Fertilization triggers localized activation of Src-family protein kinases in the zebrafish egg

Fertilization triggers activation of Src-family kinases in eggs of various species including marine invertebrates and lower vertebrates. While immunofluorescence studies have localized Src-family kinases to the plasma membrane or cortical cytoplasm, no information is available regarding the extent to which these kinases are activated in different regions of the zygote. The objective of the present study was to detect the subcellular distribution of activated Src-family kinases in the fertilized zebrafish egg. An antibody specific for the active, non-phosphorylated form of Src-family PTKs was used to detect these activated kinases by immunofluorescence. The results demonstrate that Fyn, and possibly other Src family members are activated by dephosphorylation of the C-terminal tyrosine at fertilization. The activated Src-family kinases are asymmetrically distributed around the egg cortex with an area of higher kinase activity localized adjacent to the micropyle near the presumptive animal pole. Fertilization initially caused elevation of kinase activity in the cytoplasm underlying the micropyle, but this quickly spread to involve the entire zygote cortex. Later, during egg activation, formation of the blastodisc involved concentration of active Src-family kinase in the blastodisc cortex. As cytokinesis began, activated Src-family kinases were no longer limited to the cortex, but became more evenly distributed in the clear apical cytoplasm of the blastomeres. The results demonstrate that the cortex of the zebrafish egg is functionally differentiated and that fertilization triggers localized activation of Src-family kinases at the point of sperm entry, which subsequently progresses through the entire egg cortex.     

Microtubule cycles in oocytes of the surf clam, Spisula solidissima: an immunofluorescence study

Oocytes of the surf clam, Spisula solidissima, underwent germinal vesicle breakdown and two meiotic divisions to give off polar bodies when they were fertilized or parthenogenetically activated with KCl. Fertilized eggs further proceeded to mitosis and cleaved, while parthenogenetically activated eggs remained uncleaved. We examined changes in microtubule-containing structures during meiotic divisions and subsequent mitotic processes by immunofluorescence. A monoclonal anti-tubulin antibody was applied to alcohol-fixed eggs from which the vitelline membrane had been removed by protease digestion. Up to the stage of second polar body formation, the pattern of microtubule organization in the first and second meiotic spindles was identical in both fertilized and parthenogenetically activated eggs. However, while fertilized eggs formed a sperm aster and mitotic spindles later, activated eggs formed only monaster- or ring-shaped microtubule-containing structures which underwent cycles of alternating formation and breakdown. Lactoorecin staining of parthenogenetically activated eggs revealed that the chromosome cycle could occur in these eggs, in phase with this microtubule cycle.     

Obstruction of Blastodisc Formation by Cytochalasin B in the Zebrafish, Brachydanio rerio

The blastodisc formation in the zebrafish, Brachydanio rerio , was obstructed by treatment with 1.0 μg/ml of cytochalasin B (CB), but not by 1.0 μg/ml of colchicine. The cortex in normal eggs contained a meshwork of microfilaments associated with the plasma membrane. The cortex was thicker at the vegetal pole and thinner at the animal pole of the egg. In CB treated eggs the cortex contained masses of microfilaments detached in places from the plasma membrane. Microtubules were never observed in the cortex of eggs with or without CB treatment. These results suggest that ooplasmic segregation, which results in blastodisc formation, is carried out by activity of the cortex, which contains CB sensitive microfilaments.     

Post-fertilization changes in the chorion of winter flounder, Pseudopleuronectes americanus Walbaum, eggs observed with scanning electron microscopy

Little has been reported on the fine structure of the outer membrane of fish eggs during and after fertilization. When observed in the scanning electron microscope, the unfertilized egg of the winter flounder, Pseudopleuronectes americanus , is characterized by a crisscross pattern of depressions. These depressions radiate in all directions across the membrane surface creating a wrinkled appearance. After fertilization, the surface of the chorion becomes regular with a smoother appearance. The pores of the unfertilized egg are flush with the chorion surface, but become thickened and elevated after fertilization. While the chorion of the unfertilized egg is also smooth and uniformly textured, the chorion of the fertilized egg appears granular by first cleavage of the blastodisc. Although no apparent change occurs in the distance between pores after fertilization, statistically significant decreases in pore diameter occur 5 min after fertilization. These results are compared to those on egg membranes of other species of fish and invertebrates.     

Cortical and zona reactions of heat-activated mouse eggs

Oocytes collected from superovulated Swiss albino mice were activated by heat-shock and/or fertilized in vitro. Electron microscopy showed that the cortical reaction in heat-treated eggs was incomplete. Digestion of the zona pellucida of untreated, heat-treated and fertilized eggs by pronase showed that zona pellucida hardening did occur but was weaker in heat-treated than in fertilized eggs. Fertilizability of heat-treated eggs decreased 1.5 h after heating. We concluded that, in heat-activated eggs, changes occur at the zona and plasmalemma but they are not identical with those in fertilized eggs.     

Alteration of lipid organization following fertilization of sea urchin eggs

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.     

Delayed sperm injection and fertilization in parthenogenetically activated insect egg (Athalia rosae,Hymenoptera)

Summary Mature eggs dissected from the ovary of unmated females of Athalia rosae ruficornis Jakovlev (Hymenoptera, Tenthredinidae) can be activated to develop (into haploid parthenogenetic males) simply by exposing them to distilled water. These eggs, which are primary oocytes arrested at the first meiotic metaphase, resume meiosis upon activation and reach the first meiotic telophase in 20 min. Mature eggs immediately upon dissection have previously been shown to complete karyogamy and develop as fertilized diploid females if injected with sperm. We show here that the eggs activated in water for 20 min have a much higher rate of successful fertilization if injected with sperm, and that the eggs activated for 40 min, upon sperm injection, though at a reduced frequency still develop as diploid fertilized females. Eggs left in water for 60 min, however, are no longer fertilized upon sperm injection and develop as haploid males.     

T-1, a mitotic arrester, alters centrosome configurations in fertilized sea urchin eggs

T-1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T-1-treated (1.7-8.5 microM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel-shaped. When eggs are fertilized with sperm that have been preteated with T-1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti-centrosomal monoclonal antibody reveals that T-1 modifies the structure of the centrosome so that barrel-shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T-1 induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T-1 directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T-1 can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.     

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.     

Nuclear Involvement in Localization of the Initiation Site of Surface Contraction Waves in Xenopus Eggs

The initiation site of surface contraction waves (SCWs) was examined in fertilized, parthenogenetically activated and enucleated Xenopus eggs after either rotation through 90° off the vertical axis or injection of colchicine. In enucleated eggs, SCWs always started from a top site of the egg under all conditions examined. In fertilized or activated eggs, SCWs started, depending on the experimental conditions, from either the sperm entry point, the animal pole region located sideward or the top site of the egg. Histological examinations of fertilized and activated eggs revealed that the nucleus was in most cases positioned close to the initiation site of SCWs under various experimental conditions. It is suggested from these results that the egg cytoplasm has an intrinsic capability of causing the surface to generate SCWs, and that the nucleus is generally involved in localizing the initiation site of SCWs in fertilized or activated Xenopus eggs. A possible mechanism for localizing the initiation site of SCWs in Xenopus eggs is proposed.     

Role of the vitellus in the block to polyspermy in golden hamster eggs

The block to polyspermy in golden hamster eggs is believed to operate only at the zona pellucida. However, changes in the egg vitellus also prevent further entry of capacitated sperm. When zona-free hamster eggs spontaneously activated in vitro, and in vivo fertilized eggs at pronuclear stage were inseminated with capacitated human sperm, penetration did not occur. In the case of a homologous system using hamster sperm and in vivo fertilized hamster eggs, slight attachment of sperm was observed but no penetration. The cortical granules were found to be released in spontaneously activated and in fertilized eggs as observed by phase contrast microscopy. These observations suggest that the egg vitellus plays a role in the block to poiyspermy in addition to that of the zona block.     

Partially Fertilized Sea Urchin Eggs: An Electrophysiological and Morphological Study.

Propagation of the cortical reaction in sea urchin eggs may be interrupted by a mild heat shock. In such partially fertilized eggs three distinct cortical zones may be distinguished. First, an activated area where cortical granule exocytosis is complete, the fertilization membrane is elevated, and there is a cortical meshwork of polymerized actin. Second, at the antipode an area where the cortical granules are intact, actin is not polymerized, and the surface structure in general resembles that of the virgin egg. Between the two there is a transitional zone, some 10 to 20 μm wide, where a fraction of cortical granules have exocytosed, giving rise to isolated "blebs" of elevated fertilization membrane. Partially fertilized eggs have resting potentials ranging from −20 to −80 mV, and upon re-insemination give rise to step depolarizations indicating that spermatozoa may interact and possibly fuse with the "unactivated surface".     

Hypotonic buffer induces meiosis and formation of anucleate cytoplasmic islands in the egg of the two-spotted cricket Gryllus bimaculatus

In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus.     

Ultracytochemical Modifications of Surface Carbohydrates in Fertilized Eggs of the Common Carp

The surface of the plasma membrane of unfertilized and fertilized carp eggs was examined by four cytochemical techniques, colloidal thorium, colloidal iron, ruthenium red and phosphotungstic acid stainings, to determine the carbohydrate moieties. The surface of the plasma membrane of unfertilized eggs stained only with colloidal iron, which was heterogeneously deposited: no deposits were seen on the plasma membrane near overlying cortical alveoli. In fertilized eggs, the membrane was stained by all four methods. These ultracytochemical modifications of the surface of the plasma membrane may be caused by participation of the limiting membranes of secretory organelles, probably by turnover of the inner surface of the limiting membranes. Neuraminidase treatment of fertilized eggs eliminated the deposits of colloidal iron on the surface of the plasma membrane and caused an increase in stainability with ruthenium red. Treatment with neuraminidase or trypsin prevented the staining with phosphotungstic acid.     

Insulin receptor sites as membrane markers during embryonic development. I. Data obtained with unfertilized and fertilized sea urchin eggs.

Binding of insulin to sea urchin egg plasma membrane has been studied by biochemical and immunocytochemical methods. Unfertilized and fertilized eggs as well as embryos during the first cell division have been used. 1. Competition experiments between 125I-insulin (1 nM) and an excess of native insulin (30 muM) indicate a specific hormone fixation to membrane crude extracts from unfertilized and fertilized eggs. The magnitude of "specific binding" is comparable to values recorded for mammalian cells. 2. Inhibition of insulin fixation by concanavalin A (100 mug/ml) suggests the glycoprotein composition of plasma membrane receptors. 3. An 30-min incubation of unfertilized and fertilized eggs in the presence of insulin leads to a significant increase in cyclic AMP content. 4. An immunocytochemical method demonstrates that insulin is selectively and specifically bound to the plasma membrane of eggs incubated in the presence of insulin before fixation. It can be concluded that insulin receptor sites are components of sea urchin eggs plasma membrane. Insulin binding which leads to cyclic AMP accumulation is not deeply modified by fertilization and does not include visible morphological changes in the eggs.     

The karyotype and ultrastructural characteristics of spontaneous preimplantation mouse parthenotes

Karyotypic and light and electron microscopical analyses were made of spontaneous preimplantation mouse parthenotes from the LT/Sv inbred strain. It was found that the activated oocyte and developing embryos were diploid. We believe that diploidization is achieved by the oogonium undergoing a premeiotic mitosis without cytokinesis followed by two meiotic divisions, thus producing diploid parthenotes. The developmental events with respect to membrane specialization, such as junctional complexes, were similar to those observed in fertilized embryos. A unique feature of the developing parthenote was the failure of the mitochondria to change during the morula stage. The mitochondria retained a few irregularly oriented cristae rather than many transversely oriented ones observed in morulae developing from fertilized eggs. The significance of this observation is discussed.     

Determination of cell form in cultured fibroblasts : Role of surface components and cytokinetic elements

Functions involved in the determination of the morphology of mouse embryo fibroblasts have been studied using as a system the cell rounding and reflattening related to the presence or absence of a protein component of the cell surface [18]. Cells that had undergone rounding following the removal of the surface protein gradually reflattened if their protein synthesis was not impaired but could rapidly revert to the flattened state if a fraction containing the surface protein was returned to the cells. Studies with sodium azide and potassium cyanide showed that both the rounding and reflattening were energy-dependent. When the rounded state was induced the organised appearance of the microfilaments present in the cortical layer adjacent to the plasma membrane was lost. However, microfilament structures reappeared when rounded cells respread over the glass substrate. On the other hand no reflattening occurred if microfilament function was impaired by treatment with cytochalasin B (CB), showing a requirement of microfilament integrity for the flattened form to be manifest. Microtubules also were shown to be involved in shape determination as their integrity was necessary for cells to progress from the flattened form to the rounded state, but they did not appear to be primarily involved in the process of reflattening. The distribution of macromolecular components of the plasma membrane was also shown to affect changes of cell shape as cross-linking of surface receptors by concanavalin A (ConA) or specific anti-cell immunoglobulins prevented cells from acquiring the rounded form and treatment of cells with media of low pH, which causes aggregation of intra-membrane particles, also prevented rounding. A comparison between quiescent cells and cells in S phase showed the changes of morphology to be similar in nature, but more prompt and marked in the S population. The indication of the studies presented is that determination of cell form requires the cooperative interaction between the protein component of the cell surface, integral constituents of the plasma membrane and cytokinetic organelles.     

Intracellular chloride activity and pH during polar lobe formation and cytokinesis in eggs and embryos of Ilyanassa obsoleta

The objectives of this study were to compare membrane potential, intracellular chloride activity, and intracellular pH in unfertilized and fertilized eggs of Ilyanassa obsoleta throughout polar lobe formation and cytokinesis. Membrane potential, psi m, was measured with single- and double-barrel microelectrodes containing 3 M KCl. Chloride activity, (Cl)c, was measured with single-barrel, chloride-sensitive microelectrodes. Intracellular pH, pHc, was measured with double-barrel microelectrodes, utilizing an antimony-filled barrel to monitor pHc. Unfertilized eggs having a psi m of -13.7 mV displayed an average (Cl)c of 29.9 mM, whereas at electrochemical equilibrium, (Cl)ce, it would be 155.7 mM. This suggests that Cl is pumped out of the cell in eggs that are not fertilized. Fertilized eggs had a psi m of -78.2 mV and a (Cl)c of 18.6 mM. At electrochemical equilibrium, (Cl)ce would be 12.1 mM, thus suggesting that Cl is pumped into the cell in fertilized eggs. Double-barrel microelectrodes measured a psi m of -69.0 mV and a pHc of 7.3 in fertilized eggs. The psi m, (Cl)c, and pHc remained stable as fertilized eggs underwent the cellular shape changes involved in forming first, second, and third polar lobes and first cleavage.     

小鼠受精卵微注射Cdc25b mRNA可提高卵裂率并促进受精卵发育

为了观察Cdc25B蛋白及PKA/Cdc25B 信号途径在小鼠受精卵发育中的作用,将突变型和野生型Cdc25b转录成 mRNA,显微注射到小鼠受精卵中,放入含有或不含有dbcAMP的M16中,相差显微镜下观察受精卵卵裂情况;用蛋白激酶活性测定方法检测MPF的活性;利用Western 印迹检测Cdc2-Tyr15的磷酸化状态.结果显示,未加dbcAMP的Cdc25b- S321A mRNA注射组与Cdc25b-WT组相比,能够提前使受精卵发生G ₂/M期转变,导致卵裂,并明显提高卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,Cdc25b-S321A组先于Cdc25b-WT组提前激活MPF.此外, Cdc25b-S321A mRNA注射组可以有效恢复由PKA引起的受精卵G ₂期阻滞,显著增加卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,在PKA持续激活的情况下,对比于Cdc25b-WT组,Cdc25b-S321A组提前激活MPF.因此,在小鼠受精卵发育过程中PKA主要通过磷酸化Cdc25B的321位丝氨酸,从而调控MPF的激活与失活来控制有丝分裂进程.     

Demonstration of a heterogeneous distribution of glycolytic enzymes and of pyruvate kinase isoenzymes types M1 and M2 in unfertilized hen eggs

The intracellular distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and the pyruvate kinase isoenzymes type M1 and type M2 within unfertilized hen eggs was studied. Most of glycolytic enzyme activities were found in the yolk fraction; 8-24% of total glycolytic enzyme activities were found in the vitelline membrane fraction. However, the specific activities of these enzymes in the vitelline membrane fraction are 19-72-fold higher (U/mg protein) and 45-178-fold more concentrated (U/g wet weight) than in the yolk fraction. The study of intracellular localization of pyruvate kinase isoenzymes shows that the blastodisc, latebra and vitelline membrane contain only pyruvate kinase type M2, whereas pyruvate kinase types M1 and M2 are found in the egg yolk. The exclusive occurrence of pyruvate kinase type M2 in the blastodisc is consistent with the concept that this isoenzyme is involved in the cell proliferation. The heterogeneous distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and the heterogeneous localization of the pyruvate kinase isoenzymes types M1 and M2 indicate that glycolysis is distributed heterogeneously within the unfertilized hen egg cell.