Intracellular pH and the increase in protein synthesis accompanying activation of Xenopus eggs

Metabolic activation following egg fertilization corresponds to an increase in protein synthesis and the initiation of DNA synthesis, which lead to cell division and development of the embryo. Since in several biological systems protein synthesis is regulated by intracellular pH (pHi), we have decided to investigate the situation during Xenopus egg activation. We confirmed that egg activation is accompanied by a pHi rise of 0.3 pH unit. Measurements of the rates of protein synthesis is unactivated and activated eggs, after microinjection of 3H-leucine, demonstrated that activation was followed by a 2.5-fold increase. Treatment of unactivated eggs with weak bases also increased pHi, but did not result in an increase in the rate of protein synthesis. Moreover, in vitro translation in cytoplasmic extracts was found to be pH-independent, at least between 6.8 and 8.2.     

Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs

Fertilization is accompanied by a rapid and transient calcium release in eggs, which is required for the onset of zygotic developmental program or 'egg activation'. Recently, it was found that Src family tyrosine kinase (SFK)-dependent phospholipase C (PLC) activity is necessary for the calcium transience in fertilized Xenopus eggs. The present study demonstrates that hydrogen peroxide (H2O2) stimulates protein-tyrosine phosphorylation in Xenopus eggs, which occurs primarily in the egg cortex of the animal hemisphere as revealed by indirect immunofluorescence study. Egg SFK was found to be upregulated by H2O2 while the SFK-specific inhibitor PP1 effectively blocked H2O2-induced tyrosine phosphorylation. As in fertilized eggs, PLCgamma, but not Shc, was tyrosine-phosphorylated in H2O2-treated eggs. H2O2 also caused inositol 1,4,5-trisphosphate (IP3) production and sustained calcium release. After limited application of H2O2, elevated SFK activity and tyrosine phosphorylation were quickly reversed. Under such conditions, eggs showed cortical contraction and dephosphorylation of p42 MAP kinase, both of which are indicative of egg activation. These egg activation events, as well as H2O2-induced IP3 production and calcium release, were sensitive to PP1 and PLC inhibitor U-73122. Together, the present study demonstrated that H2O2 can mimic, at least in part, early events of Xenopus egg activation that require an SFK-dependent PLC pathway.     

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 μM or 50 μM OAG for 10 min resulted in meiosis II completion in ∼80% of instances. By contrast, at a lower concentration (25 μM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 μM OAG, the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) increased transiently in all the eggs examined, whereas after the addition of 50 μM OAG, [Ca²⁺]_i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca²⁺ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway. Mol. Reprod. Dev. 48:292–299, 1997. © 1997 Wiley-Liss, Inc.     

Developmental regulation of class I major histocompatibility complex antigen expression by equine trophoblastic cells

Between days 36-38 of pregnancy equine trophoblastic cells of the chorionic girdle migrate and form endometrial cups. Just prior to invasion, the chorionic girdle cells express high levels of polymorphic, paternally inherited, major histocompatibility complex (MHC) class I antigens. Their descendents, the mature, invasive trophoblast cells of the endometrial cups, however, express low or undetectable levels of MHC class I antigens by day 44 of pregnancy. Experiments with MHC compatible pregnancies, the study of residual chorionic girdle cells that had failed to invade the endometrium and remained on the surface of a conceptus, and the study of chorionic girdle cells recovered on days 34-36 of pregnancy and then maintained in vitro for up to 24 days strongly suggest that the reduction of MHC class I antigen expression by mature invasive trophoblast cells of the endometrial cups is developmentally regulated. This phenomenon does not appear to be induced by a maternal antibody response or by other uterine factors acting after the chorionic girdle trophoblast cells invade the endometrium.     

Characterization of valine transport in sea urchin eggs

In unfertilized eggs, the mechanism of valine uptake can be summarized as follows. It is saturable over the external concentration of valine and insensitive to the presence of external sodium, depletion of cellular energy supplies and intracellular acidosis. The activation energy for the transport reaction (16.3 kcal/mol) is within the range of values reported for active transport of small molecules. In fertilized eggs, the total rate of valine uptake can be divided into two components: (i) a Na⁺-insensitive uptake which accounts for about 7% of total absorption as shown by studies in Na⁺-free medium seems to possess the same characteristics as in unfertilized eggs, (ii) a Na⁺-dependent transport of valine which constitutes the main entry is formed about 5 min after fertilization. It follows Michaelis-Menten kinetics characterized by 15-fold increase in $\text{V}{}_{\max }$ with no change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. These two mechanisms have characteristics in common, such as their insensitivity to metabolic energy supply, their energy of activation and their ability to concentrate valine. The relationship between the establishment of the Na⁺-dependent valine uptake and the ionic events triggered by fertilization is discussed.     

Multigenerational experimental simulation of climate change on an economically important insect pest

Long‐term multigenerational experimental simulations of climate change on insect pests of economically and socially important crops are crucial to anticipate challenges for feeding humanity in the not‐so‐far future. Mexican bean weevil Zabrotes subfasciatus, is a worldwide pest that attacks the common bean Phaseolus vulgaris seeds, in crops and storage. We designed a long term (i.e., over 10 generations), experimental simulation of climate change by increasing temperature and CO₂ air concentration in controlled conditions according to model predictions for 2100. Higher temperature and CO₂ concentrations favored pest''s egg‐to‐adult development survival, even at high female fecundity. It also induced a reduction of fat storage and increase of protein content but did not alter body size. After 10 generations of simulation, genetic adaptation was detected for total lipid content only, however, other traits showed signs of such process. Future experimental designs and methods similar to ours, are key for studying long‐term effects of climate change through multigenerational experimental designs.     

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.     

Biochemical characterization of lysosomes in unfertilized eggs of xenopus laevis

Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.     

大蜡螟作为试验昆虫资源的利用现状

随着对资源昆虫的不断认识,人们的目光开始逐渐转到对大蜡螟Galleria mellonella L.的开发利用方面,而不再仅仅局限于对它的防治方面。近些年来,大蜡螟逐渐被作为试验昆虫用于一些生物的研究。文章主要介绍大蜡螟被用于昆虫病原线虫、寄生蜂、新型隐球菌Cryptococcus neoformans、抗菌肽、抗菌免疫机制等方面的研究。     

Developmental potential of aged oocyte rescued by nuclear transfer following parthenogenetic activation and in vitro fertilization

Mouse oocyte aged in vitro cannot develop normally following activation. To investigate the roles of nucleus or cytoplasm elements in oocyte aged in vitro process and their subsequent development capability following activation, we reconstructed oocytes with MII chromosome spindle and cytoplasm from aged and fresh oocytes by nuclear transfer. The subsequent developmental potential after parthenogenetic activation (PA) or in vitro fertilization (IVF) was evaluated. After nuclear transfer, more than 75.6% of karyoplast and cytoplast pairs can be fused and reconstructed oocytes have a normal haploid karyotype. Following PA, aged oocytes cannot develop beyond four-cell stage, reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with a low percentage (9.1%). Instead, blastocyst formation rate of reconstructed oocyte from aged nucleus and fresh cytoplasm was higher (60.0%). Following IVF, zygote with diploid karyotype can be formed from zona pellucida (ZP)-free oocyte. After cultured in vitro, aged oocytes cannot develop beyond two-cell; reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with low percentage (15.0%). However, high blastocyst formation rate (86.2%) can be obtained from reconstructed oocytes from aged nucleus and fresh cytoplasm. Furthermore, after embryo transfer, three viable pups have been obtained, although the efficiency is very low. These observation demonstrated that cytoplasm is more crucial than nucleus to aging process. Fresh cytoplasm could partly rescue nucleus susceptibility to apoptosis from aging in vitro.     

Developmental failure of hybrid embryos originated after fertilization of bovine oocytes with ram spermatozoa

The developmental ability of hybrid zygotes, produced by in vitro fertilization of in vitro matured bovine oocytes with ram sperm, was evaluated by gross morphology, autoradiographic detection of (5-³H) uridine incorporation, and fine structure morphology. Fertilization was successful in 83% of bovine oocytes inseminated with bull sperm (control embryos) compared with 67% of bovine oocytes inseminated with ram sperm (hybrid embryos) and in both cases appeared two regularly developed pronuclei. Two-cell embryos were transferred to ewe oviducts and allowed to develop to the 8-cell stage. Although the ability of hybrid embryos to reach 8-cell stage was similar to that of control embryos, in nuclei of hybrid embryos the transition from maternal to embryonic genome control assessed according to the onset of RNA synthesis indicated the differences in the frequency of labelled nuclei and intensity of their labelling. In hybrid embryos these parameters were remarkably lower and may reflect the developmental failure of hybrid embryos. These observations are consistent with delay or inefficient reactivation of the embryonic genome in the hybrid embryos. Mol. Reprod. Dev. 48:344–349, 1997. © 1997 Wiley-Liss, Inc.     

Changes in the proteins of the vitelline membrane of hens' eggs during storage

Vitelline membranes from fresh and stored eggs were treated with either 0.5 M sodium chloride or 1% sodium dodecyl sulphate (SDS) and the resulting solutions examined by polyacrylamide gel electrophoresis. Several differences between the fresh and stored membranes were evident, the most noticeable being the loss of protein VMO I (vitelline membrane outer I) and the formation of a dimer of lysozyme during storage.     

A mathematical model of baculovirus infection on insect cells at low multiplicity of infection

The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of     

Degradation of maternal poly(A)-containing RNA during early embryogenesis of an insect (Smittia spec., chironomidae,diptera)

Summary Eggs of the chironomid midgeSmittia spec. were shown to contain maternal rRNA, tRNA and poly(A)-containing RNA. The ribonucleoprotein spectrum consisted of monosomes, ribosomal subunits, and subribosomal particles, whereas polysomes could be detected only in small amounts. Poly(A)-containing RNA was found in different regions of the RNP spectrum, mainly between 15 S and 60 S. After labelling maternal RNA by feeding tritiated uridine to the larvae, the radioactivity associated with poly(A)-containing RNA accounted for about 4% of the label in the total RNA extracted from newly deposited eggs. About half of the radioactivity in the poly(A)-containing RNA was lost between egg deposition and an advanced blastoderm stage. The loss was accompanied by both a decrease in the size of the poly(A)-containing RNA molecules and a shift of poly(A)-containing RNP particles to less dense regions in sucrose gradients. Comparison with poly(A)-containing RNA synthesized by the embryo indicates that the reduction in size of maternal poly(A)-containing RNA is not artifactual but reflects its degradation after the formation of blastoderm.     

The use of lectins to select subpopulations of insect cells

Lectins have been used in glycoprotein purification, oligosaccharide analysis, and in cell‐selection processes. Here, we utilize lectins in a rational attempt to select a subpopulation of insect cells (Estigmene acrea, EAA) with more complete glycosylation capacity by selecting cells that display more complex‐type cell‐surface oligosaccharides than the general population of cells. A lectin (ECA) from Erythrina cristagalli, specific for galactose β(1‐4)N‐acetylglucosamine, was found to be useful in recognizing a small subpopulation of Sf‐21 and EAA cells. Cell selections were performed by lectin affinity chromatography and by selective agglutination. Analysis by lectin blots of cell lysates and a quantitative agglutination assay did not reveal significant differences in regard to the level of complex glycosylation between the negatively and positively selected subpopulations of EAA cells. Statistically significant differences in binding the fluorescently labeled lectin, ECA‐TRITC were observed even 30 passages post‐selection between EAA subpopulations that were negatively and positively selected by lectin affinity chromatography. There were no differences in the two subpopulations in the ECA quantitative agglutination assay. Thus, the hypothesis that a subpopulation differing in glycosylation capacity exists and that such a subpopulation can be identified by the character of cell‐surface oligosaccharides is plausible. However, these differences appear to be too small to be of practical use. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 616–619, 1999.     

Ca2+ ionophores and the activation of human blood platelets: The effects of ionomycin,beauvericin, lysocellin,virginiamycin S,lasalocid-derivatives and McN 4308

Platelet activation is linked to an increase in the cytoplasmic Ca²⁺ concentration and consequently can also be induced by ionophores which mobilize Ca²⁺ from intracellular storage sites or transport it through the plasma membrane. The ionophores mostly used in studies on platelet activation are A 23187 and lasalocid (X-537A). The effects of eight compounds with known Ca²⁺-ionophoric activity in synthetic or natural membrane systems were studied in order to investigate the relationship between transport of Ca²⁺ and activation of platelets.Ionomycin acts as a true Ca²⁺ ionophore: it elicits rapid shape change, aggregation, the release reaction (secretion) and clot retraction (contraction). Beauvericin activates platelets too, but probably not by increasing the cytoplasmic Ca²⁺ concentration. Lysocellin does not activate platelets but induces a passive loss of serotonin. Virginiamycin S has no effect on platelets. Bromolasalocid and one epimer of dihydrolasalocid, like lasalocid, activate platelets by increasing the cytoplasmic Ca²⁺ concentration, and also induce a passive loss of serotonin. McN 4308 does not activate platelets but induces a slow uptake of ⁴⁵Ca²⁺.     

Photochemistry of platinum phosphine complexes. Perspective in C---H bond activation

The photochemistry of the diphosphino Pt(II) hydrides [LPtH₂] (L=(t-Bu)₂P(CH₂)₂P(t-Bu)₂ (7); L=(t-Bu)₂P(CH₂)₃P(t-Bu)₂ (8);L=(t-Bu)(Ph)P(CH₂)₂P(Ph)(t-Bu) (9)) is reported. The primary photoevent is the dissociation of H₂ and formation of the 14-e [LPt] species. These coordinatively unsaturated intermediates provide a versatile entry point into the C---H bond activation of hydrocarbons. [LPt] reacts with benzene in an oxidative addition reaction to yield [LPt(H)(C₆H₅)] complexes. The importance of the metal centre and ancillary ligation in the C---H bond activation is discussed.     

Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors

Protease inhibitors have been proposed as potential defense molecules for increased insect resistance in crop plants. Compensatory over-production of insensitive proteases in the insect, however, has limited suitability of these proteins in plant protection, with very high levels of inhibitor required for increased plant resistance. In this study we have examined whether combined used of two inhibitors is effective to prevent this compensatory response. We show that leaf-specific over-expression of the potato PI-II and carboxypeptidase inhibitors (PCI) results in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in homozygote tomato lines expressing high levels (#62;1 the total soluble proteins) of the transgenes. Leaf damage in hemizygous lines for these transformants was, however, more severe than in the controls, thus evidencing a compensation response of the larvae to the lower PI concentrations in these plants. Development of comparable adaptive responses in both insects suggests that insect adaptation does not entail specific recognition of the transgene, but rather represents a general adaptive mechanism triggered in response to the nutritional stress imposed by sub-lethal concentrations of the inhibitors. Combined expression of defense genes with different mechanisms of action rather than combinations of inhibitors may then offer a better strategy in pest management as it should be more effective in overcoming this general adaptive response in the insect.