Treatment Of Unfertilized Eggs Research Articles

Gamete interactions in Xenopus laevis: identification of sperm binding glycoproteins in the egg vitelline envelope.

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.

A voltage‐gated chloride channel in ascidian embryos modulated by both the cell cycle clock and cell volume.

1. Eggs of the ascidian Boltenia villosa have an inwardly rectifying Cl- current whose amplitude varies by more than 10-fold during each cell cycle, the largest amplitude being at exit from M-phase. We examined whether this current was also sensitive to changes in cell volume. 2. Cell swelling, produced by direct inflation through a whole-cell recording pipette, greatly increased the amplitude of the Cl- current at all stages of the cell cycle in activated eggs. Swelling was much less effective in unfertilized eggs. 3. The increase in Cl- current amplitude continued for 10-20 min after an increase in diameter that was complete in 10 s, suggesting the involvement of a second messenger system in the response. 4. Treatment of unfertilized eggs with 6-dimethylaminopurine (DMAP), an inhibitor of cell cycle-dependent protein kinases, increased the amplitude of the Cl- current and its sensitivity to swelling to levels characteristic of fertilized eggs. 5. Osmotically produced swelling also increased Cl- current amplitude in unfertilized eggs. 6. We propose that dephosphorylation renders the Cl- channel functional, and that swelling or activation of the egg increases the sensitivity of the channel to dephosphorylation, perhaps by disrupting its links to the cytoskeleton.

Phorbol ester treatment stimulates tyrosine phosphorylation of a sea urchin egg cortex protein.

Fertilization of the sea urchin egg results in the phosphorylation, on tyrosine, of a high molecular weight protein localized in the egg cortex. In the present study, treatment of unfertilized eggs with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulated tyrosine phosphorylation of the high molecular weight cortical protein to levels three- to fivefold higher than that occurring in response to fertilization. Experiments using agents that inhibit the egg Na+/H+ exchange system or mimic the fertilization-induced shift in cytoplasmic pHi, suggest a signal transduction pathway in which protein kinase C activates the egg Na+/H+ exchange system and the resultant cytoplasmic pHi shift promotes tyrosine phosphorylation of the high molecular weight cortical protein.

Purification and characterization of an N-acetyl-beta-D-glucosaminidase from cortical granules of Xenopus laevis eggs.

The enzyme N-acetyl-beta-D-glucosaminidase was purified from the cortical granules of Xenopus laevis eggs using affinity chromatography, gel filtration, and density gradient centrifugation. The enzyme had a molecular weight of 37,000-40,000 as determined by polyacrylamide gel electrophoresis and density gradient centrifugation, had a Km for p-nitrophenyl-beta-D-N-acetyl-glucosaminide of 0.66 mM and a Ki for glucosamine of 4.3 mM. The kinetic properties of the cortical granule enzyme were similar to the enzyme isolated from jack bean. Treatment of unfertilized eggs with the enzyme isolated from cortical granules or jack bean rendered eggs unfertilizable. Loss of fertilizability was proportional to the product of time and enzyme concentration, consistent with an enzymatic mechanism being responsible for the loss of fertilizability. The amount of enzyme present in the perivitelline space was approximately the same as that which reduced fertilizability by 50% in one hour. We suggest that the action of cortical granule N-acetyl-beta-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization.

The Suppressive Effect of Sera from Old Female Mice on in vitro Fertilization and Blastocyst Development

Superovulated, old and young female mice were either allowed to mate or were inseminated into the uterine lumen. Failure of mating occurred in the old but not in the young females. The average number of ovulations and proportion of fertilized eggs following insemination decreased significantly in the old rather than in the young females. It was found that for successful in vitro fertilization of mouse eggs, one culture medium was good for intact eggs and another one for denuded eggs, but both media were good for zona-free eggs. Treatment of unfertilized eggs and 8-celled eggs with sera of old females significantly reduced the rate of fertilization and the potential of development in culture, although treatment of zona-free eggs had no effect. Two out of 16 serum samples from old mice produced faint reaction on the zona pellucida of rat eggs by immunofluorescence test. It seems that factors which impair fertilization and early development are present in sera of old females and these factors may include an autoantibody to zona pellucida.

Effect of Dibutyryl Adenosine 3', 5'-Cyclic Monophosphate on Several Metabolic Systems in Sea Urchin Eggs

Effects of dibutyryl adenosine 3', 5'-cyclic monophosphate (db-cAMP) on glycolysis, oxygen uptake and protein synthesis were inves-tigated in unfertilized sea urchin eggs to examine the possible role of adenosine 3', 5'-cyclic monophosphate in the activation of cellular functions upon fertilization. Treatment of unfertilized eggs with 1 to 5 mM of db-cAMP caused activation of the glycolytic system but exerted no effect on oxygen uptake and protein synthesis. The influx of Ca2+ into the eggs, having been demonstrated upon fertilization, was not observed in unfertilized eggs when the eggs were treated with cAMP.

RE-EXAMINATION OF FERTILIZATION INHIBITION STUDIES WITH ANTI-FROG-EGG-JELLY ANTIBODIES: EXPERIMENTS WITH NON-PRECIPITATING ANTIBODIES.

Antisera were prepared in rabbits against oviducal and egg-jellies of the frog, Rana japonica. Gamma-globulin fractions from the antisera were degraded to a univalent, non-precipitating form by a papain digestion-reduction procedure. Agar diffusion analyses proved that the digested antibody fragments were inhibitory to the precipitin reaction of undigested, multivalent antibodies with jelly antigens. The treatment of unfertilized eggs with undigested, multivalent antibodies resulted in a significant loss of egg-fertilizability. In contrast, treatment with the univalent fragments of antibodies did not affect egg-fertilizability, similarly to the treatment with both univalent and multivalent γ-globulins from control, non-immune sera. Fertilization was inhibited in large measure when unfertilized eggs were subjected to a dual treatment with univalent antibodies and γ-globulins from anti-rabbit γ-globulin sheep serum. The inhibition of fertilization in the above experiments was always accompanied by the formation of a precipitation layer at the surface of the jelly envelopes. It is concluded that the failure of fertilization in the multivalent antibody-treated eggs results from a secondary effect rather than a specific blocking of a sperm-jelly interaction essential for fertilization.

ON THE HARDENING OF THE CHORION OF THE FISH EGG AFTER FERTILIZATION. III. THE MECHANISM OF CHORION HARDENING IN ORYZIAS LATIPES

1. Oxidizing agents, such as potassium ferricyanide, sodium tetrathionate, iodine, hydrogen peroxide, sodium periodate, chromic acid and potassium dichromate, cause hardening of the chorion. Chromic acid is less effective than any of the other agents.2. In the presence of reducing agents, such as sodium sulfide, potassium cyanide, sodium thiosulfate, sodium sulfate, sodium thioglycolate, ammonium sulfate, and potassium ferrocyanide, the chorions fail to harden.3. Treatment of unfertilized eggs with mercuric chloride or p-chloromercuribenzoate induces the inhibition of chorion hardening when they are subsequently inseminated in Ringer's solution. The same result is also obtained in the cases where both agents are applied after fertilization. Either inhibitory effect is removed if the treatment is followed by that with potassium cyanide.4. Iodoacetic acid and chloroacetophenone likewise inhibit hardening when eggs are treated after insemination.5. Neither urea nor lithium bromide has any effect on the chorion hardening.6. Aldehydes, such as formaldehyde, acetaldehyde, acrolein and benzaldehyde, are capable of hardening the chorion.7. Soft chorion is impregnated with a protein containing SH groups, together with polysaccharide which has α-glycol groups.8. It is concluded that the oxidation process is involved in the mechanism of chorion hardening. It is suggested that hardening is due to combination of SH groups with the aldehydes produced by oxidation of α-glycol groups.

ELEVATION AND RETRACTION OF THE FERTILIZATION MEMBRANE OF ECHINODERM EGGS FERTILIZED IN PAPAIN SOLUTIONS

When eggs of sea urchins and starfish are added to a suspension of sperm in papain solutions (0.02 to 0.04%), a fertilization membrane is elevated in the normal time and, after reaching full separation, retracts to the surface of the egg. This process is completed in approximately 5 minutes in the case of sea urchins (Strongylocentrotus purpuratus and Lytechinus pictus) and 25 minutes in the case of starfish (Asterias forbesii). It is not visible during subsequent development after removal of the eggs from the solution, and development to late stages is normal. Treatment of unfertilized eggs with papain prevents membrane elevation upon fertilization, as reported for other proteolytic enzymes. Treatment of eggs with fully formed fertilization membranes shows no effect on these. It is suggested that the retracted membrane, obtained by fertilization in papain solution, fuses with the egg surface, and that, in general, the question remains open as to whether or not proteolytic enzymes dissolve the vitelline m...

Vegetalization of the Sea-Urchin Egg by Dinitrophenol and Animalization by Trypsin and Ficin

ABSTRACT Herbst (1892) made the remarkable discovery that the differentiation of the sea-urchin egg was shifted in a vegetal direction when lithium ions were present in the sea-water. The vegetalization implies a failure of the apical tuft to appear, a displacement of the skeleton-forming cells in the animal direction, a reduction of the ectodermal region, and a corresponding enlargement of the endoderm, which often leads to exogastrulation. Strong lithium action may lead to complete endodermization of the egg. Many other substances have later been found to cause a change of differentiation, either in a vegetal or animal direction, e.g. a partial or complete animalization by treatment of unfertilized eggs with SCN-or 1-ions (also SO4, Br, and tartrate, Lindahl, 1936). The animal and vegetal principles are considered as two opposite, antagonistic gradients (Runnström, 1928a, b) representing different types of metabolism (Lindahl, 1936).