Characterization and miRNA-mediated posttranscriptional regulation of vitelline membrane outer layer protein I in the adult chicken oviduct.

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.

Multi-endpoint toxicities on Chinese rare minnow (Gobiocypris rarus) fed with different diets.

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.

Identification of estrogen-responsive vitelline envelope protein fragments from rainbow trout (Oncorhynchus mykiss) plasma using mass spectrometry.

Plasma peptides previously associated with exposure of juvenile male rainbow trout (Oncorhynchus mykiss) to the hormone 17ß-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.

Ovarian P450 aromatase activity in the catfish Heteropneustes fossilis: seasonal changes and effects of catecholestrogens.

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.

Flow regulates arterial-venous differentiation in the chick embryo yolk sac.

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.

Olive oil prevents the adverse effects of dietary conjugated linoleic acid on chick hatchability and egg quality.

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.

Cell adhesion mechanisms: modeling using derivatized beads and sea urchin cell systems.

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.

Effects of protease inhibitors on binding of sperm to the vitelline coat of ascidian eggs: implications for participation of a proteasome (multicatalytic proteinase complex).

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 ADP-ribosyltransferase C3 induces elevation of the vitelline coat of ascidian eggs.

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.

Meiosis reinitiation in surf clam oocytes is mediated via a 5-hydroxytryptamine5 serotonin membrane receptor and a vitelline envelope-associated high affinity binding site.

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.

Injection of Xenopus eggs before activation, achieved by control of extracellular factors, improves plasmid DNA replication after activation.

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.

Calcium-dependent phosphorylation of the amphibian oocyte plasma membrane: an early event in initiating the meiotic divisions.

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.

Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.

Rickettsial permeability. An ADP-ATP transport system.

The obligate intracellular parasitic bacterium, Rickettsia prowazeki, has a carrier-mediated transport system for ADP and ATP. The transport of nucleotides was measured by membrane filtration assays; the assay was shown not to harm the relatively labile rickettsiae. The nucleotide transport system was shown to reside in the rickettsiae, not in the contaminating yolk sac mitochondria of the preparation. The influx of nucleotide had an activation energy of 12 to 13 kcal above 22 deg-rees (an apparent transition temperature), and 30 kcal below this value. The uptake of nucleotide was independent of the Mg2+ concentration, but was markedly stimulated by the phosphate concentration. The pH optimum of the influx of nucleotide was pH 7. The specificity of the transport system was remarkable in that it required a specific moiety in each portion of the nucleotide, i.e. an adenine base, a ribose sugar, and two or three, but not one, phosphates. Of the wide variety of compounds tested, the system could transport only ADP, ATP, and (beta, gamma-methylene) adenosine 5'-triphosphate. The influx of nucleotide was a saturable process; half-maximum velocity was achieved at a nucleotide concentration of about 75 muM. ADP and ATP were competitive inhibitors of each other's transport. Although at least 95% of the labeled intracellular nucleotide was exchangeable, efflux of labeled nucleotide was observed only in the presence of unlabeled nucleotide in the medium. Half-maximum efflux was achieved at a concentration of about 75 muM. A large intracellular to extracellular concentration gradient of labeled nucleotide was maintained in the presence of metabolic inhibitors and uncouplers, which completely abolished rickettsial hemolysis. While having no effect on the steady state, KCN and DNP accelerated both influx and efflux. Measurements of the endogenous pool of adenine nucleotides in isolated rickettsiae show that is was large (5 mM), and that these unlabeled nucleotides exchanged, on approximately a 1/1 basis, with exogenously added nucleotide. These studies support the proposal that rickettsiae are not "leaky" to adenine nucleotides or to small molecules in general, and that they have a carrier-mediated transport system which allows an exchange of host and parasite ADP and ATP.

Chemosterilant (apholate)-induced ultrastructural changes during oogenesis in Aedes aegypti.

The effect of chemosterilant, apholate, on-oogenesis has been studied in Aedes aegypti. Treatment of larvae to 20 ppm of the chemical induced ultrastural changes in the presumptive and primary follicles of the adult ovary. These changes comprised condensation of chromatin, disruption of nuclear envelope and extensive degeneration as evidenced by numerous myelin figures and residual bodies. In some primary follicles, where cellular degeneration was restricted to epithelial cells, no arrest in development was observed. However, in comparison with controls, these follicles also were retarded. Larval treatment with 30 ppm apholate completely supressed ovariolar development. High incidence of autophagy was observed in tissues at both dose levels.