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In 1982, a water-soluble aminoplastic resin embedding method for electron microscopy (EM) was developed to study lipids. It involves a silylation technique for processing cured resins by converting them from hydrophilic to hydrophobic status, facilitating ultra-thin sectioning. The technique offers better preservation of lipovitellin material in frog (Rana amurensis) yolk platelets, and it shows membrane ultrastructure better than traditional epoxy embedding methods. An early (1982) pseudo-colored TEM image processing technique applied to the micrographs obtained by EM of silylated aminoplastic thin sections revealed the lipovitellin molecules, in relation to the biosynthesis of new membranes in the first cleavage of the egg, with remarkable clarity and explains the lipovitellin molecules in relation to the biosynthesis of new membranes. Because the original article was written in Chinese and published in China before its open policy, many scientists may not be aware of the publication. Thus, in this short review, the essential components of the procedure are presented in English to help guide investigators who might be interested in the use of water-soluble embedding methods for lipid research. [ABSTRACT FROM AUTHOR]

Long interspersed nuclear element‑1 (L1) is highly expressed in the early embryos of humans, rodents and fish. To investigate the molecular mechanisms underlying high expression of L1 during early embryonic development, a C1‑open reading frame (ORF)2 vector was constructed in which ORF2 of human L1 (L1‑ORF2) was inserted into a pEGFP‑C1 plasmid. C1‑ORF2 vector was injected into early zebrafish embryos (EZEs) to observe expression of EGFP reporter protein by fluorescence microscopy. RNA‑seq and RT‑qPCR were used to detect the effects of lipovitellin (LV)  on gene expression in EZEs. The binding ability of LV to L1‑ORF2 DNA was detected by electrophoretic mobility‑shift assay (EMSA). The chromatin recombinant DNase I digestion and ATAC‑seq assay were used to evaluate the accessibility of plasmid DNA. C1‑ORF2 vector induced high expression of enhanced green fluorescent protein (EGFP) reporter gene after it had been injected into 0 h post‑fertilization (hpf) zebrafish embryos, although histone octamer inhibited expression of EGFP in C1‑ORF2. SDS‑PAGE was used to show that LV was the predominant protein binding ORF2 DNA in 0 hpf zebrafish embryo lysate (ZEL). Both ZEL and purified LV from ZEL attenuated the inhibitory effects induced by histone. LV bound histone to interfere with the binding of histone to ORF2 DNA. Both in vitro chromatin reconstitution experiments and assay for transposase‑accessible chromatin with sequencing with HeLa cells were utilized to demonstrate that the interference induced by LV resulted in increased accessibility of C1‑ORF2. Transcription experiments in vitro verified that LV could enhance the mRNA levels of zebrafish early embryo expression genes grainyhead‑like transcription factor 3 (GRHL3), SRY‑box transcription factor 19a (SOX19A) and nanor (NNR) and also of the EGFP gene. LV was found to increase the expression levels of the zebrafish early embryo expression genes in liver tissue after LV had been injected into the abdominal cavity of adult male zebrafish. Taken together, the findings of the present study demonstrated that LV activates the expression of EGFP induced by ORF2 in EZEs by enhancing the accessibility of ORF2 DNA.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions. [ABSTRACT FROM AUTHOR]

Abstract Analysis of energy expense during development has achieved special interest through time on account of the crucial role of the consumption of resources required for offspring survival. Spider eggs have a fixed composition as well as some initial energy that is supplied by mothers. These resources are necessary to support the metabolic expense not only through the embryonic period but also during the post-embryonic period, as well as for post emerging activities before spiderlings become self-sustaining. Depletion of these resources would be critical for spiders since it could give rise to prey competition as well as filial cannibalism. Even though spiders represent a megadiverse order, information regarding the metabolic requirements during spiders development is very scarce. In this study, we analyse the changes in protein, lipid and carbohydrate content as well as the variation in lipovitellin reserves and hemocyanin content during Polybetes pythagoricus development. Our results show that lipovitellins and phospholipids represent the major energy source throughout embryonic and post-embryonic development. Lipovitellin apolipoproteins are gradually consumed but are later depleted after dispersion. Phosphatidylethanolamine is mainly consumed during the post-embryonic period, while triacylglycerides are consumed after juveniles’ dispersion. Finally, hemocyanin concentration starts to increase in postembryonic stages.

The increasing levels of environmental estrogens are causing negative effects on water, soil, wildlife, and human beings; label-free immunosensors with high specificities and sensitivities are being developed to test estrogeneous chemicals in complex environmental conditions. For the first time, highly fluorescent graphene quantum dots (GQDs) were prepared using a visible-Fenton catalysis reaction with graphene oxide (GO) as a precursor. Different microscopy and spectroscopy techniques were employed to characterize the physical and chemical properties of the GQDs. Based on the fluorescence resonance energy transfer (FRET) between amino-functionalized GQDs conjugated with anti-lipovitellin monoclonal antibodies (Anti-Lv-mAb) and reduced graphene oxide (rGO), an ultrasensitive fluorescent "ON-OFF" label-free immunosensor for the detection of lipovitellin (Lv), a sensitive biomarker derived from Paralichthys olivaceus for environmental estrogen, has been established. The immunosensor has a wide linear test range (0.001–1500 ng/mL), a lower limit of detection (LOD, 0.9 pg/mL), excellent sensitivity (26,407.8 CPS/(ng/mL)), and high selectivity and reproducibility for Lv quantification. The results demonstrated that the visible-Fenton is a simple, mild, green, efficient, and general approach to fabricating GQDs, and the fluorescent "ON-OFF" immunosensor is an easy-to-use, time-saving, ultrasensitive, and accurate detection method for weak estrogenic activity. [ABSTRACT FROM AUTHOR]

The increasing levels of environmental estrogens are causing negative effects on water, soil, wildlife, and human beings; label-free immunosensors with high specificities and sensitivities are being developed to test estrogeneous chemicals in complex environmental conditions. For the first time, highly fluorescent graphene quantum dots (GQDs) were prepared using a visible-Fenton catalysis reaction with graphene oxide (GO) as a precursor. Different microscopy and spectroscopy techniques were employed to characterize the physical and chemical properties of the GQDs. Based on the fluorescence resonance energy transfer (FRET) between amino-functionalized GQDs conjugated with anti-lipovitellin monoclonal antibodies (Anti-Lv-mAb) and reduced graphene oxide (rGO), an ultrasensitive fluorescent “ON-OFF” label-free immunosensor for the detection of lipovitellin (Lv), a sensitive biomarker derived from Paralichthys olivaceus for environmental estrogen, has been established. The immunosensor has a wide linear test range (0.001–1500 ng/mL), a lower limit of detection (LOD, 0.9 pg/mL), excellent sensitivity (26,407.8 CPS/(ng/mL)), and high selectivity and reproducibility for Lv quantification. The results demonstrated that the visible-Fenton is a simple, mild, green, efficient, and general approach to fabricating GQDs, and the fluorescent “ON-OFF” immunosensor is an easy-to-use, time-saving, ultrasensitive, and accurate detection method for weak estrogenic activity.

Increasing levels of estrogenic pollution in marine environments has made the development of reliable biological detection techniques urgently needed. In this study, Japanese flounder (Paralichthys olivaceus) lipovitellin (Lv) was purified and used to establish three immunological methods for the detection of vitellogenin (Vtg), a biomarker for environmental estrogens. Firstly, five different methods were employed to purify Lv, among which water-precipitation was the fastest and easiest way to purify Lv. Japanese flounder Lv was characterized as a phospholipoglycoprotein with a molecular weight of ∼369 kDa. Using purified Lv and its specific polyclonal antibody, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This assay had a working range from 7.8 to 250 ng/mL and a detection limit of 3.1 ng/mL. Furthermore, we developed an immunohistochemistry (IHC) and an immunofluorescence (IF) assay, both of which allowed visual detection of liver Vtg. Finally, Vtg induction in plasma and liver of juvenile Japanese flounders exposed to 17 β -ethinylestradiol (EE 2) was measured using these three methods. Exposure to 10 and 50 ng/L EE 2 significantly increased plasma Vtg levels, and obvious positive fluorescence signals were observed near the liver sinusoidal vessels. These results confirmed that the methods developed effectively detected estrogenic activity of exogenous chemicals. Therefore, this study provides reliable methodologies for biomonitoring of estrogenic pollution in marine environments. • Japanese flounder Lv could be easily purified by a single water precipitation method. • Vtg sandwich ELISA was established using the purified Lv and its antibody. • Vtg concentration in plasma could be quantitated by the developed ELISA. • Vtg induction in liver can be easily visualized by the developed IHC and IF methods. • This study provided reliable tools for the detection of marine estrogenic activity. [ABSTRACT FROM AUTHOR]

Nutrients are deposited in the eggs as yolk to provide energy and to build embryonic structures. Lipovitellin, the major egg-yolk protein was purified from the oocytes of Clarias gariepinus. The amino acid sequences of lipovitellin (Lv) peptides obtained by Mass spectrometry were mapped on its precursor protein, Vitellogenin (Vg). Advanced proteomic tools, PAGE {one dimensional (1-D) andtwodimensional (2-D)} and mass spectrometry were employed to characterise Vg. Similarity in sequences of peptidesre-established the precursor-product relationship between Vg and Lv. Partial mRNA transcript of vg gene was translated into amino acid sequence that corresponded to conserved domains of Vitellogenin in NCBI database. Phylogenetic analysis of the nucleotide sequence of Africancat fish vg gene revealed a close similaritywith the fishes of the order Siluriformes. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vtg) induction in carp (Cyprinus carpio) is a commonly used biomarker for oestrogenic contamination. However, the accurate quantification of Vtg was challenged because the easy degradation of Vtg standard would change the standard curves of the immunoassays. In this study, three yolk proteins were purified from carp ovarian extracts by a two-step chromatographic method. The purified proteins were characterized as phospholipoglycoproteins with molecular weights of approximately 416, 398 and 383 kDa. In SDS-PAGE, the purified proteins appeared as a major band of approximately 110 kDa and several faint bands. Based on these characteristics, purified proteins were identified as carp lipovitellin (Lv). Immunological analysis showed that anti-Vtg antiserum reacted with the purified Lv. The results of stability analysis revealed that even heated at 60°C for 60 min, the electrophoretic patterns of carp Lv in native-PAGE and SDS-PAGE did not have a significant difference compared with the Lv solution stored at 4°C. Therefore, the purified carp Lv was confirmed to have similar immunogenicity with Vtg and possess exceptionally high stability, which would be helpful for the development of robust immunoassays for accurate quantification of carp Vtg. [ABSTRACT FROM AUTHOR]

The development of Pardosa saltans wolf spiders inside an egg sac includes two periods: an embryonic period and a post-embryonic period after hatching. We investigated spiderlings’ energy expenditure to assess energetic costs during the different embryonic and post-embryonic developmental stages during which they are confined within their egg sac. We focused on the following developmental stages: egg, embryonic stages 1 and 2, and two stages, separated by a moult, during post-embryogenesis inside the egg sac: “juvenile instars 1 and 2” until emergence of 2 instar juveniles from their egg sac. We present the first biochemical characterization of the vitellus of wolf spiders’ eggs, embryos and juveniles. Lipovitellins (LV) are composed of four apolipoproteins of 116, 87, 70 and 42 kDa, respectively, and LV represent 35-45% of total protein during development. The principal LV lipids are triglycerides, phospholipids, free fatty acids and sterols. Egg caloric content averaged 127 cal/g (proteins: 91 cal/g, lipids: 33 cal/g, carbohydrates: 3 cal/g). During development from undivided egg to emerged “juvenile 2”, 67% of proteins, 51% of carbohydrates and 49% of triglycerides stocks were depleted. At the end of the post-embryonic period, at emergence from egg sac, body energy stock of “juveniles 2” was 38% of the initial calorie stocks in the eggs. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vtg) in zebrafish ( Danio rerio ) is a recommended biomarker endpoint for detecting estrogenic activity of chemicals under the OECD test guidelines. The present paper reports the development of a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the quantification of zebrafish Vtg based on monoclonal antibodies (MAbs) against lipovitellin (Lv), the major yolk protein derived from Vtg. The purified Lv was used to immunize mice and the spleen cells of mice were fused with myeloma cells. Two high-affinity MAbs (H3A8 and H4D9) were screened from hybridoma cells. Western blot analysis revealed that two MAbs were highly specific to zebrafish Vtg and recognized different antigenic epitopes because MAb H3A8 detected a main band of 143 kDa in purified Vtg, while MAb H4D9 reacted with two clear bands of purified Vtg at 117 and 102 kDa. Using MAb H3A8 as the coating antibody, HRP-labeled MAb H4D9 or HRP-labeled PAbs as the detecting antibody, two sandwich ELISAs for Vtg quantification were developed. The sandwich ELISA developed using HRP-labeled MAb H4D9 had a working range of 1.95–250 ng/mL, with a detection limit of 0.78 ng/mL, which was lower than that of the assay based on HRP-labeled PAbs. Parallelism between Lv standard curves and dilution curves of whole-body homogenates (WBH) from E 2 -treated male zebrafish confirmed the validity of the ELISAs for quantifying zebrafish Vtg. Finally, the usefulness of two assays for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to 17 β -estradiol. [ABSTRACT FROM AUTHOR]

Lipovitellin (Lv) is an apoprotein in oviparous animals. Lv consists of a heavy chain (LvH) and a light chain (LvL) which are traditionally regarded as energy reserves for developing embryos. Recently, Lv has been shown to be involved in immune defense of developing embryos in fish. However, it remains unknown if each of LvH and LvL possesses immune activity; and if so, whether or not they function similarly. Here we clearly demonstrated that recombinant LvH (rLvH) and LvL (rLvL) from zebrafish vg1 gene bound to both the Gram-negative bacteria Escherichia coli and Vibrio anguillarum and the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as the pathogen-associated molecular patterns LPS, LTA and PGN. In addition, both rLvH and rLvL were able to enhance the phagocytosis of bacteria E. coli and S. aureus by macrophages. All these data suggest that both LvH and LvL, in addition to being energy reserves, are also maternal immune-relevant factors capable of interacting with invading bacteria in zebrafish embryos/larvae. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vtg) in zebrafish ( Danio rerio ) is a core biomarker for screening environmental estrogens in test guidelines of the Organization for Economic Cooperation and Development. To accurately quantify zebrafish Vtg, lipovitellin (Lv), the main Vtg-derived yolk protein, was used as the antigen to establish a sandwich enzyme-linked immunosorbent assay (ELISA). The purified Lv was a phospholipoglycoprotein with apparent molecular weight of ~ 445 kDa, and separated into three polypeptides corresponding to ~ 117, ~ 102, and ~ 23.8 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunological analysis confirmed the specificity of the anti-Lv antibody for Vtg and the immunological similarity between Vtg and Lv. Using the purified Lv and anti-Lv antibody, a sandwich ELISA with a detection limit of 4.3 ng/mL and a detection range from 7.8 to 250 ng/mL was developed. The intra- and inter-assay coefficients of variation were both below 10%. Moreover, the Lv standard curve was nearly identical to the Vtg standard curve, and paralleled serial whole-body homogenate dilutions of male zebrafish exposed to 17 β -estradiol, demonstrating that the Lv-based ELISA could be used for quantification of zebrafish Vtg. Zebrafish Lv showed high stability during purification process, heat treatment, − 80 °C storage, and repeated freeze/thaw cycles. Additionally, the standard curve of Lv stored at − 80 °C for 3 months exhibited higher robustness than that of Vtg stored under the same conditions. Finally, the usefulness of the ELISA for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to monocrotophos. [ABSTRACT FROM AUTHOR]

The biosynthesis of neutral fat-transporting lipoproteins involves the lipidation of their nonexchangeable apolipoprotein. In contrast to its mammalian homolog apolipoprotein B, however, insect apolipophorin-II/I (apoLp-II/I) is cleaved posttranslationally at a consensus substrate sequence for furin, resulting in the appearance of two apolipoproteins in insect lipoprotein. To characterize the cleavage process, a truncated cDNA encoding the N-terminal 38% of Locusta migratoria apoLp-II/I, including the cleavage site, was expressed in insect Sf9 cells. This resulted in the secretion of correctly processed apoLp-II and truncated apoLp-I. The cleavage could be impaired by the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (decRVKRcmk) as well as by mutagenesis of the consensus substrate sequence for furin, as indicated by the secretion of uncleaved apoLp-II/I-38. Treatment of L. migratoria fat body, the physiological site of lipoprotein biosynthesis, with decRVKRcmk similarly resulted in the secretion of uncleaved apoLp-II/I, which was integrated in lipoprotein particles of buoyant density identical to wild-type high density lipophorin (HDLp).These results show that apoLp-II/I is posttranslationally cleaved by an insect furin and that biosynthesis and secretion of HDLp can occur independent of this processing step. Structure modeling indicates that the cleavage of apoLp-II/I represents a molecular adaptation in homologous apolipoprotein structures. We propose that cleavage enables specific features of insect lipoproteins, such as low density lipoprotein formation, endocytic recycling, and involvement in coagulation.

Our understanding of the function of vitellogenin (Vg) in reproduction has undergone a transformation over the past decade in parallel with new insights into the role of Vg in immunity. Initially, Vg was regarded as a female-specific reproductive protein, which is cleaved into yolk proteins such as phosvitin (Pv) and lipovitellin (Lv), stored in egg, providing the nutrients for developing embryos. Recently, Vg is shown to be an immune-relevant molecule involved in the defense of the host against the microbes including bacterium and virus. Furthermore, Pv and Lv, that both are proteolytically cleaved products of Vg, play a defense role in developing embryos. Importantly, yolk protein-derived small peptides also display antimicrobial activity. These data together indicate that Vg, in addition to being involved in yolk protein formation, plays a non-reproductive role via functioning as an immune-relevant molecule in both parent fishes and their offspring. It also shows that yolk proteins and their degraded peptides are novel players in maternal immunity, opening a new avenue to study the functions of reproductive proteins. [ABSTRACT FROM AUTHOR]

Goldfish ( Carassius auratus ) vitellogenin (Vtg) is an efficient biomarker for estrogen contamination in aquatic environments. In this study, Vtg and lipovitellin (Lv) were purified from the plasma of 17 β -estradiol (E 2 )-induced male goldfish and unfertilized eggs of females, and were used to generate polyclonal antibodies against Vtg (anti-Vtg) and Lv (anti-Lv), respectively. SDS-PAGE and Western blot were performed to confirm the specificity of the two antibodies and the immunological similarity between Vtg and Lv. As anti-Lv recognized more antigen epitopes than anti-Vtg, it was used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for goldfish Vtg with purified Lv as the standard. The detection limit of the assay was 1.82 ng/mL, and the working range was 3.9–250 ng/mL. The use of Lv instead of Vtg as the standard provided greater precision and strengthened the robustness of the sandwich ELISA. Western blot and the Lv-based ELISA were used to detect Vtg inductions in surface mucus and plasma of E 2 -induced goldfish. The surface mucus Vtg level in E 2 -induced males was significantly higher than that in the control males and E 2 -induced females, and was much closer to the plasma Vtg level in E 2 -induced males than that in E 2 -induced females. Therefore, the surface mucus Vtg level of male goldfish may be a reliable indicator of estrogenic activity in the aquatic environment. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vtg), the major egg yolk precursor protein, is traditionally thought to provide protein- and lipid-rich nutrients for developing embryos and larvae. However, the roles of Vtg as well as its derived yolk proteins lipovitellin (Lv) and phosvitin (Pv) extend beyond nutritional functions. Accumulating data have demonstrated that Vtg, Lv and Pv participate in host innate immune defense with multifaceted functions. They can all act as multivalent pattern recognition receptors capable of identifying invading microbes. Vtg and Pv can also act as immune effectors capable of killing bacteria and virus. Moreover, Vtg and Lv are shown to possess phagocytosis-promoting activity as opsonins. In addition to these immune-relevant functions, Vtg and Pv are found to have antioxidant activity, which is able to protect the host from oxidant stress. These non-nutritional functions clearly deepen our understanding of the physiological roles of the molecules, and at the same time, provide a sound basis for potential application of the molecules in human health. [ABSTRACT FROM AUTHOR]

The susceptibility of vitellogenin (Vtg) to degradation is a major problem affecting the robustness of enzyme-linked immunosorbent assay (ELISA) for goldfish ( Carassius auratus ) Vtg. In this study, a phospholipoglycoprotein with molecular mass of ∼420 kDa was purified from goldfish egg extracts and it produced a single band corresponding to ∼112 kDa in SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Additionally, the amino acid composition of the purified protein was comparable to that of lipovitellin (Lv) from other fish species. Thus, the purified protein was identified as goldfish Lv. Purified Lv and anti-Lv polyclonal antiserum were used to develop an ELISA with a detection range between 31.25 and 1000 ng mL − 1 . The intra- and inter-assay coefficients of variation were 6.45% and 7.08%, respectively. The immunological similarity between goldfish Vtg and Lv was confirmed by immunoelectrophoresis and Western blot. Goldfish Lv showed higher stability than Vtg after −80 °C storage, multiple freeze/thaw cycles, and heat treatment. Moreover, the use of treated Lv in the ELISA did not change the slopes of standard curves. Parallelism between the Lv standard curve and plasma dilution curves of vitellogenic females confirmed the validity of the assay for quantifying plasma Vtg. The Lv-based Vtg ELISA was further applied to evaluate the estrogenic activity of monocrotophos pesticide. [ABSTRACT FROM AUTHOR]

A label-free, optical waveguide lightmode spectroscopy based immunosensor was developed for frog (Bombina orientalis) vitellogenin (Vtg) determination in biological samples as a biomarker for exogenous oestrogen compounds. Antibody against Vtg was produced in rabbits immunised with purified lipovitellin (Lpv), a precursor of Vtg, from the homogenised ovary of oriental fire-bellied toads (B. orientalis). The purified protein and Lpv/Vtg-specific serum were applied in both competitive and direct immunoassay formats using optical waveguide lightmode spectroscopy immunosensor. When measuring Vtg in direct manner, the Lpv antibody (1.76 µg mL−1) was immobilised on the sensor surface, and the linear measuring range for Vtg was 0.1–10 µg mL−1. During the competitive measurement, 100 ng mL−1Lpv was applied for the immobilisation. The linear measuring range for Vtg was 0.5–50 ng mL−1. We studied the relative substrate specificity of the antibody, and it was concluded that the method is suitable for the sensitive and selective determination of Vtg levels in toads. Heart, liver and gonad samples from male animals were spiked with Vtg and were analysed using the newly developed method, and female toads and spawn samples were tested and compared to the calibration curve obtained by the spiked samples. [ABSTRACT FROM AUTHOR]

A phosphorylated protein with a molecular mass of 25,000 (pp25) is involved in Xenopus laevis vitellogenin B1 and partially overlaps with phosvitin and lipovitellin 2. The protease responsible for pp25 degradation was studied in vitro since this occurs during embryogenesis. Initially, a protease thought to be a contaminant of the purified pp25 preparation was analyzed and an antipain-sensitive protease presumed to be involved. When commercially available proteases were examined, pp25 was not degraded by calpain I or 20S proteasome, but it was degraded by cathepsin L in vitro. A survey of the protease responsible for pp25 degradation in the cytoplasm of Xenopus oocytes found partially purified pp25 was degraded in partly antipain-sensitive manner. These results suggest that an antipain-sensitive protease or cathepsin L (or a related protease) is a candidate for pp25 degradation. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vtg) is a large precursor molecule of the two major egg yolk phosphoproteins, namely; lipovitellin and phosvitin. All of the vitellogenic zinc is found in lipovitellin. Several reports had speculated into the probable relationships of Vtg with reproductive phenotype of Philippine Mallard duck (Anas platyrhynchos domesticus L.). Hence, it was hypothesized to use the Vtg profile as a nonlethal physiological index of reproductive performance in Philippine Mallard duck. Forty ducks, aged 16 weeks old, were randomly assigned into two treatment groups: 20 ducks fed with 30 ppm zinc-diet (zinc positive) and 20 ducks fed with no added zinc (zinc negative). All ducks were kept individually in cages. The circulating Vtg at sexual maturity (155.11±10.83 days old) was determined from the blood sera. The sera were assayed for Vtg in duplicate using 96-well microplate and the optical density was read at 415 nm. Results show that the circulating Vtg in the blood sera of ducks at sexual maturity was 0.69±0.07 µg Zn/dl. This circulating Vtg was identified to have negatively very weak to weak linear correlation with the majority of the laying traits (11 out of 17) in Philippine mallard duck. [ABSTRACT FROM AUTHOR]

Abstract: In oviparous species, proteins and lipids found in the vitellus form the lipoproteins called lipovitellins that are the major source of energy for the development, growth, and survival of the embryo. The energy resources provided by the lipovitellins have not yet been investigated in the Order Araneae. Using the wolf spider Schizocosa malitiosa (Lycosidae) as an experimental model, we identified and characterized the lipovitellins present in the cytosol, focusing on the energetic contribution of those lipoprotein particles in the vitellus. Two lipovitellins (LV) named SmLV1 and SmLV2 were isolated. SmLV1 is a high-density lipoprotein with 67% lipid and 3.6% carbohydrate, and SmLV2 is a very high-density lipoprotein with 9% lipid and 8.8% carbohydrate. Through electrophoresis in native conditions we observed that SmLV1 has a molecular mass of 559kDa composed of three apolipoproteins of 116, 87, and 42kDa, respectively. SmLV2 comprised several proteins composed of different proportions of the same subunits (135, 126, 109, and 70kDa). The principal lipids of these lipovitellins are sphingomyelin+lysophosphatidylcholine, esterified sterols, and phosphatidylcholine. Lipovitellin-free cytosol contains abundant phospatidylcholine and triacylglyceride related to the yolk nuclei (the vitellus organizing center). The principal fatty acids of SmLV1 and SmLV2 are 18:2 n−6, 18:1 n−9, and 16:0. Spectrophotometry detected no pigments in either the lipovitellins or the cytosol. The egg caloric content was 92cal/g, at proportions of 59.8% protein, 20.1% carbohydrate, and 19.9% lipid. SmLV1 and SmLV2 provided 19.5% and 17.1% of the calories, respectively. Both lipovitellins contribute mainly with proteins (15.8–18%), with the input of carbohydrates and lipids being lower than 1.3%. [Copyright &y& Elsevier]

Vitellogenin is a precursor of yolk protein that is necessary for embryonic development. This protein is a large multi-domain protein consisting of a signal peptide, a heavy-chain lipovitellin, a phosvitin, a light-chain lipovitellin, a von Willebrand factor type D domain (vWF-D), and a C-terminal coding region (CT), which are processed to respective domains after uptake into oocytes. It is currently believed that only lipovitellin and phosvitin domains are necessary for nutrient supply to oocytes. Thus, molecular species of vitellogenin lacking these domains are not known. Here, we show that two novel isoforms of vitellogenin, both of which possess vWF-D and CT domains but not a lipovitellin or phosvitin domain, are expressed in the gonad of the ascidian Halocynthia roretzi. In situ hybridization revealed that mRNAs of these proteins are specifically expressed in oocytes and test cells, accessory cells in the perivitelline space of ascidian eggs. Immunohistochemistry showed that these proteins are localized around the surface of test cells in immature oocytes. Immunoelectron microscopy revealed that vitellogenin associates with vesicles located beneath the vitelline coat (VC) before fertilization but that it dissociates from the VC after fertilization. These results, together with our previous results showing that vWF-D and CT domains are capable of binding to the two sperm proteases HrProacrosin and HrSpermosin, led us to propose that novel isoforms of vitellogenin, which are expressed in oocytes and test cells and released to the perivitelline space during oocyte maturation, may participate in gamete interaction upon fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 320B:118-128, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Both innate and adaptive immune-relevant factors are transferred from mother to offspring in fishes. These maternally-transferred factors include IgM, lysozymes, lectin, cathelicidin and complement components. Recently, yolk proteins, phosvitin and lipovitellin, have been shown to be maternally-transferred factors, functioning in the defense of teleost larvae against pathogens. Among these factors, the mode of action of complement components and yolk proteins has been explored, whereas that of all the other factors remains elusive. At present, the transfer mechanisms of maternally-derived immune factors are largely unknown although those of IgM and yolk protein transmission from mother to offspring have been reported in some fishes. Maternal transfer of immunity is affected by many elements, including biological factors, such as age and maturation, and environmental conditions experienced by brood fish, such as pathogens and nutritional supply. Practically, the manipulation of maternal immunity transfer can be used to enhance the survival rate of fish larvae. [Copyright &y& Elsevier]

Abstract: Label-free, optical waveguide lightmode spectroscopy (OWLS) based immunosensors were developed for vitellogenin (Vtg) determination in carp (Cyprinus carpio). Purified polyclonal serum raised in rabbits against lipovitellin (Lpv) purified from carp ovary and shown to fully cross-react with Vtg was applied both in direct and indirect measurement formats, and analytical features (limit of detection, specificity, matrix effects) were investigated. The direct OWLS immunosensor and the sandwich ELISA assay developed showed linear measuring range in 600–12,000ngmL−1 and 200–3000ngmL−1 Lpv concentrations in standard solutions, respectively. The competitive immunosensor allowed a sensitive detection range for Lpv between 3 and 100ngmL−1. Vtg content in blood and liver samples from male and female carp were determined by indirect OWLS. To reduce the matrix effect, calibration curves were obtained using Vtg-free blood and liver samples from male carp spiked with Lpv. Vtg levels in the liver of female carp was lower than determined in the blood samples, therefore, blood occurs to be a more proper tissue to monitor elevated Vtg in male carp due to possible exposure to water-contaminating endocrine disrupting chemicals. [Copyright &y& Elsevier]

Abstract: During reproduction vitellogenin (VTG) is transported to vitellogenic oocytes as a precursor of egg yolk lipovitellin (LV). As VTG synthesis is affected by environmental stressors, it is widely used as biomarker in endocrine disruption studies. However, it has seldom been employed to evaluate invertebrate hydrocarbon pollution. An ELISA with anti-LV antibody was developed to evaluate the impact of water-soluble fraction of crude oil (WSF) on Macrobrachium borellii vitellogenesis. Prawn VTG concentration was within the range reported for other crustaceans; LV values were positively correlated with gonadosomatic index (GSI). Females at different vitellogenic stages were exposed to a sub-lethal concentration of WSF for 7days. Exposed animals with GSI>7 increased their VTG and LV titer as compared to control organisms (190% and 140%, respectively). VTG levels in M. borellii were upregulated and highly sensitive to WSF exposure. This assay could be employed as a biomarker for freshwater hydrocarbon pollution. [Copyright &y& Elsevier]

Lipovitellin (Lv), a glycolipoprotein, is a major component of the egg yolk, which is usually regarded as an energy reserve of nutrients essential for growth and development. We have purified Lv from ovulated eggs of the rosy barb Puntius conchonius by two-step chromatography and characterized it by staining with periodic acid/Schiff reagent and Sudan black B, amino acid composition analysis, and peptide mass fingerprinting. The results of ligand and bacterial binding assays, an enzyme-linked immunosorbent assay (ELISA), and the phagocytosis test revealed, for the first time, that the purified native form of P. conchonius Lv acts as a pattern recognition molecule with multiple specificities capable of identifying pathogen-associated molecular patterns (PAMPs), including those of lipopolysaccharide, lipoteichoic acid, and peptidoglycan, rather than self components and that it can bind Gram-negative and -positive bacteria, such as Escherichia coli and Staphylococcus aureus. These tests also showed that the P. conchonius Lv functions as an opsonin capable of enhancing macrophage phagocytosis. Taken together, these characteristics suggest that in developing embryos/larvae of P. conchonius, the native form of Lv may be physiologically involved in the sensing of invading pathogens via interaction with PAMPs and in the recruitment of the primitive macrophages that appear in early embryos to phagocytose and digest the pathogens, thereby protecting them from pathogenic attacks. [ABSTRACT FROM AUTHOR]

Amino acids are vital for all living organisms. During early fish ontogeny, they are important fuel molecules, signalling factors and major substrates for the synthesis of a wide range of bioactive molecules and proteins. Because the majority of fish eggs are cleidoic, i.e. closed free-living systems following ovulation and activation, early development of fish depends on the maternal provision of amino acids during oogenesis. While more than 600 proteins have been identified in the growing oocytes of fish, the major vehicles for supplying amino acids to the growing oocyte before ovulation are the vitellogenins, of which many genes and multiple forms are known. Here we review the importance of amino acids for the intermediary metabolism of fish embryos and larvae, where amino acids have been shown to be the preferred catabolic substrate. Subsequently, we address the specialization of the lysosomal pathway involved in the uptake and degradation of yolk proteins. This latter pathway is specifically modified in the germline to facilitate the long-term storage of egg yolk proteins. In marine teleosts, the degradative pathway may be activated before fertilization during oocyte maturation to release free amino acids for oocyte hydration and the acquisition of egg buoyancy. In other species, including freshwater fish, a more latent activation of acid hydrolases occurs after fertilization during the four phases of yolk resorption. The developmental contributions of the yolk syncytial layer, vitelline circulation and liver are essential components of the amino acid supply during fish ontogeny. [ABSTRACT FROM AUTHOR]

Abstract: Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late). [Copyright &y& Elsevier]

Abstract: The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 µg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL1 and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs. [Copyright &y& Elsevier]

Abstract: To elucidate the utilization of major yolk nutrients in eggs and yolk-sac larvae of Japanese eel Anguilla japonica, contents of free amino acids (FAA), 380 kDa lipovitellin (oLv; the major yolk protein in ovulated eggs), and lipids were measured. All larvae hatched by the 2nd day after fertilization at a water temperature of 23 °C, and the hatched larvae absorbed almost all of the yolk mass by the 10th day. The FAA composition in ovulated eggs is unique in that glutamine (Gln) is markedly high (32% of total FAA) in comparison to other pelagic eggs of marine teleosts suggesting the importance of Gln for early development of the Japanese eel. Total FAA content decreased to 45% of the initial level by the 4th day after fertilization. Gln content showed a rapid decrease to 5% of the initial level within 2 days after fertilization. oLv content, measured by enzyme-linked immunosorbent assay using an antiserum against lipovitellin, decreased until the 4th day and was, thereafter, undetected. Total content of triacylglycerol (TG) was stable until the 2nd day, and then decreased until the 8th day. On the other hand, the content of phospholipids (PL) gradually decreased until the end of yolk sac absorption. From these data, we divided the pattern of nutrient utilization in embryos and larvae of Japanese eel into two phases: first, utilization of FAA (especially Gln) and oLv from the 1st day, followed by TG utilization from the 2nd to 4th day by which time more than 30% of the FAA and the oLv stock have been utilized. In addition, PL was utilized as a subsidiary energy source throughout development. Almost all the yolk nutrient were absorbed by the 8th day after fertilization, and it is considered that the larvae need to start feeding before this period. [Copyright &y& Elsevier]

Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate–polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species. [ABSTRACT FROM AUTHOR]

Vitellogenin (VTG) is the major protein present in the plasma of females undergoing oogenesis. In males, the VTG gene normally is suppressed; however, synthesis of VTG can be induced by exposure to xenoestrogenic compounds. In the present study, an enzyme-linked immunosorbent assay was developed and validated to evaluate VTG levels in the California halibut (Paralichthys californicus). Vitellogenin and lipovitellin (LV) were identified in the plasma of 17β-estradiol-induced females and in the ovaries of wild females, to our knowledge for the first time. Purified VTG from the plasma of induced females was obtained, and polyclonal antibodies against the LV of mature female ovaries was prepared and their specificity assessed by Western blot analysis. At Bahía Magdalena, Baja California Sur, México, quantitative measurements of VTG in the plasma of female specimens were made during one reproductive cycle. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a widespread disease caused by the deposition of lipids on arterial walls. Such lipid plaques in coronary arteries can be fatal. Although many factors related to diet, life-style, etc. contribute to the worsening of the ailment, the primary cause, the lipids in the circulatory system, come from a series of low-density lipoproteins. These lipoproteins are necessary for the transport of lipids to and from different organs. It would be valuable to medicine and the field of drug design if a more detailed understanding of the organization of lipid and protein in these molecules were available. Unfortunately because of heterogeneity in their size and lipid composition, all classes of the low-density serum lipoproteins appear to be not amenable to the most widely used method for obtaining detailed atomic data - X-ray crystallography. However there appears to be a recently identified homolog that is relatively homogeneous, and crystal structures have been obtained. Used as a molecular model, the homolog serves as a source of conformational information that might help to unravel the processes involved in the lipid loading of the low-density lipoproteins. The review attempts to give a brief summary of the structural biology of the serum low-density lipoproteins relative to the molecular model of lipovitellin. [ABSTRACT FROM AUTHOR]

To elucidate the utilization of the major yolk nutrient stocks in eggs and larvae of walleye pollock Theragra chalcogramma, the contents of free amino acids (FAA), the major yolk protein (180 kDa lipovitellin originated from vitellogenin B in ovulated eggs: oLv B), and lipids were measured. Most eggs hatched 18 days after fertilization at 5°C, and all larvae absorbed almost all their yolk mass by 28 days. The total FAA content showed no change during the first 6 days, and then decreased to 28% of the initial level by 18 days. The oLv B contents, measured by an enzyme-linked immunosorbent assay using a specific antiserum against oLv B, gradually decreased from 6 to 18 days, followed by a rapid decline. The content of phospholipids (PL) and triacylglycerols (TG) showed no marked change until hatching, and then decreased until disappearance of yolk sac. From these results, it is proposed that there are two main periods for nutrient utilization in embryos and larvae of walleye pollock. In the first period, FAA was mainly utilized until 18 days after fertilization. Active utilization of oLv B and lipids (PL and TG) instead of FAA occurred during the second period from 18 to 28 days. [ABSTRACT FROM AUTHOR]

Abstract: Embryos of the blue crab Callinectes sapidus develop in egg sacs carried on the abdomen of the female. They develop over a period of 10–13 days at 28 °C and are nutritionally dependent on yolk until they emerge from the egg sacs as free-swimming zoeae. The principal component of blue crab yolk is lipovitellin (LpII), a water-soluble lipoprotein composed of approximately equal amounts of lipid and protein. We followed changes in the concentration of apoproteins of LpII during embryogenesis by ELISA and Western blots, using monoclonal antibodies against two LpII apoprotein associated peptides identified as Protein A (107 kDa) and Protein B (75 kDa). During embryogenesis there was a decrease in Protein B but an increase in two smaller peptides (52 and 35 kDa) that reacted with the Protein B antibody. Utilization of LpII during embryogenesis was also followed morphologically by immunohistochemistry. Utilization of LpII was slow in early embryonic stages, followed by rapid utilization in late embryonic stages, such that only traces of LpII were present at the end of embryogenesis. The cells of the developing hepatopancreas appear to play an important role in the utilization of LpII. [Copyright &y& Elsevier]

Abstract: Two distinct yolk proteins (YP1 and YP2) were purified from the ovary of medaka, and specific antisera against YPs were generated to characterize YPs and reveal their relation to two vitellogenins (Vg1 and Vg2). The molecular masses of purified YP1 and YP2 on gel filtration were 270 and 380kDa, respectively. YPs were confirmed to be lipoproteins by staining with Sudan black. Amino acid compositions of YP1 and YP2 were similar to those of Vg1 and Vg2, respectively. In double immunodiffusion using anti-Vg1, a precipitin line of YP1 formed a spur against the Vg1 line. YP2 and Vg2 were reacted with anti-Vg2, and a precipitin line of YP2 formed a fuse against the Vg2 line. These biochemical and immunological analyses of purified YPs revealed that YP1 is lipovitellin 1 (Lv1) derived from Vg1 and YP2 is lipovitellin 2 (Lv2) derived from Vg2. Using specific antibodies against Lvs and Vgs, specific, high sensitivity chemiluminescent immunoassays (CLIAs) for two Vgs were developed to reveal basal Vg levels and response to exogenous estradiol-17β (E2). The measurable range of both CLIAs was from 0.975 to 1000ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (≦0.57%). When immature fish were immersed in water containing E2 for 1h, both Vgs were induced by 0.5μg/L of E2 at 24h after treatment. Vg1 increased in a concentration-dependant manner up to 100μg/L E2, while Vg2 reached a plateau at 10μg/L of E2. The ratio of Vg1:Vg2 in E2-treated fish changed in a concentration-dependent manner from 1.5:1 to 8.5:1. The results from E2-treatment suggest that differential regulation may control the expression of medaka Vgs. [Copyright &y& Elsevier]

Abstract: The ooplasm of mature oocytes of the polychaete Neanthes arenaceodentata is characteristically filled with yolk platelets. A major component of these structures is lipovitellin, which provides energy and materials required by newly hatched larvae. The lipovitellin isolated and purified from the fertilized eggs of this polychaete was a high-density lipoprotein composed of protein (57%), lipid (42%) and carbohydrate (1%). The lipid component included phospholipids (92% of lipid), triacylglycerol (3% of lipid) and cholesterol (3% of lipid), while sodium dodecyl sulfate-gel electrophoresis showed the major protein component was a 120-kDa peptide. Microscopically, mature oocytes were present in the coelom along with phagocytic eleocytes. The presence of muscle fragments and oil droplets in eleocytes suggests that eleocytes play an important role in providing the protein and lipid needed for the assembly of lipovitellin in the oocytes. [Copyright &y& Elsevier]

Confocal laser scanning microscopy (CLSM) represents a powerful, but largely unexplored ecotoxicologic tool for rapidly assessing in vivo effects of toxicants on marine invertebrate embryo quality and development. We describe here a new semiquantitative CLSM approach for assessing relative yolk quantity in marine invertebrate embryos (harpacticoid copepods) produced by parents reared from hatching to adult in the polycylic aromatic hydrocarbon chrysene. This method is based on fluorogenic labeling of embryo yolk and subsequent statistical analysis of areal pixel intensities over multiple Z-series using a general linear model (GLM)–nested analysis of variance. The fluorescent yolk-labeling method described here was able to detect statistically significant differences in yolk concentrations in marine copepod (Amphiascus tenuiremis) eggs or embryos from females exposed to ultraviolet light and chrysene-contaminated sediments. Yolk intensities in embryos from females cultured throughout their life cycles in clean sediments were statistically identical with or without UV exposure. In constrast, yolk intensities in embryos of females cultured throughout their life cycle in chrysene-contaminated sediments were significantly higher in the non-UV-exposed treatment with chrysene at 2500 ng/g sediment (65.7% higher) and the UV-exposed treatment with chrysene at 500 ng/g sediment (76.6% higher). [ABSTRACT FROM AUTHOR]

Vitellogenin (VTG) has been widely used as a biomarker of estrogenic exposure in fish, leading to the development of standardized assays for VTG quantification. However, standardized quantitative assays for invertebrate, particularly crustacean, lipovitellin (also known as vitellin [VTN]) are lacking. In this study, a fluorescence-based VTN enzyme-linked immunosorbent assay (ELISA) was developed to quantify microquantities of VTN in the estuarine, sediment-dwelling copepod Amphiascus tenuiremis. This ELISA utilizes a VTN-specific polyclonal antibody developed against amphipod (Leptocheirus plumulosus) embryo VTN and exhibits specificity toward female copepod proteins. In routine assays, the working range of the ELISA was 31.25 to 1,000 ng/ml (75-25% specific binding/maximum antibody binding [B/B0]) with a 50% B/B0 intra- and interassay variation of 3.9% (n = 9) and 12.5% (n = 26), respectively. This ELISA is capable of detecting VTN as low as 2 ng/ml, and can accurately detect VTN in as few as four copepods. The ELISA significantly discriminated positive (gravid female) and negative (male) samples, and was suitable for screening endocrine toxicity in copepods. Stage-I juvenile copepods were individually reared to adults in aqueous microvolumes of the phenylpyrazole insecticide, fipronil, and whole-body homogenate extracts were assayed for VTN levels. Fipronil-exposed virgin adult females, but not males, exhibited significantly higher levels of VTN relative to control males and females. This crustacean VTN ELISA is likely useful for evaluating endocrine activity of environmental toxicants in copepods and other crustacean species. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp. [Copyright &y& Elsevier]

Vitellogenin (Vg) is the precursor to egg yolk proteins of teleost fishes, including lipovitellin (Lv), phosvitin (Pv), and ß'-component (ß'-c). Complete Vg molecules contain the yolk protein domains arranged in linear fashion: NH2-Lv heavy chain, Pv, Lv light chain, ß'-c, C-terminal coding region (C-t)-COOH. Western blots employing an antiserum raised against a recombinant C-t polypeptide revealed that the C-t domain of Vg gives rise to a fourth yolk protein in barfin flounder oocytes. Three classes of Vg appear to exist in teleosts, including two types of complete Vg (A-type and B-type) and a smaller, incomplete Vg lacking a Pv domain (Pv-less type). By sequencing cDNA amplified from distinctive regions of each type of Vg transcript from several teleosts, we discovered that members of higher taxa (e.g.ParacanthopterygiiandAcanthopterygii) express both Vg A and Vg B, and that Pv-less Vg is widely present among teleosts. Vitellogenins A and B play distinct roles in regulation of egg buoyancy in barfin flounder through selective proteolysis of their product yolk proteins in oocytes undergoing final maturation. Protease specification procedures confirmed involvement of a cathepsin B-like enzyme in this maturation-associated proteolysis. Measurement of changing pH revealed that drastic acidification of the ooplasm accompanies hydrolysis of the yolk proteins. Thus, with respect to yolk protein hydrolysis and oocyte hydration, cytoplasmic maturation appears to be controlled by changing pH in maturing oocytes. [ABSTRACT FROM AUTHOR]

We developed an in vitro bioassay for oestrogenic compounds based on vitellogenin as a biomarker. To quantify the induction of vitellogenin in medaka (Oryzias latipes) we established a sandwich enzyme-linked immunosorbent assay (ELISA). This ELISA was carried out with two different monoclonal antibodies specific for the yolk precursor protein vitellogenin and the high molecular weight yolk proteins (lipovitellin) of medaka. Purified lipovitellin of medaka was used as a standard for the ELISA and could be quantified down to a concentration of 9 ng/ml. The calibration curve was linear up to at least 600 ng/ml and the intra-assay coefficient of variance was 3.2%. The application of the sandwich ELISA was tested using blood samples of males and females as well as primary hepatocyte cultures of medaka. Vitellogenin synthesis in cultured liver cells of males was induced by 1 and 100 nM of the xenoestrogen 17α-ethynylestradiol (EE2). The first production of vitellogenin was detected 6 days after hormone application. In contrast, isolated liver cells of sexually mature females, which were treated in the same manner, showed vitellogenin expression from the beginning of cultivation and its synthesis increased and remained high at 100 nM EE2. However, the induction of vitellogenin synthesis in male hepatocytes in vitro could be maintained for at least one month and this indicated viable and differentiated liver cells. The hepatocyte cultures of male medaka in combination with the sandwich ELISA were shown to be a suitable tool to detect and quantify oestrogenic activity of chemicals. [Copyright &y& Elsevier]

Retinoids in the eggs of four teleosts, chum salmon (Oncorhynchus keta), black porgy (Acanthopagrus schlegeli), marbled flounder (Pleuronectes yokohamae), and stingfish (Inimicus japonicus), were analyzed by high performance liquid chromatography. Retinal (RAL1) or both RAL1 and 3,4-didehydroretinal (RAL2) were major or exclusive retinoids in the eggs of every species examined. In O. keta eggs, both RAL1 and RAL2 were present at the ratio of approximately 3:4, whereas RAL1 was the only retinal in the eggs of the other three marine species. RAL1 was the exclusive retinoid in the eggs of P. yokohamae and I. japonicus, whose eggs lack lipid bodies. In the eggs of O. keta and A. schlegeli, which have lipid bodies, retinylesters were also detected, and retinals composed 69% and 93%, respectively, of total retinoids. In O. keta eggs, retinals were present mostly in the aqueous part and were bound to a protein homologous to lipovitellin 1, an amphibian yolk protein, and retinylesters were located in lipids. These results indicate that retinals are the essential mode of retinoid storage in eggs of teleosts and they are the precursors of functional retinoids, such as retinoic acid and visual pigment chromophores. Retinylesters are additional retinoids that accompany lipid accumulation. [Copyright &y& Elsevier]

Vitellogenin (Vg) and its corresponding yolk protein (YP) products, YP1, YP2 and YP3, were isolated from serum of estrogen-treated hybrid sturgeon (bester; Huso huso X Acipencer ruthenus) and eggs from untreated fish, respectively. Vitellogenin had an apparent molecular mass of 580 kDa and appeared as two major bands corresponding to 180 kDa and 120 kDa after SDS-PAGE. Apparent molecular weights of YP1, YP2 and YP3 were 370 kDa, 88 kDa and 19 kDa, respectively. After SDS-PAGE, YP1 appeared as a main band of 110 kDa, while YP2 was resolved as a single band of 94 kDa and 29 kDa band under non-reducing and reducing conditions, respectively. Yolk protein 3 appeared as a diffuse band corresponding to 16 kDa and two faint bands below 14.4 kDa after SDS-PAGE. However, the 16 kDa band alone was observed after dephosphorylation with alkaline phosphatase. The course of cleavage of yolk proteins in bester embryos and alevins was observed by SDS-PAGE and Western blotting from fertilization onward. After hatching, the main 110 kDa band of YP1 was degraded into smaller peptides during development, while YP2 hardly showed any such structural changes. The amino acid compositions of purified yolk proteins indicated that YP1, YP2 and YP3 were bester lipovitellin, β′-component, and phosvitin, respectively. [Copyright &y& Elsevier]

The present study was designed to quantify changes in serum vitellogenin (VTG) levels and to localize VTG in hepatic cells immunohistochemically during artificial maturation of the Japanese eel Anguilla japonica. Hormone treatment resulted in a gradual rise in serum VTG levels, in which the highest values were attained at the migratory nucleus stage. Hepatocytes responded to hormone treatment by increased immunoreactivity; both staining area and intensity were enhanced with ongoing gonadal development. Moreover, cytoplasmic vacuoles increased in number during artificial maturation, occupying large areas inside hepatocytes. The unusual appearance of hepatocytes may reflect the effect of artificial maturation. [ABSTRACT FROM AUTHOR]

We have cloned and sequenced a cDNA encoding a vitellogenin (Vtg) from the mummichog, Fundulus heteroclitus, an estuarine teleost. We constructed a liver cDNA library against RNA from estrogen-treated male mummichogs. Five overlapping cDNA clones totalling 5,197 by were isolated through a combination of degenerate oligonucleotide probing of the library and PCR. The cDNA sequence contains a 5,112 by open reading frame. The predicted primary structure of the deduced 1,704-amino-acid protein is 30-40% identical to other documented chordate Vtgs, establishing this Vtg as a member of the ancient Vtg gene family. Of the previously reported chordate Vtg sequences ( Xenopus laevis, Gallus domesticus, Ichthyomyzon unicuspis, and Acipenser transmontanus), all four act as precursor proteins to a yolk which is eventually rendered insoluble under physiological conditions, either as crystalline platelets or as noncrystalline granules. The yolk of F. heteroclitus, on the other hand, remains in a soluble state throughout oocyte growth. The putative F. heteroclitus Vtg contains a polyserine region with a relative serine composition that is 10-20% higher than that observed for the other Vtgs. The trinucleotide repeats encoding the characteristic polyserine tracts of the phosvitin region follow a previously reported trend: TCX codons on the 5′ end and AGY codons toward the 3′ end. Whether the difference in Vtg primary structure between F. heteroclitus and that of other chordates is responsible for the differences in yolk structure remains to be elucidated. As the first complete teleost Vtg to be reported, these data will aid in designing nucleotide and immunological probes for detecting Vtg as a reproductive status indicator in F. heteroclitus and other piscine species. [ABSTRACT FROM AUTHOR]

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age. [ABSTRACT FROM AUTHOR]