Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipens

The region of the frog egg that is receptive to fertilization was determined. As an approximation to the site of sperm entry, the start of the male pronuclear penetration path within the egg was made visible externally by bleaching fixed eggs. A bleached egg had a pigment accumulation on its surface corresponding to the start of the penetration path. The accumulation characteristically changed shape with cortical movements prior to first cleavage, and most accumulations (path starts) were within 60° of the animal pole.Localized inseminations and an analysis of the distribution of failures of fertilization at the egg plasma membrane demonstrated that few if any sperm entered the vegetal region of the egg. Localized inseminations, however, demonstrated that sperm entered between 60° from the animal pole and the animal-vegetal margin.Although sperm entry occurred throughout the animal region, most penetration paths started within 60° of the animal pole. To account for this, the sperm nucleus must move towards the animal pole prior to starting the penetration path. This movement appeared to be due to a contraction of the cortex towards the animal pole that occurred 3–4 min after activation of the egg.     

Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes.

The expression of beta-actin, gamma-actin, alpha-tubulin, and beta-tubulin mRNA during the lectin activation of human peripheral blood lymphocytes was examined with specific cDNA clones. The resting lymphocyte has a low level of both alpha- and beta-tubulin mRNAs, and these increase 10-fold after 72 h of lectin stimulation in which maximum cell transformation is achieved. Although there is a slight increase in tubulin mRNA during the first 6 h, most of the increase occurs between 6 and 24 h as the cells start to increase their RNA content and progress from G0 into G1. Both beta- and gamma-actin mRNAs are more abundant than the tubulin mRNAs in resting cells, with beta-actin mRNA being the major species. Upon activation, beta-actin mRNA increases threefold, whereas gamma-actin mRNA increases almost sixfold. Both beta- and gamma-actin mRNA are elevated 2.5-fold as early as 6 h, the gamma-actin mRNA level then increasing more than beta-actin between 6 and 24 h, resulting in the reduced beta-actin/gamma-actin mRNA ratio. The lectin-stimulated lymphocyte has a similar beta-actin/gamma-actin mRNA ratio as that of the human leukemic T-lymphoblast cell line CCRF-CEM. These increases are over and above the general increase in polyadenylated RNA content upon lectin activation. On returning to a noncycling state, the levels of these cytoskeletal mRNAs decrease. There were two beta-tubulin mRNAs present in lymphocyte cytoplasm, one of 1.8 kilobases and one of 2.8 kilobases in length. The nongrowing lymphocytes had relatively lower levels of the larger sized mRNA. Upon stimulation, the relative level of the larger mRNA was increased, and at 72 h the cells had approximately equal levels of both mRNAs as did the leukemic lymphoblasts.     

Changes in cytokine levels and NK cell activation associated with influenza

Several studies have highlighted the important role played by murine natural killer (NK) cells in the control of influenza infection. However, human NK cell responses in acute influenza infection, including infection with the 2009 pandemic H1N1 influenza virus, are poorly documented. Here, we examined changes in NK cell phenotype and function and plasma cytokine levels associated with influenza infection and vaccination. We show that absolute numbers of peripheral blood NK cells, and particularly those of CD56(bright) NK cells, decreased upon acute influenza infection while this NK cell subset expanded following intramuscular influenza vaccination. NK cells exposed to influenza antigens were activated, with higher proportions of NK cells expressing CD69 in study subjects infected with seasonal influenza strains. Vaccination led to increased levels of CD25+ NK cells, and notably CD56(bright) CD25+ NK cells, whereas decreased amounts of this subset were present in the peripheral blood of influenza infected individuals, and predominantly in study subjects infected with the 2009 pandemic H1N1 influenza virus. Finally, acute influenza infection was associated with low plasma concentrations of inflammatory cytokines, including IFN-γ, MIP-1β, IL-2 and IL-15, and high levels of the anti-inflammatory cytokines IL-10 and IL-1ra. Altogether, these data suggest a role for the CD56(bright) NK cell subset in the response to influenza, potentially involving their recruitment to infected tissues and a local production and/or uptake of inflammatory cytokines.     

Microtubules and specification of the dorsoventral axis in frog embryos

The body plan of the frog is set-up by a rearrangement of the egg cytoplasm shortly after fertilization. Microtubules play several roles in this critical developmental event.     

Electrophysiological study of the frog egg at various stages of development. IV. Changes associated with the maturation process

The I/V relationship of frog oocytes is markedly modified following maturation, lacking the mature unfertilized egg cell membrane of K-selective channels and the leakage being strongly reduced. The only kind of voltage-dependent channels still present in this membrane are activated by depolarizing steps to potentials more positive than 30-40 mV; the current flowing through these channels is carried at least partly by Na+. At difference with the immature oocyte, where several ionic mechanisms are oriented to maintain the membrane potential at a constant level, the mature egg-cell admits considerable voltage deflections. The observed variations in membrane properties could be of importance in allowing the maturation process and/or in preparing the mature egg-cell to successful fertilization.     

Root contraction in hyacinth. II. Changes in tubulin levels,microtubule number and orientation associated with differential cell expansion

Root contraction in hyacinth (Hyacinthus orientalis L.) is marked by reoriented cell growth in the cortex of the contractile region. Cellular volume of the inner cortex enlarges fourfold during root contraction. This is associated with large increases in the radial and tangential dimensions and decreases in the longitudinal dimension of the cells. In order to determine the possible role of microtubules (MTs) in these changes we compared tubulin levels and MT numbers and orientation in contracted and non-contracted regions of hyacinth roots. Tubulin content was analysed by a radioimmunoassay; MT numbers and orientation were analyzed by counting profiles in sectioned material using transmission electron microscopy. Contracted tissue was found to have significantly higher levels of tubulin on a per-cell basis than non-contracted tissue, and also increased tubulin levels relative to total protein. The spatial MT frequencies were the same in contracted and non-contracted tissues, indicating a proportional increase in MT numbers in the expanded cells. Although the absolute spatial frequency of MTs was constant, the orientation, as determined by morphometric analysis of MT profiles, was not. While in the longitudinal section plane 42% of the MTs in the non-contracted cells were oblique, in the contracted cells the percentage of MTs presenting oblique profiles increased to 87%. Additionally, a qualitative difference in MTs was observed in contracted cells; electron-opaque material was seen peripherally associated with the MTs of the inner cortex. The changes in tubulin levels and in MT numbers as well as the qualitative differences in the MTs of contracted and non-contracted root regions indicate that, in hyacinth, reoriented cellular enlargement associated with root contraction cannot be explained simply by shifts in the arrangement of preexisting cortical MT arrays, but involves more complex changes in the cytoskeleton.Abbreviations MT(s) microtubule(s) - TEM transmission electron microscopy - RIA radioimmunoassay - M_r apparent molecular mass I=Jernstedt (1984b)     

A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity

Drosophila embryo dorsoventral (DV) polarity is defined by serine protease activity in the perivitelline space (PVS) between the embryonic membrane and the inner layer of the eggshell. Gastrulation Defective (GD) cleaves and activates Snake (Snk). Activated Snk cleaves and activates Easter (Ea), exclusively on the ventral side of the embryo. Activated Ea then processes Sp?tzle (Spz) into the activating ligand for Toll, a transmembrane receptor that is distributed throughout the embryonic plasma membrane. Ventral activation of Toll depends upon the activity of the Pipe sulfotransferase in the ventral region of the follicular epithelium that surrounds the developing oocyte. Pipe transfers sulfate residues to several protein components of the inner vitelline membrane layer of the eggshell. Here we show that GD protein becomes localized in the ventral PVS in a Pipe-dependent process. Moreover, ventrally concentrated GD acts to promote the cleavage of Ea by Snk through an extracatalytic mechanism that is distinct from GD's proteolytic activation of Snk. Together, these observations illuminate the mechanism through which spatially restricted sulfotransferase activity in the developing egg chamber leads to localization of serine protease activity and ultimately to spatially specific activation of the Toll receptor in the Drosophila embryo.     

Immunoneutralization of insulin partially modifies dorsoventral patterning in developing frog embryos

Determination of anteroposterior and dorsoventral axes is an important early event in the development of vertebrates involving extensive cellular interactions including inductive events. Recently we showed that insulin plays an essential role in prepancreatic development of the frogMicrohyla ornata. In the present study we have investigated the effects of immunoneutralization of endogenous insulin on the process of pattern formation. Treatment of neurulating embryos with antiserum to insulin caused abnormal pattern formation. The defects included loss of normal architecture of the neural tube, reduction in the size of the neural tube and, most conspicuously, rotation of the dorsoventral axis of the neural tube, notochord and adjoining mesodermal elements. The effects could be alleviated partially by pretreatment of embryos with exogenous insulin. This supports our belief that insulin plays an important role in induction and pattern formation of the amphibian nervous system. In addition, using 2-deoxy-α-D-glucose, an inhibitor of glucose metabolism, it is shown that the stimulatory effects of exogenous insulin on developing frog embryos are, at least partially, through the glucose metabolism pathway. Preliminary results of this study were presented at the National Symposium on Genes and Human Environment, held at Hyderabad, February 1994 and DAE Symposium on Stress and Adaptive Responses in Biological Systems, held at Vadodara, March 1994.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H(2)O(2)) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca(2+)/Mg(2+)-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca(2+)/Mg(2+)-supplemented medium. In Ca(2+)/Mg(2+)-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 microm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H(2)O(2) (84.40+/-0.50 ng/egg) when compared to control eggs (80.46+/-1.34 ng/egg). The higher concentration of calcium ionophore (1.6 microm) induced apoptosis and pronounced generation of intracellular H(2)O(2) (92.43+/-0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H(2)O(2) level (81.20+/-1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H(2)O(2) in rat eggs.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H₂O₂) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca²⁺/Mg²⁺-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca²⁺/Mg²⁺-supplemented medium. In Ca²⁺/Mg²⁺-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H₂O₂ (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H₂O₂ (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H₂O₂ level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H₂O₂ in rat eggs.     

Changes in oviductal morphology of the skink, Lampropholis guichenoti, associated with egg production

We describe changes in the morphology of the oviductal epithelium of an oviparous skink, Lampropholis guichenoti, during the course of egg production and oviposition: to characterize the luminal epithelial changes; to provide a baseline for understanding uterine changes in viviparous species; and to establish whether the plasma membrane transformation of uterine epithelial cells is indeed a feature restricted to viviparous species. Oviducts from vitellogenic, gravid, and postgravid females were observed using scanning electron microscopy. Cellular characteristics of the oviductal epithelium previously used to determine the plasma membrane transformation were assessed morphologically. Three anatomically different areas were defined within the oviduct, but no plasma membrane transformation was observed in the oviparous skink, suggesting that this is a phenomenon particular to viviparity.     

Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.     

A highly localized activation current yet widespread intracellular calcium increase in the egg of the frog, Discoglossus pictus

Sperm entry in the egg of the painted frog, Discoglossus pictus, occurs only at a specialized region of the animal hemisphere called the animal dimple, a structure not found in other species of frog. An extracellular vibrating electrode was used to measure the activation current to determine if the ion channels that open to generate the fertilization potential are localized in this region. Eggs that were activated by microinjecting inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) exhibited activation potentials very similar to those of fertilized eggs. There was a delay between the time of Ins(1,4,5)P3 injection and the initiation of the activation potential that was proportional to the distance between the site of the activating stimulus and the animal dimple, similar to the delay previously observed in prick-activated eggs (R. Talevi, B. Dale, and C. Campanella (1985). Dev. Biol. 111, 316-323). The delay lasted 30 sec when the stimulus site was 20 degrees (300 micron) from the animal dimple and 14 min when it was 150 degrees C from the dimple. Once the activation potential was initiated, there was an excellent temporal correlation between the time of depolarization and the time of the first detectable current entering the dimple region. This inward current was typically 60 microA/cm2 in amplitude and was found only in the central 200 micron of the dimple region. The outward current was distributed over the remainder of the egg surface and was much smaller in amplitude. The activation current was carried by Cl- efflux in the animal dimple region, and was reduced by DIDS and reversed by high external Cl- or I-. The occurrence of inward current only at the dimple region indicates that Cl- channels which open to produce the activation potential are localized there. Using Ca2+-specific microelectrodes, we found that [Ca2+]i increased from 0.25 to 2 microM following both fertilization and activation and returned to the unactivated level after about 37 min. Immature oocytes of D. pictus were also studied with the vibrating probe and the inward current in these cells was much less localized than that in the activating egg. A steady transcellular current of up to 4 microA/cm2 entered the entire animal hemisphere of the oocyte and exited the vegetal hemisphere.     

Changes in brain amine levels associated with the morphological and behavioural development of the worker honeybee

Summary Changes in biogenic amine levels associated with the morphological and behavioural development of the worker honeybee are examined.A significant increase in amine levels in the head of the honeybee is associated with transition from the larval to pupal stage. Adult emergence is also accompanied by a significant increase in 5-HT levels in the brain, but no significant change in brain dopamine (DA) levels. NADA (N-acetyldopamine) levels increase during larval and pupal development, but in contrast to both DA and 5-HT, drop significantly during the transition from pupa to adult.Levels of DA in the brain of nectar and pollen forager bees, presumed to be among the oldest adults sampled, were found to be significantly higher than in nurses, undertakers or food storers. These results suggest that an age-dependent change in amine levels occurs in the brain of the worker bee. In the optic lobes, levels of DA and 5-HT were found to be significantly higher in pollen forager bees than in all other behavioural groups. Significant differences in amine levels in the optic lobes of nectar foragers and pollen foragers indicate that some differences in amine levels occur independent of worker age. The functional significance of differences in brain amine levels and whether or not biogenic amines play a direct role in the control of honeybee behaviour has yet to be established.Abbreviations DA dopamine - 5-HT 5-hydroxytryptamine or serotonin - NADA N-acetyldopamine     

Changes in the sedimentation properties of cytosolic androgen receptors associated with activation in vitro and in vivo

In cell-free systems androgen receptor (AR) labeled with (3H)DHT at 0 degrees C in the presence of 50mM molybdate remains unactivated (less than 3% binding to nuclei) and untransformed (7-8S on sucrose density gradients containing 0.4M KCl and 50mM molybdate). In the absence of molybdate, however, these complexes undergo activation and transformation even at 0 degrees C, albeit, very slowly. Incubation of unactivated, untransformed AR complexes at 18 degrees C, or at 0 degrees C in the presence of 0.4M KCl, greatly accelerated both activation and transformation. Activation and transformation are also associated with formation of high affinity (3H)DHT-receptor complexes as indicated by decreased rates of (3H)DHT dissociation from the receptor. Cytosolic AR complexes labeled with (3H)DHT in tissue slices at 37 degrees C, or in vivo, undergo rapid activation, transformation and nuclear translocation. The data suggest that activation and transformation of cytosolic AR in cell-free systems is associated with changes in the physicochemical properties of AR similar to those occurring upon hormone binding in intact cells and in vivo.