Short-term induction of vitellogenesis in the immature male yellowfin seabream ( Acanthopagrus latus) exposed to bisphenol A and 17 ß-estradiol.

Bisphenol A (BPA) is a known environmental endocrine-disrupting chemical that is widely used in plastics manufacturing. BPA enters in the aquatic environment mainly through urban and industrial sewage effluents, thereby posing a potential threat to organisms living in these ecosystems. This study was conducted to investigate the effect of BPA on VTG production with direct (sodium dodecyl sulfate-polyarylamide gel electrophoresis) and indirect (alkali-labile phosphate (ALP), total plasma calcium and protein) methods in immature male yellowfin seabream ( Acanthopagrus latus) as a marine fish model. Fish were randomly distributed into seven groups that were administered 1, 10, 50, and 100 µg g-1 week-1 of BPA and 2 µg g-1week-1 of 17ß-estradiol (E2) over a period of 2 weeks. Solvent controls received olive oil, whereas controls were not injected. The fish were sampled on days 0, 7, and 14, and their blood plasma and liver were obtained. The results showed that the hepatosomatic index of all treated fish was elevated in comparison with controls. Direct and indirect indicators showed that fish VTG protein was induced by BPA and E2 exposure. The protein was found to have two bands with molecular weights around 210 and 190 KDa. ALP, total plasma calcium and protein levels were increased in dose- and time-dependent manners. The results of this study demonstrated that short-term exposure of yellowfin seabream to BPA induced adverse effects in the reproductive system of hermaphrodite fish.

Purification and Characterization of Vitellin from the Egg of the Suminoe Oyster Crassostrea ariakensis and Cross-Reactivity of Anti-vitellin Antibody with Other Marine Invertebrate Egg Proteins.

A polyclonal antibody specific to an egg protein of Suminoe oyster Crassostrea ariakensis was previously developed in our laboratory to assess the reproductive life cycle of the oyster. The present study was undertaken to investigate vitellin of C. ariakensis (CAVt). Vitellin is an essential component of egg proteins in marine invertebrates as it provides energy and nutrients to the embryo and larvae. CAVt was purified from eggs of the oyster using ammonium sulfate precipitation followed by affinity chromatography with Concanavalin A-agarose. Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE showed that CAVt is a high molecular weight [532 kiloDaltons (kDa)] protein, with multiple subunits. Similar to other vitellin proteins, it is a phospholipoglycoprotein composed of phospholipids (12.06%), carbohydrates (mannose, 10.08% or glucose, 9.84%), and alkali-labile phosphates (4.16%). Affinity chromatography, enzyme-linked immunosorbent aasay (ELISA) and western blot analysis revealed that CAVt is only present in the ovary, and two subunits of CAVt (72 and 35 kDa) are believed to be incorporated from the hemolymph into the oocyte. The antibody specific to CAVt (anti-CAVt), raised in rabbit, strongly cross reacted with the egg proteins of oyster species and scallops, suggesting that the antigenic epitopes are highly conserved among species. Our results suggest that the anti-CAVt antibody can be used to develop a tool similar to ELISA or western blotting for investigation of the effect of microorganisms on reproduction as well as the effect of chemicals on the endocrine system in C. ariakensis.

The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease.

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.

Purification of vitellin and dynamics of vitellogenesis in the parthenogenetic tick Haemaphysalis longicornis (Acari: Ixodidae).

Vitellin (Vt) was purified from eggs of parthenogenetic bush tick Haemaphysalis longicornis by gel filtration and ion exchange chromatography. Our results revealed that only one single Vt existed in parthenogenetic bush tick, and the purified Vt was proved to be a hemoglycolipoprotein consisting of nine polypeptides with molecular weights of 203, 147, 126, 82, 74, 70, 61, 47 and 31 kDa, respectively. Polyclonal antibody and monoclonal antibody against Vt were produced using the purified Vt. The change in vitellogenin (Vg) and Vt levels over time of the parthenogenetic H. longicornis was established, and the Vg content in haemolymph and Vt in ovary at different feeding or engorgement statuses was also determined using a double antibody sandwich enzyme-linked immunosorbent assay. The Vg level in haemolymph was distinctly increased on the day of engorgement (1.785 mg/mL) and continued to increase until 2nd day post-engorgement (5.611 mg/mL). There was a slight decrease in Vg level after 4 days of engorgement, and a second peak was observed on day 2 post-oviposition (10.774 mg/mL). Subsequently, Vg content continuously decreased and reached a low level on the 10th day post-oviposition. The Vt content in ovary continuously increased once the female reached its critical weight (0.024 mg per female), and reached the maximum level on day 2 post-oviposition (1.942 mg per female). Afterwards, Vt content rapidly decreased.

Agglutinating activity and structural characterization of scalarin, the major egg protein of the snail Pomacea scalaris (d'Orbigny, 1832).

Apple snail perivitellins are emerging as ecologically important reproductive proteins. To elucidate if the protective functions of the egg proteins of Pomacea canaliculata (Caenogastropoda, Ampullariidae), involved in embryo defenses, are present in other Pomacea species we studied scalarin (PsSC), the major perivitellin of Pomacea scalaris. Using small angle X-ray scattering, fluorescence and absorption spectroscopy and biochemical methods, we analyzed PsSC structural stability, agglutinating activity, sugar specificity and protease resistance. PsSC aggluttinated rabbit, and, to a lesser extent, human B and A erythrocytes independently of divalent metals Ca(2+) and Mg(2+) were strongly inhibited by galactosamine and glucosamine. The protein was structurally stable between pH 2.0 to 10.0, though agglutination occurred only between pH 4.0 to 8.0 (maximum activity at pH 7.0). The agglutinating activity was conserved up to 60 °C and completely lost above 80 °C, in agreement with the structural thermal stability of the protein (up to 60 °C). PsSC was able to withstand in vitro gastrointestinal digestion, and showed no trypsin inhibition activity. The presence of lectin activity has been reported in eggs of other Pomacea snails, but here we link for the first time, this activity to an apple snail multifunctional perivitellin. This novel role for a snail egg storage protein is different from closely related P.canaliculata defensive proteins.

Ultrastructural study of vitellogenesis in Maritrema feliui (Digenea, Microphallidae).

During vitellogenesis in the microphallid trematode Maritrema feliui, we distinguished four stages: (I) a stem cell stage of the gonial type; (II) an early differentiation stage with the main cell activity concentrated on the initiation of protein synthetic activity and the beginning of shell globule formation; (III) an advanced differentiation stage concentrated on a rapid intensification of protein synthetic activity, the progressive fusion of individual shell globules into large shell globule clusters and the formation of saturated lipid droplets and a small amount of ß-glycogen particles in the peripheral cytoplasm, considered as a store of nutritive reserves for the developing embryos; and (IV) the mature vitellocyte. Early vitellocyte maturation is characterised by: (1) an increase in cell volume; (2) extensive development of large, labyrinth-like cisternae of GER that produce proteinaceous granules; (3) the development of Golgi complexes engaged in packaging this material; and (4) a continuous enlargement of proteinaceous granules within vacuoles and their transformation into shell globule clusters composed of the heterogeneous material observed during vitellocyte cytodifferentiation. Mature vitelline cells are very rich in two types of cell inclusions accumulated in large amounts in their cytoplasm: (1) shell globule clusters, which play an important role in eggshell formation; and (2) a few osmiophobic lipid droplets of a saturated nature that undoubtedly represent nutritive reserves for the developing embryos. In addition, there are small numbers of ß-glycogen particles in the peripheral cytoplasm of mature vitellocytes of this species. The general pattern and ultrastructure of vitellogenesis in M. feliui greatly resembles those observed in another microphallid trematode, Maritrema linguilla, in other digeneans and in some lower cestodes. Quantitative and qualitative variations in lipids (saturated and unsaturated) and glycogen (α-glycogen rosettes and ß-glycogen particles) during platyhelminth vitellogenesis between the different species of trematodes and some lower cestodes are identified and discussed.

Isolation and characterization of two vitellins from eggs of the spider Polybetes pythagoricus (Araneae: Sparassidae).

Despite vitellins being essential yolk proteins, their presence in spiders remains almost unknown. Two vitellins from the spider Polybetes pythagoricus, named LV1 and LV2, were isolated and their size, shape, lipids, fatty acids, proteins and carbohydrates moieties were determined. LV1 has a density similar to that of HDL with 49.3% lipids, and LV2 has a density similar to that of VHDL with 9.7% lipids. The major neutral lipid present in both vitellins was found to be esterified cholesterol, 16% for LV1 and 24% for LV2. The major fatty acid was 18:1n-9 in LV1 and LV2. Results from native PAGE showed a lipoprotein of 550 kDa for LV1 and three lipoproteins of 571, 400 and 257 kDa for LV2. SDS-PAGE evidenced two major apolipoproteins of 64 and 25 kDa in LV1. The three lipoproteins of LV2 were electroeluted and analyzed by SDS-PAGE, showing different proportions of the same apolipoproteins (181, 67 and 60 kDa). LVs were analyzed by spectrophotometry, immunochemical and electron microscopy, showing that the respiratory pigment hemocyanin was not present as apolipoprotein. This fact evidenced that these LVs were not related to hemolymphatic lipoproteins.

Carbohydrates and glycoforms of the major egg perivitellins from Pomacea apple snails (Architaenioglossa: Ampullariidae).

To better understand how glycans contribute to the multiple roles of perivitellins in embryo development, the carbohydrate moieties and glycoforms of the carotenoglycoproteins ovorubin and scalarin from the eggs of Pomaceacanaliculata (Lamarck, 1822) and Pomaceascalaris (d'Orbigny, 1835) were studied. All subunits of both proteins are glycosylated and appear to be glycoforms with isoelectric points ranging from approximately 5.3 to approximately 9.1. Complete deglycosylation reduced subunit heterogeneity to spots of similar molecular weight (approximately 27 and approximately 25 kDa in scalarin and ovorubin, respectively) but with varying IP. Serine phosphorylation, present in both perivitellins, explains part of the isoforms. Glycosylation patterns of scalarin were determined using biotinylated lectins, PNGaseF treatment and selective chemical deglycosylation, which revealed the presence of hybrid and oligomannose N-linked glycans in all subunits. Scalarin has terminal sialic acid residues possibly resistant to neuraminidase and O-linked residues derived from the T- and Tn antigens. Ovorubin displayed predominantly the same glycans, though in different amounts. Capillary gas chromatography (GC) showed galactose and mannose as the major monosaccharides followed by GlcNAc and fucose. An interesting feature was the important amount of sialylated and fucosylated structures found in both perivitellins determined by GC, spectroscopy and lectins. This is the first report of these structures in gastropods other than heterobranchs.

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae).

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476+/-14.5 kDa as compared to 590+/-25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.

Devitellinization of living Helobdella (leech) embryos.

Embryos of glossiphoniid leeches are enclosed in a thin vitelline envelope until "hatching" (stage 10). This protocol describes the procedure for removing the vitelline envelope from Helobdella robusta and H. sp. (Austin) embryos. This protocol is applicable to embryos of stages 1-9 but is probably more useful for early stages. With careful culturing, the devitellinized embryos can develop normally.

In situ hybridization of Helobdella (leech) embryos.

The purpose of this protocol is to reveal the localization of transcripts in the Helobdella (leech) embryo. In situ hybridization protocols for Helobdella embryos are derived from those used for zebrafish and Xenopus. The protocols are different for early- and late-stage embryos (stages 1-8 and stages 9-11, respectively) due to a difference in tissue permeability.

Vitellin of Pteromalus puparum (Hymenoptera: Pteromalidae), a pupal endoparasitoid of Pieris rapae (Lepidoptera: Pieridae): Biochemical characterization, temporal patterns of production and degradation.

Vitellin (Vt) and vitellogenin (Vg) profiles were analyzed in Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. Non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that both native Vt and Vg were likely 370 kDa in size, consisting of two subunits of approximate 206 and 165 kDa. An indirect double antibody enzyme-linked immunosorbent assay (ELISA) for monitoring hemolymph Vg and ovarian Vt levels was developed using a monoclonal antibody and a polyclonal antibody made specially against P. puparum Vt. The synthesis and uptake of Vg in this wasp was initiated immediately after adult eclosion. The hemolymph Vg and ovarian Vt reached their highest level of 0.58 and 4.51 microg per female 24 and 48 h after adult eclosion, respectively. Both Vg synthesis and uptake were in parallel with ovarian development. The Vt levels in the developing embryos decreased progressively except 12h after parasitism. Meanwhile, nine new polypeptides with sizes ranging from 59.2 to 151 kDa, possibly resulting from the limited proteolysis of Vt originally accumulated in newly laid eggs, were detected de-novo during embryonic development using Western blotting with the monoclonal antibody against Vt. These studies provide the basis for future investigation into endocrinal regulations of vitellogenesis and understanding the reproductive strategy in this wasp.

Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.

Molecular cloning and characterization of vitelline precursor protein B1 from Clonorchis sinensis.

In trematodes, vitelline precursor proteins are required for eggshell formation. A cDNA clone of Clonorchis sinensis (CsVpB1) was selected from an EST pool, encoding a polypeptide of 245 amino acids. The CsVpB1 polypeptide demonstrated homology with vitelline precursor proteins from trematodes with high sequential identities. In a phylogenic tree, CsVpB1 clustered with trematode VpB proteins. The CsVpB1 polypeptide was found to be rich in tyrosine residues, including putative predihydroxyphenyl alanine (DOPA) residues, involved in cross-linking of the precursor proteins. Mouse immune sera were raised against a recombinant CsVpB1 protein. In adult C. sinensis, CsVpB1 protein was exclusively localized in vitelline follicles. Based on these results, the CsVpB1 cDNA is believed to encode a VpB of C. sinensis.

Purification and characterization of vitellin from the estuarine mysid Neomysis integer (Crustacea; Mysidacea).

Invertebrates account for roughly 95% of all animals, yet surprisingly, little effort has been invested to understand their value in signaling potential environmental endocrine disruption. There has been, however, much recent attention on vitellogenin induction in egg-laying invertebrates and vertebrates as indicators of exposure to estrogenic xenobiotics. Mysid shrimp (Crustacea: Mysidacea) have been put forward by several researchers and regulatory bodies (e.g., US-EPA) as suitable test organisms for the evaluation of environmental endocrine disruption. In view of developing sensitive assays to study endocrine disruption in the estuarine mysid Neomysis integer, we isolated and characterized vitellin, the major yolk protein in eggs. Vitellin was purified using gel filtration and characterized by electrophoresis using different staining procedures. Specific (as shown by Western blotting) polyclonal antibodies were produced in rabbit against the purified vitellin of N. integer. These antisera will be used to develop immunoassays to study vitellogenesis in mysids and to detect potential stimulatory or inhibitory effects of endocrine disruptors on the production of vitellin.