Effects of Sublethal Concentrations of Insecticides on the Fecundity of Sogatella furcifera (Hemiptera: Delphacidae) via the Regulation of Vitellogenin and Its Receptor.

White-backed planthopper (Sogatella furcifera, Hemiptera: Delphacidae) is an important migratory pest of rice. It causes severe economic losses by reducing crop production. Vg and VgR are important proteins that help in the successful reproduction of insects and have been studied in many insects. To understand the molecular mechanisms underlying the effects of insecticides on white-backed planthopper reproduction, we studied the expression profiles of SfVg, SfVg-like, and SfVgR in white-backed planthopper exposed to insecticides. SfVg and SfVgR silencing inhibited the ovarian development, number of eggs laid by, and hatching rate of white-backed planthopper. Thiamethoxam LC10 significantly inhibited SfVg-like and SfVgR expression. In contrast, triazophos LC25 significantly promoted SfVg, SfVg-like, and SfVgR expression and increased vitellogenin content in white-backed planthopper. These results demonstrate that insecticides can regulate the reproduction of white-backed planthopper by altering the expression of SfVg and SfVgR, thereby affecting the population density of white-backed planthopper. These findings build a foundation for improving our understanding of the molecular mechanisms underlying the effects of insecticides on the reproduction and resurgence of pests.

The use of serine protease from Yarrowia lipolytica yeast in the production of biopeptides from denatured egg white proteins.

Deriving non-conventional enzymes from cheaper sources than those used for commercially available enzymes may result in the production of hydrolysates with beneficial features, while drastically reducing the cost of hydrolysis. This is especially significant for enzymatic hydrolysis as a method of protein waste utilization. We have previously described the ability of non-commercial serine protease from Yarrowia lipolytica yeast to produce/release bioactive peptides from egg white protein by-products (EP). The enzymatic hydrolysis of EP was carried out for 24 h using the serine protease at an enzyme: substrate ratio of 1:30 (w/w). The obtained hydrolysate was characterized by protein degradation of 38% and also exhibited an antioxidant and cytokine-inducing activity. The isolation procedure (ultrafiltration and RP-HPLC) of bioactive peptides from the EP hydrolysate provided peptide fractions with significant antioxidant and ACE inhibitory activities. Three homogeneous and three heterogeneous peptide fractions were identified using MALDI-TOF/MS and the Mascot Search Results database. The peptides, mainly derived from ovalbumin, were composed of 2-19 amino-acid residues. We have thus demonstrated a novel ability of serine protease from Y. lipolytica to release biopeptides from an EP by-product.

Control of transcriptional repression of the vitellogenin receptor gene in largemouth bass (Micropterus salmoides) by select estrogen receptors isotypes.

The vitellogenin receptor (Vtgr) plays an important role in fish reproduction. This receptor functions to incorporate vitellogenin (Vtg), a macromolecule synthesized and released from the liver in the bloodstream, into oocytes where it is processed into yolk. Although studies have focused on the functional role of Vtgr in fish, the mechanistic control of this gene is still unexplored. Here we report the identification and analysis of the first piscine 5' regulatory region of the vtgr gene which was cloned from largemouth bass (Micropterus salmoides). Using this putative promoter sequence, we investigated a role for hormones, including insulin and 17ß-estradiol (E2), in transcriptional regulation through cell-based reporter assays. No effect of insulin was observed, however, E2 was able to repress transcriptional activity of the vtgr promoter through select estrogen receptor subtypes, Esr1 and Esr2a but not Esr2b. Electrophoretic mobility shift assay demonstrated that Esr1 likely interacts with the vtgr promoter region through half ERE and/or SP1 sites, in part. Finally we also show that ethinylestradiol (EE2), but not bisphenol-A (BPA), represses promoter activity similarly to E2. These results reveal for the first time that the Esr1 isoform may play an inhibitory role in the expression of LMB vtgr mRNA under the influence of E2, and potent estrogens such as EE2. In addition, this new evidence suggests that vtgr may be a target of select endocrine disrupting compounds through environmental exposures.

Impact of potassium bromate and potassium iodate in a pound cake system.

This study investigates the impact of the oxidants potassium bromate and potassium iodate (8, 16, 32, 64, and 128 micromol/g dry matter of egg white protein) on pound cake making. The impact of the oxidants on egg white characteristics was studied in a model system. Differential scanning calorimetry showed that the oxidants caused egg white to denature later. During heating in a rapid visco analyzer, the oxidants caused the free sulfhydryl (SH) group levels to decrease more intensively and over a smaller temperature range. The oxidants made the proteins more resistant to decreases in protein extractability in sodium dodecyl sulfate containing buffer during cake recipe mixing and less resistant to such decreases during cake baking. We assume that, during baking, the degree to which SH/disulfide exchange and SH oxidation can occur depends on the properties of the protein at the onset of the process. In our view, the prevention of extractability loss during mixing increased the availability of SH groups and caused more such loss during baking. During cooling, all cakes baked with added oxidants showed less collapse. On the basis of the presented data, we put forward that only those protein reactions that occur during baking contribute to the formation of a network that supports final cake structure and prevents collapse.

Proteomic analysis of indium embryotoxicity in cultured postimplantation rat embryos.

Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24h in the presence of 50 microM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

Previtellogenic oocyte growth and transcriptional changes of steroidogenic enzyme genes in immature female Atlantic cod (Gadus morhua L.) after exposure to the androgens 11-ketotestosterone and testosterone.

We have studied in vivo, the effects of physiological androgen (11-ketotestosterone: 11-KT and testosterone: T) concentrations on the growth of cod previtellogenic oocytes and steroidogenic gene expression patterns. Immature female Atlantic cod were injected three times (days 0, 7 and 14) with 0.05, 0.5 and 5 mg/kg of 11-KT and T. The control group was injected with the carrier solvent (ethanol diluted 1:10 in sunflower oil). Quantitative histological analyses demonstrated growth and development of previtellogenic oocytes after exposure to androgens. The oocyte developmental effect of androgens was more pronounced in fish receiving 11-KT. Quantitative PCR analysis demonstrated dose- and androgen-specific modulation of mRNA expression for genes involved in steroidogenesis (StAR (steroidogenic acute regulatory) protein, P450scc (P450-mediated cholesterol side-chain cleavage), 20beta-HSD (20beta-hydroxysteroid dehydrogenase)) and cell growth control, namely--opioid growth factor receptor (OGF-R), progesterone receptor protein p23 (PR23P) and apoptosis-inducing TAF9-like domain 1 (TAF9). Messenger RNA species associated with the zona pelucida, namely--the zona pellucida protein A domain (ZPA) and egg envelope glycoprotein (EeG) were modulated based on dose and androgen type. Cyclin-B mRNA expression was not affected by androgen exposure. Interestingly, we showed recently that these transcripts were responsive to in vitro androgen exposure in previtellogenic cod ovary. In conclusion, the present study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, suggesting androgen control of early oocyte growth in cod. The enhanced effects of 11-KT on oocyte growth support our hypothesis that non-aromatizable androgens play significant roles in the regulation of early previtellogenic oocyte growth and development.

Toward better understanding of salt-induced hen egg white protein aggregation using field-flow fractionation.

Field-flow fractionation techniques including sedimentation field-flow fractionation (SdFFF) and flow field-flow fractionation (FlFFF) were applied to investigate hen egg white protein aggregation. The thermally induced aggregation of hen egg white protein was observed at temperatures of 60 degrees C and higher. Particle size and size distribution of hen egg white protein aggregates were characterized by SdFFF to investigate parameters affecting ZnCl 2-induced aggregation of hen egg white protein. At a fixed concentration of 1.0 M ZnCl 2 and an incubation time of 15 min, the mean particle diameters of the aggregates were determined to be 0.43, 0.67, and 0.80 mum for hen egg white protein contents of 5, 6.25, and 7.5% (w/v), respectively. With the incubation time of 15 min, increasing the concentration of ZnCl 2 from 0.5 to 1.0 and to 1.5 M caused the mean particle diameter of the aggregates to grow from 0.37 to 0.42 and to 0.68 mum, respectively at 5% (w/v) hen egg white protein. Upon prolonged contact time, larger aggregates were formed. Furthermore, FlFFF was employed as a novel approach to determine the efficiency of protein utilization for aggregation. The pH values as well as ZnCl 2 and protein concentrations influenced the efficiency of protein utilization for aggregation. With the optimum condition, that is, a protein concentration higher than 2% (w/v) and a pH greater than 5, the efficiency of protein utilization was approximately 65%.

Impact of a treatment with phospholipase A2 on the physicochemical properties of hen egg yolk.

Changes in physicochemical properties of egg yolk were investigated after a treatment with phospholipase A 2 (PLA 2), where phospholipids are converted in lyso-phospholipids. Protein solubility and protein denaturation before and after modification by PLA 2 was monitored as well as the functionality of egg yolk by means of interfacial tension. Enzymatic treatment showed a significant impact on the properties of egg yolk with regard to protein solubility and denaturation behavior. To gain a closer insight, egg yolk was separated in its water-soluble fraction called plasma and the insoluble granules. Both fractions were separately modified by PLA 2. The granule fraction shows a higher protein solubility, and the plasma proteins show very high heat stability after modification by PLA 2. Hypotheses regarding related changes in the low-density lipoprotein (LDL) particles are discussed. Results suggest that significant differences in the functional properties of untreated and PLA 2-modified egg yolk do not primarily result from the existence of lyso-phospholipids but from structural changes in egg yolk granules and LDL particles.

Proteomic analysis of selenium embryotoxicity in cultured postimplantation rat embryos.

BACKGROUND: Developmental toxicity of selenium (Se) is a nutritional, environmental and medicinal concern. Here, we investigated Se embryotoxicity by proteomic analysis of cultured rat embryos. METHODS: Rat embryos at day 9.5 or 10.5 of gestation were cultured for 48 or 24 h, respectively, in the presence of sodium selenate (100 or 150 microM) or sodium selenite (20 or 30 microM). Proteins from the embryo proper and yolk sac membrane were analyzed by two-dimensional electrophoresis for quantitative changes from those in control embryos. Proteins with quantitative changes were identified by mass spectrometric analysis. RESULTS: Growth inhibition and morphological abnormalities of cultured embryos were observed in all the Se treatment groups. By the analysis of the embryo proper, actin-binding proteins were identified as proteins with quantitative changes by selenate: increased phosphorylated-cofilin 1, increased phosphorylated-destrin, decreased drebrin E, and decreased myosin light polypeptide 3. Many proteins showed similar changes between selenate and selenite, including increased ATP-synthase, decreased acidic ribosomal phosphoprotein P0, and decreased pyrroline-5-carboxylate reductase-like. In the yolk sac membrane, antioxidant proteins were identified for protein spots with quantitative changes by selenite: increased peroxiredoxin 1 and increased glutathione S-transferase. CONCLUSION: The identified proteins with quantitative changes by selenate or selenite were considered to be candidate proteins involved in Se embryotoxicity: the actin-binding proteins for selenate embryotoxicity, proteins with the similar changes for the common Se embryotoxicity and antioxidant proteins for modification of Se embryotoxicity by redox-related treatments. These proteins may also be used as biomarkers in developmental toxicity studies.

Fibril formation of lysozyme upon interaction with sodium dodecyl sulfate at pH 9.2.

Fibril formation seems to be a general property of all proteins. Its occurrence in hen or human lysozyme depends on certain conditions, namely acidic pHs or the presence of some additives. This paper studies the interaction of lysozyme with sodium dodecyl sulfate (SDS) at pH 9.2, using UV-visible spectrophotometry, circular dichroism (CD) spectropolarimetry, electron microscopy (EM) and chemometry. Based on observations such as the strange increase in absorbance at 650nm (pH 9.2) and the presence of intermediates, it is assumed that lysozyme fibrils have been formed at pH 9.2 in the presence of SDS as an anionic surfactant. Thioflavin T emission fluorescence and an EM image confirmed this assumption. beta-cyclodextrin was then used as a turbidity inhibitor to establish its effect on the distribution of intermediates that participate in fibril formation.

Dimethyl sulfoxide is a potent modulator of estrogen receptor isoforms and xenoestrogen biomarker responses in primary culture of salmon hepatocytes.

Dimethyl sulfoxide (DMSO) has been frequently used as carrier solvent in toxicological experiments where the most compelling DMSO attributes are its exceptionally low toxicity and environmental impact. We were inspired by recent and consistent observations that ethanol and DMSO modulate endocrine-disruptor biomarker responses in both in vitro and in vivo studies in our laboratory, to take a critical evaluation of these effects. Quantitative (real-time) polymerase chain reaction (PCR) method with specific primer pairs was used in this study to measure DMSO-induced time-dependent modulation of estrogen receptor (ER) isoforms, vitellogenin (Vtg) and zona radiata-protein (Zr-protein) gene expression patterns in primary culture of salmon hepatocytes. In addition, immunochemical analysis, using indirect enzyme linked immunosorbent assay (ELISA) with monoclonal (Vtg) and polyclonal (Zr-proteins) antibodies was used to detect and measure Vtg and Zr-proteins secreted in culture media. Salmon hepatocytes were isolated by a two-step collagenase perfusion method and exposed to 0.1% or 10 microL/L of DMSO after 48 h pre-culture. Cells were harvested at 12, 24, 48 and 72 h after exposure and analysed for ERalpha, ERbeta, Vtg and Zr-protein gene expression using real-time PCR method. Media samples were collected at similar time-intervals for protein analysis. Our data show that DMSO-induced significant increase in ERalpha, ERbeta, Vtg and Zr-protein genes in a time-dependent manner. Indirect ELISA analysis showed a time-specific effect of DMSO. The use of DMSO as carrier solvent in fish endocrine disruption studies should be re-evaluated. We recommend more investigation, using other endocrine-disruptor biomarkers in order to validate the suitability of common carrier solvents used in toxicology with the aim of setting new maximum allowable concentrations. In particular, given the high sensitivity of genomic approaches in toxicology, these results may have serious consequences for the interpretation of biomarker responses.

Effects of elevated yolk androgens on perinatal begging behavior in yellow-legged gull (Larus michahellis) chicks.

Maternal hormones may represent an important pathway by which mothers can adaptively adjust offspring traits and performance to suit the prevailing environmental conditions. Earlier studies of birds have shown that egg androgens of maternal origin may enhance post-natal offspring 'begging' displays, functioning to solicit parental care. Here we investigate the effects of elevated egg androgen levels on the prenatal begging behavior of yellow-legged gull (Larus michahellis) chicks. At laying, we experimentally increased the concentration of yolk testosterone (T) within the natural range of variation, and, shortly before hatching, we compared the structural properties, rate, and loudness of vocalizations of embryos developing in T- and oil-injected (control) eggs. In addition, we compared the early post-hatch begging rate (measured as the pecking rate towards a dummy gull head) in chicks of the two experimental groups. We found that T embryos produced louder embryonic vocalizations than controls, whereas structural properties and the calling rate did not differ between T and control embryos. The post-hatch begging rate was unaffected by T treatment, but strongly decreased with increasing chick body mass, suggesting that intensity of the begging display was sensitive to chick state and may therefore reliably indicate the need of food in this species. Therefore, the results of this study show for the first time that prenatal T exposure modulates the quality of embryonic vocalizations, but are not in accordance with previous findings reporting increased post-hatching begging intensity following increased prenatal exposure to androgens.

Comparison of protein expression in plasma from nonylphenol and bisphenol A-exposed Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) by use of SELDI-TOF.

The overall objective of this study was to compare the expression of plasma proteins in juvenile cod and turbot after a 3 week exposure to two different chemicals known to be estrogenic: 4-nonylphenol (NP, 29 microg/L) and bisphenol A (BPA, 59 microg/L). ProteinChip) array technology in combination with surfaced enhanced laser desorption ionisation-time of flight (SELDI-TOF) was used to investigate general responses in plasma proteins. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) was used to analyse two specific biomarkers of estrogenic exposure, vitellogenin (Vtg) and zona radiata protein (Zrp) in plasma. Both methods revealed clear species specific responses. In cod, 67% of significantly altered proteins showed the same response (up or down regulated) in NP and BPA exposed animals (males and females combined). The rest were either specific to NP (10%), BPA (19%) or they showed opposite responses to the two chemicals (4%). In contrast, only 20% of significantly altered proteins were common for NP and BPA exposed turbot: 60% were altered only in NP and 17% only in BPA. Furthermore, in BPA exposed cod, 77% of the responses were common for male and females, whereas turbot showed only 21% similarity for the two genders. However, NP exposed male and female turbot showed 88% similarity in responses. As gender was not determined in NP exposed cod, gender specific responses could not be determined. ELISA results supported that cod responded clearly to both chemicals as a large increase was observed in Vtg and Zrp levels. Turbot responded strongly to NP, but seemed only slightly affected by BPA. Overall, the results indicated that cod are more sensitive or respond with less specificity to estrogenic chemicals than turbot. The relatively large degree of common responses in NP and BPA exposed cod may indicate that in cod BPA have similar mode of action as NP. Generally, the results show the potential of SELDI-TOF as a tool for comparing multiple responses, and for identifying exposure as well as gender specific responses.

Induction of hepatic choriogenin mRNA expression in male marine medaka: a highly sensitive biomarker for environmental estrogens.

Teleost choriogenins, precursors of the inner layer subunits of egg envelope, have been recently introduced as sensitive biomarkers for exposure to estrogenic compounds. In this study, two full-length cDNAs-ojChgH and ojChgL which encode the choriogenin H and L forms, respectively, were cloned from the marine medaka, Oryzias javanicus. The deduced protein sequences of ojChgH and ojChgL are highly similar to the corresponding homologues in the freshwater medaka (O. latipes) with identities of 77.2 and 87.6%, respectively. Phylogenetic analysis indicated that ojChgH and ojChgL are members of two different classes of liver-specific ZP-domain containing proteins (ZPB and ZPC, respectively). Computer analysis of ca. 2 kb of the 5'-flanking sequences of ojChgH and ojChgL revealed that both genes contain a number of putative estrogen response elements (EREs) and/or half-site EREs. In vivo mRNA expression patterns of the genes were examined by quantitative real-time RT-PCR. ojChgH is expressed exclusively in the liver while ojChgL is co-expressed in the liver (major) and ovary (minor). Exposure of fish to waterborne 17beta-estradiol (E2) at environmentally relevant concentrations (1, 5, 10 and 100 ng/L) resulted in dose-dependent induction of both genes in the liver, with higher sensitivity and magnitude of induction in males than in females. In the male liver, induction of ojChgH is more sensitive to E2 than that of ojChgL and two other estrogen-responsive genes, estrogen receptor alpha (ojERalpha) and vitellogenin (ojVTG). The lowest-observed-effect concentration (LOEC) of E2 on induction of hepatic ojChgH mRNA is 1 ng/L. In the ovary, expression of ojChgL is non-responsive to E2 treatment. In conclusion, the present study suggested that induction of hepatic ojChgH mRNA in male fish may be a highly sensitive biomarker for exposure to environmental estrogens.

Transcriptional modulation of brain and hepatic estrogen receptor and P450arom isotypes in juvenile Atlantic salmon (Salmo salar) after waterborne exposure to the xenoestrogen, 4-nonylphenol.

The molecular basis for vitellogenin (Vtg) and Zr-protein gene (and protein) expression shows that the Vtg and Zr-protein gene activations are receptor-mediated responses that are ligand structure-dependent interactions with ER, probably involving all isoforms, in addition to other co-activators. In addition to their effect as direct agonist to the ERs, the endocrine-disrupting effects of environmental chemicals are sometimes interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of the male and female reproductive tracts. In vertebrates, the cytochrome P450 aromatase (P450arom) is a crucial steroidogenic enzyme catalyzing the final step in the conversion of androgens to estrogens. The present study was undertaken to investigate the effects of nonylphenol on brain and liver ER and P450arom isotypes gene expressions as sensitive biomarker of effect. Fish were exposed to static and continuous waterborne nonylphenol at 5, 15 and 50 microg/L. Blood, brain and liver samples were collected after 0 (control), 3 and 7 days post-exposure. Plasma levels of Vtg and Zr-proteins were analyzed using immunoblotting and enzyme linked immunosorbent assay (ELISA) methods. A quantitative RT-PCR method was used to analyse gene expression patterns using cloned plasmid containing amplicons of interest as standards. Our data demonstrate that both ER and P450arom gene isotypes showed differential organ-specific, nonylphenol concentration- and time-dependent expression patterns after exposure to environmental relevant concentrations of nonylphenol. Liver and plasma Vtg and Zr-protein gene and protein expression showed a concentration- and time-dependent induction at day 3 and 7 post-exposure. This is the first study evaluating, in parallel, the ERs, P450aroms and protein xenoestrogen biomarkers of effect in any fish species or lower vertebrate. Therefore, the present study shows that the brain and hepatic ER and P450arom isotypes evaluated using state-of-the-art molecular approaches are sensitive xenoestrogen biomarkers of effect. These responses should be evaluated in parallel with plasma protein biomarkers such as Vtg and Zr-proteins.

Anti-hyaluronidase oligosaccharide derived from chondroitin sulfate a effectively reduces polyspermy during in vitro fertilization of porcine oocytes.

The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 microg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1-100 microg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 microg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10-100 microg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25-30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 microg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 microg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.

Effect of lidocaine on ovalbumin and egg albumin foam stability.

Foam fractionation is a simple separation process that can remove and concentrate hydrophobic molecules such as proteins, surfactants, and organic wastes from an aqueous solution. Bovine serum albumin and ovalbumin have been widely used as model proteins due to their strong foaming potential and low price. Here, we study the effect of lidocaine on albumin foam, since drugs like lidocaine are known to bind with albumin. We observed that lidocaine not only enhances the amount of foam produced but also the stability of that foam as well. The foam stability was evaluated as the decay rate constant of the foam, determined from a change in height (or volume) of the foam over a given time period.

Tyrosyl-phosphorylated proteins are involved in regulation of meiosis in the rat egg.

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg.

Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes.

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

[The antiradical activity of coumarin reductones]. / Antiradikal'naia aktivnost' kumarinovykh reduktonov.

The antiradical activity of coumarine reductones was investigated by the method of inhibition of Fe2+ induced chemiluminescence of egg-yolk lipoproteins. All coumarines studied exhibited high antioxidant activity. The dependence of chemiluminescence intensity on the antioxidant concentration shows that coumarines reductions resemble their chemical analog--ascorbic acid rather than the lipid antioxidant butilated hydroxitoluene (ionol).