Eggs Of Urechis Caupo Research Articles

A novel form of sperm detachment from eggs of Urechis caupo

Urechis sperm bind to the egg's extracellular envelope via acrosomally derived adhesive material. Sperm initially adhere tightly, but within minutes they loosen from the egg envelope. Using transmission electron microscopy, we have determined that the sperm's binding material disperses during the detachment phase. Dispersion appears to be caused by the egg and to be necessary for sperm detachment, because sperm bound to glutaraldehyde-fixed eggs do not detach, nor does their binding material disperse. The binding material also fails to disperse when sperm are artificially acrosome reacted in the absence of eggs, using an ionophore. Our results suggest a form of sperm detachment not previously described: an egg-induced removal of the binding material.

Isolation and partial characterization of Urechis caupo egg envelopes.

Eggs of Urechis caupo are surrounded by a congruent to 0.9 micrometer thick egg envelope and, attached to that, a peripheral jelly layer about 3 micrometers thick. Before fertilization, the sperm undergoes the acrosome reaction and binds to the egg envelope. As part of a study of the induction of the acrosome reaction and sperm binding in Urechis, we have developed a method to prepare an egg envelope fraction by differential centrifugation. The isolation procedure removes much of the jelly layer, but does not alter the fine structure of the envelope. When a sperm contacts an isolated envelope, it undergoes a normal acrosome reaction and binds to the envelope's outer face. Electrophoresis of the envelope fraction on sodium dodecyl sulphate (SDS)/polyacrylamide gels revealed six major components stained by Coomassie Blue, of which four are stained by the periodic acid-Schiff reagents (PAS). To measure the degree of enrichment of the envelope fraction, envelopes were isolated from eggs that had been externally radio-iodinated; the specific activity of the envelope fraction was 17 +/- 3 times greater than that of intact eggs. The amino acid composition of the envelope fraction is dominated by Gly (19 mole %), Asx (11%), Thr (11%), Ser (8%), Ala (8%) and Glx (8%). The sugars fucose, xylose, mannose, galactose, glucose, N-acetylglucosamine and N-acetylgalactosamine were detected by gas-liquid chromatography. We also investigated whether the egg envelope changes at fertilization. No change was detected in the electrophoretic 125I pattern of externally radio-iodinated eggs, and the envelope fractions prepared from unfertilized and fertilized eggs produced the same Coomassie Blue pattern on SDS/polyacrylamide gels.

Fertilization acid release in Urechis eggs : I. The nature of the acid and the dependence of acid release and egg activation on external pH

The amount of fertilization acid produced by eggs of Urechis caupo, monitored by automatically back-titrating egg suspensions with base, depends linearly on the pH of the seawater. Above pH 7.0, at which no acid is released (Paul, M., Dev. Biol. 43, 299–312, 1975) , acid release increased approximately 0.34 pmole/egg/0.1 pH unit. Activation (germinal vesicle breakdown) depended on the amount of acid release in natural seawater; it did not occur if eggs released <1.5 pmole acid/egg. When fertilization acid is released into HCO − 3-free seawater and the pH permitted to decrease, the supernatant can be tested for the presence of a volatile acid, such as CO 2, by bubbling with N 2 and comparing the increase in pH as volatile acid is driven off with experiments in which HCl or CO 2 is substituted for fertilization acid. An increase in pH of <0.2 pH units occurred on N 2 bubbling when fertilization acid or HCl was used to acidify HCO − 3-free seawater compared to an increase of >0.5 pH units when CO 2 was used. Therefore, most, if not all, of Urechis fertilization acid is not volatile, and since Paul (1975) showed that it is not a nonvolatile weak acid, it must be H +.

A new fixation procedure for preserving the ultrastructure of marine invertebrate tissues

SUMMARYAddition of 0·05% OsO4 to a conventional glutaraldehyde fixative for the first 10 min of fixation was found to improve greatly the preservation of ultrastructure in the eggs of Urechis caupo. Several workers have since confirmed this result in other marine invertebrate tissues. Specific protocols and techniques are given. We believe that the OsO4 rapidly renders the plasma membranes of cells freely permeable to glutaraldehyde, allowing faster penetration of this fixative. This method should be applicable to a wide variety of tissues that are difficult to fix.

Cytochalasin B inhibits sperm penetration into eggs of Urechis caupo (Echiura)

Cytochalasin B inhibits the penetration of sperm nuclei into Urechis eggs without inhibiting sperm-induced egg activation. The acrosome reaction appears normal, and plasma membranes of the acrosomal tubule and egg become closely apposed. It is uncertain whether or not the drug blocks fusion of these membranes; however, sperm penetration cone formation is inhibited.

The polyspermy block in eggs of Urechis caupo: Evidence for a “rapid” block

Experiments have established the presence and time course of a polyspermy block in eggs of the echiuroid worm Urechis caupo. At various times after an initial insemination the eggs were reinseminated with concentrated sperm suspensions and the polyspermy determined. The block is rapid; partial protection against polyspermy is evident within a few seconds after insemination of the eggs. Although there are other rapid responses approximately correlated in time with the beginning of the block and possibly related to it, I also consider the possibility that a relatively ‘late’ event might account for the apparent ‘rapid’ block in these eggs.

An investigation of the microtubule protein in the mature oocytes of Urechis caupo

The properties of Urechis caupo microtubule protein in mature eggs was investigated and found to be similar to that of other organisms with respect to molecular weight and colchicine-binding parameters. Selective extraction from microtubule-rich organelles and vinblastine precipitation were used to identify and purify this protein, and two distinct subunits of 54 000 and 59 000 molecular weight were resolved on SDS-polyacrylamide gels. The germinal vesicle was shown to contain little of the colchicine-binding activity of the mature egg. Minimum and maximum estimates of the percentage of microtubule protein to total mature egg protein gave values of 0.8 % and 5 %, respectively. It was concluded that a considerable pool of this protein exists in the eggs of Urechis caupo.

Kinetochore-to-pole connections during prometaphase of the meiotic divisions in Urechis eggs

Electron microscope observations of kinetochores and chromosomal spindle filaments during prometaphase of the meiotic divisions in eggs of Urechis caupo are presented. In the first meiotic division, sister kinetochores lie close together on the same side of the chromosome. By prometaphase of the second meiotic division, sister kinetochores have separated and moved to opposite sides of the chromosome. In early prometaphase of the first meiotic division, the kinetochore region of one member of a chromosome-pair may have connections to both poles, or only to one pole. In early prometaphase of the second meiotic division, sister kinetochores may be connected to the same pole or to opposite poles. A single kinetochore may have connections to both poles simultaneously. These observations strongly support Östergren's “orientation-by-pulling” hypothesis, by showing that (i) initial kinetochore-to-pole connections are made at random; (ii) the precise orientation of chromosomes by metaphase is brought about through the loss of some kinetochore-to-pole connections, and the stabilization of others; and (iii) the difference between meiosis and mitosis with respect to chromatid distribution is correlated with a difference in the structure of the kinetochore region.

THE OXIDATIVE METABOLISM OF EGGS OF URECHIS CAUPO

1. The respiration and normal development of fertilized eggs of Urechis caupo are inhibited by low concentrtaions of HCN, 5 x 10-6 M. Known concentrations of HCN were established within the manometer flasks by the use of Ca(CN)2-Ca(OH)2 mixtures in the center-wells, with and without the appropriate amounts of NaCN in the egg suspensions.2. The respiration of fertilized eggs was photo-reversibly inhibited by 95% CO in O2. The inhibition of development was not so marked at this tension as in the cyanide experiments.3. CO markedly stimulated the respiration of the eggs in the light. The occurrence of a similar action in the dark is presumed to account for the moderate degree of depression of respiration by CO in the dark.4. Spectroscopic examination of the eggs at the temperature of liquid nitrogen revealed absorption bands at 551 mµ and 580-590 mµ. Absorption bands at these wave-lengths are associated with the presence of cytochromes c and a.5. An egg brei rapidly oxidized reduced cytochrome c, but intense ...

The respiratory metabolism of the developing eggs of urechis caupo

The respiratory metabolism of the developing eggs of urechis caupo

THE INFLUENCE OF CENTRIFUGAL FORCE ON THE BILATERAL DETERMINATION OF THE SPIRALLY-CLEAVING EGG OF URECHIS

1. The eggs of Urechis caupo were ultracentrifuged for short periods with forces ranging from 6,000 to 75,000 gravities before fertilization and following fertilization at various times before cleavage.2. In the trochophores 75 per cent of the total recorded embryos had the centripetal oil in the ventral median or oblique position, in slightly more than 20 per cent it was found laterally, and in less than 5 per cent dorsally.3. The data are essentially the same for unfertilized eggs and all eggs centrifuged at various times following fertilization and before cleavage, and irrespective of the force applied.4. From these data it is concluded that the bilateral axis of the embryo is induced or influenced by the stratification of material substances in the egg. In centrifuged eggs it is unrelated to either the sperm entrance point or the first cleavage plane.5. There is no polar orientation in the centrifuge tubes before fertilization or following fertilization until the germinal vesicle breakdown. But following the germinal vesicle breakdown the eggs tend to orient in the centrifuge tubes so that the pole comes to lie most commonly near the centrifugal end.6. Normal trochophores are produced from eggs centrifuged before fertilization and at certain times following fertilization, but during some miotic and mitotic stages centrifuging suppresses polar body formation and disturbs the cleavage spindles so that few normal embryos are produced.

THE EFFECT OF ULTRA-VIOLET LIGHT UPON EARLY DEVELOPMENT IN EGGS OF URECHIS CAUPO

1. When unfertilized eggs of Urechis caupo are exposed to ultraviolet light for different lengths of time, then fertilized, polar body extrusion and first cleavage are suppressed. In addition to the suppression of these stages radiation causes an increase in the reaction rate of the egg which bears a direct relation to the length of exposure.2. Possible factors in the suppression of these stages in maturation and first cleavage are the alteration or probable injury of the egg cortex and accompanying changes in its chemical and physical properties, and viscosity changes in the egg endoplasm which inhibit internal phenomena.

FURTHER OBSERVATIONS ON THE POLARITY OF THE EGGS OF URECHIS CAUPO

The eggs of Urechis caupo, an echiuroid worm recently described by Fisher and MacGinitie (1928), are found in abundance at several readily accessible points along the coast of California. Owing to their abundance, size, and transparency, and to their unusual viability under ordinary laboratory conditions, they are proving to be valuable material, especially for the experimental embryologist. It is therefore essential that their normal embryology be known, and of first importance that a definite mark of polarity in the immature eggs, if present, be recog nized. The results of a series of checks upon the polarity of normal and centrifuged eggs of Urechis, with the conclusion that the immature egg bears no visible mark of polarity, were published by Taylor in 1931. Earlier in the same year, Tyler (1931) presented evidence that the indentation, which is characteristic of these eggs, marks the animal pole. Later, this author (Tyler, 1932) published further data and concluded that “? The main question involved in this work is whether or not the polarity of the Urechis egg is determined before fertilization, as appears to be the case for most aninial eggs. For the type fiist discussed the results clearly show that polarity is already established in the unferti lized egg, and the pole is marked by the position of the indentation. For the type in which a second indentation appears after fertilization there is no reason to doubt that polarity is established before fertiliza tion although the position of the pole is not so clearly marked by the first indentation as by the one appearing after fertilization.― According to this author, then, there are two types of eggs in Urechis as regards the point of extrusion of polar bodies: (1) those wherein this extrusion coincides with an indentation before fertilization, and (2) those in which, after fertilization, a second indentation forms and cor responds with the point of extrusion of polar bodies. Just what rela tion this second indentation has to the one in immature eggs is not made clear so that immature eggs of this second type apparently have no mark of polarity. Because of the discrepancies between our findings of 1931 and those