Changes in membrane potential, membrane resistance, and intracellular H, K, Na, and Cl activities during the progesterone-induced maturation of urodele amphibian oocytes

Changes in intracellular activities of H⁺, K⁺, Na⁺, and Cl⁻ ions were recorded with ion-selective microelectrodes during progesterone-induced maturation of full-grown oocytes of the urodele amphibians Ambystoma mexicanum and Pleurodeles waltlii. The membrane potential (E_m) and electrical resistance (R_m) were also determined. During the first hours after initiation of maturation, the oocytes slowly depolarized and R_m gradually increased. By the end of maturation of Pleurodeles oocytes E_m had stabilized at about −10 mV and R_m had increased from 410 to 1760 kΩ. The same initial pattern was observed for Ambystoma, but in most oocytes a rapid transition occurred at about the time of germinal vesicle breakdown (GVBD): E_m spontaneously shifted from about −15 to about +30 mV; simultaneously R_m dropped from 1230 down to 100 kΩ (i.e., less than the initial 270 kΩ resistance). The internal K⁺ activity did not show any important variation during maturation of Ambystoma and Pleurodeles oocytes. Na⁺ activity increased slightly at the onset of GVBD in Ambystoma; a further marked increase of Na⁺, accompanied by an increase in Cl⁻ activity, was observed as soon as E_m shifted to a positive value. In Pleurodeles sodium activity was also more elevated in matured than in immature oocytes. The average pH of Ambystoma immature oocytes was 7.48 ± 0.05 (external pH 7.5). A transient alkalinization to 7.64 ± 0.04 took place during the first 4–6 hr postprogesterone. Cytoplasmic pH was restored to 7.50 ± 0.07 between 10 and 12 hr postprogesterone, before the onset of GVBD and the shift of E_m. The difference between the measured oocyte pH and the calculated equilibrium pH decreases during the course of maturation, due partly to the depolarization of E_m.     

Rabbit oocyte maturation: changes of membrane resistance, capacitance, and the frequency of spontaneous transient depolarizations

Immature oocytes from rabbits were examined with electrophysiological techniques to determine if their membrane properties change during maturation. The input resistance increased and input capacitance decreased during maturation, although no significant change in membrane potential was observed. The changes observed were consistent with a decrease of corona radiata-oocyte electrical coupling accompanying maturation. Spontaneous transient depolarizations were recorded from immature oocytes surrounded by corona radiata, but not from mature ova. Each event consisted of a rapid depolarization, sustained for 1-100 sec, and a slow repolarization to the resting potential. Spontaneous inward currents with a time course similar to the spontaneous transient depolarizations occurred when the oocyte's membrane potential was held constant by voltage clamp. The frequency with which spontaneous transient depolarizations occurred decreased during maturation. These findings are consistent with a model in which spontaneous depolarizations originate in corona radiata cells and are detected in the oocyte via electrical coupling.     

Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Receptor-dependent translocation of diphtheria toxin across the surface membrane of Vero cells was studied using patch clamp techniques. Translocation was induced by exposing cells with surface-bound toxin to low pH. Whole cell current and voltage clamp recordings showed that toxin translocation was associated with membrane depolarization and increased membrane conductance. The conductance increase was voltage independent, with a reversal potential of approximately 15 mV. This value was unaffected by changing the Cl- gradient across the membrane and microfluorometric measurements showed that the cytosolic Ca2+ concentration was only marginally elevated by the translocation. The conductance increase is thus mainly due to monovalent cations. Exposing outside-out and cell-attached patches with bound toxin to low pH induced a new type of ion channel in the membrane. The channel current was inward at negative membrane potentials and the single channel conductance was approximately 30 pS. This value is about three times larger than for receptor-independent channels induced by diphtheria toxin or toxin fragments in artificial lipid membranes.     

Membrane depolarization facilitates sperm entry, large fertilization cone formation, and prolonged current responses in sea urchin oocytes

Depolarization of the sea urchin egg's membrane is required for two processes during fertilization: the entry of the fertilizing sperm and the block to polyspermy which prevents the entry of supernumerary sperm. In an immature sea urchin oocyte, the depolarization is very small in response to the attachment of a sperm. The purpose of this study was to determine whether the depolarization evoked by sperm attaching to an oocyte can facilitate sperm entry or induce the block to polyspermy. Individual oocytes of the sea urchin with diameters which ranged from 86 to 102% that of the average diameter for mature eggs from the same female were examined. The oocytes have a membrane potential of -73 +/- 6 mV (SD, n = 80) and a very low input resistance compared to that of mature eggs. Single sperm, following attachment to an oocyte, elicit a brief, small depolarization with a maximum amplitude of 8 +/- 1.4 mV (SE, n = 15), frequently followed by the formation of tiny filament-like fertilization cones, but the sperm fail to enter. If oocytes are voltage-clamped at membrane potentials more negative than -20 mV, following attachment of the sperm small transient inward currents occur, similar filament-like cones form, and the sperm do not enter. When many sperm attach to an oocyte which is not voltage clamped, the depolarizations sum to create a large depolarization with an amplitude of 60 to 80 mV, which shifts the oocyte's membrane potential to a value between -10 and +5 mV; more positive values are not attained. At such membrane potentials, whether the potential is maintained by the summed depolarizations of many attached sperm or by voltage clamp, large fertilization cones form, the sperm enter, and the oocytes can become highly polyspermic. In oocytes voltage clamped at +20 mV, however, both sperm entry and fertilization cone formation are suppressed. Therefore, both types of voltage-dependence for sperm entry are present in oocytes, although the depolarization caused by a single sperm is not large enough to permit its entry, nor is the depolarization caused by many sperm sufficient to prevent the entry of supernumerary sperm.     

Calcium currents correlate with oocyte maturation during the reproductive cycle in Octopus vulgaris

Using the whole-cell voltage clamp technique, we have studied the Ca2+ currents and the steady-state conductance during different oocyte growth stages and during the reproductive cycle of the female of Octopus vulgaris. Evidence is presented that L-type Ca2+ currents are high in small pre-vitellogenic oocytes (80-150 microm diameter) and significantly lower in early vitellogenic oocytes (180-300 microm diameter). Similarly, a significant decrease of the steady-state conductance occurred from the pre to early- vitellogenic oocytes.Octopus oocytes showed larger Ca2+ currents in the reproductive rather than non-reproductive periods. These data indicates that ion and L-type Ca2+ currents play a role in oocyte growth and cytoplasmic maturation, and possibly in preparing the plasma membrane to the interaction with the spermatozoon. By using fluorescent microscopy, we show that oocytes from 80 to 400 microm diameter have the large germinal vesicle characteristic of the immature oocytes. In subsequent stages of growth (up to 1000 microm diameter) the nucleus is no more visible and the metaphase spindle appears. These data demonstrate that Octopus vulgaris oocytes are arrested in the first meiotic prophase up to the early-vitellogenic stage and resume meiosis at this stage up to a second block presumably in metaphase I. We discuss a possible role for progesterone as the hormonal stimulus for the first prophase-metaphase meiotic transition.     

Maturation involves suppression of voltage-gated currents in the frog oocyte

Voltage- and time-dependent currents having slow kinetics have been studied in plasma membranes of immature oocytes of the european frog, Rana esculenta. IK, corresponding to an outward flow of K+, is activated at potentials more positive than about -40 mV, and subserves outward rectification; Iir, corresponding to an outward flow of Cl-, is activated at potentials more negative than about -80 mV and subserves inward rectification. Such currents can act as negative feedback mechanisms in the control of membrane potential in the immature oocyte and limit to a somewhat restricted range its possible deviations from resting values. Besides IK, membrane depolarizations to potentials more positive than about +30 mV are capable of activating INa, corresponding to outflow of Na+. By contrast, the frog mature egg-cell has a single voltage- and time-dependent current, IM, activated at potentials more positive than +30 mV, with properties similar to INa. The disappearance of IK and Iir along with remarkable reduction in leakage lowers impedance in the egg membrane. It seems reasonable to suggest that the observed changes in membrane permeability reflect changes which have taken place along the maturation process and are of importance for successful fertilization.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

Membrane currents in the oocyte of the toad Bufo arenarum

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes.     

FORMATION OF THE FERTILIZATION MEMBRANE BY INSEMINATION OF IMMATURE STARFISH OOCYTES PRETREATED WITH CALCIUM-FREE SEAWATER

When immature oocytes of the starfish, Asterina pectinifera , were treated with calcium-free seawater for 1 hr and then inseminated in normal seawater, they formed several blisters, indicative of polyspermy, and raised fertilization membranes. These oocytes continued to have intact germinal vesicles. Electron microscopic study revealed that the egg surface remained virtually unchanged after the treatment with calcium-free seawater. Upon insemination, however, the cortical granules broke down and the fertilization membrane was formed. These immature oocytes with ferilization membranes underwent maturation (germinal vesicle breakdown) after treatment with 1-methyladenine.
The treatment with calcium-free seawater seems to bring about some physiological change on the surface of immature oocyte, which bestows some attributes of maturation but is insufficient to mature the oocytes completely.     

Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation in vivo and in vitro

Heterologous intercellular communication was determined qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites and electrical current in hamster oocyte-cumulus complexes during meiotic maturation in vitro and in vivo. In addition, changes in cell resting potentials during maturation were recorded. Significantly less time was required for germinal vesicle breakdown (GVBD) in oocytes matured in vitro than in oocytes stimulated in vivo (1.81 +/- 0.06 hr, N = 13 vs 2.46 +/- 0.07 hr, N = 18, respectively, P less than 0.001). Resting potentials of the oocyte (RP-o) and cumulus cells (RP-c) significantly increased contemporaneously with GVBD in vitro (RP-o: from -18.9 +/- 3.2 mV to -33.2 +/- 2.9 mV, P less than 0.001; RP-c: from -16.3 +/- 1.9 mV to -27.5 +/- 2.6 mV, P less than 0.001) and in vivo after hCG injection (RP-o: from -16.8 +/- 5.9 mV to -30.1 +/- 3.9 mV, P less than 0.001; RP-c: from -15.5 +/- 3.8 mV to -26.3 +/- 3.2 mV, P less than 0.001). RP-o and RP-c progressively increased with time of culture up to 7 hr (maximum time examined) while the values reached maxima in in vivo matured oocytes 4.5 hr post-hCG and subsequently declined concomitant with the onset of cumulus expansion. Cumulus to oocyte coupling decreased progressively with time after release from meiotic arrest both in vitro and in vivo, as assessed by a progressive reduction in transfer of either uridine marker or lucifer yellow from the cumulus cell to the oocyte. By 4.5 hr after hCG injection, cumulus expansion had begun in 100% of complexes examined. Expansion was extensive by 7 hr post-hCG and spread of lucifer yellow from a cumulus cell was limited to very few adjacent cumulus cells. Oocyte to cumulus cell metabolic coupling also decreased progressively with time in both treatment groups. Examination of the extent of heterologous ionic coupling revealed that ionic coupling exhibited biphasic and, bidirectionally parallel, increases during meiotic maturation. While these temporal changes were observed in both groups, the coupling ratios were much greater in those complexes matured in vitro than in vivo. These results show that dye, metabolic, and electrical coupling exist between the immature hamster oocyte and its surrounding cumulus cells but that during the early stages of meiosis, metabolic and dye coupling decrease, while electrical coupling increases biphasically.     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

Maturation of pig oocytes: observations on membrane potential

The membrane-potential changes of pig oocytes during maturation are described. Cumulus-enclosed oocytes have a resting potential of -41.81 +/- 0.60 mV; the removal of cumulus cells caused this potential to drop to -30.95 +/- 0.43 mV. Adding LH to the culture medium did not influence the potential of denuded oocytes but depolarized the potential of cumulus-enclosed oocytes to -32.90 +/- 0.43 mV. FSH did not affect the membrane potential of denuded or cumulus-enclosed oocytes, but significantly reduced the amplitude of the depolarization induced by LH. The effect of gonadotropins on cultured granulosa cells was also investigated. Plated granulosa cells have a resting potential of -45.21 +/- 0.72 mV, similar to that of cumulus-enclosed oocytes. As recorded in cumulus-enclosed oocytes, LH depolarized granulosa cell membrane potential (-30.33 +/- 0.69 mV) and FSH reduced this effect. To evaluate if oocyte maturation in vivo is accompanied by membrane-potential depolarization, follicular growth and oocyte maturation were induced in 6 prepubertal gilts by using an eCG-hCG treatment. Twenty hours after the beginning of oocyte maturation in vivo (induced by hCG), the membrane potential of the oocyte was depolarized to -28.84 +/- 1.01 mV, a value similar to that observed in vitro. These data indicate that both LH and FSH can influence the membrane potential of follicular somatic cells and, consequently, that of the oocyte. The electrical coupling between somatic cell and oocyte may represent a means by which the gonadotropin message is passed to the germinal cell by the somatic compartment.     

Changes in voltage-dependent currents and membrane area during maturation of starfish oocytes: species differences and similarities

Full grown starfish oocytes are arrested at meiotic prophase I in the ovary. The natural hormone 1-methyladenine triggers oocyte maturation which involves meiosis reinitiation along with a variety of morphological, biochemical, and electrical changes. In studying oocytes of two species, Henricia leviuscula and Asterina miniata, using the voltage-clamp technique, we found interesting differences and similarities in the electrophysiological changes which occurred during maturation. Oocytes of both species have three major voltage-dependent currents: an inward Ca2+ current, an inwardly rectifying K+ current, and a transient outward K+ current (A-current). The Ca2+ current and the A-current were similar in the two species but the inward rectifier in Henricia had activation kinetics that were more than 10-fold slower than in Asterina. Nonetheless, all three currents were affected similarly during maturation: the inward Ca2+ currents remained constant in both species, while the two K+ currents decreased in amplitude. In Henricia the membrane surface area decreased substantially during maturation, while in Asterina it remained constant. This may be explained by the more highly infolded state of the membrane in the immature Henricia oocyte. The selective loss of K+ current followed the time course of the area decrease in Henricia, but the same percentage decrease in current occurred in Asterina without a net membrane loss.     

Ionic Blockage of Sodium Channels in Nerve

Increasing the hydrogen ion concentration of the bathing medium reversibly depresses the sodium permeability of voltage-clamped frog nerves. The depression depends on membrane voltage: changing from pH 7 to pH 5 causes a 60% reduction in sodium permeability at +20 mV, but only a 20% reduction at +180 mV. This voltage-dependent block of sodium channels by hydrogen ions is explained by assuming that hydrogen ions enter the open sodium channel and bind there, preventing sodium ion passage. The voltage dependence arises because the binding site is assumed to lie far enough across the membrane for bound ions to be affected by part of the potential difference across the membrane. Equations are derived for the general case where the blocking ion enters the channel from either side of the membrane. For H⁺ ion blockage, a simpler model, in which H⁺ enters the channel only from the bathing medium, is found to be sufficient. The dissociation constant of H⁺ ions from the channel site, 3.9 x 10^-6 M (pK_a 5.4), is like that of a carboxylic acid. From the voltage dependence of the block, this acid site is about one-quarter of the way across the membrane potential from the outside. In addition to blocking as described by the model, hydrogen ions also shift the responses of sodium channel "gates" to voltage, probably by altering the surface potential of the nerve. Evidence for voltage-dependent blockage by calcium ions is also presented.     

Quantification and distribution of equine oocyte cortical granules during meiotic maturation and after activation

In vitro fertilization (IVF) is being routinely used in humans and several domestic species, however, limited success has been achieved in the horse. Although immature equine oocytes are capable of completing meiosis in vitro, subsequent fertilization, and embryonic development of those oocytes are questionable. The lack of development of these oocytes could be attributed to an impaired cytoplasmic maturation. In the horse, the study of oocyte cytoplasmic maturation and post-fertilization development has been hindered by the lack of progress in IVF. In mammalian oocytes, migration of cortical granules (CG) has been used as an important criterion to evaluate cytoplasmic maturation. The aim of this study was to describe and quantify the CG distribution of equine oocytes during in vitro meiotic maturation and to assess activation of oocytes with calcium ionophore based upon fluorescein isothiocyanate (FITC)-labeled Lens culinaris agglutinin (LCA) and laser confocal microscopy. The results of this study indicate that CG are distributed throughout the cytoplasm of oocytes at the germinal vesicle (GV) stage (immature). As maturation proceeds, a progressive centripetal migration of CG to the oocyte cortex occurs with the formation of a monolayer adjacent to the plasma membrane starting by the end of a 30 hr incubation period and increasing significantly after 36 hr. After activation, significant reduction in the number of CG was observed (P < 0.001) suggesting that oocytes cultured under the present conditions possess the ability to release CG in response to the elevation of intracellular free calcium.     

The development of calcium and potassium currents during oogenesis in the starfish, Leptasterias hexactis

The development of membrane electrical properties of oocytes of the starfish Leptasterias hexactis during oogenesis was studied using voltage- and current-clamp techniques. Two voltage-dependent K currents--the fast transient and inwardly rectifying--are present early in oogenesis, before the rapid growth phase, and are maintained throughout oogenesis at the same current density and kinetics. The inward current, which is composed of a Ca current and a slower Ca-dependent inward sodium current, is also present early in oogenesis, but at very low current density. Late in oogenesis, after the oocyte has grown to full size, the inward current increases in amplitude by about fivefold, and undergoes major changes in kinetics. These changes are closely associated with the migration of the germinal vesicle to the cell periphery. The relationship of these events to electrophysiological changes during subsequent maturation and fertilization of the oocytes is discussed.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

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.     

Maturation of pig and rat oocytes transplanted into surrogate pig follicles in vitro

Pig oocytes obtained from slaughterhouse material and rat oocytes obtained from PMSG-treated immature females were incubated as isolated oocytes or injected into explanted pig follicles (5–8 mm). Free oocytes of both species, with or without their cumulus investment or gonadotropins during culture, matured at high rates after 30 hr or 9–10 hr of culture, respectively. Gonadotropic stimulation was necessary for maturation of both the native and injected cumulus-intact pig oocytes in follicle culture. Cumulus-free pig oocytes injected into follicle failed to mature in response to gonadotropic stimulation, suggesting an inability to perceive or respond to stimulation. Injected rat oocytes, however, matured irrespective of cumulus investment or gonadotropic stimulation. Their maturation was delayed and reduced at 9 hr. These results in the rat suggest that the pig follicular environment is incapable of regulating rat oocyte maturation but rather presents a permissive or supportive environment for their maturation. The explanted surrogate follicles from the pig or other species may provide a useful model for the study of oocyte-follicle interactions in oocyte maturation within or between species.