Observations on Eye Movements in Amphibian Larvae

Observations have shown that amphibian larvae, contrary to what has been stated in the literature, have different kinds of eye movements. All of the anurans observed (Xenopus, Rana, Bufo, Hyperoleus and Bombina) have some kind of reflex eye movements effected through stimuli from the labyrinth organs as a response to movements of the body, while only Rana and Bufo have spontaneous scanning movements. Of the urodelians (Ambystoma and Triturus), larvae of Ambystoma have no observable eye movements, while Triturus larvae have reflex eye movements associated with movements of the neck, as well as spontaneous scanning movements; both kinds of movements differ from those seen in Anura.     

鸡蛋胚下表层卵黄DNA的提取方法

利用Ficoll-400不连续密度梯度离心将受精和未受精鸡蛋的胚下表层卵黄进行纯化, 显微镜观察表明卵黄球形态良好, 没有胚细胞的存在.然后利用较高浓度的蛋白酶K消化,较长时间的酚抽提,最后提取了DNA. 电泳显示DNA条带清晰.该方法简便快速,从每个鸡蛋的胚下表层卵黄可回收10 ng DNA.     

Stimulation of Unfertilized Eggs of the Echiuroid, Urechis unicinctus by Polyamines

Unfertilized eggs of the echiuroid, Urechis unicinctus , were activated by polyamines, such as putrescine, spermidine and spermine at concentrations above 10 μM. Fertilization membrane elevated and germinal vesicle disappeared in unfertilized eggs kept for several min in sea water containing these polyamines. Following the addition of these polyamines, a decrease of pH value in the egg suspension, occurred in a similar manner as observed following fertilization. Several sec after the addition of polyamines to the egg suspension, the respiratoy rate increased very slightly and the sensitivity of the respiration to 2, 4-dinitrophenol, which was lower in unfertilized eggs than in fertilized eggs, became as high as in fertilized ones. Irregular cleavage occurred in the eggs stimulated by polyamines. The incorporation of [³H]-deoxyadenosine into DNA was initiated by adding polyamines in the unfertilized eggs preloaded with the isotope. The rate of [³H]-leucine incorporation into protein in the preloaded unfertilized eggs was also enhanced by polyamines, in almost the same manner as observed following fertilization.     

Melittin, a Component of Bee Venom, Activates Unfertilized Sea Urchin Eggs

Melittin, which is known to stimulate phospholipase A , in many cells, caused as much elevation of fertilization membranes and increase in respiration of unfertilized eggs of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus as normal fertilization.
In melittin-activated eggs, amino acid transport was decreased to less than that of unfertilized eggs, nucleoside transport was only slightly, activated, protein synthesis was rather inhibited and neither DNA synthesis nor cleavage was observed. It is concluded that although melittin induces the cortical reaction and activation of respiration in unfertilized eggs, its cytotoxicity prevents any "late changes".     

Remodeling of Human Sperm Chromatin Mediated by Nucleoplasmin from Amphibian Eggs

The molecular events associated with decondensation of human sperm nuclei were analyzed by incubating sperm with egg extracts from an amphibian, Bufo japonicus . Acid-urea-Triton polyacrylamide gel electrophoresis (AUT-PAGE) showed that the nuclear basic proteins of human sperm consist mainly of protamines (HPI, HPII) with minor amounts of nucleosomal histones. On incubation of lysolecithin (LC)- and dithiothreitol (DTT)-treated human sperm with the egg extract, the nuclei lost HPI and HPII within 15 min in association with extensive nuclear decondensation, and the acquirement of a whole set of nucleosomal histones. Incubation of LC-DTT-sperm with nucleoplasmin purified from Bufo eggs also induced nuclear decondensation and loss of protamines within 30 min. Native-PAGE and Western blot analyses of incubation medium indicated tight association of the released protamines to nucleoplasmin, strongly suggesting that protamines are removed from sperm nuclei not enzymatically but by their specific binding to nucleoplasmin. On incubation of LC-DTT-sperm with nucleoplasmin and exogenous nucleosomal core histones, micrococcal nuclease-protected DNA fragments were released, although their unit repeat length was slightly less than that of somatic nucleosomes. Thus remodeling of human sperm during fertilization can be mimicked under defined conditions with nucleoplasmin and exogenous histones.     

Sperm Nuclear Transformation and Aster Formation Related to Metabolic Behaviour in Amphibian Eggs

Bufo arenarum oocyte maturation is related to important modifications in the metabolism of carbohydrates. Such changes involve a different relative participation of the main oxidative routes and are under the influence of seasonal variations of the pituitary activity.
Ovulated coelomic oocytes obtained during the winter period, were unable to initiate cleavage after injection of a sperm suspension. The extent of sperm head transformation and aster formation in the cytoplasm of oocytes with a different metabolic behaviour (obtained during the winter and summer periods) were studied.
Our results show that sperm nuclear transformation and DNA synthesis were quite similar in both types of oocytes. In contrast to summer oocytes, in which the pronucleus was sourranded by an aster, no aster structure was formed in winter ooctyes notwithstanding pronucleus formation occurred.
These results suggest that the failure to develop aster may explain the lack of cleaving in winter oocytes. It appears that the metabolic changes, aster formation and the capability to cleave are closely related and could be dissociated from oocyte nuclear maturation in this species.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Some Properties of the Hardening Process in Chorions Isolated from Unfertilized Eggs of Medaka, Oryzias latipes

Chorions isolated from unfertilized eggs of medaka, Oryzias latipes , harden during incubation with Ca²⁺ ions (Masuda et al. , 1991). In this process, i.e. in vitro Ca²⁺-hardening, the amounts of the major proteins of unfertilized egg chorions (83 K, 78 K and 51 K, corresponding to ZI-1, 2 and 3 of oocyte chorions reported by Hamazaki et al , 1987) decreased and new proteins having molecular weights of 148 K or more appeared. Immunoblotting analysis using anti-ZI-1, 2 antisera and anti-ZI-3 antisera showed that the 148 K protein was an intermediate formed during polymerization of the original proteins.
The mechanism of in vitro Ca²⁺-hardening was studied by examining the decrease in ZI-1, 2, and 3, the formation of 148 K protein, and the change in solubility of chorions in 6% sodium dodecylsulfate-1% 2-mercaptoethanol-15% glycerol-0.2 M Tris-HCl (pH 6.8). In vitro Ca²⁺-hardening was inhibited at temperatures higher than 70°C and its optimum pH was about 5.5. It was inhibited by neither aminotriazole nor cadaverine. The results suggested that in vitro Ca²⁺-hardening was generated by some factor(s) other than ovoperoxidase and transglutaminase.     

Immunoelectron Microscopic Demonstration of Cortical Granule Lectins in Coelomic, Unfertilized and Fertilized Eggs of Xenopus laevis

Immunoelectron microscopic studies demonstrated cortical granule lectins (CGLs) in coelomic, unfertilized and fertilized eggs of Xenopus laevis . An antiserum raised against purified cortical granule lectin 1 specifically reacted with the CGLs in immunoblotting and agar diffusion tests. When ultrathin sections were treated with the antiserum and protein A-gold solution, gold particles, indicating antigenic sites, were seen over cortical granules of coelomic and unfertilized eggs, and over the perivitelline space, the vitelline coat and the condensed region of the fertilization layer of fertilized eggs. The pre-fertilization layer immediately adjacent to the outer margin of the vitelline coat in unfertilized eggs was free from gold particles. These observations suggest that released CGLs permeate through the vitelline coat of fertilized eggs and interact with the pre-fertilization layer mainly at the outer margin of the vitelline coat, resulting in formation of the fertilization layer which acts as a block to polyspermy.     

Effect of Microtubular Poisons on Cleavage Furrow Formation and Induction of Furrow-like Dent in Amphibian Eggs

The effects of the microtubular poisons colchicine, vinblastine and nocodazole, on cleavage furrow formation and induction of furrow-like dents in eggs of the newt, Cynops pyrrhogaster , were examined.
Solutions of the poisons were injected beneath the cortex around the small initial furrow, or around the advancing tip of the furrow of eggs during the first cleavage. This resulted in prompt block of the progress of the furrow at the injection site, and subsequent total regression of the furrow or incomplete cleavage.
The ability of the cortex of a cleavage-arrested blastomere to form a furrow-like dent was tested by inhibiting furrow formation of one blastomere of two-cell embryos by injection of the microtubular poisons, and then transplantation of the blastomere under the cortex of the animal half with furrow-inducing cytoplasm (FIC) taken from normally cleaving eggs. No dent was formed. Moreover, FIC from eggs treated with a poison had no ability to induce a dent on the surface of normally cleaving eggs.
These results show that microtubule structures are directly involved in formation of a cleavage furrow.     

Movements of Echinochrome Granules during the Early Development of Sea Urchin Eggs

ECHINOCHROME pigment granules in unfertilized eggs of the sea urchin Arbacia punctulata undergo randomly-directed saltatory movements^1,2. After fertilization, nearly all these granules migrate to the egg cortex and become embedded. Subsequent pigment granule movements may represent mass cortical changes rather than independent granule movements^2,3. At the fourth cleavage, a quartet of micromeres containing little or no pigment forms at the vegetal pole. By the two or four-cell stage, pigment granules have begun to move out of this region, leaving a “clear area” on each blastomere (Fig. 1 and refs. 4, 5). To investigate possible mechanisms for these movements and their relation to cortical events     

Origin of a Meloidogyne incognita Surface Coat Antigen

The surface coat (SC) of plant nematodes is thought to originate either from the living hypodermis or from secretory glands associated with the excretory system or nervous system. In this study, we investigated the origin of the SC of Meloidogyne incognita by immunolocalization with a monoclonal antibody raised against the surface coat of the preparasitic juveniles (J2). Under the electron microscope, strong labeling was found on the cuticular surface and in the rectal dilation of the J2, while labeling was absent in other parts of the nematode, including the hypodermis, excretory system, nervous system, and digestive system. Because the rectal glands are known to be the origin of the gelatinous egg matrix produced by adult females of Meloidogyne, we also examined sections of mature females from monoxenic cultures of Arabidopsis thaliana. Labeling of the female occurred in the rectal glands and in the gelatinous matrix exuded from the anus. At the ultrastructural level, gold particles were mainly deposited in multivesicular bodies that appeared to be associated with the Golgi bodies of the rectal glands. Our results suggest that at least one component of the J2 SC originates from the rectal gland cells and that the SC of the J2 shares common epitopes with the gelatinous egg matrix of mature females.     

Electrophoretic Properties, Cell Surface Morphology and Calcium in Amphibian Gastrulation

The process of gastrulation is characterized by extensive morphogeneticmovements, cell shape changes and intercellular rearrangements.This paper presents the results and inferences of experimentalanalysis of these events. Cell electrokinetic mobility, whichis a measure of net cell surface charge density, cell surfacemorphological changes, and the role of calcium are aspects ofgastrular events which we believe play a significant role. Ourhypothesis is that these parameters are interrelated and weoffer suggestions with respect to the interrelationships andhow these aspects mediate morphogenetic movements.     

Steroid-Cell Surface Interactions in the Induction of Meiotic Maturation in Amphibian Oocytes

The involvement of steroid-cell surface interactions, in the induction of oocyte maturation, was investigated. A simple in vitro method was developed for the application of steroids or other substances to isolated portions of the surface of individual amphibian ovarian follicles or oocytes. The technique involved placing follicles in conical tubes between two reservoirs containing culture media. With the use of dye or radioactive tracer studies, the extent of exchange between reservoirs could be monitored and the proportion of the surface treated could be calculated.
Follicular oocytes remained viable and underwent nuclear and cytoplasmic maturation while maintained in conical tubes after a short exposure to progesterone over their total surface. Restriction of the percentage of the oocyte surface exposed to progesterone markedly reduced the incidence of meiotic maturation. Reduced responsiveness to locally applied progesterone occurred even in the presence of elevated steroid concentrations. The results suggest that the amount of follicle surface exposed to steroid is an important determinant in initiation of nuclear-cytoplasmic interactions associated with the meiotic maturation process. The procedures and results described should provide a new approach to investigating regional differences in oocyte and follicle surfaces and to studying their differentiation.     

Phorbol Ester Induces Elevation of the Vitelline Coat of Eggs of the Ascidian Halocynthia roretzi

Elevation of the vitelline coat of eggs of the ascidian, Halocynthia roretzi , was induced by 12-O-tetradecanoylphorbol-13-acetate or phorbol-12, 13-didecanoate, but not by their 4-epimers. After elevation of the vitelline coat in this way, eggs were unable to undergo cleavage on addition of sperm. This elevation of the vitelline coat by phorbol esters was inhibited by specific inhibitors of trypsin-like enzyme, calmodulin, phospholipase A₂, and protein kinase C. In association with elevation of the vitelline coat, a trypsin-like enzyme was released from the eggs. The properties of this enzyme were similar to those of the enzyme released by calcium ionophore, which also induced elevation of the vitelline coat, suggesting that similar exocytosis of intracellular granules occurred in both cases. Calmodulin, possibly involved in the elevation process, was isolated from eggs and characterized.     

Expression of Surface Coat Glycoconjugates by Bacteria-Fed Tetrahymena1

ABSTRACT. We have shown that bacteria-fed Tetrahymena express at their surface and excrete into the medium a glycoconjugate absent in axenically grown cells. A preliminary analysis of the purified molecule is given. Immunolabeling of blotted surface extracts and fixed cells shows that bacteria-fed Tetrahymena build up a surface coat whose material originates totally or in part in the mucocysts. The glycoconjugate is located externally on the coat and mediates cell immobilization and immunolabeling by the serum. The results also indicate that axenic cells are probably devoid of surface coat.     

Immunoelectophoretic Identification of Jelly Coat Ligands Bound by the Cortical Granule Lectin from Xenopus laevis Eggs

The formation of the fertilization layer in the Xenopus laevis egg fertilization envelope involves a lectin-ligand interaction and establishes a block to polyspermy in the extracellular matrix of the egg. The cortical granule lectin participating in the formation of the fertilization layer has been isolated but its ligand has not. We identified three jelly coat ligands bound by the cortical granule lectin using immunoelectrophoretic analyses. Two antigens were detected with anti-jelly serum and a third was identified using anti-envelope serum. All three antigenic ligands were associated with the innermost jelly coat layer, J₁, and two of the three antigenic ligands contained sulfate. One or more of these jelly coat ligands may function in establishing a block to polyspermy at fertilization in Xenopus laevis .