ANALYSIS OF SODIUM TRANSPORT IN THE AMPHIBIAN OOCYTE BY EXTRACTIVE AND RADIOAUTOGRAPHIC TECHNIQUES

The transport of Na⁺ in mature Eurycea oocytes was studied by quantitative radioautography of ²²Na⁺ using techniques suitable for localization of diffusible solutes, together with conventional extractive techniques. Intracellular Na⁺ consisted of three kinetic fractions: a cytoplasmic fast fraction of about 8.5 µeq/ml H₂O; a cytoplasmic slow fraction of about 58.7 µeq/ml H₂O; and a nuclear fast fraction of about 11.1 µeq/ml H₂O. A nuclear slow fraction, if it exists, does not exceed 5% of the cytoplasmic. The fast fractions represent freely diffusible Na⁺ in the two compartments; the nuclear solvent space is 1.3 times the cytoplasmic. The flux of both fast fractions is determined by the permeability of the cortical membrane, with neither the nuclear membrane nor diffusion in the cytoplasm detectably slowing the flux. The cytoplasmic slow fraction is interpreted to represent Na⁺ bound to nondiffusible constituents which are excluded from the nucleus; these may be yolk platelets, although the widespread observation of Na⁺ binding in other cells, and the high Na⁺/K⁺ selectivity, argues against simple ion-binding to the yolk phosphoprotein.     

Protein synthesis during meiotic maturation of mouse oocytes in vitro: Synthesis and phosphorylation of a protein localized in the germinal vesicle

Isolated fully grown mouse oocytes, arrested in dictyate of the first meiotic prophase, synthesize a protein with an apparent molecular weight of 28,000 which is localized in the germinal vesicle of the oocyte (germinal vesicle-associated protein; GVAP). Analyses of the distribution of GVAP have been carried out on SDS-polyacrylamide gels using oocytes cultured in vitro in the presence of [³⁵S]methionine or [³H]lysine and germinal vesicles isolated individually from these cultured oocytes. The results of such analyses show that GVAP contains only about 2% of the total radiolabel incorporated into mouse oocyte proteins, but as much as 40% of the total radiolabel incorporated into proteins associated with isolated germinal vesicles. These measurements indicate that GVAP is at least 1000-fold more concentrated in the germinal vesicle than in the cytoplasm of the oocyte. Furthermore, the synthesis and phosphorylation of GVAP are apparently terminated at a time which coincides with germinal vesicle breakdown during spontaneous meiotic maturation of mouse oocytes in vitro. Although the exact nature of GVAP is not known as yet, it appears to be an example of a protein that is selectively sequestered in the germinal vesicle of the oocyte during oogenesis and whose synthesis and modification are dependent upon the presence of an intact germinal vesicle.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Two components of auxin transport

The transport of indoleacetic acid-1-¹⁴C out of sunflower stem sections has been analyzed by a compartmental analysis procedure in which the radioactivity moving out of the tissue (log per cent) is plotted against time. The analysis indicates that indoleacetic acid is transported via a fast transport system (t_½ of about 30 minutes) and a slow transport system (t_½ about 10 hours). While we do not know the sources of these two pools, by analogy with ion transport studies, the fast efflux is characteristic of transport from the cytoplasm across the plasmalemma and the slow efflux is characteristic of transport across the tonoplast and thus out of the vacuole. Both components of transport are inhibited by 2,3,5-triiodobenzoic acid.     

THE PERMEABILITY OF THE AMPHIBIAN OOCYTE NUCLEUS, IN SITU

Ultralow temperature radioautography, suitable for the quantitative localization of diffusible solutes, was used to study the permeability of the nuclear envelope in the intact amphibian oocyte Sucrose-³H solutions were injected into mature oocytes, in volumes of 0 016–0 14% of that of the cell, and the subsequent movement of the solute was recorded. The resultant radioautographs show diffusion gradients in the cytoplasm and nucleus, and concentration gradients across the nuclear envelope Analysis of these gradients discloses that the nuclear envelope is as permeable as a comparable structure composed of cytoplasm, and is about 10⁸ times more permeable than the oocyte plasma membrane The diffusion coefficient of sucrose in cytoplasm is 2 x 10^-6 cm²/sec, or about one-third its diffusivity in pure water. This reduction can probably be accounted for by an effective lengthening of the diffusional path because of obstruction by cytoplasmic inclusions. The nuclear: cytoplasmic sucrose concentration ratio at diffusional equilibrium is about 3 05, or 1.6 times as great as expected from the water content of the two compartments This asymmetry is attributed to an unavailability of 36% of the cytoplasmic water as solvent Finally, sucrose entry into oocytes from a bathing solution was monitored by whole cell analysis and radioautography. These and the microinjection results are consistent with a model in which sucrose entry into the cell is entirely limited by the permeability of the plasma membrane. The results are inconsistent with cell models that hypothesize a short-circuit transport route from the extracellular compartment to the nucleus, and with models in which cytoplasmic diffusion is viewed as limiting the rate of solute permeation.     

The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans

Summary Externally applied membrane permeable cAMP derivatives and the injection of cAMP induce oocyte maturation in several species of hydrozoans. This technique for inducing oocyte maturation has been used to study ion permeability changes, maturation promoting factor activity and surface tension changes during maturation. Oocyte membrane potential remains constant during maturation. Cyclic AMP induced maturation proceeds in the absence of external Ca²⁺, K⁻, Mg²⁺ or Na⁺. Cytoplasm from maturing oocytes that induces oocyte maturation when it is injected into untreated oocytes is produced during cAMP induced maturation. Surface tension, as measured by the application of a standardized force that mechanically deforms individual oocytes, declines during the first part of maturation. This is followed by a sharp rise and fall of surface tension at first and second polar body formation that accompanies a slow rise in the resistance of oocytes to deformation during the last part of maturation. The production of maturation promoting factor activity and some of the changes in surface tension during maturation can occur in the absence of germinal vesicle material. Two early developmental events that follow oocyte maturation are the production of sperm chemoattractant and calcium channel function. Neither of these events occurs in eggs that have undergone maturation in the absence of germinal vesicle material. The addition of germinal vesicle contents from oocytes to eggs that have undergone maturation in the absence of germinal vesicle material initiates calcium channel function. This experiment indicates that the germinal vesicle contains factors that are necessary for post-maturation developmental events.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

THE GLYCEROL PERMEABILITY OF THE PLASMALEMMA OF THE HALOTOLERANT GREEN ALGA DUNALIELLA PARVA (VOLVOCALES)1

The glycerol permeability of the plasmalemma of the green alga Dunaliella parva Lerche was investigated by efflux studies with labelled glycerol, by enzymatic determination of glycerol leakage, and the determination of the reflection coefficient from osmotically induced volume changes (zero flow method). All results indicate that the plasmalemma of D. parva does not exhibit a special low permeability towards glycerol as would be expected from a glycerol accumulating alga. Rather, significant amounts of glycerol diffuse continuously into the medium following the glycerol concentration gradient between the cells and the medium. Efflux rates vary between 0.1 and 2 μmoles glycerol·mg^?1 chlorophyll·h^?1 depending on the external NaCl concentration. After one day up to 25% of the total glycerol of the algal suspension was found in the medium. Within 10 days this value can increase to 60%, depending on the growth constant of the culture. The reflection coefficient σ was determined to be 0.87, the permeability coefficient 2800 × 10^?11 m·sec^?1. To maintain a proper endogenous glycerol level corresponding to the external osmotic pressure, glycerol efflux in D. parva has to be balanced by a continuous synthesis of glycerol. D. parva follows the strategy of “glycerol efflux tolerance” instead of “glycerol efflux avoidance”. The alga has to pay the energetic costs of this strategy of tolerance.     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids Permeability and 13C-nuclear magnetic resonance measurements

¹³C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of ¹³C-NMR relaxation enhancement measurements using cholesterol enriched with ¹³C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-¹³C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether-phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated ⁴⁵Ca²⁺ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether-phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for ⁴⁵Ca²⁺ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether-, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. These results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether-phosphatidylcholine bilayers, the 4-¹³C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.     

Na+ and K+ uptake and exchange by the amphibian oocyte during the first meiotic division

The uptake and efflux of ²²Na and ⁴²K were studied in denuded Rana pipiens oocytes following progesterone induction of the resumption of meiotic maturation. Coincident with the breakdown of the large nucleus, or germinal vesicle, there is a virtual disappearance of K⁺ permeability of the oocyte plasma membrane. Only about 1–2% of the total [K⁺]_i is exchanged by completion of nuclear breakdown (8–10 hr) and accounts for the finding that there is no detectable change in total [K⁺]_i during the first meiotic division (20–24 hr). In the case of Na⁺, influx, exchange, and efflux kinetics were unchanged during the first meiotic division, with 20 and 35% of the total oocyte Na⁺ exchanging by the completion of nuclear breakdown and first meiotic division, respectively. Removal of Na⁺ from the incubation medium produced and earlier nuclear breakdown, whereas a K-free medium delayed breakdown. There was no effect of 10 μm/ml tetrodotoxin or 10^?5M strophanthidin on the time course of nuclear breakdown. Thus one action of progesterone appears to be a selective turning off of “K channels” in the oocyte plasma membrane. The disappearance of K selectivity of the oocyte plasma membrane coincides with plasma membrane depolarization, as well as nuclear swelling and breakdown.     

Glycerolization of Chinese hamster ovary cells in monolayer culture

CHO monolayer cultures were used as a model system to examine the kinetics of cell glycerolization and deglycerolization. Both influx and efflux of [³H]glycerol appear to be first-order processes, the rate of flux being proportional to glycerol concentration. When net flux ceases, the intracellular and extracellular concentrations of glycerol appear to be identical. Influx can be described by a single first-order curve, whereas efflux requires at least two such functions. Flux rates are sensitive to changes in osmolality, the fast efflux component accelerating and the slow component decelerating with increased application of osmotic stress. The rate of influx slows as the temperature drops and as the viscosity of the medium increases. Cells can tolerate a much steeper deglycerolizing gradient at 37 °C than at 0 °C.     

Compartmental efflux analysis and removal of extracellular cadmium from roots

Profiles of ¹⁰⁹Cd efflux from roots into three solutions were determined for young intact plants of Agrostis gigantea and maize. The solutions were (a) nutrient culture medium containing 3 micromolar Cd at room temperature, (b) ice-cold 5 millimolar CaCl₂, and (c) ice-cold 5 millimolar PbCl₂. Efflux profiles were clearly resolved into three easily discernible components having fast, medium, and slow exchange rates. These results were unexpected for the situation where some intracellular Cd was present both as extractable Cd-binding peptide and in electron-dense granules within the cytoplasm and the vacuoles. Adding a fourth compartment to the curve-fitting model produced a splitting of the fast exchanging component. Use of these efflux kinetics to estimate Cd fluxes through membranes was inappropriate. However, they were useful in determining optimal washing times for the removal of extracellular Cd. A 10 minute wash in ice-cold 5 millimolar CaCl₂ is recommended for this purpose for Agrostis and maize roots.     

The development of oocytes in the ovary of a parthenogenetic tick, Haemaphysalis longicornis

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10?days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens.     

Changes in Cell Membrane Permeability in Sunflower Hypocotyls Infected with Sclerotinia sclerotiorum

Influx and efflux of water and urea and electrolyte leakage are less for sunflower (Helianthus annuus) hypocotyl sections above lesions caused by Sclerotinia sclerotiorum than for those from healthy plants. Urea uptake by sections above lesions is reduced (celery, squash, and tomato) or unchanged (bean) in other hosts after Sclerotinia infection. Efflux of urea from sunflower hypocotyls is biphasic, suggesting diffusion in series from two cellular compartments (cytoplasm and vacuole). Efflux during the fast phase was 7 to 20 times greater than that during the slow phase. No difference was noted in urea efflux from healthy and diseased tissues during the slow phase. However, efflux during the fast phase from diseased tissues was slower than from healthy tissues, suggesting that the increased resistance to diffusion of urea in host cells above lesions resides in the plasmalemma. Water movement across cell membranes of healthy and diseased sunflower hypocotyls was reduced when tissues were treated with p-hydroxymercuribenzoate.     

Acetylcholine receptor-mediated sodium ion efflux after rapid hypoosmotic loading of radiotracer

Torpedo membrane vesicles (microsacs) enriched in acetylcholine receptors can be loaded with radiotracer permeants such as ²²Na⁺ and [³H]sucrose in only a few minutes. The fast loading technique employs exposure of vesicles to hypoosmotic solutions containing the radiotracers. Subsequent to loading of permeants, their efflux from the vesicles can be determined by a Millipore filtration technique. Efflux rates following hypoosmotic loading are comparable to those following the conventional loading technique which employs isotonic solutions. Hence, efflux measurements can be begun immediately after vesicle preparation. Furthermore, simultaneous loading of ²²Na⁺ and [³H]sucrose allows a normalization of ²²Na⁺ efflux with respect to vesicle volume, since ²²Na⁺, but not [³H]sucrose, is released specifically by cholinergic effectors. Such double-isotope measurements increase the reliability of the data derived.     

A theoretical model of the pinocytotic vesicular transport process in endothelial cells

A new theoretical model for vesicular transport in single endothelial cells is described using a kinetic molecular approach in which the vesicle diffusion process is coupled with the vesicle attachment/detachment process occurring at the cell plasmalemmal boundaries. Rate constants k^d_i, k_i characterizing a two stage reaction sequence in the attachment/detachment region and the vesicle diffusion coefficient D are obtained by comparison of the theory with the results of tracer studies. For the condition of rapid vesicle loading/discharge of macromolecules it is found that the permeability of endothelial cells to macromolecules tends to be controlled by the vesicular attachment/detachment process rather than the vesicle diffusion process. The rate limiting step in the vesicle attachment/detachment process tends to be the reaction process involving the rate at which a vesicle and the plasmalemmal membrane are brought into/separated from intimate contact rather than that involving the rate of formation/dissolution of the membrane diaphragm of an attached vesicle. Estimated relaxation times for processes occurring in the attachment/detachment region and in the diffusion region, the vesicle transit time in the diffusion region, and the viscosity of the cytoplasm in the diffusion region are deduced. Fair agreement is obtained between the predicted and the observed temperature dependence of the permeability.     

A procedure for the simultaneous determination of small quantities of hyaluronate and isomeric chondroitin sulfates by chondroitinases

A simple, commercially available apparatus is described which can be used for injection of nanoliter quantities of aqueous solutions into the germinal vesicle or the cytoplasm of Xenopus laevis oocytes.     

Nature of progesterone action on amino acid uptake by isolated full-grown oocyte of Xenopus laevis

l-leucine uptake into full-grown oocytes of Xenopus laevis is a saturable process which is Na⁺ dependent and presumably coupled to Na⁺ gradient. Our results indicate that progesterone (10^?6 M) blocks abruptly, around the germinal vesicle breakdown, the saturable transport of l-leucine. $\text{p-}\text{Chloromercuribenzoate}$ (10^?4 M) induces maturation and after a short lag of time strongly inhibits l-leucine uptake. Cycloheximide prevents progesterone-induced maturation and permeability changes.     

Functional symmetry of the β-galactoside carrier in escherichia coli

Cytoplasmic membrane vesicles with either normal or inverted orientation were prepared from Escherichia coli. The lactose transport activity of these vesicle preparations was compared. The parameters measured were net efflux, counterflux, and K⁺/valinomycin-induced active uptake of lactose. With membrane vesicles derived from both wild-type and cytochrome-deficient strains the right-side-out and inverted membrane preparations showed similar rates of lactose flux in all assays. According to these criteria, the activity of the β-galactoside transport protein is inherently symmetrical.One major difference was observed between the native and inverted vesicle preparations: the inverted vesicles had approximately twice the specific activity of native vesicles in the counterflux and K⁺/valinomycin-induced uptake assays. This difference can be largely ascribed to the presence in the normal vesicle preparation of vesicles with a high passive permeability to lactose. Such vesicles are apparently absent from the inverted vesicle preparations.     

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian,Styela

The regional distribution of poly(A)⁺ RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [³H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [³H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [³H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [³H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [³H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [³H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A)⁺ RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela.