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K Swann

Publications and source records attributed to K Swann.

At least 55 records · Page 3Linked to original sources

Nuclei from fertilized mouse embryos have calcium-releasing activity.

During mammalian fertilization, the sperm triggers a series of intracellular Ca2+ oscillations which initiate oocyte activation and the formation of pronuclei. Oocyte activation can be induced artificially by a variety of chemical and physical stimuli which elevate intracellular calcium. We show that the transfer of nuclei from 1- and 2-cell-stage fertilized mouse embryos to unfertilized oocytes stimulates the completion of meiosis and the formation of pronuclei. Nuclei from embryos that had developed to the 4-cell stage did not stimulate meiotic resumption. The ability to cause oocyte activation was specific to nuclei transferred from fertilized embryos as nuclei from parthenogenetic embryos or cytoplasts from fertilized or parthenogenetic embryos did not induce activation. Nucleus-induced oocyte activation was associated with the generation of intracellular Ca2+ transients, which were seen after nuclear envelope breakdown of the transferred nuclei. Treatment of the oocyte with the intracellular Ca2+ chelator, BAPTA, prior to nuclear transfer inhibited intracellular Ca2+ transients and oocyte activation. The specific Ca(2+)-releasing activity of the nucleus was not caused by sperm-induced protein synthesis since similar activity was present in nuclei originating from embryos exposed to cycloheximide throughout fertilization. The specific ability of nuclei from fertilized embryos to stimulate Ca2+ transients and oocyte activation was also found in nuclei from embryos parthenogenetically activated by the injection of a partially purified cytosolic sperm factor. The results suggest that the fertilizing sperm introduces Ca(2+)-releasing activity which becomes associated with the nucleus of early mammalian embryos.

Animals↗

Regenerative and non-regenerative calcium transients in hamster eggs triggered by inositol 1,4,5-trisphosphate.

1. Inositol 1,4,5-trisphosphate (InsP3) injected into unfertilized golden hamster eggs elicits a hyperpolarizing response (HR) that is due to stimulation of calcium-activated potassium channels in the egg plasma membrane. 2. A single injection of InsP3 gave a single HR above a threshold value of 0.3 nM. At 5 nM and above, InsP3 induced HRs with no detectable latency. At concentrations between these two values a latency was observed. The amplitude of the HR was independent of InsP3 concentration. 3. A second HR could be elicited by injection of InsP3, but five times more InsP3 was required to trigger a second HR, and 10-100 times more to give an HR of similar magnitude to the first, and there was no latency. 4. The increase in [Ca2+]i in response to an initial injection of 1 nM InsP3 could be resolved into two distinct components: a slow, early rise immediately after InsP3 injection (phase I) followed by a larger and more rapid increase (phase II). The initiation of an HR coincided with the second component of the [Ca2+]i increase. 5. Further injection of InsP3 resulted only in slow, smaller increases in [Ca2+]i that resembled phase I and often did not cause an HR. Phase II appeared to be absent. However, 100-fold greater InsP3 concentrations gave slow, larger Ca2+ transients (and HRs) with no detectable latency. 6. If large amounts of InsP3 were allowed to leak into the eggs constantly from a pipette, repetitive calcium transients were seen. Unlike the sustained repetitive responses seen at fertilization, they were often smaller than the initial transient and less well sustained. However, a subsequent transient could still be elicited on injection of very large concentrations of InsP3. 7. InsP3 can induce regenerative, all-or-none [Ca2+]i increase (CICR) in hamster eggs, often with a long latency, as well as non-regenerative increases. InsP3 injections desensitize CICR and cannot mimic all the features of Ca2+ signalling at fertilization in the hamster egg, in particular, the sensitization of CICR caused by the sperm.

Animals↗

Ca2+ oscillations and sensitization of Ca2+ release in unfertilized mouse eggs injected with a sperm factor.

The mechanisms by which a sperm factor caused Ca2+ oscillations in unfertilized mouse eggs was investigated by monitoring the fluorescence of intracellular Fluo-3. Injecting sperm extracts triggered Ca2+ oscillations that varied in number and frequency rather than amplitude. All the sperm factor-induced Ca2+ transients showed a characteristic rapid increase and decline. The oscillations persisted for variable times in eggs bathed in Ca2+ free media and were distinct in character from those caused by inserting pipettes containing inositol 1,4,5-trisphosphate (InsP3). InsP3-induced Ca2+ oscillations were always of high frequency but varied considerably in form and amplitude depending upon InsP3 concentration. The oscillations produced by injecting Ca2+ into unfertilized eggs were similar to those produced with low doses of InsP3 but different from those caused by sperm extract injection. After injection of sperm extracts further injection of InsP3, or Ca2+, produced high frequency large amplitude Ca2+ transients. It is concluded that the sperm factor mediates its effects by sensitizing Ca2+ release mechanisms.

Animals↗

A cytosolic sperm factor triggers calcium oscillations and membrane hyperpolarizations in human oocytes.

Intracellular free Ca2+ concentrations ([Ca2+]i) and membrane potentials were measured in mature human oocytes. Injection of cytosolic extracts made from human or hamster spermatozoa triggered oscillations in [Ca2+]i in human oocytes similar to those described previously in mouse and hamster oocytes. In contrast, injection of carrier buffer caused no [Ca2+]i increase and injection of Ca(2+)-containing solutions caused only a single [Ca2+]i transient. Injection of human sperm extracts also triggered [Ca2+]i oscillations in mature mouse oocytes. The [Ca2+]i oscillations in human oocytes were accompanied by hyperpolarizations in membrane potential. Perfusing oocytes with the sulphydryl reagent thimerosal also caused oscillations in the free [Ca2+]i concentration simultaneously with membrane potential hyperpolarizations. These data suggest that human oocytes possess a similar mechanism for generating [Ca2+]i oscillations to those described in other mammalian oocytes and a membrane potential response similar to that seen previously specifically in hamster oocytes. The data also support the view that human oocytes are activated at fertilization by diffusion of a protein from the spermatozoa into the ooplasm after gamete membrane fusion.

Animals↗

Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes.

Calcium oscillations occur during meiotic maturation of mouse oocytes. They also trigger activation at fertilization. We have monitored [Ca2+]i in oocytes at different stages of growth and maturation to examine how the calcium release mechanisms alter during oogenesis. Spontaneous calcium oscillations occur every 2-3 minutes in the majority of fully grown (but immature) mouse oocytes released from antral follicles and resuming meiosis. The oscillations last for 2-4 hours after release from the follicle and take the form of global synchronous [Ca2+]i increases throughout the cell. Rapid image acquisition or cooling the bath temperature from 28 degrees C to 16 degrees C did not reveal any wave-like spatial heterogeneity in the [Ca2+]i signal. Calcium appears to reach highest levels in the germinal vesicle but this apparent difference of [Ca2+] in nucleus and cytoplasm is an artifact of dye loading. Smaller, growing immature oocytes are less competent: about 40% are able to resume meiosis and a similar proportion of these oocytes show spontaneous calcium oscillations. [Ca2+]i transients are not seen in oocytes that do not resume meiosis spontaneously in vitro. Nonetheless, these oocytes are capable of [Ca2+]i oscillations since they show them in response to the addition of carbachol or thimerosal. To examine how the properties of calcium release change during meiotic maturation, a calcium-releasing factor from sperm was microinjected into fully grown immature and mature oocytes. The sperm-factor-induced oscillations were about two-fold larger and longer in mature oocytes compared to immature oocytes. Calcium waves travelling at 40-60 microns/second were generated in mature oocytes, but not in immature oocytes. In some mature oocytes, successive calcium waves had different sites of origin. The modifications in the size and spatial organization of calcium transients during oocyte maturation may be a necessary prerequisite for normal fertilization.

Animals↗

The role of calcium in mammalian oocyte maturation and egg activation.

The maturation of the immature oocyte and the fertilization of a mature egg are two absolute prerequisites for mammalian embryo development. There is increasing evidence in mammals that both oocyte maturation and egg activation at fertilization are controlled by changes in intracellular free Ca2+ levels. The role of Ca2+ changes at fertilization is clear in that they are both required and sufficient for egg activation. However, it is not established how the sperm causes Ca2+ changes in eggs at fertilization, nor how different patterns of Ca2+ change affect embryo development. The role of Ca2+ in triggering oocyte maturation is less clear, although preventing intracellular Ca2+ changes can inhibit meiotic maturation at specific stages. Studies on how Ca2+ regulates meiosis and fertilization in mammals may provide new insights into the causes of failed fertilization in human IVF procedures.

Animals↗

Different triggers for calcium oscillations in mouse eggs involve a ryanodine-sensitive calcium store.

Relative intracellular free Ca2+ concentrations ([Ca2+]i) were monitored in mature unfertilized mouse eggs by measuring fluorescence of intracellular fluo3. A number of different agents were found to cause sustained repetitive transient [Ca2+]i oscillations. These were microinjection of a cytosolic sperm factor, sustained injection of Ins-(1,4,5)P1, or extracellular addition of the thiol reagent thimerosal. Stimulating G-protein activity by injection of guanosine 5'-[gamma-thio]triphosphate plus application of carbachol also caused [Ca2+]i oscillations, but less reliably than other stimuli. A role for Ca(2+)-induced Ca2+ release and a ryanodine-sensitive Ca2+ channel in mouse eggs was suggested by the finding that microinjection, or external addition, of ryanodine also caused [Ca2+]i increases. Furthermore, ryanodine, along with thimerosal, increased the sensitivity of eggs to Ca(2+)-induced [Ca2+]i oscillations. When ryanodine was added to eggs oscillating in response to the sperm factor, InsP3 or thimerosal, it caused a decrease in amplitude of oscillations and eventually a block of [Ca2+]i oscillations associated with a sustained elevation of [Ca2+]i. These data suggest that a ryanodine-sensitive Ca(2+)-release mechanism exists in mouse eggs and that a ryanodine-sensitive Ca2+ store plays a role in generating intracellular [Ca2+]i oscillations.

Animals↗

Spontaneous cytosolic calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes.

Immature mouse oocytes undergo spontaneous meiotic maturation when released from antral follicles into culture media. The first sign of meiotic resumption is germinal vesicle breakdown (GVB). Cytosolic free Ca2+ was measured in mouse oocytes during spontaneous maturation by monitoring fluorescence of indo-1 or fluo-3. The majority of oocytes showed a series of Ca2+ oscillations that continued for 1-3 h. Repetitive Ca2+ increases occurred every 1-3 min and lasted for 10-60 s. The Ca2+ oscillations appeared to be caused by an increase in inositol 1,4,5-trisphosphate (InsP3) because once they ceased, similar oscillations were triggered by injection of exogenous InsP3. Also, injection of the InsP3 receptor antagonist heparin (final concentration, 100 micrograms/ml) blocked the spontaneous Ca2+ oscillations. In contrast, Ca2+ oscillations induced by thimerosal were not inhibited by heparin. Treating oocytes with media containing 20 microM BAPTA/AM abolished Ca2+ oscillations in oocytes but did not affect the rate of GVB. The data show that cytosolic Ca2+ oscillations apparently caused by polyphosphoinositide turnover occur during mammalian oocyte maturation. However, the spontaneous oscillations do not appear to trigger GVB. Also, the data indicate that there are two separate Ca2+ release mechanisms in mouse oocytes, one sensitive to InsP3, the other to thimerosal.

Animals↗

Sperm-induced currents at fertilization in sea urchin eggs injected with EGTA and neomycin.

Membrane currents were measured in single voltage-clamped sea urchin eggs (Lytechinus pictus and Lytechinus variegatus) that were injected with either EGTA or neomycin and inseminated. Although egg activation and the fertilization calcium wave were prevented by injection of either of these compounds, sperm attached and still elicited inward currents. Sperm-induced currents in EGTA-injected eggs had an abrupt onset, quickly reached a maximum, and then slowly declined in amplitude. Sperm incorporation occurred readily in EGTA-injected eggs. Similar results were obtained with another calcium chelator, BAPTA. In neomycin-injected eggs, sperm-induced currents generally had an abrupt onset and, in contrast to EGTA-injected eggs, the currents usually cut off rapidly. Sperm failed to enter the neomycin-injected eggs and the duration of sperm-induced currents in neomycin-injected eggs was markedly dependent upon the voltage-clamp holding potential, with shorter duration currents occurring at -70 than at -20 mV. The lability of the initial interaction between sperm and egg at negative holding potentials may explain why activation often fails when the egg membrane is voltage clamped at these potentials (Lynn et al., Dev. Biol. 128, 305-323, 1988).

Animals↗

Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.

Adenosine Diphosphate Ribose↗

Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.

We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca2+]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca2+]i increase and activation are prevented in eggs in which the InsP3-sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor.

Animals↗

Thimerosal causes calcium oscillations and sensitizes calcium-induced calcium release in unfertilized hamster eggs.

Calcium-induced-calcium-release (CICR) was assayed in unfertilized golden hamster eggs by injecting Ca2+ and monitoring Ca2(+)-dependent hyperpolarizing responses (HRs) and Ca2(+)-sensitive fluo-3 fluorescence. Incubating eggs in the sulfhydryl reagent thimerosal caused [Ca2+]i oscillations as monitored by Ca2(+)-dependent HRs and decreased approximately 10-fold the Ca2+ injection current required to generate an HR and cause a large intracellular Ca2+ increase. Thimerosal also enhanced the sensitivity of eggs to Ca2+ injection in a calcium-free medium. The effects of thimerosal on CICR were prevented by dithiothreitol and were not mimicked by injecting inositol 1,4,5-trisphosphate. The data suggest that thimerosal may be an alternative agent for studying CICR in caffeine-insensitive cells.

Animals↗

Synergistic activation by serotonin and GTP analogue and inhibition by phorbol ester of cyclic Ca2+ rises in hamster eggs.

1. Synergistic activation of a GTP-binding protein (G protein) by external serotonin (5-hydroxytryptamine, 5-HT) and internally applied guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) in hamster eggs was demonstrated by the facilitation of repetitive increases in cytoplasmic Ca2+ as measured by their associated hyperpolarizing responses (HRs) and by aequorin luminescence. 2. Rapid application of 70 nM-5-HT caused a single HR of 10-12 s duration and with a delay of 80 s. The critical concentration of 5-HT to cause an HR was 50 nM. 3. With 10 microM-5-HT four to six HRs were often elicited with a delay to the first HR of 8-30 s. HRs disappeared after prolonged or repeated application of 5-HT, indicating an apparent desensitization. 4. 5-HT-induced HRs were completely inhibited by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (TPA) (100 nM). Conversely, the PKC inhibitor sphingosine (2 microM) enhanced the series of HRs by shortening the delay to the first HR (3-9 s) and by causing more HRs. 5. Ionophoretic injection of GTP gamma S into the egg usually produced a large HR with a delay of 120-240 s followed by a series of much smaller HRs. When 5-HT was applied within 1 min of injection of GTP gamma S. 70 nM-5-HT induced a number of large HRs and even 1 nM-5-HT could induce HR(s). In contrast, when 5-HT was applied after the size of GTP gamma S-induced HRs had declined, as much as 10 microM-5-HT could only elicit a single large HR. Thus, GTP gamma S apparently caused a sensitization and then a desensitization of the action of 5-HT. 6. GTP gamma S-induced Ca2+ transients were facilitated when injected in the presence of 5-HT concentrations as low as 0.1 nM. The time delay to the first HR was 65 s in 0.1 nM-5-HT or 4 s in 100 nM-5-HT whereas it was 170 s without 5-HT (mean values). The magnitude as well as frequency of HRs succeeding the first HR was enhanced by 5-HT at concentrations above 0.01 nM. 7. TPA (100 nM) blocked the GTP gamma S-plus-5-HT-potentiated HRs after the first four to five HRs. Sphingosine (2 microM) augmented the series of HRs.(ABSTRACT TRUNCATED AT 400 WORDS)

Aequorin↗

A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs.

Microinjection of cytosolic sperm extracts into unfertilized golden hamster eggs caused a series of increases in cytoplasmic free calcium, Ca2+i, and membrane hyperpolarizing responses, HRs. These HRs and Ca2+i transients are similar to those seen during in vitro fertilization of hamster eggs. The sperm factor that is responsible for causing these effects appears to be of high molecular weight and protein based. Injection of sperm factor activated eggs and mimicked fertilization in causing repetitive HRs in the presence of phorbol esters and in sensitizing the egg to calcium-induced calcium release. Since these effects cannot be mimicked by injecting G-protein agonists or calcium-containing solutions, it seems unlikely that a receptor-G-protein signalling system is involved at fertilization. These data instead suggest a novel signal transduction system operates during mammalian fertilization in which a protein factor is transferred from the sperm into the egg cytoplasm after gamete membrane fusion.

Animals↗

Second messengers at fertilization in sea-urchin eggs.

The activation of the sea-urchin egg at fertilization is triggered by two ionic changes in the egg cytoplasm. First there is a wave of increase in cytoplasmic free calcium concentration (Ca2+i) that travels across the egg within the first minute of fertilization. The calcium wave is followed by a sustained rise in intracellular pH (pHi). Fertilizing spermatozoa cause these two ionic changes by stimulating the hydrolysis of phosphatidylinositol 1,4,5-bisphosphate (PtdInsP2). PtdInsP2 hydrolysis produces inositol 1,4,5-trisphosphate, which releases calcium from intracellular stores, and diacylglycerol, which stimulates a plasma membrane sodium/hydrogen antiporter causing the rise in pHi. A positive feedback cycle of intracellular calcium release and InsP3 production appears to be part of the mechanism by which the calcium wave is propagated. It has been suggested that the spermatozoon triggers the calcium wave by acting on a cell surface receptor that couples to the egg phosphoinositidase C via a G-protein. We discuss evidence for an alternative hypothesis of egg activation in which a spermatozoon triggers the calcium wave by introducing a soluble activating factor into the egg cytoplasm.

Animals↗