Elevation Of Fertilization Envelope Research Articles

Tyrosine Kinase-Dependent Activation of Phospholipase Cγ Is Required for Calcium Transient in Xenopus Egg Fertilization

In a previous study (K.-I. Sato et al., 1999, Dev. Biol. 209, 308–320), we presented evidence that a Src-related protein-tyrosine kinase (PTK), named Xyk, may act upstream of the calcium release in fertilization of the Xenopus egg. In the present study, we examined whether PTK activation of phospholipase Cγ (PLCγ) plays a role in the fertilization-induced calcium signaling. Immunoprecipitation studies show that Xenopus egg PLCγ is tyrosine phosphorylated and activated within a few minutes after fertilization but not after A23187-induced egg activation. Consistently, we observed a fertilization-induced association of PLCγ with Xyk activity that was not seen in A23187-activated eggs. A Src-specific PTK inhibitor, PP1, blocked effectively the fertilization-induced association of PLCγ with Xyk activity and up-regulation of PLCγ, when microinjected into the egg. In addition, a PLC inhibitor, U-73122, inhibited sperm-induced inositol 1,4,5-trisphosphate production and the calcium transient and subsequent calcium-dependent events such as cortical contraction, elevation of fertilization envelope, and tyrosine dephosphorylation of p42 MAP kinase, all of which were also inhibited by PP1. On the other hand, A23187 could cause the calcium response and calcium-dependent events in eggs injected with PP1 or U-73122. These results support the idea that Xenopus egg fertilization requires Src-family PTK-dependent PLCγ activity that acts upstream of the calcium-dependent signaling pathway.

Sperm-induced local [Ca2+]i rise separated from the Ca2+ wave in sea urchin eggs in the presence of a gamete fusion inhibitor, jaspisin .

An increase in intracellular Ca2+ concentration ([Ca2+]i) at a focal plane was recorded simultaneously with sperm-egg binding and membrane current upon insemination of sea urchin Hemicentrotus pulcherrimus eggs. No change in current and [Ca2+]i occurred in the presence of jaspisin, a novel substance that inhibits metallo-endoproteinase and sperm-egg membrane fusion (S. Ikegami, H. Kobayashi, Y. Myotoishi, S. Ohta and K. H. Kato (1994) J. Biol. Chem. 269, 23262-23267). With low doses of jaspisin, a spermatozoon first produced a step inward current (I(on)) as an indication of gamete membrane fusion and then induced a local [Ca2+]i rise at the site of sperm attachment 6-10 seconds after I(on). The sperm, however, soon detached from the egg. Increasing inward current was abruptly cut off (I(off)) within 9-15 seconds and the local [Ca2+]i rise began to decline 1-3 seconds after I(off). In most cases, no further responses or an elevation of fertilization envelope (FE) occurred. In some cases, [Ca2+]i at the sperm attachment site increased again even after the sperm detached and triggered a Ca2+ wave which caused an activation current and FE formation. This recording of a gamete membrane-fusion-induced local [Ca2+]i rise, separated from the Ca2+ wave, is a key phenomenon for elucidating the initial sperm stimulation of the egg at fertilization.

Proteases Stimulate Fertilization-like Responses in Starfish Eggs

Application of proteases to eggs of the starfish, Asterina miniata, caused several responses like those seen at fertilization. Cortical granule exocytosis and fertilization envelope elevation occurred within about 1 min after exposure to trypsin, chymotrypsin, or pronase; protease inhibitors prevented these responses. Kallikrein caused cortical granule exocytosis and fertilization envelope elevation, but this response required more time (∼30 min). Exocytosis was also seen in response to a recombinant trypsin, but not to a point-mutated trypsin without proteolytic activity. The extent of exocytosis was similar to that seen at fertilization, as measured by the fluorescent dye FM 1-43. In addition to causing exocytosis, application of trypsin, chymotrypsin, or pronase caused an increase in intracellular free calcium, detected by calcium green dextran, and stimulation of DNA synthesis, detected by incorporation of bromodecxyuridine. Exocytosis also occurred when trypsin or chymotrypsin was applied in artificial sea water in which the free calcium was reduced to a low level (40-70 n M) such that Ca influx would be reduced by >10,000-fold; this indicated that the proteases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extracellularly exposed protein that when proteolyzed can induce fertilization-like responses; this protein may be a receptor that transduces a signal from the sperm to initiate egg activation at fertilization.

Tyrosine phosphorylation of the egg receptor for sperm at fertilization

Gamete interaction triggers a variety of responses within the egg, collectively referred to as egg activation. In addition to the hallmarks of calcium release and fertilization envelope elevation, there are cytoskeletal rearrangements, protein tyrosine phosphorylation, and an increase in pH, among others. The ultimate goal of these concerted activation events is entry of the newly fertilized egg into the cell cycle. However, the molecular mechanisms which promote downstream cell activation events remain poorly understood. One model suggests that sperm deliver an "activating factor" upon fusion with the egg plasma membrane, while a second model proposes that the egg receptor for sperm transduces a signal that mediates a cascade of subsequent events. It also is possible that multiple pathways are activated. As a first step toward testing the hypothesis of receptor-mediated signal transduction, we have investigated the tyrosine phosphorylation state of the sea urchin egg receptor for sperm using specific antibodies. The present work indicates that the sperm receptor is phosphorylated by an egg cortical tyrosine kinase in response to sperm or purified ligand (bindin) binding. Maximal phosphorylation was reached within 20 sec. These data support the hypothesis that the sperm receptor is a gamete recognition protein which responds to ligand binding and focus attention on the question of the role of this tyrosine phosphorylation signal in egg activation.

Immunolocalization of the Sea Urchin Sperm Receptor in Eggs and Maturing Ovaries1

The sperm receptor from Strongylocentrotus purpuratus eggs is a high molecular weight proteoglycan-like molecule that inhibits fertilization species-specifically in a competition bioassay. A preparation of highly active sperm receptor that contained one major N-terminal sequence was used to generate polyclonal antibody in rabbits. This antibody species-specifically inhibited fertilization at low concentrations without interfering with fertilization envelope elevation. On immunofluorescence microscopy, the antibody recognized determinants on the mature egg cell surface and in the cortical granules just beneath the surface. Preabsorption of the antibody with the calcium-soluble fraction of the exudate from cortical granules rendered the antibody specific for the cell surface in mature eggs and still able to inhibit fertilization at the same concentrations as before treatment with cortical granule exudate. With antibody preabsorbed with cortical granule and by counting antibody-gold particles viewed by electron microscopy, sperm receptor was almost undetectable on the cell surface of immature oocytes in preseason ovaries, present on the cell surface and intracellularly in immature oocytes of ovaries collected at the beginning or at the height of the spawning season, and present only on the cell surface of mature oocytes in the lumen of the ovaries. Our results indicate that receptor is synthesized early in oogenesis and is rapidly moved to the egg cell surface.

Inositol trisphosphate, inositol phospholipid metabolism, and germinal vesicle breakdown in surf clam oocytes

Others have reported that microinjection of inositol 1,4,5-trisphosphate (InsP 3) releases stored intracellular Ca 2+ and causes fertilization envelope elevation, part of the activation process normally initiated by fertilization in deuterostome eggs. In the protostome, Spisula solidissima, germinal vesicle breakdown (GVBD) is the first visible response of the egg to fertilization. To test the effects of InsP 3 on egg activation in this organism, we microinjected the compound into oocytes. Microinjection of 0.4−7.0 × 10 −21 moles of InsP 3 (equivalent to 5–80 p M if distributed throughout the cell) elicited GVBD in a dose-dependent manner, demonstrating that increased oocyte InsP 3 can mimic part of the activation process in this protostome. Synthesis of InsP 3 occurs in vivo when phosphatidylinositol 4,5-bisphosphate (PtdInsP 2) is hydrolyzed by phospholipase C. To determine whether stimulus-induced synthesis of InsP 3 occurs after fertilization of Spisula oocytes, we labeled oocyte lipids with [ 32P]orthophosphate and measured the radioactivity in phospholipids after insemination. Fertilization resulted in a rapid, transient loss of radioactivity from PtdInsP 2. Because the radioactivity in phosphatidylinositol 4-phosphate and other phospholipids did not change, the loss of radioactivity from PtdInsP 2 is most likely due to its hydrolysis, yielding InsP 3 and diacylglycerol. The latter compound activates protein kinase C which has also been shown to be involved in regulating Spisula oocyte GVBD. Since both of these compounds appear to be early products of fertilization, they could coordinately activate Ca 2+- and protein kinase C-dependent processes involved in Spisula oocyte GVBD. These data indicate that egg activation in this protostome includes pathways similar to those found in deuterostome eggs and in other eukaryotic cells.

Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores.

In sea urchin eggs fertilization is accompanied by cortical granule exocytosis, a secretory event thought to be initiated by release of intracellularly sequestered calcium. We have examined the effect of two drugs on this process: chlortetracycline (CTC), a known chelator of intracellular calcium, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release in both skeletal and smooth muscle. Preincubation of eggs for 10 min with either CTC or TMB-8 blocked sperm entry, inhibited the burst of 45Ca2+ efflux normally seen postinsemination, and prevented fertilization envelope elevation. Half-maximal inhibition occurred with 200 microM CTC and 60 microM TMB-8. Electron microscopy confirmed that cortical granule exocytosis had been blocked, although inhibition was not due to a direct effect on exocytosis. CTC and TMB-8 had no effect on Ca2+-stimulated granule fusion in isolated egg cortices. Rather, these drugs block the early events in egg activation: sperm incorporation and triggering of exocytosis. These two effects appear to be independent since addition of either drug just before insemination permits sperm entry but inhibits calcium release and cortical granule exocytosis.

Sequestered calcium triggers oocyte maturation in Chaetopterus

In order to test the involvement of Ca 2+ in maturation and activation of eggs, we have treated Chaetopterus eggs with ionophore A23187 and quercetin, an inhibitor of Ca 2+-dependent ATPase. Ionophore A23187 induced rapid germinal vesicle breakdown (GVBD) and activated eggs as evidenced by fertilization envelope elevation at a wide range of concentrations. Higher concentrations of A23187 induced GVBD in Ca 2+-free artificial sea water, demonstrating the independence of GVBD in Chaetopterus from external Ca 2+. At high concentrations of ionophore, eggs became ameboid and underwent the initial phases of differentiation without cleavage. Low concentrations of quercetin induced GVBD with or without external Ca 2+. This treatment did not activate eggs, but allowed them to be fertilized and undergo some development. The results of these experiments indicate 1) that Ca 2+ fluxes regulate GVBD and activation in Chaetopterus, 2) that these fluxes can be internally generated, and 3) that Ca 2+ sequestration by Ca 2+-dependent ATPase may have a role in maintaining the intact germinal vesicle.

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization.

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.

FORMATION OF THE CORTICAL CONCAVITY AT FERTILIZATION IN THE SEA URCHIN EGG.

During the initial stages of fertilization envelope elevation in eggs of Strongylocentrotus pur puratus and S. droebachiensis a large concavity of the egg cortex was observed in the light microscope. This concavity corresponded in shape and size with the elevating fertilization envelope. However, after the vitelline layers of eggs were disrupted and the eggs inseminated, the concavity failed to develop although the eggs were fertilized and developed normally. We propose that the concavity is formed owing to increased hydrostatic pressure within the perivitelline space. To further support this hypothesis we measured total egg protein secreted during fertilization, and found that 98% was retained within the perivitelline space. Furthermore, 80% of the total protein was contributed by the hyaline layer. Presumably, colloidal osmotic pressure and/or hydration of fertilization product, trapped beneath the fertilization envelope, is responsible for increased hydrostatic pressure within the perivitelline space, and therefore promotes not only fertilization envelope elevation, but the cortical concavity as well.