Major Yolk Protein Precursor Research Articles

Identification of two cationic amino acid transporters required for nutritional signaling during mosquito reproduction

The defining characteristic of anautogenous mosquitoes is their requirement for a blood meal to initiate reproduction. The need for blood drives the association of vector and host, and is the primary reason why anautogenous mosquitoes are effective disease vectors. During mosquito vitellogenesis, a key process in reproduction, yolk protein precursor (YPP) gene expression is activated specifically in the fat body, the insect analogue of the vertebrate liver. We have demonstrated that blood meal derived amino acids (AAs) activate YPP genes via the target of rapamycin (TOR)-signal transduction pathway. Here we show, by stimulating fat bodies with balanced AA solutions lacking individual AAs, that specific cationic and branched AAs are essential for activation of the vitellogenin (vg) gene, the major YPP gene. Treatment of fat bodies with AA uptake inhibitors results in a strong inhibition of AA-induced vg gene expression proving that an active transport mechanism is necessary to transduce the AA signal. We identified two cationic AA transporters (CATs) in the fat body of Aedes aegypti females--Aa slimfast and iCAT2. RNAi knockdown of slimfast and iCAT2 results in a strong decrease in the response to AAs by the vg gene similar to that seen due to TOR inhibition. These data demonstrate that active uptake of specific AAs plays a key role in nutritional signaling during the onset of vitellogenic gene expression in mosquitoes and it is mediated by two cationic AA transporters.

Effects of cyclic nucleotides, calcium ionophore, and phorbol ester on vitellogenin mRNA levels in incubated ovarian fragments of the kuruma prawn Marsupenaeus japonicus

In crustaceans, vitellogenin (VTG, the precursor of major yolk protein) is synthesized in the ovary and/or hepatopancreas, and its synthesis is considered to be under the negative control of the vitellogenesis-inhibiting hormone (VIH), a neuropeptide secreted from the X-organ/sinus gland complex in the eyestalks. In the present study, the effects of pharmacological agents on VTG mRNA levels in incubated ovarian fragments of the kuruma prawn Marsupenaeus japonicus were examined to determine the intracellular signalling pathways for VTG synthesis. After 24 h incubation, A23187 (calcium ionophore), dibutyl-cAMP (cAMP analogue), dibutyl-cGMP (cGMP analogue), forskolin (adenylate cyclase activator), 3-isobutyl-1-methylxanthine (IBMX, phosphodiesterase inhibitor), and phorbol 12-myristate 13-acetate (PMA, protein kinase C activator) decreased VTG mRNA levels in the ovarian fragments. This result suggests that cyclic nucleotides, Ca 2+, and protein kinase C are involved in the signalling pathways for the regulation of VTG mRNA levels in the ovaries. Furthermore, the inhibitory effects of sinus gland extract and the pharmacological agents on VTG mRNA were larger in previtellogenic ovaries than in vitellogenic ovaries. This result suggests that the degree of responsiveness to VIH changes during ovarian development and that the changes in responsiveness to VIH involve maturity-related changes in cellular signalling mechanisms in the ovaries.

GATA Factor Translation Is the Final Downstream Step in the Amino Acid/Target-of-Rapamycin-mediated Vitellogenin Gene Expression in the Anautogenous Mosquito Aedes aegypti

Ingestion of blood is required for vector mosquitoes to initiate reproductive cycles determining their role as vectors of devastating human diseases. Nutritional signaling plays a pivotal role in regulating mosquito reproduction. Transcription of yolk protein precursor genes is repressed until mosquitoes take blood. Previously, we have shown that to signal the presence of blood in the gut, mosquitoes utilize the target-of-rapamycin (TOR) pathway. The TOR signaling pathway transduces the amino acid signal activating the major yolk protein precursor gene, vitellogenin (Vg). Here we report the identification of a GATA factor (AaGATAa) that is synthesized after a blood meal and acts as a transcriptional activator of Vg. We showed that AaGATAa bound specifically to GATA-binding sites present in the proximal promoter region of the Vg gene and positively regulated Vg expression in transfection assays. RNA interference-mediated knock down of AaGATAa transcript resulted in a significant inhibition of Vg expression in both fat-body tissue culture and blood-fed mosquitoes. AaGATAa mRNA accumulated in the fat body prior to blood feeding. However, translation of GATA was activated by blood feeding because the GATA protein increased dramatically in the fat body of blood-fed mosquitoes. This increase was also reproduced in the fat-body culture stimulated with amino acids. GATA translation was inhibited by rapamycin and cycloheximide as well as by RNA interference-mediated knock down of S6 kinase. These experiments have revealed that the TOR signaling pathway induced by nutritional signaling regulates the translation of a GATA factor, which is the specific transcriptional activator of the Vg gene.

Cloning and characterization of vitellogenin cDNA from the common Japanese conger ( Conger myriaster) and vitellogenin gene expression during ovarian development

The major yolk protein precursor, vitellogenin (VTG) was detected in plasma from vitellogenic females and estradiol-17β (E 2)-treated immature females, but not in males and immature females by Western blotting in common Japanese conger Conger myriaster. Its molecular mass was approximately 180 kDa under denaturing and reducing conditions. The common Japanese conger VTG cDNA was cloned from the liver of vitellogenic female. It contains 5110 nucleotides including an open reading frame that encodes 1663 amino acids. The deduced amino acid sequence of the common Japanese conger VTG shares 80% identity with that of eel Anguilla japonica VTG-1, and 45–55%, 32–34% and 27–29% identity with the deduced amino acid sequences of other fish, amphibian and avian VTG with polyserin domain, respectively. In female common Japanese conger, VTG gene was highly expressed in the liver of this species similar with other oviparous vertebrates. The expression levels of VTG gene in the liver increased from the oil droplet stage to the tertiary yolk globule stage and were maintained until the migratory nucleus stage.

The early gene Broad is involved in the ecdysteroid hierarchy governing vitellogenesis of the mosquito Aedes aegypti

The broad (br) gene, encoding a family of C2H2 type zinc-finger DNA-binding proteins, has been shown to act as a crucial member of the 20-hydroxyecdysone (20E) regulatory hierarchy in the fruitfly, Drosophila melanogaster and the moth, Manduca sexta. In this study, we have shown that the br gene is involved in the 20E-regulatory hierarchy controlling vitellogenesis in the mosquito, Aedes aegypti. Unlike E74 and E75 early genes, expression of br was activated in previtellogenic females, during a juvenile hormone (JH)-dependent period. The levels of Z1, Z2 and Z4 isoform mRNA were elevated in the fat body of 2-day-old females after in vitro exposure to JH III. However, JH III repressed 20E activation of br in 3-to 5-day-old females, indicating a switch in hormonal commitment. Expression of Z1, Z2 and Z4 was stimulated after blood feeding in both vitellogenic tissues, the fat body and the ovary, corresponding to peaks of ecdysteroid titers. In the fat body, the mRNA profiles of these three isoforms correlated well with those of yolk protein precursor (YPP) genes. These BR isoforms were activated by 20E in fat bodies cultured in vitro and behaved as early genes, with a self-repressive autoregulatory loop that can be blocked by the protein inhibitor, cyclohexamide. Multiple binding sites for all four BR isoforms were present in the 5'-regulatory region of the major YPP gene, vitellogenin (Vg). Effects of BR isoforms on the expression of Vg have been demonstrated by cell transfection analysis. In particular, BR isoforms by themselves had no effects on the Vg promoter. However, Z1 and Z4 each repressed Aedes aegypti ecdysone receptor (EcR)/Ultraspiracle (USP)-mediated 20E activation of the Vg promoter, while Z2 enhanced activation of the Vg promoter by AaEcR/AaUSP in the presence of 20E. Z3 had no obvious effect in the same experiment. These results suggested that BR isoforms are essential for proper activation and termination of the Vg gene in response to 20E. Overall, our study implicated br in the regulation of mosquito vitellogenesis.

CREB isoform represses yolk protein gene expression in the mosquito fat body

In mosquitoes, the steroid 20-hydroxyecdysone (20E) is the main regulator of yolk protein precursor (YPP) gene expression. However, peptide hormones have also been implicated. To investigate involvement of the cAMP-mediated signal-transduction cascade in regulation of mosquito vitellogenic events, we cloned an Aedes aegypti cAMP response element binding protein (AaCREB). The AaCREB contained the domains characteristic to members of the cAMP response element binding protein (CREB) family of transcription factors: a kinase inducible domain region and a bZIP domain responsible for DNA binding and protein dimerization. In the mosquito fat body (site of YPP gene expression), the AaCREB gene was constitutively expressed and produced a transcript of 3.5–4 kb. In vitro fat body organ culture experiments demonstrated that elicitors of the cAMP signal-transduction pathway attenuated 20E-stimulated YPP gene expression. Cell transfection analysis indicated that AaCREB served as a potent repressor of transcription (designated AaCREBr). The role of AaCREBr as a transcriptional repressor supported the electrophoretic mobility shift assay (EMSA) with nuclear extracts from vitellogenic fat bodies. This analysis detected CREB-specific band-shift complexes in nuclear extracts at 24 and 36 h post-blood meal (PBM), when YPP gene expression reaches its peak then terminates. Examination of the regulatory regions of two major YPP genes, vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP), revealed the presence of putative CREB response elements (CREs). These elements competed with the CRE consensus sequence for binding of in vitro-expressed AaCREBr. We propose that AaCREBr functions as a repressor of YPP gene expression at the time of vitellogenesis termination in the fat body.

The major yolk proteins of higher Diptera are homologs of a class of minor yolk proteins in lepidoptera.

In most oviparous animals, including insects, vitellogenin (Vg) is the major yolk protein precursor. However, in the higher Diptera (cyclorrhaphan flies), a class of proteins homologous to lipoprotein lipases called yolk polypeptides (YP) are accumulated by oocytes instead of Vg, which is not produced at all. Lepidopterans (moths) produce Vg as the major yolk protein precursor, but also manufacture a class of minor yolk proteins referred to as egg-specific proteins (ESP) or YP2s. Although the lepidopteran ESP/YP2s are related to lipoprotein lipases, previous attempts to directly demonstrate their homology with higher-dipteran YPs were unsuccessful. In this paper, a multiple alignment of amino acid sequences was constructed using a shared lipid binding motif as an anchor, to demonstrate that lepidopteran ESP/YP2s, higher-dipteran YPs, and lipoprotein lipases are indeed homologous. Phylogenetic analyses of the aligned sequences were performed using both distance-based and parsimony strategies. It is apparent that the higher dipterans did not requisition a lipoprotein lipase to replace Vg as a yolk protein precursor, but instead utilize a class of proteins with an evolutionary history of use as minor constituents of yolk in other insects.

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins.

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.

Molecular characterization of putative yolk processing enzymes and their expression during oogenesis and embryogenesis in rainbow trout (Oncorhynchus mykiss).

Vitellogenin is the major yolk protein precursor in fish, but little is known about its processing pathway in the oocyte, nor about mobilization of yolk proteins during embryogenesis. In this study we cloned three putative yolk processing enzymes; specifically, cathepsin B and L, and lipoprotein lipase (LPL), from the rainbow trout ovary and determined their patterns of gene expression, together with cathepsin D, during oogenesis and embryogenesis using reverse transcription-polymerase chain reaction. The approximate sizes of both cathepsin B and cathepsin L transcripts were estimated as 1.7-1.8 kilobases by Northern blot analysis. Cathepsin D mRNA and cathepsin L mRNA were expressed constitutively throughout vitellogenesis and embryogenesis, showing the highest levels of expression at around fertilization. Cathepsin B and LPL were expressed exclusively during oogenesis. Quantitatively, expression of cathepsin D mRNA was higher than cathepsin B, cathepsin L, and LPL mRNA throughout the period studied. The different patterns of expression for these genes during oogenesis and embryogenesis signify specific temporal roles in yolk protein processing.

Cloning of cDNA encoding vitellogenin and its expression in red sea urchin, Pseudocentrotus depressus

Both male and female red sea urchins, Pseudocentrotus depressus, accumulate a large quantity of the major yolk protein (MYP) in the nutritive phagocytes of immature gonads before the initiation of game-togenesis. To examine the accumulation mechanism of this protein in the gonad, we cloned full-length cDNA encoding vitellogenin (Vg; the MYP precursor in the coelomic fluid), and investigated its expression in various tissues of immature adults. The nucleotide sequence of Vg contains an open reading frame of 4050 bp encoding 1349 amino acids. The deduced amino acid sequence near the N-terminal showed 25% homology to the vertebrate transferrin family. Vitellogenin mRNA was detected in the ovary, testis, stomach, intestine and rectum by Northern blot analysis, with the highest level of mRNA expression in the gonad. Weak expression was also detected in the esophagus and coelomocytes by RT-PCR. In situ hybridization demonstrated that nutritive phagocytes, which exclusively fill the lumina of the immature gonad, contained Vg mRNA. These results suggested that the MYP stored in the immature gonads is synthesized and accumulated mainly within the nutritive phagocytes.

Molecular characteristics of insect vitellogenins and vitellogenin receptors

The recent cloning and sequencing of several insect vitellogenins (Vg), the major yolk protein precursor of most oviparous animals, and the mosquito Vg receptor (VgR) has brought the study of insect vitellogenesis to a new plane. Insect Vgs are homologous to nematode and vertebrate Vgs. All but one of the insect Vgs for which we know the primary structure are cleaved into two subunits at a site [(R/K)X(R/K)R or RXXR with an adjacent β-turn] recognized by subtilisin-like proprotein convertases. In four of the Vgs, the cleavage site is near the N-terminus, but in one insect species, it is near the C-terminus of the Vg precursor. Multiple alignments of these Vg sequences indicate that the variation in cleavage location has not arisen through exon shuffling, but through local modifications of the amino acid sequences. A wasp Vg precursor is not cleaved, apparently because the sequence at the presumed ancestral cleavage site has been mutated from RXRR to LYRR and is no longer recognized by convertases. Some insect Vgs contain polyserine domains which are reminiscent of, but not homologous to, the phosvitin domain in vertebrate Vgs. The sequence of the mosquito VgR revealed that it is a member of the low-density lipoprotein receptor (LDLR) family. Though resembling chicken and frog VgRs, which are also members of the LDLR family, it is twice as big, carrying two clusters of cysteine-rich complement-type (Class A) repeats (implicated in ligand-binding) instead of one like vertebrate VgRs and LDLRs. It is very similar in sequence and domain arrangement to the Drosophila yolk protein receptor (YPR), despite a non-vitellogenin ligand for the latter. Though vertebrate VgRs, insect VgR/YPRs, and LDLR-related proteins/megalins all accommodate one cluster of eight Class A repeats, fingerprint analysis of the repeats in these clusters indicate they are not directly homologous with one another, but have undergone differing histories of duplications, deletions, and exon shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.

Juvenile hormone acid: evidence for a hormonal function in induction of vitellogenin in larvae of Manduca sexta.

In the tobacco hornworm (Manduca sexta), vitellogenin (Vg), the major yolk protein precursor, and its mRNA are first detectable in the prepupal stage; and production of both can be enhanced by methoprene, a juvenile hormone (JH) analog. Competence to respond to methoprene is acquired after ecdysteroid-initiated commitment for metamorphosis. Here we show that acquisition of competence requires prior exposure to JH-II acid in addition to ecdysteroid. Application of 20-hydroxyecdysone or RH5992, an ecdysteroid analog, to isolated abdomens from feeding larvae (precommitment) results in exposure of the dorsal vessel (EDV), a sign of metamorphic commitment--but such abdomens do not make Vg in response to methoprene. However, injection of JH-II acid along with 20-hydroxyecdysone into isolated abdomens causes Vg production in response to methoprene. Methoprene acid similarly induces competence to respond to methoprene. Northern blot analysis confirmed that Vg transcripts are present in fat body only if isolated abdomens were pretreated with both ecdysteroid, and JH-II acid or methoprene acid. The latter two can induce competence even in precocious prepupae resulting from removal of the corpora allata (the glands that produce JH) from early penultimate larvae. JH-III acid and related metabolites such as farnesol, farnesoic acid, and methyl farnesoate do not induce competence. Hitherto, JH acids have been regarded as precursors or catabolites of JHs. Here we show for the first time that JH acid has a hormonal function that cannot be performed by JH itself.

Internalization and recycling of vitellogenin receptor in the mosquito oocyte.

The major yolk protein precursor in mosquito oocytes, vitellogenin (Vg), is internalized by a 205-kDa membrane-bound receptor (VgR). Recently, VgR has been isolated permitting the production of polyclonal anti-VgR antibodies. To elucidate the pathway of VgR internalization and recycling in mosquito oocytes during Vg uptake, we carried out an immunogold electron-microscopic study, labeling both Vg and VgR in ultrathin frozen sections of ovarian tissue. VgR immunolabeling demonstrated that the oocyte plasma membrane was subdivided into microdomains, with VgR being located between and at the lower portions of the oocyte microvilli. During the early stages of internalization, Vg and VgR were observed together in coated pits, coated vesicles, and early endosomes. Fusion of early endosomes created transitional yolk bodies (TYB) in which Vg and VgR became segregated. VgR label was present in the numerous tubular compartments that protruded from the TYBs. These tubular organelles extended to and fused with the plasma membrane, suggesting that they represented the vehicle for VgR recycling. Vg label was not observed in the tubular compartments. Instead, Vg accumulated in the core of the TYB, a region free of VgR label. Mature yolk bodies (MYB) were heavily labeled for Vg, but completely lacked any VgR label, indicating that MYB are storage compartments that do not participate in receptor recycling. Thus, our immunocytochemical data clearly visualize the steps in Vg/VgR internalization, dissociation, sorting, and recycling of the receptor to the plasma membrane.

Mosquito vitellogenin receptor: Purification, developmental and biochemical characterization

Vitellogenin receptors (VgRs) play a critical role in egg development of oviparous animals by mediating endocytosis of the major yolk protein precursor, vitellogenin. A modification of the method for extracting the mosquito (Aedes aegypti) VgR from ovary membranes resulted in an 11-fold higher yield and 56-fold increase in relative purity of the VgR, in turn permitting purification, antibody production, and microsequencing. A K d of 15 nM was estimated from binding assays for the enriched VgR, indicating a very high affinity for its ligand. Immunoprecipitation of [ 14C]VgR using anti-VgR polyclonal antibodies followed by SDS-PAGE under reducing conditions and fluorography demonstrated that the 205 kDa VgR does not consist of subunits held together with disulfide bonds. However, an immunoblot of the native VgR suggests that it exists as a ∼390 kDa noncovalent homodimer in its native state. Immunoblot assays confirmed that the VgR is present only in ovarian tissue. A quantitative immunoassay of VgR extracts showed that VgR was present in previtellogenic ovaries on the day of emergence, increasing from 2 ng to more than 10 ng per ovary by day 5. After initiation of vitellogenesis and onset of Vg uptake, VgR quantity increased rapidly between 8 and 24 h after a blood meal, then began to decline between 24 and 36 h. Immunocytochemistry confirmed the presence of substantial amounts of the VgR in 4-day-old previtellogenic oocytes. In both previtellogenic and vitellogenic ovaries, the VgR was present only in the oocyte, primarily in the cortex.

Vitellogenin gene of the silkworm, Bombyx mori: Structure and sex-dependent expression

Vitellogenin of Bombyx mori is a precursor of major yolk protein synthesized in the female fat body at larval—pupal ecdysis. The gene for B. mori vitellogenin is composed of seven exons interspersed by six introns. Developmental profile of the primary transcript of the gene indicated that the biosynthesis of B. mori vitellogenin is regulated transcriptionally in a sex- and stage-dependent manner in the fat body. The Arg-X-Arg-Arg sequence, which conforms to the recognition site of mammalian furin, occurs in a region just upstream of the putative proteolytic cleavage site of B. mori previtellogenin.

Structure and expression of mRNA for vitellogenin in Bombyx mori

Vitellogenin, a precursor of major yolk protein of the silkworm, Bombyx mori is a tetramer composed of each two molecules of heavy and light subunits. We cloned mRNA sequence for the B. mori vitellogenin and analyzed its structure. Sequence alignment of several overlapping cDNA clones indicated that the vitellogenin mRNA is approx. 5.7 kb, containing an open reading frame for a peptide with 1782 amino acid residues. By comparing the deduced amino acid sequence with the amino-terminal primary structures of vitellogenin subunits, it is suggested that the heavy and light subunits of the B. mori vitellogenin are encoded by a single contiguous mRNA. The primary translation product of the vitellogenin mRNA was detected in the microsomal fraction prepared from the fat body of vitellogenic females. Northern blot analysis of the fat body RNA demonstrated that the biosynthesis of vitellogenin in B. mori is regulated in a tissue-, sex- and stage-specific manner at the level of mRNA. Possible cause for discrepancy between the present results and our previous proposal (Izumi, S. and Tomino, S. (1983) Insect Biochem. 13, 81–85) on the biosynthesis of B. mori vitellogenin is also discussed.

Stored forms of vitellogenin mRNA in fat body cells of Locusta migratoria

Vitellogenin (Vg), a major yolk protein precursor for locust ( Locusta migratoria) eggs, is synthesized by female fat body. At the time of oviposition Vg production decreases greatly and Vg mRNA is stored for reinitiation of Vg synthesis in the next reproductive cycle. Sedimentation analysis of fat body extracts and dot-hybridization analysis of RNA have revealed at least three stored forms of Vg mRNA. Two of these sediment in the preribosome zone and one in the postribosome zone. CsCl density gradient analysis has revealed that all of them have buoyant density in the range 1.51–1.57 g/cm 3. In contrast to Vg, synthesis of apolipophorin (apoLp-III) is relatively constant in locust fat body. Unlike Vg mRNA, the stored form of ApoLp-III mRNA has density and sedimentation characteristics close to those of stored mRNA forms from other systems. This supports the conclusion that the unusual stored forms of Vg mRNA found are not an experimental artifact but have a function in the locust's reproductive cycle.

Characterization of the solubilized mosquito vitellogenin receptor

In this study we solubilized and characterized the receptor for the major egg yolk protein precursor, vitellogenin (Vg), from the yellow fever mosquito, Aedes aegypti. The receptor was solubilized from vitellogenic ovary membranes using octyl-β- d-glucoside (OG). Under equilibrium binding conditions, [ 35S]Vg bound with high affinity ( K d = 2.8 × 10 −8 M) to a single class of binding sites in solubilized ovary extracts. The solubilized receptor was present in ovarian extracts and bound selectively A. aegypti Vg and its storage form, vitellin (Vn). The receptor preparation was heat and trypsin sensitive. Binding of Vg to its receptor could be inhibited as well dissociated with suramin. The receptor was visualized by ligand-blotting as a 205 kDa protein under non-reducing conditions. It did not share immunological cross-reactivity with antibodies to chicken and locust Vg receptors. Vitellogenin, Vn and its purified subunits competed for binding to the receptor in the order, Vg ≈ Vn > Vn large subunit > Vn small subunit. Binding of dephosphorylated Vg was significantly reduced. Deglycosylated Vg, on the other hand, formed high molecular weight aggregates resulting in artifactually high binding which indicates importance of glycosylation for the stability of Vg molecule. During egg maturation, the number of receptor binding sites in ovaries correlated with the rate of Vg uptake and peaked between 24–30 h after which it reduced to no binding by 48 h post blood meal.