Biomedical subjects

D Kline

Publications and source records attributed to D Kline.

At least 19 recordsLinked to original sources

PubMed1996

Redistribution and increase in cortical inositol 1,4,5-trisphosphate receptors after meiotic maturation of the mouse oocyte.

Mouse oocytes develop sensitivity to inositol 1,4,5-trisphosphate (IP3) during oocyte maturation. We recently reported that a change in the organization of the endoplasmic reticulum (ER) during oocyte maturation may contribute to this enhanced sensitivity (Mehlmann et al., 1995, Dev. Biol. 170, 607-615). Here, we investigated whether there is an increase in the number of available IP3 receptors after maturation and whether there is a redistribution of IP3 receptors similar to the redistribution of the ER that occurs during maturation. Western blot analysis of the IP3 receptor in oocytes and eggs demonstrated a 1.8-fold increase in immunoreactive mass of the IP3 receptor following oocyte maturation. Microinjection of the function-blocking monoclonal antibody 18A10 inhibited IP3-induced Ca2+ release in a concentration-dependent manner in both eggs and oocytes. More antibody was required to inhibit Ca2+ release to the same extent in eggs compared to oocytes when both were injected with the same concentration of IP3, suggesting that eggs contain a greater number of functional IP3 receptors. Immunolocalization of the IP3 receptor revealed that receptors were present in large clusters, 1-2 microm in diameter, in the cortex of the mature egg except in a ring-shaped band of cortex adjacent to the meiotic spindle. In contrast, receptor clusters were located around the entire cortex of the immature oocyte and were much smaller (<1 microm); larger patches were sometimes seen, but they did not display the same spherical organization as those in eggs. These results suggest that the number of cortical IP3 receptors increases during mouse oocyte maturation and that this increase may contribute to enhanced Ca2+ release at fertilization.

Animals↗

PubMed1995

Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly.

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca2+]i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca2+]i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca2+]i, and despite disruption of the spindle.

Animals↗

PubMed1995

Maintenance of metaphase in colcemid-treated mouse eggs by distinct calcium- and 6-dimethylaminopurine (6-DMAP)-sensitive mechanisms.

In mouse eggs, the arrest at meiotic metaphase II is released by the fertilization-induced increase in intracellular calcium. When eggs treated with the microtubule inhibitor colcemid are fertilized or exposed to the calcium ionophore A23187, normal calcium increases occur, but the eggs remain at metaphase. However, when colcemid-treated eggs are fertilized or A23187-treated and then exposed to the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), they enter interphase. Although colcemid-treated eggs require a calcium signal and exposure to 6-DMAP, colcemid-treated embryonic cells are released from metaphase by treatment with 6-DMAP, but not by exposure to A23187. These results suggest that two distinct mechanisms maintain metaphase: one is the calcium-sensitive mechanism involving cytostatic factor, which normally maintains metaphase II arrest in eggs; the other mechanism, which may require the activity of 6-DMAP-sensitive kinases, maintains metaphase in the absence of spindle assembly in both mitotic cells and eggs.

Adenine↗

PubMed1995

Reorganization of the endoplasmic reticulum during meiotic maturation of the mouse oocyte.

The endoplasmic reticulum (ER) of live metaphase II mouse eggs and prophase I-arrested oocytes was compared using the fluorescent, lipophilic dicarbocyanine dye, DiI. DiL, dissolved in soybean oil, was microinjected into oocytes and eggs; the dye diffused throughout the cytoplasm to label the ER, which was imaged by confocal microscopy. The mature egg had a fine reticular network of ER throughout the cell and numerous dense accumulations of membrane in the cortex. These ER accumulations, 1-2 microns in diameter, were generally absent deeper in the cytoplasm. A similar staining pattern was observed when the eggs were fixed within 1 min of injection, providing evidence that the cortical accumulations of membrane are part of a continuous ER membrane system, since membrane trafficking could not occur in a fixed egg. Cortical ER accumulations were localized to the same region of the egg as the cortical granules and were not observed in the cortical granule-free region adjacent to the meiotic spindle. In contrast, ER accumulations were rarely found in the cortex of the immature, prophase I-arrested oocyte, but larger and less well-defined membrane clusters were found throughout the deeper cytoplasm of the oocyte. The appearance of ER clusters in the egg cortex following oocyte maturation correlates with an increased ability of the mature egg to release calcium at fertilization. Since the ER is a calcium store, structural reorganization of the ER may be necessary to permit the large release of calcium and resulting cortical granule exocytosis at fertilization.

Animals↗

PubMed1995

Calcium-independent, meiotic spindle-dependent metaphase-to-interphase transition in phorbol ester-treated mouse eggs.

Mouse eggs, arrested at metaphase II of meiosis, form pronuclei as a result of fertilization or exposure to parthenogenetic agents, such as the phorbol ester phorbol myristate acetate (PMA). Exposure of eggs to the microtubule inhibitor colcemid caused the disappearance of the meiotic spindle and prevented the PMA-induced release from metaphase. However, colcemid- and PMA-treated eggs which lacked spindles formed nuclei when treated in addition with the protein synthesis inhibitor cycloheximide or the protein kinase inhibitor 6-dimethlyaminopurine. To prevent an increase in intracellular calcium concentration ([Ca2+]i), eggs were exposed to the cell permeant acetoxymethyl ester (AM) form of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA). Nuclei formed in 45% of eggs treated with BAPTA AM and PMA and in 90% of eggs treated with BAPTA AM, PMA, and cycloheximide, suggesting that an increase in [Ca2+]i is not necessary for the PMA-induced release from metaphase. The [Ca2+]i did not change in eggs which formed nuclei in response to PMA, providing additional evidence that PMA activates eggs without elevating the [Ca2+]i.

Animals↗

PubMed1995

Absence of an intracellular pH change following fertilisation of the mouse egg.

The intracellular pH of the mouse egg was measured during fertilisation to determine whether an increase in pH accompanies activation of this mammalian egg. The pH-sensitive dye BCECF [2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein] was introduced into the mouse egg by incubation in BCECF-AM or by microinjection of dextran-conjugated BCECF. The cells were also loaded with the DNA-specific fluorochrome Hoechst 33342 to confirm fertilisation by observation of Hoechst-stained, decondensing sperm heads in the cytoplasm. The ratio of emission intensities for the dye (494/440 nm excitation wavelengths) was monitored continuously with a photon-counting photomultiplier tube. There was no change in pH during or after fertilisation. Control eggs displayed the expected increase in pH when exposed to NH4Cl. In other experiments, intracellular pH and intracellular Ca2+ were monitored simultaneously during fertilisation. The eggs were injected with BCECF dextran and Fura dextran. Fluorescence emission was recorded at excitation wavelengths of 495 nm (BCECF, pH-sensitive wavelength) and 385 nm (Fura, Ca(2+)-sensitive wavelength). A decrease in emission intensity at 385 nm excitation clearly marked the repetitive Ca2+ transients at egg activation. There was no change in the fluorescence emitted at 495 nm excitation, indicating an absence of any change in intracellular pH. These results indicate that intracellular alkalinisation of the cytoplasm does not accompany activation of this vertebrate egg.

Animals↗

PubMed1995

Activation of hamster zona-free oocytes by homologous and heterologous spermatozoa.

Spermatozoa of a wide variety of species can fuse with zona-free hamster oocytes. Zona-free hamster oocytes were inseminated with spermatozoa of homologous (hamster) and other (mouse, guinea-pig and human) species, and their responses were closely examined to determine whether such interspecific sperm-oocyte fusion always induces normal oocyte activation. While guinea-pig and human spermatozoa could activate hamster oocytes as efficiently as hamster spermatozoa, mouse spermatozoa could not. Mouse spermatozoa fused readily with hamster oocytes, yet most oocytes remained inactivated at least during the first 1.5-2 h. The amount of M-phase (metaphase) promoting factor was reduced in hamster oocytes fused with one or several mouse spermatozoa; however, repetitive Ca2+ transients failed to occur unless oocytes were inseminated with a concentrated sperm suspension and penetrated by very many spermatozoa. These observations suggest that sperm-oocyte membrane fusion per se is not sufficient to trigger oocyte activation, and that putative sperm-derived oocyte activating factors show some degree of species specificity.

Animals↗

PubMed1994

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

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.

Animals↗

PubMed1994

Regulation of intracellular calcium in the mouse egg: evidence for inositol trisphosphate-induced calcium release, but not calcium-induced calcium release.

Fertilization of the mammalian egg initiates transient and repetitive release of Ca2+ from intracellular stores. The mechanism by which these Ca2+ transients are produced is not completely known. We examined the role of two principal Ca2+ release mechanisms, inositol trisphosphate-induced Ca2+ release and Ca(2+)-induced Ca2+ release, in altering intracellular Ca2+ in the mouse egg. Microinjection of inositol 1,4,5-trisphosphate (IP3) transiently elevated intracellular Ca2+ and, at higher concentrations, produced repetitive Ca2+ transients. Addition of 100 microM thimerosal, a sulfhydryl reagent, caused repetitive Ca2+ transients. IP3 and thimerosal responses were inhibited by prior injection of heparin, a competitive antagonist of IP3-induced Ca2+ release. Addition of caffeine or injection of caffeine, ryanodine, or cyclic ADP-ribose, which are known to initiate or modulate Ca(2+)-induced Ca2+ release in sea urchin eggs and other cells, produced no change in intracellular Ca2+. The response to injection of Ca2+ was not altered by prior injection of ryanodine. The magnitude of the Ca2+ transients produced by injection of IP3 was not changed by prior injection of cyclic ADP-ribose or external caffeine. We found no evidence of Ca(2+)-induced Ca2+ release from ryanodine- or caffeine-sensitive stores. It is most likely that release of Ca2+ from intracellular stores in the mouse egg is dependent on IP3-induced Ca2+ release.

Adenosine Diphosphate Ribose↗

PubMed1994

Regulation of intracellular calcium in the mouse egg: calcium release in response to sperm or inositol trisphosphate is enhanced after meiotic maturation.

Fertilization of the immature, prophase I-arrested mouse oocyte produces multiple Ca2+ transients similar to those of the mature, metaphase II egg; however, the first Ca2+ transient is much lower in amplitude and shorter in duration. In contrast to prophase I-arrested oocytes, maturing oocytes fertilized after germinal vesicle breakdown have first Ca2+ transients similar to those of mature fertilized eggs. Immature, prophase-arrested oocytes release less Ca2+ in response to injection of inositol 1,4,5-trisphosphate (IP3) than eggs. At high concentrations, the sulfhydryl reagent, thimerosal (200 microM), causes Ca2+ oscillations in eggs and produces similar oscillations in oocytes. A lower concentration of thimerosal (25 microM) does not cause Ca2+ oscillations, but does sensitize IP3-induced Ca2+ release in both eggs and oocytes, since IP3-induced Ca2+ release is enhanced in the presence of 25 microM thimerosal. Incubation of oocytes in 25 microM thimerosal before injection of 2.2 microM IP3 causes oocytes to release as much Ca2+ as is released in eggs injected with 2.2 microM IP3. These results indicate that immature mouse oocytes possess intracellular stores of releasable Ca2+ similar in size to Ca2+ stores in eggs; however, these stores are less sensitive to IP3. Development of the IP3-induced Ca2+ release mechanism may be an important component of maturation; at fertilization of the egg, Ca2+ must be elevated to levels sufficient to activate further development and establish a block to polyspermy. Mouse oocytes appear to develop an increased sensitivity to IP3 during the course of oocyte maturation.

Animals↗

PubMed1992

Thapsigargin activates a calcium influx pathway in the unfertilized mouse egg and suppresses repetitive calcium transients in the fertilized egg.

At fertilization, the sperm initiates development of the mouse egg by inducing a large transient increase in the intracellular Ca2+ concentration ([Ca2+]i), which is followed by repetitive transient increases in [Ca2+]i. To determine how the repetitive Ca2+ transients are produced, thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, was used to deplete intracellular Ca2+ stores within the egg. In the unfertilized egg, thapsigargin (1-50 microM) caused a slowly rising and falling transient increase in [Ca2+]i with or without extracellular Ca2+. An influx pathway for Ca2+ is activated by thapsigargin, since an immediate increase in [Ca2+]i occurred when Ca2+ was added to eggs after thapsigargin treatment in a Ca2+, Mg(2+)-free medium. This suggests that Ca2+ entry in the mouse egg may be coupled to the emptying of an intracellular store. The magnitude of the first Ca2+ transient at fertilization was reduced by as much as 84% in eggs pretreated with thapsigargin. Reduction of extracellular Ca2+, by addition of a Ca2+ chelator, suppressed the repetitive Ca2+ transients following fertilization. The Ca2+ transients also require filling of an intracellular store; they were suppressed when thapsigargin was added before or after fertilization. These results support the hypothesis that the first sperm-induced Ca2+ transient at fertilization depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and that the enhanced Ca2+ influx causes repetitive Ca2+ transients due to the periodic filling and emptying of an intracellular Ca2+ store.

Adenosine Triphosphatases↗

PubMed1992

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.

The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.

Animals↗

PubMed1991

Evidence for the involvement of a pertussis toxin-insensitive G-protein in egg activation of the frog, Xenopus laevis.

Activation responses of the frog egg at fertilization include the release of calcium from intracellular stores and the opening of calcium-dependent chloride channels, which produce the fertilization potential. To investigate the presence of guanine nucleotide-binding proteins (G-proteins), and their role in initiation of these events in the egg of the frog Xenopus laevis, we assayed for pertussis and cholera toxin substrates, and applied activators and inhibitors of G-proteins. Pertussis toxin catalyzed the [32P]ADP ribosylation of a Mr 40,000 component, but no cholera toxin substrates were demonstrated. Injection of greater than or equal to 25 pmole of guanosine-5'-O-(3-thiotriphosphate) GTP-gamma-S), an activator of G-proteins, produced a change in membrane potential that mimicked the fertilization potential and also caused cortical granule exocytosis and cortical contraction. Injections of up to 600 pmole of guanosine 3':5'-cyclic monophosphate or 9 nmole of guanosine-5'-(beta-gamma-imido)triphosphate did not active eggs. The membrane potential response to GTP-gamma-S injection showed the same peak and chloride dependence as the fertilization potential, although the duration of the GTP-gamma-S response was somewhat greater. GTP-gamma-S did not activate eggs if the calcium rise was prevented by prior injection of the calcium chelator BAPTA. Injection of up to 200 ng of cholera toxin did not activate eggs. However, eggs were activated by applying 1 nM serotonin to eggs that had been injected with a specific mRNA for the serotonin 1c receptor, a member of the class of receptors that act by way of G-proteins. Egg activation in response to either sperm or serotonin was not inhibited by pertussis toxin, under experimental conditions where approximately 80-90% of the toxin substrate was ADP-ribosylated. These results support the hypothesis that sperm activate Xenopus eggs at fertilization by way of a pertussis and cholera toxin-insensitive G-protein.

Adenosine Diphosphate Ribose↗

PubMed1990

Morphology of polyanhydride microsphere delivery systems.

Scanning electron microscopy (SEM) was used to elucidate the mechanism of polymer degradation and drug release in polyanhydride microspheres. Three different fabrication methods--solvent removal, solvent evaporation, and hot melt microencapsulation--were used to prepare polyanhydride microspheres containing a variety of drugs. The morphology of these microspheres releasing drug in vitro and in vivo was studied by SEM and compared with degradation and release data measured by conventional methods. Microspheres prepared by the three techniques were shown to have distinctive morphological characteristics induced by the nature of the fabrication method. In addition, SEM analysis could be used to explain the drug release profiles and polymer degradation behavior seen in vitro as well as the in vivo effects of insulin-loaded microspheres on diabetic rats. This study has shown SEM to be an important and powerful tool for analyzing the effects of microsphere fabrication method on drug release.

Anhydrides↗

PubMed1988

Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus eggs.

Fertilization initiates in the egg a dramatic increase in intracellular calcium that opens ion channels and causes exocytosis. To explore the possibility that these events might involve a receptor-mediated pathway, receptors for serotonin or acetylcholine (M1 muscarinic) were expressed in the Xenopus egg; serotonin or acetylcholine then could initiate a series of responses similar to those normally initiated by sperm. Thus, there may be an endogenous receptor in the egg membrane that is activated by sperm, and the serotonin or M1 muscarinic receptor may replace the sperm receptor in this pathway.

Animals↗

PubMed1988

Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronuclei.

Eggs of Xenopus laevis were injected with a calcium buffer before insemination, to examine the effect of preventing or suppressing the sperm-induced increase in intracellular calcium on the fertilization potential, exocytosis, and pronuclear formation. Microinjection of BAPTA [(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)] at concentrations between 0.2 and 0.7 mM usually suppressed the fertilization potential to a series of transient depolarizations. The fertilization potential was completely inhibited when the final concentration of BAPTA in the egg was greater than 0.7 mM. These observations support the hypothesis that activation of the chloride conductance responsible for the fertilization potential depends on an increase in intracellular calcium. Exocytosis of cortical granules and elevation of the fertilization envelope were prevented by injecting BAPTA at concentrations greater than 0.2 mM. Injection of BAPTA to suppress the rise in calcium did not inhibit sperm entry and BAPTA-injected eggs were highly polyspermic. Examination by light and electron microscopy revealed that sperm decondensation and pronuclear formation were prevented by injection of the calcium buffer before insemination.

Animals↗

PubMed1988

The cortical reaction in the egg of Discoglossus pictus: a study of the changes in the endoplasmic reticulum at activation.

In Discoglossus pictus previous ultrastructural observations have shown that at the animal dimple, where sperm fuse with the egg, cortical granules (CG), vacuoles, and tightly packed clusters of small cisternae are present. At fertilization the clusters open (i.e., become loose) and give rise to longer cisternae arranged in whorls and chains which migrate toward the plasma membrane. The vacuoles fuse to form cisternae and exocytose along with the CG. In the rest of the egg periphery, while exocytosis occurs, the clusters do not open as a result of activation (C. Campanella, R. Talevi, U. Atripaldi, and L. Quaglia (1986). In "Molecular and Cellular Biology of Fertilization" (J.L. Hedrick, Ed.). Plenum, New York). We have recently conducted electrophysiological studies which have detected inward currents at the dimple center, outward current at the rest of the egg surface, and an eightfold increase in [Ca2+]i which propagates from the site of activation throughout the egg (R. Nuccitelli, D. Kline, W. Busa, R. Talevi, and C. Campanella (1988). Dev. Biol. 130, 120-132). In this paper we have asked whether the anionic current and the Ca2+ increase could be causally related to the changes of the smooth endoplasmic reticulum (SER) at activation. The results obtained by activating the eggs in ion-substituted Ringers indicate that (1) the migration of cisternae is not dependent on the polarity of the activation current crossing the dimple, but is strongly impaired, together with CG exocytosis, by 5 x Cl- Ringer; (2) TMB-8, a drug which partially blocks calcium release (C. Y. Choiu and M. J. Malagodi (1975). Brit. J. Pharmacol. 53, 279-288), partially inhibits opening of cisternae clusters and the formation of an SER network in the dimple. This suggests a causal relationship between the Ca2+ rise and the cluster transformation at activation.

Animals↗

PubMed1988

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.

Animals↗