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The aim of the experiment was to evaluate the potential use of citric acid as a modifier of quality changes in table eggs during their storage. About 780 table hen eggs were collected on the same day. They were numbered individually and placed on trays 30 pcs on each. Control group (CA0) consisted of eggs unmodified with any additional substances. In experimental groups CA10 and CA15, eggshells were sprayed with the aqueous solution of citric acid (10 and 15% concentration, respectively). At the start of the experiment, only quality traits of eggs from the control group were analyzed. The remaining eggs were stored at 14°C and 70% RH (typical storage conditions). Their quality was evaluated after 7, 14, 21, and 28 d. The depth of the air cell, egg weight and specific gravity, traits of shell (permeability, strength, weight, thickness, density), and egg content (pH of yolk and albumen, Haugh units, yolk weight and color) were evaluated each time. The use of citric acid decreased the severity of qualitative changes. Citric acid-treated eggs demonstrated smaller weight loss, shallower air cell, higher structural albumen, less-intensive water diffusion from albumen to yolk indicating the improved resistance of the vitelline membrane. Owing to the fact that citric acid is accepted and recognized as a safe food preservative is a relatively cheap and available substance, it seems that it can be used to inhibit quality changes in table eggs during their storage., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Stereotyped left-right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the ascidian, Halocynthia roretzi. Neurula embryos rotate along the anterior-posterior axis in a counterclockwise direction, and the rotation stops when the left side of the embryo is oriented downwards, resulting in contact of the left-side epidermis with the vitelline membrane at the bottom of perivitelline space. Then, such contact induces the expression of nodal and its downstream Pitx2 gene in the left-side epidermis. Vitelline membrane is required for the promotion of nodal expression. Here, we showed that a chemical signal from the vitelline membrane promotes nodal gene expression, but mechanical stimulus at the point of contact is unnecessary since the treatment of devitellinated neurulae with an extract of the vitelline membrane promoted nodal expression on both sides. The signal molecules are already present in the vitelline membranes of unfertilized eggs. These signal molecules are proteins but not sugars. Specific fractions in gel filtration chromatography had the nodal promoting activity. By mass spectrometry, we selected 48 candidate proteins. Proteins that contain both a zona pellucida (ZP) domain and epidermal growth factor (EGF) repeats were enriched in the candidates of the nodal inducing molecules. Six of the ZP proteins had multiple EGF repeats that are only found in ascidian ZP proteins. These were considered to be the most viable candidates of the nodal-inducing molecules. Signal molecules are anchored to the entire vitelline membrane, and contact sites of signal-receiving cells are spatially and mechanically controlled by the neurula rotation. In this context, ascidians are unusual with respect to mechanisms for specification of the left-right axis. By suppressing formation of epidermis monocilia, we also showed that epidermal cilia drive the neurula rotation but are dispensable for sensing the signal from the vitelline membrane., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, we aimed to evaluate the effect of canthaxanthin (CX) and iodine (I) on the production of laying hens, on counteracting debilitation of the vitelline membrane, and on inhibiting Salmonella growth in eggs stored at 30°C. Three hundred hens were reared in cages. Birds were divided into six feeding groups (10 hens × 5 repetitions) that were administered 0, 3 or 6 ppm of CX and 1 or 10 ppm of I with their diets. Laying rate, egg weights, and feed conversion ratios were controlled. The quality of fresh eggs was assessed in wks 25-26, 48-50 and 62-63 of hens lives. An additional batch of eggs was incubated at the temperature of 30°C, and egg quality changes were monitored on days 3, 6 and 9 of storage. Additionally, eggs collected from four experimental groups of hens whose diets had been iodated with 1 or 10 ppm of I and supplemented with 0 or 6 ppm of CX were infected under laboratory conditions with Salmonella, and incubated for 5 and 10 d. The laying rate, egg weights, and feed conversion ratio were significantly improved. Dietary inclusion of CX contributed to a higher resistance of the vitelline membrane of egg yolks, but only for fresh eggs. Vitelline membrane degradation during egg storage at 30°C was significantly counteracted by dietary inclusion of I at a dose of 10 ppm. The same I dose resulted in the complete inhibition of Salmonella growth until day 10 of incubation, but exclusively for eggs collected from 40-week-old hens. Dietary supplementation with 10 ppm of I was found to impart high level of resistance to the vitelline membrane against the growth of Salmonella in case of eggs stored at 30°C; therefore, I was found to be more beneficial by ensuring longer preservation than that of CX. However, dietary supplementation with CX was found to increase the resistance of vitelline membrane in fresh eggs.

An in vitro study has been carried out to monitor changes to the female reproductive system in adult triclabendazole (TCBZ)-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh ("Mirazid"). Flukes were immersed for 6 h and 24 h in myrrh extract at a concentration of 200 µg/ml, then processed for histological and transmission electron microscope (TEM) examination of the uterus, Mehlis' gland, ovary and vitellaria. Egg production had become abnormal at 6 h post-treatment (pt), with the uterine lumen being filled with free vitelline cells and masses of shell protein material; few eggs were present. At 24 h pt, no eggs were present. Distinct changes to the ovary and Mehlis' gland were only observed after 24 h incubation in Mirazid. The ovary contained numbers of apoptotic oogonia and oocytes. In the Mehlis' gland, the S1 cells were disorganised and the processes from them were vacuolated, although the disruption was not significant. More severe changes were observed in the vitelline cells and follicles. After 6 h incubation in Mirazid, although the gross organisation of the vitelline follicles appeared to be normal, nuclear changes indicative of the early stages of apoptosis were observed in the stem cells and shell protein production by the mature cells had decreased. At 24 h pt, a distinct shift in cell population was evident, with the follicles containing mainly mature cells and spaces were present between the cells. The shell globule clusters in the mature cells were disorganised. In more severely-affected follicles, cells were seen to be breaking down, with karyolytic nuclei and disintegrating cytoplasm. Overall, the results have shown that exposure to Mirazid treatment had a severe impact on egg production by TCBZ-resistant flukes, an effect that was mediated by disruption of the vitelline cells and of the mechanism co-ordinating egg formation in the ootype.

Background: Triamcinolone is a long acting corticosteroid used in the treatment of arthritis, eczema, psoriasis and similar conditions which cause inflammation. Triamcinolone has half-life of 88min. Prolonged oral use is associated with gastrointestinal adverse effects as peptic ulcer, abdominal distention and ulcerative esophagitis as described in various patents. Microemulgel offers advantage of better stability, better loading capacity and controlled release especially for drug with short half life., Objective: Objective of the present study was to optimize microemulgel based transdermal delivery of triamcinolone., Method: Saturated solubility of triamcinolone in various oils, surfactants and co-surfactants is estimated. Pseudo-ternary phase diagrams were constructed to determine the region of transparent microemulsion. Microemulsion was evaluated for globule size (FE-SEM, zetasizer), % transmittance, pH, viscosity, conductivity etc. Design of experiment was used to optimize microemulsion based gel. Carbopol 971P and HPMC K100M were used as independent variables. Microemulsion based gel was evaluated for in-vitro as well as ex-vivo parameters., Results: Microemulsion was formulated with oleic acid, lauroglycol FCC and propylene glycol. PDI 0.197 indicated microemulsion is mono-disperse. 32 factorial design gave batch F8 as optimized. Design expert suggested drug release; gel viscosity and bio-adhesive strength were three significant dependant factors affecting the transdermal delivery. F8 showed drug release 92.62.16±1.22% through egg membrane, 95.23±1.44% through goat skin after 8hr and Korsmeyer-Peppas release model was followed., Conclusion: It can be concluded that a stable, effective controlled release transdermal microemulgel was optimised for triamcinolone. This would be a promising tool to deliver triamcinolone with enhanced bioavailability and reduced dosing frequency., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Gd(3+) is increasingly used in EPR spectroscopy due to its increased intracellular stability and signal-to-noise ratios. Here we present the incorporation of Gd(3+)-DOTA into internal positions in DNA. Distance measurements via pulsed Electron Paramagnetic Resonance (EPR) spectroscopy in vitro and in cellula proved enhanced stability and efficiency compared to nitroxide labels.

The laying hen is the best model for oviduct growth and development. The chicken oviduct produces the egg components, including the egg white and eggshell. However, the mechanism of egg component production during oviduct development requires further investigation. Vitelline membrane outer layer protein 1 (VMO-1) is found in the outer layer of the vitelline membrane of avian eggs. Comparison of the chicken VMO-1 protein-coding sequence and the human, mouse, rat, and bovine VMO-1 proteins via multiple sequence alignment analysis revealed high degrees of homology of 55%, 53%, 48%, and 54%, respectively. Although the avian homologue of VMO-1 is highly expressed in the magnum of the oviduct, little is known about the transcriptional and posttranscriptional regulation of VMO-1 during oviduct development. The results of this study revealed that estrogen induces VMO-1 messenger RNA (mRNA) expression in oviduct cells in vitro. The expression of genes interacting with VMO-1 by RNA interference (RNAi) functional analysis revealed that ovomucin expression was decreased by VMO-1 silencing. In addition, gga-miR-1623, 1552-3p, and 1651-3p influenced VMO-1 expression via its 3'-UTR, suggesting the posttranscriptional regulation of VMO-1 expression in chickens. Collectively, these results suggest that VMO-1 is an estrogen-induced gene that is posttranscriptionally regulated by microRNAs (miRNAs). The present study may contribute to an understanding of egg component production during chicken oviduct development.

To compare the effects of three diets, rare minnow was fed with three diets from 30 dph to mature period. The activities of EROD, PROD, SOD and GST were measured in the WBHs as well as Vtg and TBARS concentrations at 60 dph. The rest fish were fed until adulthood for breeding studies. The group A served as the control group. It was found that Vtg, GST and EROD were significantly increased in the groups B and C, but SOD, TBARS and PROD levels were significantly increased only in the group C. In the adulthood, Vtg was significantly induced in the males in the group C. In generation F1, inhibition of CAT D activities and decrease of reproductive success were only found in pellet A group, but not in pellet B group. These findings indicate that the selection of diet is extremely important to assure veracity of the experiment results., (Copyright © 2010. Published by Elsevier B.V.)

Plasma peptides previously associated with exposure of juvenile male rainbow trout (Oncorhynchus mykiss) to the hormone 17β-estradiol (E2) were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Specifically, plasma peptides of interest were fractionated and subsequently identified via spectra obtained by MALDI QqTOF MS/MS and LC-MALDI TOFTOF MS/MS analysis, de novo sequencing and database matching. The two peptide masses were identified as significant matches for fragments of the C-terminal propeptides from rainbow trout vitelline envelope protein (VEP)α and VEPγ isoforms. Our findings document the presence of the C-terminal propeptides from rainbow trout VEPα and VEPγ proteins in the bloodstream of juvenile male rainbow trout exposed to E2 via MALDI-TOF-MS detection. We provide three possible explanations for the presence of C-terminal propeptides in the bloodstream, as well as compare previously obtained hepatic transcriptomic results with the plasma proteomic results obtained in the present study., (Published 2010 Wiley-Liss, Inc.)

To improve toxoid preparation, the effects of selective heat denaturation were assessed on Deinagkistrodon acutus venom. The venom and its fractions (peak 1 and peak 2 separated by gel filtration chromatography) were heated to various temperatures (45-70 degrees C) for 30 min, after which protein concentration, immunoreactivity, lethality, myotoxicity and hemorrhagic and membrane lysis activities of the samples were determined. In addition, the synergistic effects of the venom fractions were evaluated by separate or simultaneous intramuscular injection in mice. The results showed that the peak 1 fraction consisted primarily of proteins in the range of 18 to 105 kDa, while the peak 2 fraction consisted primarily of proteins smaller than 21 kDa. The hemorrhagic activity, immunoreactivity, and protein concentration of heated samples were gradually reduced as the temperature increased from 25 degrees C to 70 degrees C. Bioactivities significantly decreased but immunoreactivity was retained when the crude venom, peak 1 fraction, or peak 2 fraction were heated to the critical temperatures of 60 degrees C, 55 degrees C, or 60 degrees C, respectively. Synergistic effects of two kinds of heated fractions were observed in toxicity and antibody production after the peak 1 and peak 2 injected simultaneously or respectively. The results suggest that venom fractions heated and injected separately could significantly reduce their toxicity and enhance the neutralization of antiserum induced by them., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The development of a species-specific protocol for dechorionation and permeabilization of insect eggs is a necessary prerequisite to cryopreserve the embryos. Here we tested different procedures based on heptane or the surfactant Tween 80 as an alternative to alkane, evaluating their efficacy and toxicity on the early (24 h post-oviposition) and late (75 h post-oviposition) stage embryos. Heptane efficiently permeabilized the eggs of G. mellonella but the hatching rate ranged from 0.1 to 4.2 percent in the early stage and from 4.3 to 11.2 percent in the late stage. The embryos treated with 1.25 percent NaOCl + 0.08 percent Tween 80 for 2 min showed the same shrinkage and reswelling percentages as eggs exposed to heptane for 10 sec, with a significantly higher hatching percentage in the early (68.2 +/- 1.5 percent) and late stages (22.4 +/- 3.7 percent). Thus, 0.08 percent Tween 80 allows sufficient permeabilization of G. mellonella embryos without the high toxicity of alkane.

Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed "egg water" (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca(2+) stores (induced by thapsigargin) promoted [Ca(2+)](i) rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca(2+) chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni(2+) and mibefradil prevented [Ca(2+)](i) rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca(2+) channel.

Ovarian microsomal aromatase (P450arom) activity was studied in relation to season and incubation of follicles with catecholestrogens [(2-hydroxyestradiol-17beta (2-OHE2) and 2-methoxyestradiol-17 beta (2-methoxyE2)] using a product (estradiol-17 beta) assay. Peak P450arom activity was noticed in late preparatory phase (April) and it decreased significantly in pre-spawning, spawning and post-spawning phases to give the lowest value in resting phase. Apparent Km and Vmax of the enzyme varied significantly and the values were high in the preparatory (vitellogenic) phase (Km 74.62+/-1.73 nM, Vmax 0.81+/-0.01 pmol/mg protein/min) and low in the spawning (post-vitellogenic) phase (Km 62.01+/-1.68 nM, Vmax 0.69+/-0.002 pmol/mg protein/min). The incubation of the ovarian microsomes with 2-OHE2 elicited significant biphasic effects on enzyme activity. In the vitellogenic phase, concentrations of the steroid up to 1 microM inhibited enzyme activity significantly with the highest inhibition at 10nM. However, in the post-vitellogenic ovary, the highest inhibition was registered at 100 nM. The higher concentrations (10 microM or 100 microM) did not elicit any significant change compared to the control groups. A comparison of the aromatase inhibition index (AI50, indicates 50% inhibition of aromatase activity) of fadrozole, a known aromatase inhibitor and 2-OHE2 shows that the AI50 was 4.4 nM for fadrozole and 0.864 nM (vitellogenic phase) and 1.31 nM (post-vitellogenic phase) for 2-OHE2 indicating higher potency of the latter. The incubation of the ovarian microsomes with 2-methoxyE2 increased enzyme activity only at the higher concentrations (1-100 microM). The results show seasonality in the potential of the ovary to synthesize E2 and the potent enzyme inhibiting activity of 2-OHE2, which is reported for the first time.

Embryogenesis in vertebrates and marine invertebrates begins when a mature oocyte is fertilized, resulting in a rise in intracellular calcium (Ca(2+)) that activates development. Insect eggs activate without fertilization via an unknown signal imparted to the egg during ovulation or egg laying. One hypothesis for the activating signal is that deformation of eggs as they pass through a tight orifice provides a mechanical stimulus to trigger activation. Ovulation could produce two forms of mechanical stimulus: external pressure resulting from the passage of oocytes from the ovary into the narrow oviducts, and osmotic pressure caused by hydration-induced swelling of the oocyte within the oviducts. Ovulation could also trigger activation by placing the oocyte in a new environment that contains an activating substance, such as a particular ion. Here, we provide the first evidence that Drosophila oocytes require Ca(2+) for activation, and that activation can be triggered in vitro by mechanical stimuli, specifically osmotic and hydrostatic pressure. Our results suggest that activation in Drosophila is triggered by a mechanosensitive process that allows external Ca(2+) to enter the oocyte and drive the events of activation. This will allow exploitation of Drosophila genetics to dissect molecular pathways involving Ca(2+) and the activation of development.

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro., (2006 Wiley-Liss, Inc.)

Formation of the yolk sac vascular system and its connection to the embryonic circulation is crucial for embryo survival in both mammals and birds. Most mice with mutations in genes involved in vascular development die because of a failure to establish this circulatory loop. Surprisingly, formation of yolk sac arteries and veins has not been well described in the recent literature. Using time-lapse video-microscopy, we have studied arterial-venous differentiation in the yolk sac of chick embryos. Immediately after the onset of perfusion, the yolk sac exhibits a posterior arterial and an anterior venous pole, which are connected to each other by cis-cis endothelial interactions. To form the paired and interlaced arterial-venous pattern characteristic of mature yolk sac vessels, small caliber vessels of the arterial domain are selectively disconnected from the growing arterial tree and subsequently reconnected to the venous system, implying that endothelial plasticity is needed to fashion normal growth of veins. Arterial-venous differentiation and patterning are controlled by hemodynamic forces, as shown by flow manipulation and in situ hybridization with arterial markers ephrinB2 and neuropilin 1, which show that expression of both mRNAs is not genetically determined but plastic and regulated by flow. In vivo application of ephrinB2 or EphB4 in the developing yolk sac failed to produce any morphological effects. By contrast, ephrinB2 and EphB4 application in the allantois of older embryos resulted in the rapid formation of arterial-venous shunts. In conclusion, we show that flow shapes the global patterning of the arterial tree and regulates the activation of the arterial markers ephrinB2 and neuropilin 1.

The lectin Concanavalin A (ConA) has long been known to potentiate current responses of native and recombinant ionotropic glutamate receptors (iGluRs), apparently by inhibition of receptor desensitization. We compared the effects of a broad range of lectins with different carbohydrate specificities on recombinant AMPA (GluR1) and kainate receptors (GluR6) expressed in Xenopus oocytes. Interestingly, the extent of inhibition of desensitization appears to depend on the sugar preference of lectins at kainate (KA) receptors, but not at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. None of the lectins potentiated current responses at non-glycosylated GluRs produced in tunicamycin-treated oocytes, demonstrating the requirement of lectin interaction with carbohydrate moieties of the receptors. At AMPA receptors, potentiation of current responses afforded by ConA and the well-known inhibitor of desensitization, cyclothiazide (CTZ), are additive, suggesting that the lectin and CTZ act independently. Current amplitudes of GluR1(L479Y), a nondesensitizing mutant, however, could not be further increased by ConA.

Self-sterility of solitary ascidians is a typical example of the allogeneic recognition, though its molecular mechanism remains an open question. In this paper we analyze the fertility between siblings from selfed and crossed eggs to understand the genetic basis of self-sterility in the ascidian, Ciona intestinalis. First, we show that the self-sterility is strict and stable, and the individuality expressed in gametes is highly diversified in the wild population that we used. Secondly, we show one-way cross-sterility and reciprocal cross-sterility within the siblings that are self-sterile but fertile with non-siblings. Thirdly, we show self-sterility and cross-sterility share some natures and both are closely related to the sperm capacity not to bind to the vitelline coat of the autologous eggs or the eggs sterile to the sperm concerned. In all, this paper shows that the self-sterility is genetically governed by a multiple-locus system, and that most probably individual-specific determinants are haploid expression in sperm and diploid expression in eggs, given they recognize self but not non-self.

Dietary conjugated linoleic acid (CLA) decreases yolk 18:1(n-9), induces chick embryonic mortality and alters egg quality. A study was conducted to determine whether olive oil would prevent these adverse effects of CLA. Hens (15 per treatment) were fed diets containing 0.5 g corn oil/100 g (CO), 0.5 g CLA/100 g (CLA), 0.5 g corn oil plus 10 g olive oil/100 g (CO + OO) or 0.5 g CLA plus 10 g olive oil/100 g (CLA + OO). After 74 d of feeding, hens were placed on CO for 10 d. Hens were artificially inseminated weekly. For hatchability studies, fertile eggs were collected daily, stored at 15 degrees C for 24 h and then incubated. After 6 d of feeding, embryonic mortality rates were 15, 100, 8 and 16% in the CO, CLA, CO + OO and CLA + OO groups, respectively. When CLA-fed hens were fed the CO diet, hatchability improved to that of the CO group within 7 d. For fatty acid analysis, three eggs were obtained at the 7 d of feeding. Relative CLA levels of yolk from CO-, CLA-, CO + OO- and CLA + OO-fed hens were 0.11 +/- 0.01, 1.91 +/- 0.16, 0.08 +/- 0.04 and 0.69 +/- 0.07 g/100 g fatty acids, respectively. The ratios of 16:0/16:1(n-7) and 18:0/18:1(n-9) of yolk from CLA-fed hens were approximately 1- and approximately 1.5-fold greater, respectively, compared with those fed CO. OO prevented CLA-induced increases in 16:0 and 18:0 and the decrease in 18:1(n-9) in yolk. Fertile eggs were stored at 4 degrees C for 2 or 10 wk and analyzed for pH or mineral levels. Dietary CLA caused abnormal pH changes of albumen and yolk when eggs were stored at 4 degrees C. The pH of yolk and albumen from CO-fed hens after 10 wk of storage was 6.12 +/- 0.12 and 9.06 +/- 0.03, respectively, versus 7.89 +/- 0.25 and 8.32 +/- 0.16, respectively, in eggs from CLA-fed hens. OO prevented CLA-induced abnormal changes in the pH of albumen and yolks. Eggs from CLA-fed hens had greater iron, calcium and zinc concentrations and lower magnesium, sodium and chloride concentrations in albumen relative to those from hens fed CO. OO prevented CLA-induced mineral exchange between yolk and albumen, presumably by reducing the yolk saturated fatty acids, which are believed to disrupt the vitelline membrane during cold storage. This study suggests that the adverse effects of CLA may be due to the increased level of saturated fatty acids. However, because the addition of olive oil also lowered egg CLA content, the direct role of egg CLA on egg hatchability and quality cannot be ruled out.

Oocyte activation is a critical component of the current animal cloning scheme. This study was designed to examine surface characteristics of bovine oocytes by scanning electron microscopy (SEM) after activation by calcium ionophore A23187 (A23187) and electric pulse combined with cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) treatments. In vitro matured (IVM) oocytes were activated then harvested at 0 to 19 hours after the onset of treatments for SEM processing. The zona pellucida (ZP) of untreated IVM oocytes exhibited an open mesh structure. The ZP surface showed little changes after A23187 alone, but dramatically changed to a less porous surface 3 hours after combined treatments with CHX or 6-DMAP. The vitelline membrane of IVM oocytes was covered with well-developed microvilli (MV). The MV became shorter (0.83 vs. 1.35 microm, p < 0.01) 8 hours after A23187 treatment alone. The vitelline membrane was altered in all oocytes examined 3 hours after incubation with A23187 plus CHX or 6-DMAP. A 1.5-fold increase in the diameter of MV in the CHX group and a higher incidence of large cytoplasmic protrusions (more than 1 microm width) in the 6-DMAP group were observed. After removal of inhibitors, the surface morphologies of the ZP and vitelline membrane were returned nearly to those of untreated IVM oocytes in both groups. The present study clearly showed that surface characteristics of the bovine oocyte were more profoundly changed by a combination of agents for parthenogenetic stimulation, and that the ultrastructural effects were reversible.

The solitary ascidians Ciona intestinalis and Ciona savignyi co-occur in southern California harbors, but no hybrids have been recognized in nature. Numerous differences in their egg morphology were detected. Homologous (normal outcross) fertilization yielded 96-99% cleavage, where autologous (self) fertilization showed 3% and heterologous (hybrid) fertilization showed 0-1%. Acid treatment (pH 3.2) removed the block to selfing (P < 0.0001) but not hybridization for both species. Heterologous sperm bind to the vitelline coat (VC), but fail to penetrate. Enzymatic removal of the VC resulted in 91-97% cleavage with autologous and heterologous sperm (P < 0.0001). The vitelline coats of the two species differ in lectin binding to surface glycosides. Fertilization in both species is significantly inhibited by the lectins, fucose binding protein (P < 0.0001) and concanavalin A (P < 0.0001), and wheat germ agglutinin inhibits fertilization in C. intestinalis (P < 0.0001) but is without effect on C. savignyi fertilization. Self and hybrid blocks employ different mechanisms including glycoside composition and acid sensitivity., (Copyright 2000 Wiley-Liss, Inc.)

Hallmark signs of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in rainbow trout sac fry, are yolk sac edema, hemorrhage, craniofacial malformation, and growth retardation culminating in mortality. Our objective was to determine the role of cardiovascular dysfunction in the development of this toxicity. An embryotoxic TCDD dose (385 pg/g egg) caused a progressive reduction in blood flow in rainbow trout sac fry manifested first and most dramatically in the 1st and 2nd branchial arches and vessels perfusing the lower jaw. Blood flow was reduced later in the infraorbital artery and occipital vein of the head as well as segmental vessels and caudal vein of the trunk. Reduced perfusion occurred last in gill branchial arteries involved with oxygen uptake and the subintestinal vein and vitelline vein involved with nutrient uptake. Although heart rate throughout sac fry development was not affected, heart size at 50 days post-fertilization (dpf) was reduced far more than body weight or length, suggesting that the progressive circulatory failure caused by TCDD is associated with reduced cardiac output. Craniofacial development was arrested near hatch, giving rise to craniofacial malformations in which the jaws and anterior nasal structures were underdeveloped. Unlike the medaka embryo, in which TCDD causes apoptosis in the medial yolk vein, endothelial cell death was not observed in rainbow trout sac fry. These findings suggest a primary role for arrested heart development and reduced perfusion of tissues with blood in the early-life stage toxicity of TCDD in trout.

Aim: To observe the ultrastructural changes in the body wall and the vitelline cells of Paragonimus westermani in vitro and in vivo before and after triclabendazole treatment., Methods: The worms were obtained from in vitro and in vivo tests. All of the samples were processed by conventional techniques, and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM)., Results: The external plasma membrane and matrix were cracked or disappeared after the treatment. The necrosis of the muscular layer differed. The cell membranes of cortex and vitelline cells were damaged. Nuclear membrane was damaged partially, heterochromatin solidified and condensed to brim and dissolved. The Golgi complex disappeared, endoplasmic reticulum expanded, mitochodria denatured and dissolved. The damage was more serious in vivo than in vitro., Conclusion: Triclabendazole is remarkablely effective against Paragonimus westermani by damaging the body wall and vitelline cells, mainly affecting the nuclei, membrane structures and microtubular system.

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6)M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate, (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

The surface of mature eggs of the freshwater oligochaete Tubifex exhibits numerous microvilli. Upon activation, microvilli become narrower at their base and separated from the ooplasmic surface. Here it is shown that concanavalin A (Con A) reversibly inhibits the separation of microvilli from activated Tubifex eggs. The Con A-treated eggs undergo meioses and mitoses at a normal rate. Microvilli on these eggs change their length in a meiotic cycle-dependent manner; their core bundles of microfilaments elongate significantly during the second meiosis. The Con A-treated eggs fail to complete polar body formation, ooplasmic segregation and cleavages. Treatment with Con A of eggs that have accomplished microvillar separation does not exert any inhibitory effect on their development. Succinyl-Con A, a dimeric derivative of Con A, does not prevent microvillar separation, suggesting that the tetravalent form of Con A is essential for Con A to exert its inhibitory effect on microvillar separation.

Agarose beads derivatized with amino acids, peptides, carbohydrates and lectins were used to systematically determine what types of molecules, isolated from all others, can make adhesive bonds strong enough to hold cell-like beads together. The results indicated that strong adhesion occurred when at least one of the two members of certain bead pairs was derivatized with molecules that were dimers or trimers but not monomers. Also, beads derivatized with phosphorylated amino acids, but not their non-phosphorylated counterparts, adhered to beads derivatized with positively charged peptides. Adhesion was sensitive to ionic strength and pH of the medium. It was proposed that adhesion occurred between the phosphate groups of the phosphoamino acids and amino and guanidinium groups of the peptides. Cooperative bonding can explain the stability of the adhesion observed in this system. Information gained from the bead modeling work was used to design experiments to examine the role of phosphorylated molecules in modulating adhesion in sea urchin systems. Phosphoamino acids inhibited sperm-egg interaction, but not reaggregation of blastula cells. Inhibitors of alkaline phosphatase, however, did inhibit reaggregation. The results suggest that cell surface phosphorylated molecules may modulate cellular adhesiveness, in some systems promoting, while in others inhibiting adhesion.

Maturation of the starfish oocyte cortex to produce an effective cortical granule reaction and fertilisation envelope is believed to develop in three phases: (1) pre-methyladenine (1-MA) stimulation; (2) post-1-MA stimulation, pregerminal vesicle breakdown; and (3) post-germinal vesicle breakdown. The present study was initiated to identify what each of these phases may encompass, specifically with respect to structures associated with the oocyte cortex, including cortical granules, microvilli and vitelline layer. 1-MA treatment brought about an orientation of cortical granules such that they became positioned perpendicular to the oocyte surface, and an approximately 4-fold decrease in microvillar length. A-23187 activation of immature oocytes treated with (10 min; pregerminal vesicle breakdown) or without 1-MA resulted in a reduction in cortical granule number of 21% and 41%, respectively (mature oocytes underwent a 96% reduction in cortical granules). Elevation of the fertilisation envelope in both cases was significantly retarded compared with activated mature oocytes. In activated mature oocytes, the vitelline layer elevated 20.0 +/- 5.4 mu m from the egg's surface, whereas in immature oocytes treated with just A-23187 or with 1-MA (10 min) and A-23187, it lifted 0.35 +/- 0.1 and 0.17 +/- 0.04 mu m, respectively. The fertilisation envelopes of activated (or fertilised) immature oocytes also differed morphologically from those of mature oocytes. In activated, immature oocytes, the fertilisation envelope was not uniform in its thickness and possessed thick and thin regions as well as fenestrations. Additionally, it lacked a complete electron-dense stratum that characterised the fertilisation envelopes of mature oocytes. The nascent perivitelline space of immature oocytes was also distinguished by the presence of numerous vesicles which appeared to be derived from microvilli. Differences in the morphology of cortices from activated (fertilised) and non-activated, immature and mature oocytes substantiate previous investigations demonstrating three phases of cortical maturation, and are consistent with physiological changes that occur during oocyte maturation, involving ionic conductance of the plasma membrane, establishment of slow and fast blocks to polyspermy and elevation of a fertilisation envelope.

In Drosophila developmental biological studies, X-gal staining is commonly employed to study the spatio-temporal expression of the lacZ reporter gene in the transformed flies or their embryos. Study of the lacZ pattern in embryos often suffers from the lack of an efficient and high yielding technique for devitellinization of X-gal stained embryos. Devitellinization techniques employed during antibody staining, in situ hybridization or embryonic cuticular preparations generally do not give satisfactory results when used for similar purpose in X-gal stained embryos. This results in the flaky appearance of the blue stain. We present here an improved chemical devitellinization technique which gives a high yield of devitellinized embryos and a better resolution of the X-gal staining pattern.

The organic solvent octane has been used routinely to permeabilize the hydrophobic vitelline membrane surrounding the Drosophila embryo, thereby allowing the movement of small molecules into the egg. We present evidence that hexane is a more effective permeabilizing agent than octane and compare the effects of these solvents on uniformity of permeabilization and embryonic viability. The ability of each solvent to make the embryo accessible to a range of biological stains was compared. The effect of octane versus hexane permeabilization on subsequent embryonic viability was measured at seven different stages during early embryogenesis. We found that although hexane is a superior solvent for permeabilizing the vitelline membrane, it decreases the viability of embryos exposed between 0 and 3 hr of age. Older embryos treated with either hexane or octane are usually viable. We also showed that molecules with a molecular mass of 984 Daltons or more did not diffuse into the embryo following treatment with either hexane or octane. Results presented here challenge a phase-partition model that has been proposed previously to explain the molecular basis of permeabilization of the Drosophila egg. An alternative model is described as well as an optimized protocol for permeabilizing and staining Drosophila embryos at any stage during early embryogenesis while maintaining viability for subsequent culture.

Among various protease inhibitors, chymostatin (an inhibitor of sperm chymotrypsin-like protease) strongly inhibited the binding of sperm to the vitelline coat of glycerinated eggs of the ascidian Halocynthia roretzi, whereas leupeptin (an inhibitor for sperm acrosin), antipain, and soybean trypsin inhibitor had no significant inhibitory effects. Dansyl-Val-Pro-argininal (an inhibitor of the sperm trypsin-like protease, spermosin) had an inhibitory effect on the binding of sperm that was much smaller than its effects on fertilization. Since the sperm chymotrypsin-like protease that is involved in ascidian fertilization has been identified as a proteasome (multicatalytic proteinase complex), we tested the effects of several peptidyl argininals, inhibitors of the activities of proteasomes, on this binding process. The ranking of the inhibitory effects of these compounds on the binding of sperm was the same as that of their effects on the chymotrypsin-like activity of the proteasome, reported previously. The potent inhibitors of binding used in these studies had no or minimal effects on sperm motility. These results suggest that a sperm chymotrypsin-like protease (most probably the chymotrypsin-like protease in the proteasome) plays a key role in binding of sperm to the vitelline coat of the ascidian egg.

Botulinum exoenzyme C3 ADP-ribosylates a 23 kDa protein of unfertilized eggs of the ascidian, Halocynthia roretzi. Microinjection of C3 into the eggs induced elevation of the egg vitelline coat. Co-injection of heparin or EGTA with C3 inhibited the inducing effect of C3. The vitelline coat of eggs which had been previously co-injected with heparin and C3 was elevated by addition of calcium ionophore, but not by insemination. C3 also induced an increased formation of inositol 1,4,5-triphosphate (IP3) in ascidian egg membranes. Thus the ADP-ribosylation of small GTP-binding protein by C3 seems to be responsible for elevation of the vitelline coat of ascidian eggs through IP3 formation and intracellular calcium mobilization.

The neurohormone serotonin (5-hydroxytryptamine, 5HT) triggers meiosis reinitiation in prophase-arrested oocytes of Spisula solidissima. The original pharmacological profile of this response prompted us to examine whether it involved a novel type of 5HT receptor. In order to characterize these putative receptors, we performed [3H]5HT binding assays. Given the complexity of oocyte surface architecture, [3H]5HT-specific binding was measured in both plasma membrane and vitelline envelope fractions. Our data reveal the existence of a single class of 5HT-specific binding sites in each fraction. These binding sites appear distinct by their kinetic properties: 1) plasma membrane binding sites are of lower affinity but are more numerous than vitelline envelope sites; 2) [3H]5HT binding is more rapid in membrane fraction. The pharmacological profiles of both binding sites were defined in competition experiments by using sixteen 5HT-related and unrelated compounds. Both membrane and vitelline envelope sites displayed novel profiles. However, only the pharmacological profile determined for the plasma membrane sites corresponds to that observed for 5HT-induced meiosis reinitiation. These new 5HT5 binding sites are therefore the physiological receptors that mediate the effects of 5HT in Spisula oocytes. The putative role of vitelline envelope sites is discussed.

In the viviparous guppy, fertilization and gestation are intrafollicular. Fully developed embryos are ovulated at the end of gestation just prior to parturition. The metabolism in vitro of various radiolabeled steroid precursors by isolated ovarian follicles at various stages of the reproductive cycle and extrafollicular (EF) tissue of the guppy was investigated. While estradiol-17 beta was one of the end products of metabolism in vitellogenic follicles, 17 alpha, 20 beta-P and several 5-reduced metabolites were synthesized by postvitellogenic follicles. The yield of 17 alpha, 20 beta-P, however, was much lower than some 5 beta-reduced metabolites synthesized by postvitellogenic follicles. Gestation stage follicles rapidly converted the precursors into 5-reduced and polar 7-hydroxylated steroids, and their glucuronides. Although postpartum follicles showed very poor potential for steroid metabolism, they synthesized estradiol-17 beta from testosterone. These results demonstrate distinct changes occurring in the steroidogenic potential of the follicles during the reproductive cycle. Unlike in other viviparous vertebrates, no particular steroid seems to be involved in maintaining gestation in the guppy; all the steroid precursors are converted into highly polar metabolites and their conjugates during gestation, thereby facilitating their excretion. The EF ovarian tissue also synthesized 7-hydroxylated steroids and their glucuronides, providing evidence for the first time that the teleost ovarian EF tissue plays a role in steroidogenesis. The possible physiological significance of the synthesis of the novel polar steroids by the follicles and the EF tissue is discussed.

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.

Blocks to polyspermy may act either at the level of the egg plasma membrane to prevent gamete fusion or at the level of egg surface coats to prevent gamete attachment. The present study was undertaken to determine what type(s) of block(s) to polyspermy exist in Chaetopterus. The results showed the existence of both types. A rapid block acts at the plasma membrane level based on independence from detectable changes in the vitelline layer and is dependent on external sodium ions. A vitelline layer block had been predicted on morphological evidence and is supported here by demonstrating an increase in polyspermy following chemical disruption of the vitelline layer. However, the vitelline layer of the fertilized egg retained its ability to initiate the acrosome reaction in sperm and attach sperm which had undergone the acrosome reaction. The vitelline layer block resulted from the retraction of egg microvilli from the vitelline layer, and not from elevation of the vitelline layer per se. Thus the vitelline layer of the fertilized egg could be involved in preventing sperm penetration into the egg without being altered structurally or functionally.

Caissarone (mol. wt 229.5; melting point 285-290 degrees C) is a novel purine isolated and purified from the sea-anemone Bunodosoma caissarum. The purine inhibits the detachment of the vitelline layer from the sea-urchin egg plasma membrane after fertilization and this effect leads to polyspermy. Various abnormalities were detected at various embryonic stages, from multipolar egg division through unequal cleavages and exogastrulation up to teratogenic effects on the sea-urchin larvae (echinopluteus).

The analysis of sea urchin egg fertilization shows that several phenomena common to other biological systems are involved: cell recognition, cell fusion, exocytosis and initiation of mitotic activity. Both the role of calcium ions in cell fusion and exocytosis and the function of the cell surface in the initiation of mitotic activity appear to have general applicability. The study of fertilization can contribute to the elucidation of these processes and, reciprocally, progress in this field can help to advance our understanding of the mechanisms of fertilization in sea urchins and other organisms.

Injection of molecular probes into unfertilized Xenopus eggs requires suppression of activation. But the unfertilized egg is poised for activity, and pricking, like sperm penetration, triggers the start of the first cell cycle. Methods of suppressing activation generally rely on introduction of drugs into the cell, but some of these techniques are irreversible. I report here that injection without activation can also be accomplished by simply limiting extracellular free Ca2+ to 1-2 microM. The site of injection heals, but the cortex does not contract. Gentle modification of the vitelline envelope, which causes it to become tougher, improves the rate of healing to about 100%. Healed eggs are stable for hours and can be activated when needed. Injection of a plasmid derived from type 1 bovine papilloma virus revealed that replication occurs only after activation, but preloading the DNA markedly increased the efficiency of first-round replication. DNA interaction with the unactivated egg cytoplasm may therefore be required for efficient replication of exogenous DNA. The new procedures described here are likely to be of general utility.

The mechanism and site of teratogenic action of trypan blue on mammalian embryos was reinvestigated. The experiments to be presented include (1) an analysis of the effect of trypan blue treatment on the morphology of the early mouse egg cylinder, (2) a demonstration of the effect of dye treatment on the enzyme acid phosphatase of yolk sac epithelium using histochemical procedures. Results obtained from these experiments indicate that trypan blue injected into mothers on day 7 of gestational age leads, within 12 to 24 hours after treatment, to dramatic abnormalities in 90-95% of egg cylinders examined. The frequency of gross malformations obtained by this treatment is considerably less when litters are examined at later stages of gestation. Acid phosphatase activity in yolk sac epithelium is depressed by the dye treatment, but there is no difference between enzymatically depressed yolk sacs of malformed embryos and yolk sacs surrounding normally appearing litter mates both obtained from trypan blue treated mothers. The hypothesis that trypan blue may exert its teratogenic effect by the direct exposure of egg cylinder stages to the dye, and that some of the egg cylinders affected may subsequently repair, is recommended for further testing.

The electrical membrane potential (E(m)) and electrical membrane resistance (R(m)) were measured continuously during the first cleavage of Xenopus eggs, using intracellular microelectrodes. A sharp hyperpolarization of E(m) and decrease in R(m) can be observed from 6 to 8 min after the onset of cleavage. This moment coincides with the onset of the insertion of new membrane (Bluemink and de Laat, 1973) leading to the formation of the interblastomeric membrane during normal cleavage. Removal of the vitelline membrane or exposure to cytochalasin B (CCB) leads to exposure of the entire surface area of the membrane newly formed during cleavage. These conditions allow for a direct measurement of the permeability properties of the new membrane. It was found that under these conditions E(m) reaches values about 3 times more negative and R(m) reaches values about 1.5-3 times smaller than during normal cleavage. The extent of reduction of R(m) can be correlated with the surface area of the newly formed membrane. We conclude that the new membrane has different ionic permeability properties than the pre-existing membrane (most probably a relatively high permeability for K(+) ions). Its mean specific resistance is 1-2 kOmega.cm(2), as against 74 kOmega.cm(2) for the pre-existing membrane. No influence of CCB on the permeability properties of the pre-existing or new membrane could be detected.

Plasma membranes isolated from Rana oocytes showed a 7-10 fold increase in the Ca2+-dependent phosphorylation of endogenous protein following exposure to meiotic stimuli (progesterone, insulin) either in vivo or in-vitro. Exogenous phosphatidylmonomethylethanolamine (PME) was effective in stimulating Ca2+-dependent membrane phosphorylation and also induced meiosis. Induction of phosphorylation was blocked by the protease inhibitor leupeptin, as are all other responses to meiotic stimuli. Phosphatidylserine was inactive when added to intact oocytes, but stimulated membrane phosphorylation nearly 15-fold when added to isolated membranes. The results indicate a link between phospholipid methylation and protein kinase C activation.