eggs as an important antioxidant food. PMID:26404361

Nematode development after removal of egg cytoplasm: absence of localized unbound determinants.

PubMed

Laufer, J S; von Ehrenstein, G

1981-01-23

Embryos of Caenorhabditis elegans develop into fertile adults after cell fragments, containing presumptive cytoplasm of somatic and germ line precursors, are extruded from uncleaved eggs or early blastomeres through laser-induced holes in the eggshells. This suggests that the determinate development of this worm is not dependent on the prelocalization of determinants in specific regions of the egg cytoplasm.

Numerical modeling of heat transfer and pasteurizing value during thermal processing of intact egg.

PubMed

Abbasnezhad, Behzad; Hamdami, Nasser; Monteau, Jean-Yves; Vatankhah, Hamed

2016-01-01

Thermal Pasteurization of Eggs, as a widely used nutritive food, has been simulated. A three-dimensional numerical model, computational fluid dynamics codes of heat transfer equations using heat natural convection, and conduction mechanisms, based on finite element method, was developed to study the effect of air cell size and eggshell thickness. The model, confirmed by comparing experimental and numerical results, was able to predict the temperature profiles, the slowest heating zone, and the required heating time during pasteurization of intact eggs. The results showed that the air cell acted as a heat insulator. Increasing the air cell volume resulted in decreasing of the heat transfer rate, and the increasing the required time of pasteurization (up to 14%). The findings show that the effect on thermal pasteurization of the eggshell thickness was not considerable in comparison to the air cell volume.

Inactivation kinetics and steady-state current noise in the anomalous rectifier of tunicate egg cell membranes.

PubMed Central

Ohmori, H

1978-01-01

1. Inward K current through the anomalous rectifier in the tunicate egg (Halocynthis roretzi, Drashe) was studied under voltage clamp. The transient inward current in response to a step change of membrane potential was measured. The steady-state current fluctuations were analysed using the power density spectrum (p.d.s.). 2. The inward current showed time-dependent changes, which were described by a pair of the first order kinetic parameters, n and s for activation and inactivation, respectively. The steady-state channel open probability due to the activation process (n infinity) was assumed to be 1.0 for V more negative than about--100 mV, but that of the inactivation process (s infinity) and the time constant of inactivation (taus) were membrane potential dependent in the same potential range; both decreased with increasing hyperpolarization. 3. The inward currents in Na-free choline medium did not inactivate, but were decreased in size. In Na-free Li medium, inactivation was very small; the steady-state conductance was not affected significantly. 4. After exposure to high Ca media, an increase of the conductance was observed. This effect is probably caused by an increase of intracellular Ca due to Ca ions entering through the Na channels. Mg ions slightly decreased the conductance. 5. In the hyperpolarized membrane (-160 less than or equal to V less than or equal to -80mV), steady-state current noise was recorded and analysed using p.d.s. A p.d.s. of the 1/[1 + (f/fc)2] type as well a p.d.s. of the 1/f type was observed; f, frequency, fc, cut-off frequency. 6. fc was translated into time constant tauN (= 1/2pIfC) and compared with the time constant of inactivation, taus. There was a significant correlation betwen these values with a regression coefficient of 0.82. 7. Changing from 400 mM-Li abloshied inactivation and changed the p.d.s. from the 1/[1 + (f/fc)2] into the 1/f type. These results (paragraphs 5--7)suggest that the fluctuations in the steady-state currents originatte in the inactivation gatin kinetics of the an ofthe anomalous rectifier. 8. The number of anomalous rectifier channels and the unit channel conductance were estimated from the 1/[1 + (f/fc)2] type current noise according to the formula : (see text), where I infinity = gamma Nninfinity s infinity (V--VK), gamma the unit channel conductance, N the maximum number of channels that can be opened by a hyperpolarizing pulse per egg. The unit conductance was 6 pmho in standard artificial sea water and the channel density was 0.028/micrometer2. 9. The unit channel conductance (gamma) was dependent upon external K concentration, but the number ofchannels (N) was not. 10. The increase in chord conductance evoked by higher Ca concentrations was due to the increase of the channel number. By contrast, Mg ions seem to decrease the unit channel conductance slightly. PMID:568176

Regioselective cis-trans isomerization of arachidonic double bonds by thiyl radicals: the influence of phospholipid supramolecular organization.

PubMed

Ferreri, Carla; Samadi, Abdelouahid; Sassatelli, Fabio; Landi, Laura; Chatgilialoglu, Chryssostomos

2004-02-04

Trans unsaturated fatty acids in humans may be originated by two different contributions. The exogenous track is due to dietary supplementation of trans fats and the endogenous path deals with free-radical-catalyzed cis-trans isomerization of fatty acids. Arachidonic acid residue (5c,8c,11c,14c-20:4), which has only two out of the four double bonds deriving from the diet, was used to differentiate the two paths and to assess the importance of a radical reaction. A detailed study on the formation of trans phospholipids catalyzed by the HOCH2CH2S* radical was carried out on L-alpha-phosphatidylcholine from egg lecithin and 1-stearoyl-2-arachidonoyl-L-alpha-phosphatidylcholine (SAPC) in homogeneous solution or in large unilamellar vesicles (LUVET). Thiyl radicals were generated from the corresponding thiol by either gamma-irradiation or UV photolysis, and the reaction course was followed by GC, Ag/TLC, and 13C NMR analyses. The isomerization was found to be independent of cis double bond location (random process) in i-PrOH solution. In the case of vesicles, the supramolecular organization of lipids produced a dramatic change of the isomerization outcome: (i) in egg lecithin, the reactivity of arachidonate moieties is higher than that of oleate and linoleate residues, (ii) in the linoleate residues of egg lecithin, the 9t,12c-18:2 isomer prevailed on the 9c,12t-18:2 isomer (3:1 ratio), and (iii) a regioselective isomerization of SAPC arachidonate residues occurred in the 5 and 8 positions. This effect of "positional preference" indicates that thiyl radicals entering the hydrophobic region of the membrane bilayer start to isomerize polyunsaturated fatty acid residues having the double bonds nearest to the membrane surfaces. We propose that arachidonic acid and its trans isomers can function as biomarkers in membranes for distinguishing the two trans fatty acid-forming pathways.

Infection of the reproductive tract and eggs with Salmonella enterica serovar pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity.

PubMed

Wigley, Paul; Hulme, Scott D; Powers, Claire; Beal, Richard K; Berchieri, Angelo; Smith, Adrian; Barrow, Paul

2005-05-01

Salmonella enterica serovar Pullorum causes persistent infections in laying hens. Splenic macrophages are the main site of persistence. At sexual maturity, numbers of bacteria increase and spread to the reproductive tract, which may result in vertical transmission to eggs or chicks. In this study we demonstrate that both male and female chickens may develop a carrier state following infection but that the increases in bacterial numbers and spread to the reproductive tract are phenomena restricted to hens, indicating that such changes are likely to be related to the onset of egg laying. The immunological responses during the carrier state and through the onset of laying in hens were determined. These indicate that chickens produce both humoral and T-cell responses to infection, but at the onset of laying both the T-cell response to Salmonella and nonspecific responses to mitogenic stimulation fall sharply in both infected and noninfected birds. The fall in T-cell responsiveness coincided with the increase in numbers of Salmonella serovar Pullorum and its spread to the reproductive tract. Three weeks after the onset of egg laying, T-cell responsiveness began to increase and bacterial numbers declined. Specific antibody levels changed little at the onset of laying but increased following the rise in bacterial numbers in a manner reminiscent of a secondary antibody response to rechallenge. These findings indicate that a nonspecific suppression of cellular responses occurs at the onset of laying and plays a major role the ability of Salmonella serovar Pullorum to infect the reproductive tract, leading to transmission to eggs. The loss of T-cell activity at the point of laying also has implications for Salmonella enterica serovar Enteritidis infection and transmission to eggs, along with its control by vaccination offering a "window of opportunity" in which infection may occur.

Melatonin protects against clomiphene citrate-induced generation of hydrogen peroxide and morphological apoptotic changes in rat eggs.

PubMed

Tripathi, Anima; PremKumar, Karuppanan V; Pandey, Ashutosh N; Khatun, Sabana; Mishra, Surabhi Kirti; Shrivastav, Tulsidas G; Chaube, Shail K

2011-09-30

The present study was aimed to determine whether clomiphene citrate-induces generation of hydrogen peroxide in ovary, if so, whether melatonin could scavenge hydrogen peroxide and protect against clomiphene citrate-induced morphological apoptotic changes in rat eggs. For this purpose, forty five sexually immature female rats were given single intramuscular injection of 10 IU pregnant mare's serum gonadotropin for 48 h followed by single injections of 10 IU human chorionic gonadotropin and clomiphene citrate (10 mg/kg bw) with or without melatonin (20 mg/kg bw) for 16 h. The histology of ovary, ovulation rate, hydrogen peroxide concentration and catalase activity in ovary and morphological changes in ovulated eggs were analyzed. Co-administration of clomiphene citrate along with human chorionic gonadotropin significantly increased hydrogen peroxide concentration and inhibited catalase activity in ovary, inhibited ovulation rate and induced egg apoptosis. Supplementation of melatonin reduced hydrogen peroxide concentration and increased catalase activity in the ovary, delayed meiotic cell cycle progression in follicular oocytes as well as in ovulated eggs since extrusion of first polar body was still in progress even after ovulation and protected against clomiphene citrate-induced egg apoptosis. These results clearly suggest that the melatonin reduces oxidative stress by scavenging hydrogen peroxide produced in the ovary after clomiphene citrate treatment, slows down meiotic cell cycle progression in eggs and protects against clomiphene citrate-induced apoptosis in rat eggs. Copyright © 2011 Elsevier B.V. All rights reserved.

Topology of the germ plasm and development of primordial germ cells in inverted amphibian eggs

NASA Technical Reports Server (NTRS)

Wakahara, M.; Neff, A. W.; Malacinski, G. M.

1984-01-01

Inverted Xenopus eggs have reduced numbers of primordial germ cells (PGCs). The extent of the reduction varies from spawning to spawning. Histologic examination revealed that PGC counts were lowest in inverted eggs which displayed the greatest amount of shift in the vegetal mass of large yolk platelets, although the germ plasm itself always remained localized in the egg's original vegetal hemisphere. Even at blastulation the germ plasm continued to be localized in the egg's original vegetal hemisphere. In many cases, however, it was confined to the periphery of the embryo, which probably accounts for the reduced PGC number in some tadpoles. In other cases it may have been dispersed and therefore not detectable in histologic analyses. Although the altered site of involution in inverted embryos did not influence PGC development, subsequent cell movement patterns apparently did. Those embryos which displayed the largest degree of pattern reversal at the tail-bud stage also exhibited the most extreme reduction in PGC numbers. A brief cold shock (4 degrees C, 10 min) prior to first cleavage leads to a further reduction in PGC numbers in inverted embryos, probably as a result of the displacement of the germ plasm away from its original vegetal pole location.

Gravity and positional homeostasis of the cell

NASA Technical Reports Server (NTRS)

Nace, G. W.

1983-01-01

The effect of gravity upon cytoplasmic aggregates of the size present in eggs and upon cells is investigated. An expression is developed to describe the tendency of torque to rotate the egg and reorganize its constituents. This expression provides the net torque resulting from buoyancy and gravity acting upon a dumbbell-shaped cell, with heavy and light masses at either end and floating in a medium. Torques of approximately 2.5 x 10 to the -13th to 0.85 dyne-cm are found to act upon cells ranging from 6.4 microns to 31 mm (chicken egg). It is noted that cells must expend energy to maintain positional homeostasis against gravity, as demonstrated by results from Skylab 3, where tissue cultures used 58 percent more glucose on earth than in space. The implications for developmental biology, physiology, genetics, and evolution are discussed. It is argued that at the cellular and tissue levels the concept of gravity receptors may be unnecessary.

Electrophysiological Evidence for the Presence of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Mouse Sperm

PubMed Central

Dulce, Figueiras Fierro; José, Acevedo Juan; Pablo, Martínez; Escoffier, Jessica; Sepúlveda, Francisco V.; Enrique, Balderas; Gerardo, Orta; Pablo, Visconti; Alberto, Darszon

2014-01-01

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/PKA pathway and involves increases in intracellular Ca2+, pH, Cl−, protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole-cell patch-clamp recordings from testicular mouse sperm now reveal a Cl− selective component to membrane current that is ATP-dependent, stimulated by cAMP, cGMP and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTRinh-172, two well-known CFTR antagonists. Furthermore, the Cl− current component activated by cAMP and inhibited by CFTRinh-172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss-of-function mutation, indicating that CFTR is responsible for this component. A Cl− selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTRinh-172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation. PMID:22833409

Electrogenesis in the lower Metazoa and implications for neuronal integration

PubMed Central

Meech, Robert W.

2015-01-01

Electrogenic communication appears to have evolved independently in a variety of animal and plant lineages. Considered here are metazoan cells as disparate as the loose three-dimensional parenchyma of glass sponges, the two-dimensional epithelial sheets of hydrozoan jellyfish and the egg cell membranes of the ctenophore Beroe ovata, all of which are capable of generating electrical impulses. Neuronal electrogenesis may have evolved independently in ctenophores and cnidarians but the dearth of electrophysiological data relating to ctenophore nerves means that our attention is focused on the Cnidaria, whose nervous systems have been the subject of extensive study. The aim here is to show how their active and passive neuronal properties interact to give integrated behaviour. Neuronal electrogenesis, goes beyond simply relaying ‘states of excitement’ and utilizes the equivalent of a set of basic electrical ‘apps’ to integrate incoming sensory information with internally generated pacemaker activity. A small number of membrane-based processes make up these analogue applications. Passive components include the decremental spread of current determined by cellular anatomy; active components include ion channels specified by their selectivity and voltage dependence. A recurring theme is the role of inactivating potassium channels in regulating performance. Although different aspects of cnidarian behaviour are controlled by separate neuronal systems, integrated responses and coordinated movements depend on interactions between them. Integrative interactions discussed here include those between feeding and swimming, between tentacle contraction and swimming and between slow and fast swimming in the hydrozoan jellyfish Aglantha digitale. PMID:25696817

Stability studies of immobilized lipase on rice husk and eggshell membrane

NASA Astrophysics Data System (ADS)

Abdulla, R.; Sanny, S. A.; Derman, E.

2017-06-01

Lipase immobilization for biodiesel production is gaining importance day by day. In this study, lipase from Burkholderia cepacia was immobilized on activated support materials namely rice husk and egg shell membrane. Both rice husk and eggshell membrane are natural wastes that holds a lot of potential as immobilization matrix. Rice husk and eggshell membrane were activated with glutaraldehyde. Lipase was immobilized on the glutaraldehyde-activated support material through adsorption. Immobilization efficiency together with enzyme activity was observed to choose the highest enzyme loading for further stability studies. Immobilization efficiency of lipase on rice husk was 81 as compared to an immobilization efficiency of 87 on eggshell membrane. Immobilized lipase on eggshell membrane exhibited higher enzyme activity as compared to immobilized lipase on rice husk. Eggshell membrane also reported higher stability than rice husk as immobilization matrix. Both types of immobilized lipase retatined its activity after ten cycles of reuse. In short, eggshell membrane showed to be a better immobilization platform for lipase as compared to rice husk. However, with further improvement in technique of immobilization, the stability of both types of immobilized lipase can be improved to a greater extent.

Removal of Salmonella Enteritidis from commercial unpasteurized liquid egg white using pilot scale cross flow tangential microfiltration.

PubMed

Mukhopadhyay, Sudarsan; Tomasula, Peggy M; Luchansky, John B; Porto-Fett, Anna; Call, Jeffrey E

2010-09-01

Effectiveness of a cross flow microfiltration (MF) process for removal of a cocktail of Salmonella enterica serovar Enteritidis species from commercial unpasteurized liquid egg white (LEW) from a local egg breaking plant, while maintaining its functional properties was evaluated. To facilitate MF, LEW was wedge screened, homogenized and then diluted (1:2 w/w) with distilled water containing 0.5% sodium chloride. Diluted unpasteurized LEW was inoculated with five strains of S. Enteritidis (ATCC 4931, ATCC BAA-708, ATCC 49215, ATCC 49218, and ATCC BAA-1045) to a level of approximately 10(7)CFU/mL of LEW and microfiltered using a ceramic membrane. Process parameters influencing egg white functional properties and pathogen removal efficiency were evaluated. Average permeates flux increased by almost 126% when pH of LEW was adjusted from pH 8 to pH 7 at 25 degrees C. Microbial removal efficiency was at least, on average, 6.8Log(10)CFU/mL (limit of detection < or =0.5Log(10)CFU/mL). Functional property analysis indicated that the MF process did not alter the foaming power of LEW. Published by Elsevier B.V.

A stable planar bilayer membrane of phospholipid supported by cellulose sheets.

PubMed

Setaka, M; Yamamoto, T; Sato, N; Yano, M; Kwan, T

1982-01-01

A new method is reported for preparing a thin planar membrane of 1,2-distearoylsn-glycero-3-phosphocholine and egg yolk lecithin-cholesterol (molar ratio of 1:1) between a pair of cellulose sheets. This technique, developed from the method of the multilayer planar membrane preparation (Setaka, M., et al. (1979) J. Biochem. 86, 355-362; 1619-1622; (1980) J. Biochem. 88, 1819-1829), consisted of three experimental processes. First, a phospholipid monolayer was prepared at an air-water interface, then taken up on a stretched cellulose sheet. A thin lipid membrane, supported from both sides by cellulose sheets, was constructed by combining two of these lipid monolayer-cellulose sheets. The permeability coefficient of the thin lipid membrane was estimated by removing the effect of two outer cellulose sheets, and this permeability was found to be larger than those of other model membranes of a lipid bilayer, indicating that the present lipid membrane is not a perfect single lipid bilayer. However, certain experimental evidence suggests that the bulk of the phospholipids formed a bilayer between the two cellulose sheets. Since this lipid membrane is particularily stable, larger membranes can be prepared by the present method than other planar bilayer membranes of lipid, which are usually constructed inside a pin hole in a thin teflon sheet.

Evolution and development of fetal membranes and placentation in amniote vertebrates.

PubMed

Ferner, Kirsten; Mess, Andrea

2011-08-31

We review aspects of fetal membrane evolution and patterns of placentation within amniotes, the most successful land vertebrates. Special reference is given to embryonic gas supply. The evolution of fetal membranes is a prerequisite for reproduction independent from aquatic environments. Starting from a basically similar repertoire of fetal membranes - the amnion, chorion, allantois and yolk sac, which form the cleidoic egg - different structural solutions for embryonic development have evolved. In oviparous amniotes the chorioallantoic membrane is the major site for the exchange of respiratory gases between fetus and outer environment. The richly vascularised yolk sac and allantois in concert with the chorion play an important role in the evolution of placentation in various viviparous amniotes. Highly complex placentas have evolved independently among squamate sauropsids and in marsupial and placental mammals. In conclusion, there seems to be a natural force to improve gas exchange processes in intrauterine environments by reducing the barrier between the blood systems and optimising the exchange areas. Copyright © 2011 Elsevier B.V. All rights reserved.

Picking the right tool for the job – phosphor-proteomics of egg activation

PubMed Central

Wessel, Gary M.

2016-01-01

Eggs are the rarest cell in the human body, yet their study is essential for the fields of fertility, reproduction, and fetal health. Guo et al use a “surrogate” animal to discover the phophoproteomic pathways involved in egg activation. With datasets of several thousand phophosites on 2500 different proteins, these investigators have defined new pathways, connections to pathways, and priorities in searches for how eggs are activated at fertilization. These results in a sea urchin are now transposable to mammals for testing on a per candidate strategy. PMID:26573262

High diadenosine tetraphosphate (Ap4A) level in germ cells and embryos of sea urchin and Xenopus and its effect on DNA synthesis.

PubMed

Weinmann-Dorsch, C; Grummt, F

1985-09-01

Ap4A levels in sperms, eggs and different developmental stages of sea urchin (Psammechinus miliaris) and (Xenopus laevis) were determined by a method based on ATP measurement with luciferin/luciferase after splitting diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) into ATP and AMP. Appreciable storage pools of Ap4A were found in unfertilized eggs of Psammechinus and Xenopus as well as in sea urchin sperms. The actual Ap4A concentration of 28 microM in sperm represents the highest Ap4A level so far observed in eukaryotic cells. Upon fertilization an instant onset of de novo synthesis of Ap4A was demonstrated. Ap4A levels during early embryogenesis of P. miliaris and X. laevis (2.5-4 microM) are higher than those in exponentially growing mammalian culture cells and mammalian fetuses. Microinjection of Ap4A into unfertilized eggs of Psammechinus miliaris caused a 3-7 fold increase of DNA synthesis in comparison with mock-injected eggs.

Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle.

PubMed

Chang, Jeremy B; Ferrell, James E

2013-08-29

Despite the large size of the Xenopus laevis egg (approximately 1.2 mm diameter), a fertilized egg rapidly proceeds through mitosis in a spatially coordinated fashion. Mitosis is initiated by a bistable system of regulatory proteins centred on Cdk1 (refs 1, 2), raising the possibility that this spatial coordination could be achieved through trigger waves of Cdk1 activity. Using an extract system that performs cell cycles in vitro, here we show that mitosis does spread through Xenopus cytoplasm via trigger waves, propagating at a linear speed of approximately 60 µm min(-1). Perturbing the feedback loops that give rise to the bistability of Cdk1 changes the speed and dynamics of the waves. Time-lapse imaging of intact eggs argues that trigger waves of Cdk1 activation are responsible for surface contraction waves, ripples in the cell cortex that precede cytokinesis. These findings indicate that Cdk1 trigger waves help ensure the spatiotemporal coordination of mitosis in large eggs. Trigger waves may be an important general mechanism for coordinating biochemical events over large distances.

Voltage-clamp study of the activation currents and fast block to polyspermy in the egg of Xenopus laevis.

PubMed

Glahn, David; Nuccitelli, Richard

2003-04-01

Voltage-clamped mature, jelly-intact Xenopus eggs were used to carefully examine the ionic currents crossing the plasma membrane before, during, and after fertilization. The bulk of the fertilization current was transient, of large amplitude, and reversed at the predicted Cl- reversal potential. However, the large amplitude fertilization current was preceded by a small, step-like increase in holding current. This small increase in holding current is referred to in this paper as Ion to acknowledge its qualitative similarity to the Ion current previously described in the sea urchin. It was observed in both fertilized and artificially activated eggs, and was found to be unaffected by 10 mm tetra-ethyl ammonium (TEA), a concentration found to block K+ currents in Rana pipiens. Current-voltage relationships are presented for the large fertilization potential, and show that the fertilization currents have a marked outward rectification and are voltage sensitive. These properties are in contrast to the total lack of rectification and slight voltage sensitivity seen before or after the fertilization currents. The time required for sperm to fertilize the egg was found to be voltage dependent with a relatively more depolarized voltage requiring a longer time for fertilization to occur. The percentage of eggs blocked with varying potential levels was determined and this information was fitted to a modified Boltzmann equation having a midpoint of -9 mV.

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Que, Emily L.; Duncan, Francesca E.; Bayer, Amanda R.

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as ‘zinc sparks.’ The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as ‘hardening’, which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using x-ray fluorescence microscopy,more » transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. Finally, this adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.« less

Spawn in two deep-sea volute gastropods (Neogastropoda: Volutidae) from southwestern Atlantic waters

NASA Astrophysics Data System (ADS)

Penchaszadeh, Pablo E.; Teso, Valeria; Pastorino, Guido

2017-12-01

The gastropods Odontocymbiola pescalia and Provocator corderoi and their egg capsules were collected by the R/V Puerto Deseado from the Mar del Plata Submarine Canyon ( 37°53‧S, at depths of 291-1404 m) and from Burdwood Bank ( 54°27‧S, 128-785 m). Odontocymbiola pescalia egg capsules measured 15.67 ± 3.38 mm in diameter. They were subspherical in shape with an external calcareous layer. Each egg capsule contained 3-5 embryos and white material as extra embryonic food. Embryos grew to a size of up to 9.3 ± 1.1 mm in mean shell length before hatching as crawling juveniles. The spawn of P. corderoi consisted of a single dome shaped egg capsule of 14.17 ± 1.5 mm in diameter, attached to hard substrata by a basal membrane with a rounded outline. A curved semilunar furrow (seam) on one side of the capsules was always present. The number of embryos per capsule was 2-6. Embryos hatched as crawling juveniles with a shell length of 5.9 ± 0.6 mm. The size and number of whorls in the hatchling shell suggested a slow rate of development, akin to many other deep-sea invertebrates. The egg capsules and reproductive development strategies of both species were compared with those from other congeneric representatives.

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

DOE PAGES

Que, Emily L.; Duncan, Francesca E.; Bayer, Amanda R.; ...

2017-01-19

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as ‘zinc sparks.’ The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as ‘hardening’, which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using x-ray fluorescence microscopy,more » transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. Finally, this adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.« less

Atomic Force Microscope Studies of the Fusion of Floating Lipid Bilayers

PubMed Central

Abdulreda, Midhat H.; Moy, Vincent T.

2007-01-01

This study investigated the fusion of apposing floating bilayers of egg L-α-phosphatidylcholine (egg PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Atomic force microscope measurements of fusion forces under different compression rates were acquired to reveal the energy landscape of the fusion process under varied lipid composition and temperature. Between compression rates of ∼1000 and ∼100,000 pN/s, applied forces in the range from ∼100 to ∼500 pN resulted in fusion of floating bilayers. Our atomic force microscope measurements indicated that one main energy barrier dominated the fusion process. The acquired dynamic force spectra were fit with a simple model based on the transition state theory with the assumption that the fusion activation potential is linear. A significant shift in the energy landscape was observed when bilayer fluidity and composition were modified, respectively, by temperature and different cholesterol concentrations (15% ≤ chol ≤ 25%). Such modifications resulted in a more than twofold increase in the width of the fusion energy barrier for egg PC and 1,2-dimyristoyl-sn-glycero-3-phosphocholine floating bilayers. The addition of 25% cholesterol to egg PC bilayers increased the activation energy by ∼1.0 kBT compared with that of bilayers with egg PC alone. These results reveal that widening of the energy barrier and consequently reduction in its slope facilitated membrane fusion. PMID:17400691

Atomic force microscope studies of the fusion of floating lipid bilayers.

PubMed

Abdulreda, Midhat H; Moy, Vincent T

2007-06-15

This study investigated the fusion of apposing floating bilayers of egg L-alpha-phosphatidylcholine (egg PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Atomic force microscope measurements of fusion forces under different compression rates were acquired to reveal the energy landscape of the fusion process under varied lipid composition and temperature. Between compression rates of approximately 1000 and approximately 100,000 pN/s, applied forces in the range from approximately 100 to approximately 500 pN resulted in fusion of floating bilayers. Our atomic force microscope measurements indicated that one main energy barrier dominated the fusion process. The acquired dynamic force spectra were fit with a simple model based on the transition state theory with the assumption that the fusion activation potential is linear. A significant shift in the energy landscape was observed when bilayer fluidity and composition were modified, respectively, by temperature and different cholesterol concentrations (15% < or = chol < or = 25%). Such modifications resulted in a more than twofold increase in the width of the fusion energy barrier for egg PC and 1,2-dimyristoyl-sn-glycero-3-phosphocholine floating bilayers. The addition of 25% cholesterol to egg PC bilayers increased the activation energy by approximately 1.0 k(B)T compared with that of bilayers with egg PC alone. These results reveal that widening of the energy barrier and consequently reduction in its slope facilitated membrane fusion.

Morphodifferentiation of Gené's organ in engorged Amblyomma sculptum Berlese, 1888 female ticks (Acari: Ixodidae).

PubMed

Dos Santos, Marcelo Francisco; Maltauro Soares, Magna Aparecida; Lallo, Maria Anete; Barros-Battesti, Darci Moraes; de Lima-Netto, Solange; Spadacci-Morena, Diva Denelle

2018-03-01

The Gené's organ (GO) secretes a waxy substance on eggs that reduces water loss and has antimicrobial properties. The current study evaluated morphological and histochemical aspects of GO in Amblyomma sculptum from the period of post-feeding - when ticks detach from the host - to the stage just before oviposition. In this species, GO is composed of a corpus and two pairs of glands, namely, cranial and caudal. Glandular cells are joined laterally by a system of interdigitating membranes with junctional complexes. Histochemistry showed that lipid droplets became more evident as GO developed, while glycogen gradually disappeared, and proteins were detected only near the onset of oviposition. The ultrastructural results revealed a marked distension of the cuticle filled with an amorphous material. Glandular cells showed poor endoplasmatic reticulum, many mitochondria mainly in the basal cell poles and a very developed basal labyrinth. We concluded that the development of GO in A. sculptum ticks was continuous and progressive, and it started after detachment from the host. Additionally, the ultrastructure study suggests that gland cells have an important absorption ability and a low synthetic activity, which indicates that the majority of wax precursors are derived from haemolymph. Copyright © 2018 Elsevier GmbH. All rights reserved.

Multiciliated Cells in Animals.

PubMed

Meunier, Alice; Azimzadeh, Juliette

2016-12-01

Many animal cells assemble single cilia involved in motile and/or sensory functions. In contrast, multiciliated cells (MCCs) assemble up to 300 motile cilia that beat in a coordinate fashion to generate a directional fluid flow. In the human airways, the brain, and the oviduct, MCCs allow mucus clearance, cerebrospinal fluid circulation, and egg transportation, respectively. Impairment of MCC function leads to chronic respiratory infections and increased risks of hydrocephalus and female infertility. MCC differentiation during development or repair involves the activation of a regulatory cascade triggered by the inhibition of Notch activity in MCC progenitors. The downstream events include the simultaneous assembly of a large number of basal bodies (BBs)-from which cilia are nucleated-in the cytoplasm of the differentiating MCCs, their migration and docking at the plasma membrane associated to an important remodeling of the actin cytoskeleton, and the assembly and polarization of motile cilia. The direction of ciliary beating is coordinated both within cells and at the tissue level by a combination of planar polarity cues affecting BB position and hydrodynamic forces that are both generated and sensed by the cilia. Herein, we review the mechanisms controlling the specification and differentiation of MCCs and BB assembly and organization at the apical surface, as well as ciliary assembly and coordination in MCCs. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

[Embryonic stem cells and therapeutic cloning].

PubMed

Sunde, A; Eftedal, I

2001-08-30

Increased interest in the therapeutic use of human stem cells has emerged following significant progress in ongoing research. The cloning of a sheep, the isolation of human embryonic stem cells, and the discovery that adult stem cells may be reprogrammed taken together give substance to hopes that novel principles of treatment may be developed for a variety of serious conditions. Embryonic stem cells are derived from pre-embryos at the blastocyst stage and may give rise to all bodily tissues and cells. Animal models have demonstrated that embryonic stem cells when transplanted into adult hosts may differentiate and develop into cells and tissues applicable for treatment of a variety of conditions, including Parkinson's disease, multiple sclerosis, spinal injuries, cardiac stroke and cancer. Transplanted embryonic stem cells are exposed to immune reactions similar to those acting on organ transplants, hence immunosuppression of the recipient is generally required. It is, however, possible to obtain embryonic stem cells that are genetically identical to the patient's own cells by means of therapeutic cloning techniques. The nucleus from a somatic cell is transferred into an egg after removal of the egg's own genetic material. Under specific condition the egg will use genetic information from the somatic cell in organising the formation of a blastocyst which in turn generates embryonic stem cells. These cells have a genetic composition identical to that of the patient and are suitable for stem cell therapy.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

PubMed

Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

2013-04-01

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013 Elsevier B.V. All rights reserved.

The in ovo feeding administration (gallus gallus)-an emerging in vivo approach to assess bioactive compounds with potential nutritional benefits

USDA-ARS?s Scientific Manuscript database

In recent years, the in ovo feeding in fertilized broiler (Gallus gallus) eggs approach was further developed and currently is widely applied in the evaluation process of the effects of functional foods (primarily plant origin compounds) on the functionality of the intestinal brush border membrane, ...

Chitin synthases are required for survival, fecundity and egg-hatch in the red flour beetle, Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

The synthesis of chitin, the Beta-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, ...

Beyond the Drake Equation: On the Probability of the Nature of Extraterrestrial Life Forms in Our Galaxy Today

NASA Astrophysics Data System (ADS)

Geller, Harold A.

2014-01-01

I will discuss my research into the issues associated with the nature of any extraterrestrials that may be encountered in the future in our galaxy. This research was sparked by statements made by Stephen Hawking in 2010 regarding his fear of emitting radiation from our Earth so that an extraterrestrial intelligent civilization may be alerted to our existence in the galaxy today. While addressing issues of extraterrestrial altruism, a probabilistic equation was developed which addresses the number of extraterrestrial intelligent life forms that may exist in our galaxy today, who could use our bodies for nourishment or reproductive purposes. The equation begins with the results from a Drake Equation calculation, and proceeds by addressing such biochemical parameters as the fraction of ETIs with: dextro sugar stereo-isomers; levo amino acid stereo-isomers; similar codon interpretation; chromosomal length and, similar cell membrane structure to allow egg penetration.

Development of the follicle complex and oocyte staging in red drum, Sciaenops ocellatus Linnaeus, 1776 (Perciformes, Sciaenidae).

PubMed

Grier, Harry J

2012-08-01

Pelagic egg development in red drum, Sciaenops ocellatus, is described using tiered staging. Based on mitosis and meiosis, there are five periods: Mitosis of Oogonia, Active Meiosis I, Arrested Meiosis I, Active Meiosis II, and Arrested Meiosis II. The Periods are divided into six stages: Mitotic Division of Oogonia, Chromatin Nucleolus, Primary Growth, Secondary Growth, Oocyte Maturation and Ovulation. The Chromatin Nucleolus Stage is divided into four steps: Leptotene, Zygotene, Pachytene, and Early Diplotene. Oocytes in the last step possess one nucleolus, dispersed chromatin with forming lampbrush chromosomes and lack basophilic ooplasm. The Primary Growth Stage, characterized by basophilic ooplasm and absence of yolk in oocytes, is divided into five steps: One-Nucleolus, Multiple Nucleoli, Perinucleolar, Oil Droplets, and Cortical Alveolar. During primary growth, the Balbiani body develops from nuage, enlarges and disperses throughout the ooplasm as both endoplasmic reticulum and Golgi develop within it. Secondary growth or vitellogenesis has three steps: Early Secondary Growth, Late Secondary Growth and Full-Grown. The Oocyte Maturation Stage, including ooplasmic and germinal vesicle maturation, has four steps: Eccentric Germinal Vesicle, Germinal Vesicle Migration, Germinal Vesicle Breakdown and Resumption of Meiosis when complete yolk hydration occurs. The period is Arrested Meiosis II. When folliculogenesis is completed, the ovarian follicle, an oocyte and encompassing follicle cells, is surrounded by a basement membrane and developing theca, all forming a follicle complex. After ovulation, a newly defined postovulatory follicle complex remains attached to the germinal epithelium. It is composed of a basement membrane that separates the postovulatory follicle from the postovulatory theca. Arrested Meiosis I encompasses primary and secondary growth (vitellogenesis) and includes most of oocyte maturation until the resumption of meiosis (Active Meiosis II). The last stage, Ovulation, is the emergence of the oocyte from the follicle when it becomes an egg or ovum. Copyright © 2012 Wiley Periodicals, Inc.

Recommendations for administering the triple viral vaccine and antiinfluenza vaccine in patients with egg allergy.

PubMed

Piquer-Gibert, M; Plaza-Martín, A; Martorell-Aragonés, A; Ferré-Ybarz, L; Echeverría-Zudaire, L; Boné-Calvo, J; Nevot-Falcó, S

2007-01-01

Actually, food allergy is an emerging pathology; and egg allergy is the most frequent in childhood. The recommendations for measles, mumps and rubella (MMR) and influenza vaccination are increasing each year. This implementation increases the exposure of patients with egg allergy to such vaccines. In Spain, since 2004 the only available vaccine for MMR is grown in cultures of fibroblast from chick embryos; previously, patients with egg allergy were vaccinated with an alternative vaccine cultivated in diploid human cells which is no longer commercialized. Influenza vaccines grow in chick egg and the final product contains egg proteins (large variation in egg protein content has been reported). As controversy exist, the Food Allergy Committee of Spanish Society of Clinical Immunology and Pediatric Allergy decided to report some recommendations for the safe administration of MMR and influenza vaccines in patients with egg allergy. In summary, MMR vaccine is safe for children with egg allergy, only in patients with severe anaphylactic reaction after egg ingestion is recommended the administration in his reference hospital. Influenza vaccine is contraindicated in patients with severe anaphylactic reaction after egg ingestion. The rest can receive influenza vaccine in a 2-dose protocol with a vaccine that contains no more than 1.2 mcg of egg protein for mL.

Effects of salinity and sea salt type on egg activation, fertilization, buoyancy and early embryology of European eel, Anguilla anguilla.

PubMed

Sørensen, Sune Riis; Butts, Ian Anthony Ernest; Munk, Peter; Tomkiewicz, Jonna

2016-02-01

Improper activation and swelling of in vitro produced eggs of European eel, Anguilla anguilla, has been shown to negatively affect embryonic development and hatching. We investigated this phenomenon by examining the effects of salinity and sea salt type on egg dimensions, cell cleavage patterns and egg buoyancy. Egg diameter after activation, using natural seawater adjusted to different salinities, varied among female eels, but no consistent pattern emerged. Activation salinities between 30-40 practical salinity unit (psu) produced higher quality eggs and generally larger egg diameters. Chorion diameters reached maximal values of 1642 ± 8 μm at 35 psu. A positive relationship was found between egg neutral buoyancy and activation salinity. Nine salt types were investigated as activation and incubation media. Five of these types induced a substantial perivitelline space (PVS), leading to large egg sizes, while the remaining four salt types resulted in smaller eggs. All salt types except NaCl treatments led to high fertilization rates and had no effect on fertilization success as well as egg neutral buoyancies at 7 h post-fertilization. The study points to the importance of considering ionic composition of the media when rearing fish eggs and further studies are encouraged.

TRPV3 channels mediate strontium-induced mouse egg activation

PubMed Central

Carvacho, Ingrid; Lee, Hoi Chang; Fissore, Rafael A.; Clapham, David E.

2014-01-01

SUMMARY In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a Transient Receptor Potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, as TrpV3−/− eggs failed to permeate Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is the major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. PMID:24316078

Optimization of mosquito egg production under mass rearing setting: effects of cage volume, blood meal source and adult population density for the malaria vector, Anopheles arabiensis.

PubMed

Mamai, Wadaka; Bimbile-Somda, Nanwintoum S; Maiga, Hamidou; Juarez, José Guillermo; Muosa, Zaynab A I; Ali, Adel Barakat; Lees, Rosemary Susan; Gilles, Jeremie R L

2017-01-24

Anopheles arabiensis is one of the major malaria vectors that put millions of people in endemic countries at risk. Mass-rearing of this mosquito is crucial for strategies that use sterile insect technique to suppress vector populations. The sterile insect technique (SIT) package for this mosquito species is being developed by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. To improve mass-rearing outcomes for An. arabiensis, the question of whether the egg production by females would be affected by the size of the adult holding cages, the source of the blood meal and the total number of pupae that could be loaded into the cages was addressed and finally the impact of adding additional pupae to the cage daily to maintain adult numbers on egg productivity assessed. Mass production cages of two different volumes, two different sources of blood meal (bovine and porcine) and two different population densities (cages originally loaded with either 15,000 or 20,000 pupae) were tested and evaluated on the basis of eggs produced/cage or per female. Males and females pupae with a ratio of 1:1 were added to the cages at day 1 and 2 of pupation. The emerging adults had constant access to 5% sugar solution and blood fed via the Hemotek membrane feeding system. Eggs were collected either twice a week or daily. A generalized linear model was used to identify factors which gave significantly higher egg production. Neither cage volume nor blood meal source affected egg production per cage or per female. However, increasing population density to 20,000 pupae had a negative effect on eggs produced per cage and per female. Although high density negatively impacted egg production, adding 1000 daily additional pupae compensating for daily mortality resulted in a substantial increase in egg production. Moreover, in all tests the first and the third egg batches collected were significantly higher than others eggs batches. With the equipment and protocols described here and routinely used at the Insect Pest Control Laboratory (IPCL), it was possible to produce up to 120,000 eggs/cage/day. These results demonstrated that 15,000 is the optimal number of pupae to be loaded into the Anopheles Mass production cages. Under this condition, an average of 40 eggs per female was obtained for five gonotrophic cycles. However, an improvement in egg production can be achieved by daily addition, to the original 15,000 pupae, of one thousand pupae a day. Interestingly, feeding females with bovine or porcine blood using both large and small versions of the mass production cage did not affect egg productivity.

Lake trout reproductive behavior: influence of chemosensory cues from young-of-the-year by-products

USGS Publications Warehouse

Foster, Neal R.

1985-01-01

Chemosensory cues, particularly those emanating from substrate areas occupied by previously hatched young, may play an important role in the reproductive behavior of lake trout Salvelinus namaycush. Support for this hypothesis was obtained in laboratory experiments. Adults were placed in a large circular pool with four experimental reefs. Egg membranes and feces obtained from young that had hatched earlier were placed in polyester fiber in screen envelopes and positioned on selected reefs. Females approached and males contacted ('cleaned') the treated reefs but not the untreated reefs. Of 6,858 eggs recovered, 92% were from treated reefs. Some of these eggs had been fertilized and provide the first record of volitional spawning by lake trout under artificial conditions. A second experiment was less definitive, because no spawning occurred, but visual observations and analysis of videotaped behavior sequences showed that, again, adults were attracted more to reefs treated with feces of young of the year than to untreated reefs.

The Molecules of the Cell Membrane.

ERIC Educational Resources Information Center

Bretscher, Mark S.

1985-01-01

Cell membrane molecules form a simple, two-dimensional liquid controlling what enters and leaves the cell. Discusses cell membrane molecular architecture, plasma membranes, epithelial cells, cycles of endocytosis and exocytosis, and other topics. Indicates that some cells internalize, then recycle, membrane area equivalent to their entire surface…

A bio-NanoRobot design for drosophila therapeutic cloning

NASA Astrophysics Data System (ADS)

Chang, Chia-Pin; Szu, Harold

2006-04-01

To investigate Somatic Cell Nuclear Transfer (SCNT), we choose the Drosophila cloning based on a recent experiment (Haigh, MacDonald, Lioyd, Gen. V.169,1165, 2005) to be improving the adulthood rate in 2-week turn-around time. Original 1% success rate might be due to three less certain key steps: (i) The double membranes of a nucleus has at its pore led to the attached Rough Endoplasmic Reticulum (ER), passing the genetic instruction to assemble amino acids, proteins and lipid at its smooth end. Also, any mismatch of nucleus with mitochondria (MT) having own small genome for energy production had led to reprogramming failure. (D. Wallace, UC Irvine, Nature,Vol. 439, pp.653). We ask "whether a guest DNA shall come with its servants, ER, MT, etc or not." It seemed to be logical to have a whole package replaced the embryonic host cell, equipped with all housekeeping, energy production and mitosis functionalities except the genetic information. To answer this hypothesis, we design a bio-NanoRobot having a surgical precision in removing the desired nucleus with or without its attached ER and MT material. The design is based on a real-time multiplexing principle of combining both the soft-contact-vision of the Nobel Laureate Binning called Atomic Force Microscope (AFM) and the hard-grasp-action called NanoRobot TM by Xi and Szu, 2004. However, applying it, we must re-design a new bio-NanoRobot, consisting of two parts: (a) multiple resolution analysis (MRA) using AI to control a dual-resolution vision system: the soft-contact-vision AFM co-registered with a on-contact high resolution imaging; and (b) two cantilever arms capable to hold and enucleate a cell. The calibration and automation are controlled by AI Case-Based reasoning (CBR) together with AI Blackboard (BB) of the taxonomy, necessary for integrating different tool's tolerance and resolution at the same location. Moreover, keeping the biological sample in one place, while a set of tools rotates upon it similar to a set of microscopic lenses, we can avoid the non-real-time re-imaging, and inadvertent contamination. Applying an imposing electrical field, we can take the advantage of structure differences in smooth nuclear membranes inducing Van der Waal's forces versus random cytoplasm. (ii) The re-programming of transplanted cells to the ground state is unclear and usually relies on electrochemical means tested systematically in a modified 3D Caltech micro-fluidics. (iii) Our real-time MRA video-manipulator can elucidate the mitosis's tread-mill assembly mechanism in the development course of pluripotent stem cell differentiation into specialized tissue cell engineering. Such a combination bio-NanoRobot and micro-fluidic massive parallel assembly-line approach might not only replace the aspirating pipette with a self- enucleating Drosophila embryonic eggs, but also genetically reproduce a large amount of cloning embryonic eggs repeatedly for various re-programming hypotheses.

The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition.

PubMed

Purdy, P H; Fox, M H; Graham, J K

2005-08-01

Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 degrees C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium-p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 degrees C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 degrees C (P<0.05), however, adding cholesterol to the cells induced membranes to become more rigid (P<0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 degrees C and cryopreservation (P<0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 degrees C and after cryopreservation.

Sperm as microswimmers - navigation and sensing at the physical limit

NASA Astrophysics Data System (ADS)

Kaupp, Ulrich B.; Alvarez, Luis

2016-11-01

Many cells and microorganisms have evolved a motility apparatus to explore their surroundings. For guidance, these biological microswimmers rely on physical and chemical cues that are transduced by cellular pathways into directed movement - a process called taxis. Only few biological microswimmers have been studied as detailed as sperm from sea urchins. Sperm and eggs are released into the seawater. To enhance the chances of fertilization, eggs release chemical factors - called chemoattractants - that establish a chemical gradient and, thereby, guide sperm to the egg. Sea urchin sperm constitute a unique model system for understanding cell navigation at every level: from molecules to cell behaviours. We will outline the chemotactic signalling pathway of sperm from the sea urchin Arbacia punctulata and discuss how signalling controls navigation in a chemical gradient. Finally, we discuss recent insights into sperm chemotaxis in three dimensions (3D).

An Extensive Description of the Peptidomic Repertoire of the Hen Egg Yolk Plasma.

PubMed

Arena, Simona; Scaloni, Andrea

2018-03-28

Hen egg is a raw material widely used for the preparation of food,  pharmaceutical and cosmetoceutical products. Dedicated proteomic studies were accomplished on eggshell membrane, egg white, and yolk, identifying the most abundant proteins. No similar peptidomic studies have been performed so far. Only preliminary investigations on bioactive peptides in egg fractions and digestates were accomplished through functional screening assays, characterizing antioxidant, antibacterial, antiviral, immunomodulatory, and antihypertensive preparations and isolated components. This study fills this gap and provides a comprehensive picture of the peptides present in the yolk plasma of different hen egg types after 24 and 264 h of laying, taking advantage of a procedure based on a two-step fractionation followed by combined MALDI-TOF-TOF-MS- and nanoLC-ESI-Q-Orbitrap-MS/MS-based analysis. Six hundred and twenty-eight peptides were characterized as deriving from the proteolytic processing of larger protein components after the physiological action of chicken chymotrypsin-like and pepsin-like enzymes. Structural details on their post-translational modifications were also provided. Identified peptides were subjected to bioinformatic analysis and further compared with available data from the literature, ascertaining 198 peptides associable with putative antihypertensive, antimicrobial, anticancer, antiviral, antibiofilm, anorectic, calcium-binding, and anti-inflammatory activities. This analysis was often confirmative of previous experimental evidence on functional properties of unfractionated preparations or isolated molecules. These results further emphasize the bioactive action of yolk-derived peptides as related to egg consumption, and the potential use of these molecules as additive ingredients in the preparation of functional foods and cosmetics.

Tick vitellogenin receptor reveals critical role in oocyte development and transovarial transmission of Babesia parasite.

PubMed

Boldbaatar, Damdinsuren; Battsetseg, Badgar; Matsuo, Tomohide; Hatta, Takeshi; Umemiya-Shirafuji, Rika; Xuan, Xuenan; Fujisaki, Kozo

2008-08-01

A cDNA encoding the vitellogenin receptor of the ixodid tick, Haemaphysalis longicornis Neumann (HlVgR) was cloned and characterized. The full-length cDNA is 5631 bp, including an intact ORF encoding an expected protein with 1782 amino acids. The deduced amino acid sequence of the HlVgR cDNA revealed two ligand-binding domains with four class A cysteine-rich repeats in the first domain and eight in the second domain similar to those of insect VgRs. The immunoblot analysis detected approximately 197 kDa protein in both tick ovary and egg. The developmental expression profile demonstrated that HlVgR mRNA exists throughout the ovarian development, and the transcriptional level is especially high in the previtellogenic period. Immuno electron microscopy analysis demonstrated that the localization of HlVgR is detected on the external surface of oocyte plasma membrane. RNAi showed that eggs of HlVgR dsRNA-injected adult ticks had not developed into fully mature oocytes and laid abnormal eggs. The Babesia parasite DNA was not detected in the eggs of HlVgR dsRNA-injected tick that fed on Babesia gibsoni infected dog, whereas it was detected in the eggs of PBS-injected ticks and noninjected ticks. Expression of HlVgR was increased by the vitellogenic hormone 20-hydroxyecdysone. These results indicate that HlVgR, which is produced by the developing oocytes, is essential for Vg uptake, egg development in the H. longicornis tick, and transovarial transmission of Babesia parasites.

Effect of microwave exposure on the ovarian development of Drosophila melanogaster.

PubMed

Panagopoulos, Dimitris J

2012-06-01

In the present experiments the effect of GSM radiation on ovarian development of virgin Drosophila melanogaster female insects was studied. Newly emerged adult female flies were collected and divided into separate identical groups. After the a lapse of certain number of hours-different for each group-the insects (exposed and sham-exposed) were dissected and their intact ovaries were collected and photographed under an optical microscope with the same magnification. The size of the ovaries was compared between exposed and sham-exposed virgin female insects, during the time needed for the completion of oogenesis and maturation of the first eggs in the ovarioles. Immediately after the intact ovaries were photographed, they were further dissected into individual ovarioles and treated for TUNEL and acridine-orange assays to determine the degree of DNA damage in the egg chamber cells. The study showed that the ovarian size of the exposed insects is significantly smaller than that of the corresponding sham-exposed insects, due to destruction of egg chambers by the GSM radiation, after DNA damage and consequent cell death induction in the egg chamber cells of the virgin females as shown in previous experiments on inseminated females. The difference in ovarian size between sham-exposed and exposed virgin female flies becomes most evident 39-45 h after eclosion when the first eggs within the ovaries are at the late vitellogenic and post-vitellogenic stages (mid-late oogenesis). More than 45 h after eclosion, the difference in ovarian size decreases, as the first mature eggs of the sham-exposed insects are leaving the ovaries and are laid.

Effects of egg storage on hatchability, chick quality, performance and immunocompetence parameters of broiler chickens.

PubMed

Goliomytis, Michael; Tsipouzian, Theofania; Hager-Theodorides, Ariadne L

2015-09-01

Pre-incubation egg storage is a necessity for the poultry industry. This study evaluated the effects of pre-incubation storage length of broiler eggs on hatchability, 1-day-old chick quality, subsequent performance, and immunocompetence. To this end, a total of 360 hatching eggs were stored for 4, 12, or 16 d prior to incubation. Hatchability and chick quality were assessed at hatch, and growth performance and immunocompetence parameters were assessed during a 35 d rearing period. Hatchability of set and fertile eggs, and embryonic mortality, were not affected by egg storage. On the contrary, 1-day-old chick BW and length were linearly negatively correlated with egg storage length (P-linear<0.05). Nevertheless, BW corrected for egg weight prior to setting was unaffected, and corrected chick length was positively affected by storage length. One-day-old chick Tona score, navel quality, and post-hatch growth performance (BW at 7 and 35 d, cumulative feed intake, and feed conversion ratio at 35 d) were unaffected by egg storage (P, P-linear>0.05). Lymphoid organ weights at 2 and 35 d, the titre of maternal anti-NDV antibodies, most of the thymocyte subpopulations defined by CD3, CD4, and CD8 cell surface expression in the thymus of 2-d-old chicks, cellular responses to the PHA skin test, humoral responses to primary SRBC, and NDV immunizations were also not influenced by length of storage (P, P-linear>0.05). On the contrary, the length of egg storage was found to negatively influence the abundance of CD3+CD4-CD8- thymocytes that represent the majority of γδ-T cells in the thymus of 2-day-old chicks, as well as the humoral response to booster NDV immunization of the birds. In brief, pre-incubation storage of broiler hatching eggs for up to 16 d did not affect most developmental and growth parameters investigated, except for BW and length at hatch. Egg storage was found to suppress some aspects of the immunocompetence of the birds, particularly aspects of acquired immunity. © 2015 Poultry Science Association Inc.

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

PubMed Central

Stith, Bradley J.

2015-01-01

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation.

PubMed

Moraes, E A; Matos, W C G; Graham, J K; Ferrari, W D

2015-07-01

This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15min at 22°C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15min at 22°C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5°C over a 2h period. Loaded into 0.25ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P<0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency. Copyright © 2015 Elsevier B.V. All rights reserved.

Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

PubMed

Ubilla, A; Valdebenito, I; Árias, M E; Risopatrón, J

2016-05-01

In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell membrane disruption stimulates cAMP and Ca2+ signaling to potentiate cell membrane resealing in neighboring cells.

PubMed

Togo, Tatsuru

2017-12-15

Disruption of cellular plasma membranes is a common event in many animal tissues, and the membranes are usually rapidly resealed. Moreover, repeated membrane disruptions within a single cell reseal faster than the initial wound in a protein kinase A (PKA)- and protein kinase C (PKC)-dependent manner. In addition to wounded cells, recent studies have demonstrated that wounding of Madin-Darby canine kidney (MDCK) cells potentiates membrane resealing in neighboring cells in the short-term by purinergic signaling, and in the long-term by nitric oxide/protein kinase G signaling. In the present study, real-time imaging showed that cell membrane disruption stimulated cAMP synthesis and Ca 2+ mobilization from intracellular stores by purinergic signaling in neighboring MDCK cells. Furthermore, inhibition of PKA and PKC suppressed the ATP-mediated short-term potentiation of membrane resealing in neighboring cells. These results suggest that cell membrane disruption stimulates PKA and PKC via purinergic signaling to potentiate cell membrane resealing in neighboring MDCK cells. © 2017. Published by The Company of Biologists Ltd.

Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

PubMed

Lee, Seungki; Katayama, Naoto; Yoshizaki, Goro

2016-09-23

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of cholesterol depletion on membrane nanostructure in MCF-7 cells by atomic force microscopy

NASA Astrophysics Data System (ADS)

Wang, Yuhua; Jiang, Ningcheng; Shi, Aisi; Zheng, Liqin; Yang, Hongqin; Xie, Shusen

2017-02-01

The cell membrane is composed of phospholipids, glycolipids, cholesterol and proteins that are dynamic and heterogeneous distributed in the bilayer structure and many researches have showed that the plasma membrane in eukaryotic cells contains microdomains termed "lipid raft" in which cholesterol, sphingolipids and specific membrane proteins are enriched. Cholesterol extraction induced lipid raft disruption is one of the most widely used methods for lipid raft research and MβCD is a type of solvent to extract the cholesterol from cell membranes. In this study, the effect of MβCD treatment on the membrane nanostructure in MCF-7 living cells was investigated by atomic force microscopy. Different concentrations of MβCD were selected to deplete cholesterol for 30 min and the viability of cells was tested by MTT assay to obtain the optimal concentration. Then the nanostructure of the cell membrane was detected. The results show that an appropriate concentration of MβCD can induce the alteration of cell membranes nanostructure and the roughness of membrane surface decreases significantly. This may indicate that microdomains of the cell membrane disappear and the cell membrane appears more smoothly. Cholesterol can affect nanostructure and inhomogeneity of the plasma membrane in living cells.

Comparison of semen variables, sperm DNA damage and sperm membrane proteins in two male layer breeder lines.

PubMed

M, Shanmugam; T R, Kannaki; A, Vinoth

2016-09-01

Semen variables are affected by the breed and strain of chicken. The present study was undertaken to compare the semen quality in two lines of adult chickens with particular reference to sperm chromatin condensation, sperm DNA damage and sperm membrane proteins. Semen from a PD3 and White Leghorn control line was collected at 46 and 47 weeks and 55 weeks of age. The semen was evaluated for gross variables and sperm chromatin condensation by aniline blue staining. Sperm DNA damage was assessed by using the comet assay at 47 weeks of age and sperm membrane proteins were assessed at 55 weeks of age. The duration of fertility was studied by inseminating 100 million sperm once into the hens of the same line as well as another line. The eggs were collected after insemination for 15days and incubated. The eggs were candled on 18th day of incubation for observing embryonic development. The White Leghorn control line had a greater sperm concentration and lesser percentage of morphologically abnormal sperm at the different ages where assessments occurred. There was no difference in sperm chromatin condensation, DNA damage and membrane proteins between the lines. Only low molecular weight protein bands of less than 95kDa were observed in samples of both lines. The line from which semen was used had no effect on the duration over which fertility was sustained after insemination either when used in the same line or another line. Thus, from the results of the present study it may be concluded that there was a difference in gross semen variables between the lines that were studied, however, the sperm chromatin condensation, DNA damage, membrane proteins and duration over which fertility was sustained after insemination did not differ between the lines. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell Membrane Softening in Cancer Cells

NASA Astrophysics Data System (ADS)

Schmidt, Sebastian; Händel, Chris; Käs, Josef

Biomechanical properties are useful characteristics and regulators of the cell's state. Current research connects mechanical properties of the cytoskeleton to many cellular processes but does not investigate the biomechanics of the plasma membrane. We evaluated thermal fluctuations of giant plasma membrane vesicles, directly derived from the plasma membranes of primary breast and cervical cells and observed a lowered rigidity in the plasma membrane of malignant cells compared to non-malignant cells. To investigate the specific role of membrane rigidity changes, we treated two cell lines with the Acetyl-CoA carboxylase inhibitor Soraphen A. It changed the lipidome of cells and drastically increased membrane stiffness by up regulating short chained membrane lipids. These altered cells had a decreased motility in Boyden chamber assays. Our results indicate that the thermal fluctuations of the membrane, which are much smaller than the fluctuations driven by the cytoskeleton, can be modulated by the cell and have an impact on adhesion and motility.

Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

PubMed

Lete, Marta G; Byrne, Richard D; Alonso, Alicia; Poccia, Dominic; Larijani, Banafshé

2017-01-15

Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P 3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P 3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes. © 2017. Published by The Company of Biologists Ltd.

Insect water-specific aquaporins in developing ovarian follicles of the silk moth Bombyx mori: role in hydration during egg maturation.

PubMed

Maruyama, Mariya; Kambara, Kohei; Naka, Hideshi; Azuma, Masaaki

2015-08-01

Egg formation in terrestrial insects is an absorptive process, accommodated not only by packing proteins and lipids into yolk but also by filling chorions with water. An osmotic swelling of ovarian follicles takes place during oocyte maturation. This study investigated the role of the aquaporin (AQP) water channel in the osmotic uptake of water during oogenesis in the silk moth Bombyx mori Linnaeus, 1758. Using the antibodies that specifically recognize previously characterized AQPs, two water-specific subtypes-AQP-Bom1 and AQP-Bom3-belonging to the Drosophila integral protein (DRIP) and Pyrocoelia rufa integral protein (PRIP) subfamilies of the insect AQP clade, respectively, were identified in the developing ovaries of B. mori. During oocyte growth, Bombyx PRIP was distributed at the oocyte plasma membrane, where it likely plays a role in water uptake and oocyte swelling, and may be responsible for oocyte hydration during fluid absorption by ovarian follicles. During the transition from vitellogenesis to choriogenesis during oocyte maturation, Bombyx DRIP expression became abundant in peripheral yolk granules underlying the oocyte plasma membrane. The restricted DRIP localization was not observed in non-diapause-destined follicles, where DRIP was evenly distributed in medullary yolk granules. There was no difference in PRIP distribution between diapause- and non-diapause-destined follicles. The diapause-destined oocytes encase DRIP protein in the peripheral yolk granules, where DRIP might be inert. This would be reflected in the metabolic arrest associated with diapause after fertilization and egg oviposition. © 2015 Marine Biological Laboratory.

Combretastatin A-4 derived 5-(1-methyl-4-phenyl-imidazol-5-yl)indoles with superior cytotoxic and anti-vascular effects on chemoresistant cancer cells and tumors.

PubMed

Mahal, Katharina; Biersack, Bernhard; Schruefer, Sebastian; Resch, Marcus; Ficner, Ralf; Schobert, Rainer; Mueller, Thomas

2016-08-08

5-(1-Methyl-4-phenyl-imidazol-5-yl)indoles 5 were prepared and tested as analogs of the natural vascular-disrupting agent combretastatin A-4 (CA-4). The 3-bromo-4,5-dimethoxyphenyl derivative 5c was far more active than CA-4 with low nanomolar IC50 concentrations against multidrug-resistant KB-V1/Vbl cervix and MCF-7/Topo mamma carcinoma cells, and also against CA-4-resistant HT-29 colon carcinoma cells. While not interfering markedly with the polymerization of tubulin in vitro, indole 5c completely disrupted the microtubule cytoskeleton of cancer cells at low concentrations. It also destroyed real blood vessels, both in the chorioallantoic membrane (CAM) of fertilized chicken eggs and within tumor xenografts in mice, without harming embryo or mouse, respectively. Indole 5c was less toxic than CA-4 to endothelial cells, fibroblasts, and cardiomyocytes. In highly vascularized xenograft tumors 5c induced distinct discolorations and histological features typical of vascular-disrupting agents, such as disrupted vessel structures, hemorrhages, and extensive necrosis. In a first preliminary therapy trial, indole 5c retarded the growth of resistant xenograft tumors in mice. © 2016 Elsevier Science Ltd. All rights reserved. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

XCTK1: A Xenopus C-terminal Kinesin-like Protein

NASA Technical Reports Server (NTRS)

Winfree, Seth; Wilhelm, Heike; Sawyer, Alan; Karsenti, Eric; Mitchison, Tim; Walczak, Claire; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

2002-01-01

XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.