Xenopus Laevis - Review & Journal
Writer’s comment: Although both these papers were written for English 104E (Scientific Writing), the focus of each grew out of topics currently under research in Dr. Richard Nuccitelli’s lab at UC Davis. During the past year I have been fortunate to assist Dr. David Glahn, a researcher in this lab, in the collection of the previously unpublished data presented in my journal submission. I would like to thank him for allowing me to shape my paper around these results. I chose to review articles on IP3 in an attempt to write something original: there are no previous reviews tracing the development of IP3 research in the frog Xenopus laevis.
- David Corman
Instructor’s comment: David wrote these two papers for English 104E: Scientific Writing. Together they demonstrate how different genres of scientific writing—the literature review and the research report—can grow out of the same research area. Both papers move quickly through fairly technical territory, but David’s explanations are clear, connected, and easy to follow (at least for someone with some scientific background). It’s quite amazing that David was able to review relevant findings of seventeenpapers so fluidly.
- Jared Haynes, English Department
INOSITOL TRISPHOSPHATE AND THE CA2+ FERTILIZATION WAVE IN XENOPUS EGGS
FAST BLOCK TO POLYSPERMY: VOLTAGE-CLAMP CHARACTERIZATION IN EGGS OF XENOPUS LAEVIS
The Role of Ca2+ in the Cell
Since the middle of this century it has been known that Ca2+ acts as an intracellular messenger in a myriad of different cell types. Through the use of several different signaling mechanisms, Ca2+ is a clearly ubiquitous, versatile and important ion in all cellular processes (see Berridge et al.1999 for review). One such process is the activation of eggs at the time of fertilization, an event accompanied in all species thus far studied by an increase in the intracellular free calcium concentration ([Ca2+]i) (Jaffe, 1990; Nuccitelli, 1991; Whitaker and Swann 1993). One of the most well studied of these species is the frog Xenopus laevis.As in many other species, the transient rise in [Ca2+]i in Xenopuseggs manifests itself as a wave of increased Ca2+ which spreads from the sperm entry site across the egg to the opposite pole (the antipode) (Fontanilla and Nuccitelli 1998). This wave has been well characterized by several methods, including Ca2+ sensitive microelectrodes (Busa and Nuccitelli 1985), fluorescence ratio imaging (Nuccitelli et al.1993), and confocal microscopy (Fontanilla and Nuccitelli 1998). This tide of Ca2+ that sweeps across the egg is important because it appears to be responsible for other events accompanying fertilization, among them liftoff of the fertilization envelope, exocytosis of the cortical granules and a change in intracellular pH. Correspondingly, some of the most important research in recent years regarding the Ca2+ wave at activation has been to determine the biochemical pathway leading to the release of calcium.
Mechanisms of Calcium Release in Cells
Several methods that cells use to release calcium have been well documented. Two are particularly relevant to the Ca2+ wave.
One common mechanism for Ca2+ release is the hydrolysis of phosphatidylinositol biphosphate (PIP2), which produces two products, inositol trisphosphate (IP3) and diacylglycerol (DAG). Both of these products act as secondary messengers to stimulate another event. In particular, IP3 diffuses through the cytosol and binds to IP3 gated Ca2+ channels in the endoplasmic reticulum, causing the release of Ca2+.
Another mechanism of calcium release is known as calcium induced calcium release (CICR). It is thought that an increase in [Ca2+]i itself may induce further calcium release in a self-generative manner. Evidence such as that reported by Cross (1981), that eggs of the frog Rana pipiens can be activated by Ca2+ injection, support this idea. One major proponent of this theory has been Jaffe (1990), who suggested that the sperm might contribute an initial input of calcium to begin wave propagation.
Implication of IP3 in Xenopus Fertilization
Initial Signs of IP3 Involvement at Fertilization
The first indications of the involvement of IP3 in the events of fertilization came not from Xenopus,but from a study of sea urchin eggs. In the 1980’s an increase in PIP2 was detected in the sea urchin egg within twenty seconds after fertilization (Turner et al. 1984). Around this same time it was discovered that micro-injecting IP3 could cause artificial activation in Xenopuseggs (Busa et al. 1985). To further these results Stith et al. (1993) monitored the mass of IP3 found in the Xenopusegg from the time of sperm-egg fusion to well after fertilization. Although earlier studies (LePeuch et al.1985, Busa 1988) had attempted to find similar data using label incorporation instead of IP3 mass, the results were not entirely consistent. What all studies did show however was a sharp, transient increase in IP3 following activation.
Mechanism of Calcium Release at Fertilization
Although these results very clearly point to Ca2+ release by PIP2 hydrolysis, they do not rule out the possibility that CICR is also involved. Given the evidence for the two different theories of calcium release, it seemed likely that some combination of both mechanisms was at work. Nuccitelli et al.(1993) helped to resolve this problem. Using microinjections of several blockers and inducers and fluorescence ratio imaging, they demonstrated that PIP2 hydrolysis was not only necessary, but also sufficient to allow the Ca2+ wave. They also found, however, that CICR is involved in the propagation of the Ca2+ wave, apparently through the IP3 receptor channel.
Also at issue was the fact that both IP3 and calcium are present in the sperm as well as in the egg, allowing the possibility that calcium release in the egg is triggered by a contribution of one of these factors from the sperm. Nuccitelli et al.(1993) also helped to resolve this issue by reporting that neither the Ca2+ nor the IP3 found in the sperm were sufficient to activate the egg when a PIP2 antibody was injected. This demonstrated that PIP2 hydrolysis is actually taking place inside the egg. Further, they showed the dominance of the IP3 mechanism over CICR by showing that activation was not impeded when CICR blockers were injected into the eggs before fertilization. Even with these results, the contribution of the sperm has not been entirely resolved. When the level of IP3 following fertilization was compared in sperm fertilized eggs and artificially activated eggs, the fertilized eggs showed an IP3 increase of greater magnitude than did eggs activated by other means (Snow et al.1996). Although not sufficient on its own, this shows that the sperm does offer some contribution to IP3 production and initiation of the Ca2+ wave, even though the primary source of the rise of IP3 is clearly PIP2 hydrolysis.
Correlation of IP3 Changes with the Calcium Wave
The endogenous production of IP3 following fertilization has been better characterized using a competitive binding assay, first by Stith et al. (1993), and then more accurately by Snow et al. (1996). This characterization allowed a measurement of the temporal rise and fall of IP3, and also avoided the problems associated with radiolabeling. The increases in IP3 after fertilization or activation were compared with the calcium rise, and maximum concentrations were found just as the wave reached the antipode of its course across the egg. Also, PIP2 was shown to unexpectedly increase during this time, even though IP3 levels were also increasing. This rise in PIP2 suggests that PIP2 synthesis, and not just hydrolysis, also increases after fertilization in order to help provide sufficient IP3.
Increases in DAG at Fertilization
If in fact the hydrolysis of PIP2 is taking place at fertilization, then DAG, the other by-product of this process, should also show a demonstrable increase. In 1997, Stith et al. used a DAG mass assay to demonstrate that this was in fact the case. Interestingly, DAG increased more than IP3, revealing that other sources of DAG are present in addition to PIP2 hydrolysis, and suggesting a greater role in fertilization for this secondary messenger.
Direction of IP3 Research
Since there is still much to be understood in the pathway leading to the fertilization wave and the role that IP3 plays in this pathway, research is ongoing. One direction being pursued was first suggested during the 1993 investigation by Nuccitelli et al. They observed small, local increases in [Ca2+]i following sperm-induced activation in eggs which had been pre-injected with heparin, a blocker of IP3 induced calcium release. When eggs were treated with approximately half of the dose of heparin needed to block fertilization 100% of the time, these local increases, termed ‘hot spots’, followed addition of sperm. The hot spots were non-propagating, although partial waves were also sometimes observed. Because of this clear link with IP3 production following fertilization, several other observations have been made of these hot spots. In one of these observations, confocal imaging of the effects of blocking IP3 (using heparin) was performed (Fontanilla and Nuccitelli, 1998), and not only revealed hot spots once again, but also demonstrated that in the cases where partial Ca2+ waves were induced, they were slower moving and lesser in magnitude than the normal, fully propagating waves. Nuccitelli and Fontanilla are currently investigating this phenomenon more fully. In another of these observations, hot spots were observed in eggs injected with lavendustin A, a tyrosine kinase inhibitor (Glahn et al. 1999). Since these hot spots were qualitatively similar in nature to those previously seen, tyrosine kinase may be somehow implicated in the production of IP3.
A better grasp of the role that IP3 plays in the fertilization of Xenopus eggs has clearly helped to understand the pathway leading to the fertilization wave of increased [Ca2+]i. There are still, however, many unknown details in this pathway that must be learned. Continuing research on the involvement of IP3, like the closer examination of the hot spot phenomenon, may eventually help to fill in some of the missing pieces.
Berridge, M., Lipp, P., Bootman, M. (1999). Calcium signaling. Curr. Bio. 9, R157-159.
Busa, W.B. (1988). Roles for the phosphatidylinositol cycle in early development. Philos. Trans. R. Soc. Lond. B Biol. Sci. 320, 415-426.
Busa, W.B., Ferguson, J.E., Joseph, S.K., Williamson, J.R., Nuccitelli, R. (1985). Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of calcium release from intracellular stores. J. Cell. Bio. 101, 677-682.
Busa, W.B., Nuccitelli, R. (1985). An elevated free cytosolic calcium wave follows fertilization in eggs of the frog Xenopus laevis. J. Cell. Bio. 100, 1325-1329.
Cross, N.L. (1981). Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog, Rana pipiens. Dev. Bio. 85, 380-384.
Fontanilla, R.A., Nuccitelli, R. (1998). Characterization of the Sperm-Induced Calcium Wave in Xenopus Eggs using Confocal Microscopy. Biophys. Soc. 75, 2079-2087.
Glahn, D., Mark, S.D., Behr, R.K., Nuccitelli, R. (1999). Tyrosine Kinase Inhibitors Block Sperm-Induced Egg Activation in Xenopus laevis. Dev. Bio. 205, 171-180.
Jaffe, L.F. (1990). The roles of intermembrane Ca2+ in polarizing and activating eggs. In “Mechanisms of Fertilization: Plants to Humans,” pp. 389-418. Springer-Verlag, Berlin.
LePeuch, C.J., Picard, A., Doree, M. (1985). Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs. FEBS Lett. 187, 61-64.
Nuccitelli, R. (1991). How Do Sperm Activate Eggs? Curr. Top. in Dev. Bio. 25, 1-16.
Nuccitelli, R., Yim, D.L., Smart, T. (1993). The Sperm-Induced Ca2+ Wave Following Fertilization of the Xenopus Egg Requires the Production of Ins(1,4,5)P3. Dev. Biol. 158, 200-212.
Snow, P. Yim, D.L., Leibow, J.D., Saini, S., Nuccitelli, R. (1996). Fertilization Stimulates an Increase in Inositol Trisphosphate and Inositol Lipid Levels in Xenopus Eggs. Dev. Bio. 180, 108-118.
Stith, B.J., Goalstone, M., Silva, S., Jaynes, C. (1993). Inositol 1,4,5-Trisphosphate Mass Changes from Fertilization Through First Cleavage in Xenopus laevis. Mol. Bio. Cell 4, 435- 443.
Stith, B.J., Woronoff, K., Espinoza, R., Smart, T. (1997). sn-1,2-Diacylglycerol and Choline Increase after Fertilization in Xenopus laevis. Mol. Bio. Cell 8, 755-765.
Turner, P.R., Sheetz, M.P., Jaffe, L.A. (1984). Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature, 310, 414-415.
Whitaker, M., Swann, K. (1993). Lighting the fuse at fertilization. Dev. 117, 1-12.
FAST BLOCK TO POLYSPERMY: VOLTAGE-CLAMP CHARACTERIZATION IN EGGS OF Xenopus Laevis
In Xenopus laevis the fast-block to polyspermy is executed with a rapid increase in the egg’s membrane potential. This increase is due to an efflux of Cl– ions. Voltage clamping the eggs allows monitoring of the fertilization potential through current measurements. By fertilizing the eggs at different holding potentials, the effect of the fast-block can be tracked as a function of membrane potential. The ability of the sperm to fertilize the egg is greatly reduced for even small steps above the resting potential of the egg (typically –12mV).
During the mating of the frog Xenopus laevis, thousands of sperm are deposited onto the surface of the egg. Out of these many possibilities, however, only one sperm may be admitted if the egg is to develop normally. Like many other species, the Xenopus egg must have some very rapid mechanism to prevent more than one sperm from fusing with it. This mechanism, called the fast-block to polyspermy, is a depolarization of the membrane potential of the egg, a voltage change termed the fertilization potential. The advantage of such a system is that it may be executed very rapidly and it encompasses the entire cell surface. Although the fertilization potential is well documented in frog eggs (see for example, Kline and Nuccitelli 1985), and clearly established as the fast-block (Grey et al. 1981), a fully documented relationship between membrane potential and ability to block sperm has never been characterized in Xenopus. We have set out to obtain this data using a voltage-clamp technique.
The fertilization potential is due to an efflux of Cl– ions out of the egg through Ca2+-mediated Cl– channels (Cross 1981, Miledi 1982, Barish 1983, Kline 1988). By holding the egg at an assigned voltage, however, changes in membrane channel activity normally leading to voltage fluctuations may be monitored as changes in the current necessary to maintain that assigned voltage. This technique is referred to as voltage-clamping. Since current is measured as a movement of positive charges, and Cl- carries a negative charge, the fertilization current is an inward current. Two approaches are of interest when examining this phenomenon. The first method is to hold the egg at a low voltage (preventing the depolarization at fertilization), and watch for polyspermy. The second is to hold the egg at a high voltage (replicating the depolarization at fertilization), and watch for the inability of the sperm to fertilize the egg. A combination of these methods was used to characterize the fast block in Xenopus.
II. Materials and Methods
Female Xenopus were injected with 250-500 IU of human chorionic gonadotropin 12-16 hours before experiments. The eggs were then removed from the frog by gentle stimulation of the abdomen, and placed directly onto the lid of a virgin 60mm culture dish. The lid was placed on a culture dish partially filled with distilled water to prevent dehydration of the eggs. Eggs were stored in this state for no more than an hour, after which fresh eggs were obtained.
Egg Preparation and Impalement
To voltage clamp the eggs, 3-5 were placed on a clean 60mm culture dish. The adhering strength of the jelly was enough to hold the eggs in place during impalement. The dish was then set on to the stage of a Zeiss IM-35 inverted microscope, and filled approximately half–way with Ca2+-free OR-2 (in mM: 82.5 NaCL, 2 KCl, 5 HEPES, 20 MgCl2, 0.1 EGTA; pH 7.4). Microelectrodes (approximately 3mm shank, 5-10mm tip opening, 2-5MW resistance when filled with 3M KCl) were pulled using a Flaming-Brown P-95 horizontal pipette puller. The position of the stage was fine tuned by viewing an egg through the inverted microscope, and lining it up with a predetermined point. The microelectrodes were placed into two head stages mounted in micro-manipulators at 3 and 9 o’clock relative to the culture dish.
The microelectrodes were then brought close to the surface of one of the eggs (but remained outside of the egg’s jelly coat) and the voltage reading was set to zero. Each electrode was then impaled through the surface of the egg until the expected resting potential (normally –12mV) was observed, and then backed out as far as possible to minimize physical disturbances to the egg.
Voltage Clamping and Fertilization
The eggs were allowed a few minutes to heal during which time the voltage clamp was activated using a Gene-Clamp 500 (Axon Instruments, Foster City, CA) amplifier. The desired voltage was then obtained, and recordings were considered usable only if a change of 50mV from the resting potential required no more than 200nA of current. Typically, eggs were depolarized to some voltage, sperm (from macerated testis) were added, and a fertilization current was watched for. If no fertilization potential was observed at the test voltage, then the eggs were dropped to -50mV to make sure they were fertilizable. Fertilization was confirmed by watching for other signs such as liftoff of the vitelline envelope.
When microelectrodes are first inserted into the cell, the initial voltage reading may be taken as the cell’s resting potential. The resting potential was normally found to –12mV, although it was occasionally –13 or –14mV. This voltage is therefore taken as the starting reference point. Although the fast-block relies on voltages higher than the resting potential, experiments were also done to confirm ability to fertilize at lower voltages, down to –50mV. Figure 1 shows a typical fertilization current in an egg clamped at low voltage. A holding potential of –50mV also serves as a clear example because of the high amplitude of the peak current as compared to less negative holding potentials. This high amplitude is due to the fact that more Cl- ions will efflux from the egg when the voltage is lower. The time shown between adding sperm to the egg (marked by an arrow on the graph) and the drop in membrane potential is the time necessary for the sperm to penetrate the jelly coat and outer layer of the egg.
The results for the percent of eggs fertilizing at each voltage are shown in Figure 2. The numbers next to each data point are the number of eggs tested at that voltage. Aside from the odd data point at –2mV the curve is relatively smooth, and it is clear that after stepping above the resting potential of –12mV the ability for the sperm to penetrate the egg is greatly diminished.
While the results shown suggest a curve with a very steep drop roughly centered around –9mV, there is still considerable room to refine the data to achieve more exact results. The apparent increase in fertilizability at –2mV needs to be corrected as it does not fit in with the scheme believed to be correct. A greater number of samples in the range of –10-0mV need to be collected, but as the fast-block takes effect, it becomes more difficult to view the fertilization current. The trend that is suggested by the data, however, fits very well with what might be expected, and provides reasonable estimates of how many eggs might fertilize at given holding potentials.
Barish, M.E. (1983). A transient calcium-dependent chloride current in the immature Xenopus oocyte. J. Physiol., 342, 309-325.
Cross, N.L. (1981). Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog, Rana pipiens. Dev. Biol., 85, 380-384.
Grey, R., Bastiani, M.J., Webb, D.J., Scherterl, E.R. (1981). An electrical Block is Required to Prevent Polyspermy in Eggs Fertilized by Natural Mating of Xenopus laevis. Dev. Biol., 89, 475-484.
Kline, D. (1988). Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronucleii. Dev. Biol., 111, 471-487.
Kline, D., Nuccitelli, R. (1985). The Wave of Activation Current in the Xenopus Egg. Dev. Biol., 111, 471-487.
Miledi, R. (1982). A calcium-dependent transient outward current in Xenopus laevis oocytes. Proc. R. Soc. Lond. [Biol.], 215, 491-497.