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.

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.

Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea).

Mysid crustaceans have been put forward by several regulatory bodies as suitable test organisms to screen and test the potential effects of environmental endocrine disruptors. Despite the well-established use of mysid reproductive endpoints such as fecundity, egg development time, and time to first brood release in standard toxicity testing, little information exists on the hormonal regulation of these processes. Control of vitellogenesis is being studied intensively because yolk is an excellent model for studying mechanisms of hormonal control, and vitellogenesis can be chemically disrupted. Yolk protein or vitellin is a major source of nourishment during embryonic development of ovigorous egg-laying invertebrates. The accumulation of vitellin during oocyte development is vital for the production of viable offspring. In this context, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for vitellin of the estuarine mysid Neomysis integer. Mysid vitellin was isolated using gel filtration, and the purified vitellin was used to raise polyclonal antibodies. The ELISA was sensitive within a working range of 4 to 500 ng vitellin/mL. Serial dilutions of whole body homogenates from female N. integer and the vitellin standard showed parallel binding curves, validating the specificity of the ELISA. The intra- and interassay coefficients of variation were 8.2% and 13.8%, respectively. Mysid vitellin concentrations were determined from ovigorous females and eggs at different developmental stages. The availability of a quantitative mysid vitellin ELISA should stimulate further studies on the basic biology of this process in mysids. Furthermore, it could provide a means to better understand and predict chemically induced reproductive effects in mysids.

Yolk protein profiles of three prawn (Macrobrachium) species during reproductive cycle.

Ovarian yolk protein (vitellin) of adult freshwater prawns Macrobrachium malcolmsonii, M. rosenbergii and M. lamarreii was characterized, and relationship between the gonadosomatic index (GSI) and the appearance of specific polypeptides in their ovary during different stages (early-mature, mature and spent) of reproductive cycle was studied. Mature ovaries of all the three species showed increased GSI values, compared to the respective early-mature ovaries. Protein profiles at the three stages of ovarian maturation were analysed by SDS-PAGE. Polypeptides of low molecular mass (20 to 70 kDa) were prominent in the early-mature ovary, whereas in mature ovary, 89 and 100 kDa polypeptides were predominant. Immunodiffusion studies, using antiserum raised against purified vitellin from mature ovary of M. malcolmsonii indicated antigenic similarities of vitellins among the three species.

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.

Purification of vitellin from the ovary of Chinese mitten-handed crab (Eriocheir sinensis) and development of an antivitellin ELISA.

Vitellin was purified from ovaries of mature female Chinese mitten-handed crab (Eriocheir sinensis) using gel filtration chromatography. Analysis by native PAGE showed the vitellin had a native molecular mass of 520 kDa, while denaturing SDS-PAGE revealed two subunits of 97 and 74 kDa. Purified vitellin was used to raise polyclonal antisera, with which an enzyme-linked immunosorbent assay (ELISA) was developed. The ELISA was sensitive and could effectively detect vitellin in the range of 7.8-500 ng. Furthermore, vitellin levels in various developmental stages of oogenesis were measured with the ELISA assay. The results indicated that levels of vitellin increased significantly from 0.22 mg/ovary at Stage II to 360.31 mg/ovary at Stage IV.