Effects of bisphenol A and DDT on mRNA expression of vitellogenin II in liver of quail embryos

This study was conducted to reveal the estrogenic effects of bisphenol A and o, p'-DDT on quail embryos. Thirteen fertilized eggs were used as control [injected with 20 microl corn oil], 15 eggs were injected with estradiol 17beta [0.04 mg dissolved in 20 microl corn oil], 20 eggs were injected with BPA [2 mg dissolved in 20 microl corn oil] and 20 eggs were injected with o, p'-DDT [2 mg dissolved in 20 microl corn oil] at day 13 of incubation. Two days later the livers of the embryos were collected. The DNA was extracted from the liver for molecular sexing, while total RNA was extracted for vitellogenin II [VTGII] mRNA expression in embryos. In female embryos, BPA and o, p'-DDT induced variable levels of VTGII mRNA expression, while in male embryos, o, p'-DDT induced a slightly VTGII mRNA expression. In contrast, there was no expression of VTGII after BPA injection. In conclusion, the estrogenicity of BPA was lower than o, p'-DDT and both of them were lower than the estradiol 17beta

Single and combined effects of estrone and 17β-estradiol on male goldfish / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To assess the single and combined effects of estrone (E1) and 17β-estradiol (E2) on goldfish (Carassius auratus).</p><p><b>METHODS</b>Batch tests were conducted. Serum levels of vitellogenin (VTG) and E2, gonadosomatic indices (GSI), gonadal DNA damage and liver 7-ethoxyresorufin-O-deethylase (EROD) activity were measured after exposure for 14 days.</p><p><b>RESULTS</b>The VTG level increased significantly in a concentration-dependent manner. The serum E2 level was significantly higher and the GSI level was significantly lower in goldfish after exposed to the 3 drugs. DNA damage occurred in treated samples and EROD activity was significantly suppressed 7 days after exposure. The joint effect of E1 and E2 was additive with regard to VTG induction.</p><p><b>CONCLUSION</b>The results of our study highlight a series of effects of steroidal estrogens on goldfish. Further study is needed to confirm their effect as a whole.</p>

Development of an Enzyme-linked Immunosorbent Assay Using Vitellin for Vitellogenin Measurement in the Pale Chub, Zacco platypus

OBJECTIVES: Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. METHODS: Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. RESULTS: The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was 99.5+/-5.5%. CONCLUSIONS: A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

La langosta de agua dulce Cherax quadricarinatus es una especie tropical de gran interés para la acuicultura. Se midió la concentración de vitelogenina (Vg) en hemolinfa, ovario y hepatopáncreas de hembras adultas de esta especie, por medio de ELISA. Las mediciones fueron hechas en los cuatro períodos del ciclo reproductivo: pre-reproductivo, reproductivo medio, reproductivo tardío y post-reproductivo. Se detectaron variaciones significativas en los niveles de Vg tanto en hemolinfa como en hepatopáncreas, se observó el mayor valor durante el período reproductivo medio. Además, tales variaciones se correlacionaron positivamente con el índice hepatosomático. Se observó además una correlación positiva de los niveles de Vg entre hepatopáncreas y ovario. Estos resultados apoyan evidencias previas sobre el papel central del hepatopáncreas como sitio de síntesis de Vg, en esta especie, y también enfatizan la importancia de la hemolinfa para el transporte de la Vg del hepatopáncreas al ovario. Para propósitos de acuicultura, la medición de Vg en hemolinfa podría ser utilizada como un método no lesivo, con el fin de constatar la actividad reproductiva de hembras de C. quadricarinatus.

Strategy for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin / 生物工程学报

We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.

Purification and clinical application of egg yolk immunoglobulins / 中华男科学杂志

IgY antibodies, also called egg yolk immunoglobulins, are the only immunoglobulins in egg yolk and transferred in the female from serum to egg yolk to confer passive immunity to embryos and neonates. Using hens instead of mammals as the immunization host brings a number of advantages: Eggs are cheap and readily available; antibody levels in yolks are high; IgY isolation is fast and simple; and what is more; IgY neither binds the rheumatoid factor nor reacts to the mammalian complement factor. All these differences make IgY technology more widely applicable, such as in the production of polyclonal antibodies against various antigens, immunodiagnostics and immunotherapy, and many medical areas in both animals and human. IgY also has a good prospect in human immunocontraception.

Histological ultrastructural aspects fat body virgin physogastric queens Melipona quadrifasciata anthidioides lepeletier, 1836 (HYMENOPTERA, APIDAE, MELIPONINI)

Variations in the morphology and biochemical content of insect fat body have been associated withmetabolic activity and the reproductive cycle (synthesis of vitellogenin). In social insects such as bees,the functional traits of fat body also differ between workers and queens. In this work, we used light andtransmission electron microscopy to examine the morphological features of fat body trophocytes of virginand physogastric mated queens of the stingless bee Melipona quadriafasciata anthidioides before and duringvitellogenesis. Virgin queens had few, small fat body cells in which lipid deposits predominate, and showedno evidence of biosynthetic activity or the uptake of exogenous substances. In contrast, the fat body cells ofphysogastric queens were almost completely devoid of lipids, exhibit a well-developed rough endoplasmicreticulum with an obvious intraluminal product, and contained Golgi stacks that release numerous vesicles.These ultrastructural findings were suggestive of proteosynthesis. However, there was no evidence for theaccumulation of synthesized material in the form of secretory granules. We conclude that the trophocytes ofvirgin and physogastric queens differ basically in their switch from a storage role in the former to a syntheticrole in the latter. In addition, the high level of vitellogenesis seen in egg-laying queens suggests that themain material synthesized is vitellogenin.

Oogenesis and egg development in triatomines: a biochemical approach

Em triatomíneos, assim como em outros insetos, o acúmulo de vitelo é um processo no qual um tecido extraovariano, o corpo gorduroso, produz proteínas que são empacotadas no interior de um ovo. A principal proteína, sintetizada pelo corpo gorduroso, que é acumulada no interior de um ovócito, é a vitelogenina. Este processo é também conhecido por vitelogênese. Existem crescentes evidências em triatomíneos, que além do corpo gorduroso, o ovário também produz proteínas de vitelo. A forma como estas proteínas de vitelo entram nos ovócitos será aqui comentada. O vitelo é um material complexo composto por proteínas, lipídeos, carboidratos e outros compostos minoritários que são empacotados de uma maneira organizada no interior dos ovócitos. A fertilização dispara a embriogênese, um processo que culmina com o desenvolvimento do embrião. Durante a embriogênese o vitelo será utilizado para a construção de um novo indivíduo, a ninfa de primeiro estádio. O desafio para a próxima década é entender onde e como estas proteínas de vitelo são utilizadas junto com os seus componentes não protéicos, em compasso com o programa genético do embrião, que comanda a diferenciação celular (fase inicial da embriogênese) e diferenciação do embrião (fase final da embriogênese) no interior do ovo.

Seasonal effects of melatonin on ovary and plasma gonadotropin and vitellogenin levels in intact and pinealectomized catfish, Clarias batrachus (Linn).

Effects of daily administration of melatonin for 15 days were evaluated with respect to ovarian activities and plasma gonadotropin (GtH II) and vitellogenin (Vg) levels in intact (INT) and pinealectomized (Px) female catfish, C. batrachus, during preparatory (April), prespawning (May and June), spawning (July) and post-spawning (September) periods. Px (saline control groups) caused a stimulatory effect during preparatory (with respect to Vg synthesis and incorporation) and prespawning (with respect to Vg synthesis) periods whereas no effect was observed during spawning and post-spawning periods with respect to the reproductive parameters studied. During April, melatonin-treatment significantly decreased plasma GtH II levels and percentage of vitellogenic oocytes without any significant changes in plasma Vg levels and gonadosomatic index (GSI). During early prespawning period, in May, 50microg melatonin brought about a significant reduction in plasma GtH II levels in INT group, whereas 100microg caused a decrease in all parameters; on the other hand, in Px groups both dose levels proved to be inhibitory. In June (late prespawning period) melatonin-treatment could not bring about any change in GSI and plasma Vg levels compared to the control groups regardless of Px but plasma GtH II and mean number of yolky oocytes were significantly reduced in melatonin-treated INT group. During spawning period (July) melatonin inhibited the GSI, mean number of yolky oocytes and plasma GtH II levels without affecting plasma Vg levels. In September (post-spawning period), melatonin did inhibit both GSI and plasma GtH II levels. The results, thus, indicate that melatonin showed variable effects (inhibitory and/or no effect) to GSI, mean number of yolky oocytes and plasma Vg levels but a consistent inhibiton of plasma GtH II levels indicating that melatonin may control the reproduction by blocking the GtH II release from the pituitary via affecting the hypothalamo-hypophysial axis.

A better method for standardizing vitellogenin content of fish tissues.

Vitellogenin (vtg) concentrations were measured in plasma and liver samples from 12 hybrid Tilapia oreochromis niloticus x O. aureus to compare concentrations in these tissues. The results were calculated under two different normalizations: volume per gram of sample used (similar to normalization usually published in the literature and typically used for ELISA) and volume per total protein (similar to normalization used in polyacrylamide gel electrophoresis; PAGE). It was observed that the normalization procedure used in PAGE (per gram total protein) minimized the method detection limit by about 1000 and 2500 times in plasma and liver respectively, compared to the normalization usually reported in the literature. It was also observed that normalizing per gram total protein makes it possible to eliminate a potential problem of accidental dilution of plasma samples during sample collection. Moreover, the normalization on a per gram of total protein makes it possible even to compare results from the two different methods namely PAGE and ELISA. It also allows comparison between different tissues. Using the normalization procedures as used in PAGE (per gram total protein) for liver and the normalization method as reported in literature for ELISA (per volume of sample used), it was observed that liver samples had higher vtg levels (mean: 62 microg vtg/g) compared to the corresponding plasma samples (mean: 0.24 microg vtg/ml). However, when both results were normalized per gram total protein all but one liver sample were lower (62 microg vtg/g) than the corresponding plasma concentrations (mean = 246 microg vtg/g).

Characters that differ between diploid and haploid honey bee (Apis mellifera) drones

Diploid males have long been considered a curiosity contradictory to the haplo-diploid mode of sex determination in the Hymenoptera. In Apis mellifera, 'false' diploid male larvae are eliminated by worker cannibalism immediately after hatching. A 'cannibalism substance' produced by diploid drone larvae to induce worker-assisted suicide has been hypothesized, but it has never been detected. Diploid drones are only removed some hours after hatching. Older larvae are evidently not regarded as 'false males' and instead are regularly nursed by the brood-attending worker bees. As the pheromonal cues presumably are located on the surface of newly hatched bee larvae, we extracted the cuticular secretions and analyzed their chemical composition by gas chromatograph-mass spectrometry (GC-MS) analyses. Larvae were sexed and then reared in vitro for up to three days. The GC-MS pattern that was obtained, with alkanes as the major compounds, was compared between diploid and haploid drone larvae. We also examined some physical parameters of adult drones. There was no difference between diploid and haploid males in their weight at the day of emergence. The diploid adult drones had fewer wing hooks and smaller testes. The sperm DNA content was 0.30 and 0.15 pg per nucleus, giving an exact 2:1 ratio for the gametocytes of diploid and haploid drones, respectively. Vitellogenin was found in the hemolymph of both types of imaginal drones at 5 to 6 days, with a significantly lower titer in the diploids.

Immunological identification of two female-specific proteins from the plasma of Indian freshwater murrel, Channa punctatus (Bloch).

Existence of a non-phosphorylated female-specific protein (FS II), in addition to phosphorylated vitellogenin (FS I), in the plasma of murrel by exogenous administration of estradiol-17beta is reported. Polyspecific rabbit antibodies were raised against estrogen-inducible murrel plasma proteins. This antiserum was absorbed with normal male serum in order to obtain female-specific antiserum (FSAS). Radial immunodiffusion studies suggested that both the proteins (FS I and FS II) were present in the plasma of E2-treated and normal vitellogenic females and in the ovarian homogenate from gravid females, but absent in normal male plasma. Autoradiographic experiments demonstrated that phosphorus moiety was attached with FS I only. Further, immunoelectrophoretic analysis and peptide maps supported the observation that FS I and FS II were discrete, unrelated female-specific proteins.

Purification of vitellogenin from the plasma of Indian freshwater murrel, Channa punctatus (Bloch) by different methods: a comparative study.

Plasma from estrogenized, [32P] NaH2PO4-injected murrel, Channa punctatus was collected in the presence of proteolytic inhibitors and subjected to different separation procedures singly or in combination, viz., gel filtration chromatography on Ultrogel AcA 34, ion-exchange chromatography on DEAE sephacel, or selective precipitation with dimethylformamide or with Mg2+: EDTA in order to isolate vitellogenin from other plasma proteins. The results show that chromatography on Ultrogel or DEAE sephacel yields intact vitellogenin whereas prior precipitation with DMF or with Mg2+: EDTA results in either co-precipitation of other plasma proteins or in the cleavage of phosvitin-like material from the native vitellogenin molecule.

Demonstration of two forms of vitellogenin in serum of estradiol-17 beta-treated Indian major carp, Labeo rohita.

Vitellogenin (Vg) synthesis was induced in the male and non-vitellogenic female Rohu, the Indian major carp, by estradiol-17 beta(E2) where effect was more in female. A crude preparation of Vg was isolated in the second peak after gel filtration on Ultrogel AcA 34 from the sera of vitellogenic female Rohu and E2-treated male and female Rohu. Estimation of alkali-labile phosphorus was shown to be used as an index of Vg. Native-PAGE analysis has revealed the presence of two forms of Vg (Vg1: 430,000 dalton and Vg2:240,000 dalton) in Vg fraction obtained after gel filtration as well as in the sera of E2-treated male and female Rohu. Immunological cross-reaction studies between antiserum to yolk protein and Vg fractions as well as the sera from E2-treated male and female Rohu further indicates the presence of two precipitin lines (not clearly visible as the two lines fused to form a thick line) suggesting the occurrence of two forms of Vg in the Rohu.

Structures and properties of vitellin isolated from Triatominae species

Se realizaron estudios comparativos de los pesos moleculares composicion quimica e identidad inmunológica de la vitelina (VN) de distintas especies de triatominos. Se determinó que la VN es una lipoglicofosfoproteina con 12.76% de hidratos de carbono 12.53% de lipidos y 0.6% de fósforo unido a mas aproteica. La VN nativa muestra por filtración en gel una única banda de 310 KDa. La VN delipidizada muestra por PAGE SDS una banda principal de 175 KDa coincidente con la banda hallada en la vitellogenina (VG). La composición de aminoácidos fue similar en las cuatro especies ensayadas

The effect of pH and metal chelators upon the sedimentation velocity of Odontophrynus americanus vitellogenin

In the tetraploid amphibian Odontophrynus americans the selective precipitation of vitellogenin by Mg2+ from plasma treated with ethylene diaminetetraacetic acid (EDTA) or ethylene bis (oxyethylenenitrilo)-tetraacetic acid (EGTA) is a pH-dependent phenomenon. Utilizing sucrose gradient centrifugation of whole plasma we have shown that under standardconditions (pH 7.0) the estimated apparent sedimentation coefficient of vitellogenin is 17s.At pH 8.0 and in the presence of EDTA or EGTA there is a decrease of the vitellogenin sedimentation coefficient.This behavior is restricted to vitellogenin as othewr plasma proteins show no alteration in their sedimentation coefficient after similar treatment. The treatment with EDTA at pH 8.0 also induces changes in the vitellogenin molecule which can be detected by partial proteolysis with chymotripsin A

The vitellogenin gene family of Aedes aegypti

We have been interested in identifying genes that play a role in reproduction of the mosquito Aedes aegypti. Our interests are currently focused on the vitellogenin genes which in the mosquito are expressed only in the fat body in response to the insect steroid hormone, 20-hydroxyecdysone. Four of the five vitellogenin genes in the genome have been cloned. We have examined the relationships between these genes and find that they form a small gene family exhibiting different levels of relationship.

Vitellin and vitellogenin characterization of Triatoma infestans and related species

La vitelogenina-vitelina (VG-VN) de Triatoma infestans es una glicolipoproteína con un peso molecular de 220 000, es una lipoproteína de alta densidad (1.18-1.2 gm/ml) con poca movilidad electroforética a pH 8.2. Hembras, machos, ninfas y huevos comparten numerosas proteínas pero ninguna de ellas es glicolipoproteica. La proteína común más importante posee un peso molecular de 43 000, es anódica y se encuentra a lo largo de todo el gradiente salino. Machos y hembras poseen una glicolipoproteína con una movilidad electroforética similar a l VG-VN, pero es una proteína de baja densidad. Otra VG-VN, catódica, no glicolipoproteica, se encuentra en huevos y hemolinfa de hembra. La VG-VN principal puede ser aislada por ultracentrifugación en gradiente de BrNa o por cromatografía en DEAE-celulosa. Varias especies dentro del género Triatoma demostraron identidad inmunológica entre sus VG-VN. Las hembras y los huevos de Panstrogylus megistus y las hembras, los huevos y los machos de Rhodnius prolixus poseen identidad inmunológica parcial con las VG-VN de T. infestans