Vertebrate vitellogenin gene duplication in relation to the "3R hypothesis": correlation to the pelagic egg and the oceanic radiation of teleosts

The spiny ray-finned teleost fishes (Acanthomorpha) are the most successful group of vertebrates in terms of species diversity. Their meteoric radiation and speciation in the oceans during the late Cretaceous and Eocene epoch is unprecedented in vertebrate history, occurring in one third of the time for similar diversity to appear in the birds and mammals. The success of marine teleosts is even more remarkable considering their long freshwater ancestry, since it implies solving major physiological challenges when freely broadcasting their eggs in the hyper-osmotic conditions of seawater. Most extant marine teleosts spawn highly hydrated pelagic eggs, due to differential proteolysis of vitellogenin (Vtg)-derived yolk proteins. The maturational degradation of Vtg involves depolymerization of mainly the lipovitellin heavy chain (LvH) of one form of Vtg to generate a large pool of free amino acids (FAA 150-200 mM). This organic osmolyte pool drives hydration of the ooctye while still protected within the maternal ovary. In the present contribution, we have used Bayesian analysis to examine the evolution of vertebrate Vtg genes in relation to the "3R hypothesis" of whole genome duplication (WGD) and the functional end points of LvH degradation during oocyte maturation. We find that teleost Vtgs have experienced a post-R3 lineage-specific gene duplication to form paralogous clusters that correlate to the pelagic and benthic character of the eggs. Neo-functionalization allowed one paralogue to be proteolyzed to FAA driving hydration of the maturing oocytes, which pre-adapts them to the marine environment and causes them to float. The timing of these events matches the appearance of the Acanthomorpha in the fossil record. We discuss the significance of these adaptations in relation to ancestral physiological features, and propose that the neo-functionalization of duplicated Vtg genes was a key event in the evolution and success of the teleosts in the oceanic environment.     

Goldsinny wrasse (Ctenolabrus rupestris) is an extreme vtgAa-type pelagophil teleost

During oocyte maturation in the goldsinny wrasse (Ctenolabrus rupestris) extensive proteolysis of yolk proteins generates a large pool of free amino acids that drive hydration of the pelagic egg. By cloning hepatic vitellogenins (vtg) and using mass spectrometry, N-terminal microsequencing, and Western-immunoblotting to identify the yolk proteins (Yp), we show that multiple forms of vitellogenin mRNAs (vtgAa, vtgAb, and vtgC) are expressed in the liver, but only a single major class of the Yps derived from vtgAa predominates in the oocytes. Some Yps derived from vtgAb and vtgC appear also to be incorporated in the oocytes and eggs, but only at background levels. During oocyte hydration the vtgAa-derived lipovitellin heavy chain (LvH-Aa) and its cleavage variants are completely degraded leaving only a processed lipovitellin light chain (LvL-Aa) fragment as the major yolk protein for embryonic development. The maturational cleavage site of the LvL-Aa is identified as two amino acids downstream from the conserved Tyr(1168) of VtgAa in Atlantic halibut. In addition, although a beta'-component (approximately 18 kDa) is present in the oocytes, it is not fully degraded during the hydration process.     

Vertebrate yolk complexes and the functional implications of phosvitins and other subdomains in vitellogenins

In nonplacental or nontrophotenic vertebrates, early development depends on the maternal provision of egg yolk, which is mainly derived from large multidomain vitellogenin (Vtg) precursors. To reveal the molecular nature of the protein pools in vertebrate oocytes, published data on the N-termini of yolk proteins has been mapped to the deduced primary structures of their parent Vtgs. The available evidence shows that the primary cleavage sites of Vtgs are conserved, whereas the cleavage products exist as multidomain variants in the yolk protein pool. The serine-rich phosvitin (Pv) domains are linearly related to the molecular masses of the lipovitellin heavy chain. The 3-D localization of Pv maps to the outer edges of the Vtg monomer, where it is proposed to form amphipathic structures that loop up over the lipid pocket. At this locus, it is proposed that Pv stabilizes the nascent Vtg while it receives its lipid cargo, thereby facilitating the hepatic loading and locking of lipid within the Vtg (C-sheet)-(A-sheet)-(LvL) cavity, and enhances its solubility following secretion to the circulating plasma. The C-terminal regions of Vtgs are homologous to human von Willebrand factor type D domains (Vwfd), which are conserved cysteine-rich molecules with homologous regions that are prevalent in Vtgs, lipophorins, mucins, integrins, and zonadhesins. Unlike human VWFD, lower vertebrate Vwfds do not contain RGD motifs, which are associated with extracellular matrix binding. Although its function in Vtg is unknown, the lubricant properties associated with mucins and the cell adhesion properties associated with integrins and zonadhesins implicate Vwfd in the genesis of hemostatic platelet aggregation. Similarly, the proteolytic inhibitory properties associated with the binding of factor VIII in humans suggest that Vwfd stabilizes Vtg during passage in the systemic circulation.     

Phosvitins in Fundulus oocytes and eggs. Preliminary chromatographic and electrophoretic analyses together with biological considerations

Vitellogenin serves as the plasma precursor for the yolk proteins, lipovitellin and phosvitin, in nonmammalian vertebrates. 32P-Vitellogenin was isolated from the plasma of the teleost, Fundulus heteroclitus, and was used both to label phosvitin in the ovary and to indicate the phosvitin region in preparative chromatographs of ovarian extracts on DEAE-cellulose. Crude [32P]phosvitin could be resolved further into two labeled components with shallow gradients on DEAE-cellulose and into eight labeled components by electrophoresis on 12% polyacrylamide gels. Only the two largest electrophoretically resolved components could be correlated with Coomassie Blue staining bands, but several of the smaller components could be indicated with the cationic carbocyanine dye, Stains-all. Stains-all-dyed components were also generally indicated as multiple bands. The ovary of a reproductively active female contains vitellogenic oocytes, postvitellogenic oocytes undergoing maturation prior to ovulation, and ovulated eggs. Examination of various types of follicles and eggs on polyacrylamide gels revealed that during maturation, the largest phosvitin components formed during vitellogenesis either disappear or diminish, while smaller phosvitin components appear. The transformation of phosvitin components can also be achieved in vitro by incubating prematurational follicles in a saline medium containing deoxycorticosterone. These preliminary results demonstrate that a complex array of phosvitin-like components are present within a single ovary of F. heteroclitus. We also postulate that one reason for the anomalous yolk proteins generally found thus far in teleost eggs is that some of the proteins derived from vitellogenin during vitellogenesis undergo further proteolysis during oocyte maturation.     

Derivation of major yolk proteins from parental vitellogenins and alternative processing during oocyte maturation in Fundulus heteroclitus

Various Coomassie blue-staining yolk proteins (YPs) present in oocytes and eggs of Fundulus heteroclitus, a teleost that produces low hydrated, demersal eggs (benthophil species), were subjected to N-terminal microsequencing. Four YPs were N-terminally blocked, while five yielded sequence information. Of the latter, four corresponded to internal sequences of vitellogenin 1 (Vg1), whereas a fifth band corresponded to the N-terminal sequence of Vg2. Phosphorylated YPs (phosvitins and phosvettes) derived from the polyserine domain of Vg were not successfully sequenced. The major N-terminally blocked 122-and 103-kDa YPs both represented the lipovitellin heavy chain of Vg1 (LvH1), and thus most of the oocyte YPs were derived from Vg1. During oocyte maturation in vivo and in vitro, the LvH1 122 is degraded, concomitant with an increased enzymatic activity of cathepsin B, while the 45-kDa YP is converted to a 42-kDa YP. The LvH1 122 was found to contain a consensus site for proteolytic degradation (PEST) near its C-terminus, which is missing from its stable, but truncated twin sequence, LvH1 103. We suggest that this site becomes exposed to cathepsin B during the hydration process that accompanies oocyte maturation and renders the LvH1 122 susceptible to proteolysis. PEST sites are found in Vg sequences from other benthophil fish, whereas, interestingly, they are missing in marine teleosts that spawn highly hydrated, pelagic eggs (pelagophil species), displaying a different pattern of Vg incorporation into YPs and LvH1 and LvH2 processing to that found in F. heteroclitus. Thus, different models of Vg/YP precursor/product relationship and further processing during oocyte maturation and hydration are proposed for pelagophil and benthophil teleosts.     

Fundulus heteroclitus vitellogenin: The deduced primary structure of a piscine precursor to noncrystalline,liquid-phase yolk protein

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.Abbreviations AGY AG(T or C) - TCX TC(AGC or T) - Lv lipovitellin - Pv phosvitin - Vtg vitellogenin Correspondence to: G.J. LaFleur, Jr.     

The crystalline yolk-platelet proteins and their soluble plasma precursor in an amphibian, Xenopus laevis

A single lipophosphoprotein complex, vitellogenin, was isolated and purified from the plasma of oestrogen-stimulated female toads by preparative ultracentrifugation and chromatography on TEAE-cellulose (triethylaminoethylcellulose). The protein contains 12% lipid, 1.5% phosphorus, 1.6% calcium and smaller amounts of carbohydrates and biliverdin. In amino acid composition it is identical with total yolk-platelet protein. The platelet protein, however, is fractionated on TEAE-cellulose into two components, a high-molecular-weight lipovitellin and a smaller phosvitin. Analyses of the soluble plasma vitellogenin suggest that it is a complex of two phosvitin molecules covalently bound to one lipovitellin dimer, and that it is the immediate precursor of the yolk proteins, into which it is converted by a molecular rearrangement. Uptake of vitellogenin from the plasma into the growing oocyte, and its subsequent crystallization as a yolk platelet, appear to be enhanced by gonadotrophic hormones.     

Conserved and Variant Molecular and Functional Features of Multiple Egg Yolk Precursor Proteins (Vitellogenins) in White Perch (Morone americana) and other Teleosts

Three complete cDNAs encoding different forms of vitellogenin (Vtg) were isolated from a white perch (Morone americana) liver cDNA library and characterized with respect to immunobiochemical and functional features of the three Vtgs and their product yolk proteins (YPs) in this species and in the congeneric striped bass (Morone saxatilis). The two longest cDNAs encoded Vtgs with a complete suite of yolk protein domains that, based on comparisons with vtg sequences from other species, were categorized as VtgAa and VtgAb using the current nomenclature for multiple teleost Vtgs. The shorter cDNA encoded a Vtg that lacked a phosvitin domain, had a shortened C-terminus, and was categorized as VtgC. Mapping of peptide sequences from the purified Vtgs and their derived YPs to Vtg sequences deduced from the cDNAs definitively identified the white perch VtgAa, VtgAb, and VtgC proteins. Detailed comparisons of the primary structures of each Vtg with partial or complete sequences of Morone yolk proteins or of Vtgs from other fishes revealed conserved and variant structural elements of teleost Vtgs with functional significance, including, as examples, signal peptide cleavage sites, dimerization sites, cathepsin D protease recognition sites, and receptor-binding domains. These comparisons also yielded an interim revision of the classification scheme for multiple teleost Vtgs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Final oocyte maturation in vivo and in vitro in marine fishes with pelagic eggs; yolk protein hydrolysis and free amino acid content

The role of free amino acids (FAA) in oocyte hydration during final maturation has been studied in plaice Pleuronectes platessa and lemon sole Microstomus kitt by in vivo and in vitro measurements. In vitro final maturation was initiated by the administration of human chorionic gonadotropin on large vitellogenic oocytes. The eggs produced in vitro had the same fraction of their total amino acid pool present in the free form as the in vivo hydrated eggs, regardless of whether FAA had been present in the incubation medium or not. The FAA pool in the mature egg was increased 10–15 times that of the oocyte, and the two FAA pool profiles differed strongly. The FAA profiles of the egg groups (intra- as well as interspecific) were almost identical except that the taurine content was lower in eggs in vitro . A major protein band of about 100 kDa was present on SDS electrophoretic gels of oocytes but missing on gels of hydrated eggs. This protein, presumably a lipovitellin, is the most likely origin of the egg FAA pool. We suggest that marine fishes with pelagic eggs share a common mechanism for oocyte hydration whereby partial hydrolysis of specific yolk proteins to FAA creates a major part of the osmotic potential needed for the water influx.     

Vitellogenin-derived yolk proteins of white perch,Morone americana: purification,characterization, and vitellogenin-receptor binding

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.     

Lipovitellins derived from two forms of vitellogenin are differentially processed during oocyte maturation in haddock (Melanogrammus aeglefinus)

In the process of cloning vitellogenin (Vtg) cDNAs from haddock (Melanogrammus aeglefinus), two related, but distinct, mRNAs were identified. Full-length cDNA sequences were determined for both Vtg types (Had1 and Had2), and the deduced amino acid sequences were found to be 54% identical to each other and 48-58% identical to other teleost Vtgs. To investigate the expression of the two Vtg mRNAs, proteins from prehydrated oocytes and fertilized eggs were separated on SDS-polyacrylamide gels. Only a single lipovitellin I band was detected in each sample, and the egg lipovitellin I was smaller (97 vs. 110 kDa) than the oocyte protein, indicative of proteolytic processing during oocyte hydration. Mass spectrometric (MALDI-TOFMS and tandem mass spectrometry) analyses of tryptic fragments from the haddock oocyte and egg lipovitellin I revealed that the lipovitellin I from prehydrated oocytes contained tryptic fragments that matched the sequences of both types of Vtg, suggesting that there were two proteins in this band, while the egg lipovitellin I contained tryptic fragments that only matched the Had1 cDNA sequence, indicating that the Had2 lipovitellin had been degraded during hydration. Physiological data from haddock oocytes and eggs demonstrate that, as in other marine fish that spawn pelagic eggs, the free amino acid content increases during oocyte hydration and apparently contributes to hydration by driving the osmotic uptake of water. The correlation of the disappearance of one lipovitellin I with the increase of free amino acids in the oocyte suggests that this protein is a major source of the free amino acids for oocyte hydration.     

Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs

Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa beta'-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs.     

Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional Vg genes and their circulating and yolk protein products

The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17beta (E(2)) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa beta'-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E(2) treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.     

Free amino acids in Japanese eel eggs obtained by hormonal inducement

Restriction proteolysis of the yolk proteins during the final oocyte maturation appears to result in the low content of free amino acids (FAA) and the unique FAA profile in Japanese eel Anguilla japonica eggs obtained by hormonal inducement.     

Reversible response of protein localization and microtubule organization to nutrient stress during Drosophila early oogenesis

The maturation of animal oocytes is highly sensitive to nutrient availability. During Drosophila oogenesis, a prominent metabolic checkpoint occurs at the onset of yolk uptake (vitellogenesis): under nutrient stress, egg chambers degenerate by apoptosis. To investigate additional responses to nutrient deprivation, we studied the intercellular transport of cytoplasmic components between nurse cells and the oocyte during previtellogenic stages. Using GFP protein-traps, we showed that Ypsilon Schachtel (Yps), a putative RNA binding protein, moved into the oocyte by both microtubule (MT)-dependent and -independent mechanisms, and was retained in the oocyte in a MT-dependent manner. These data suggest that oocyte enrichment is accomplished by a combination of MT-dependent polarized transport and MT-independent flow coupled with MT-dependent trapping within the oocyte. Under nutrient stress, Yps and other components of the oskar ribonucleoprotein complex accumulated in large processing bodies in nurse cells, accompanied by MT reorganization. This response was detected as early as 2 h after starvation, suggesting that young egg chambers rapidly respond to nutrient stress. Moreover, both Yps aggregation and MT reorganization were reversed with re-feeding of females or the addition of exogenous insulin to cultured egg chambers. Our results suggest that egg chambers rapidly mount a stress response by altering intercellular transport upon starvation. This response implies a mechanism for preserving young egg chambers so that egg production can rapidly resume when nutrient availability improves.     

Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein

The gene encoding the major vitellogenin from chicken has been completely sequenced and its exon-intron organization has been established. The gene is 20,342 base-pairs long and contains 35 exons with a combined length of 5787 base-pairs. They encode the 1850-amino acid pre-peptide of vitellogenin, which is the precursor of the mature yolk proteins, the serine-rich and heavily phosphorylated phosvitin and the lipovitellin. The 217-amino acid phosvitin polypeptide occupies an internal position (residue 1112 through 1328) within the vitellogenin molecule. The 125,000 and 30,000 Mr lipovitellin polypeptides are encoded by the sequences at the N-terminal and the C-terminal sides of the phosvitin section, respectively. The main features of the gene and protein sequences, and the evolutionary implications, are discussed.     

Identification and characterization of proteases involved in specific proteolysis of vitellogenin and yolk proteins in salmonids.

A pepstatin A-sensitive enzyme involved in yolk formation was purified from the masu salmon (Oncorhynchus masou) ovary using in vitro generation of yolk proteins from purified vitellogenin to assay enzymatic activity. Purification of the enzyme involved precipitation of ovarian extracts by water and ammonium sulfate followed by five steps of column chromatography. After SDS-PAGE and Western blotting, the purified enzyme appeared as a single approximately 42 kDa band that was immunoreactive to anti-human cathepsin D. The course of proteolytic cleavage of the three major yolk proteins (lipovitellin, beta'-component, and phosvitin) in fertilized masu salmon and Sakhalin taimen (Hucho perryi) eggs and embryos was visualized by SDS-PAGE and Western blotting using specific antisera. Major yolk protein bands appeared in positions corresponding to 92 kDa, 68 kDa, and 22 kDa (lipovitellin-derived peptides), as well as 17 kDa (beta'-component). During embryo development, the 92 kDa and 22 kDa bands gradually decreased in intensity, becoming undetectable in alevins. The 68 kDa band and a minor 24 kDa band became more intense after the eyed stage. Two additional peptides, corresponding to 40 and 28 kDa, newly appeared in alevins. During embryonic growth, the beta'-component band (17 kDa) persisted and phosvitin appeared to be progressively dephosphorylated. In vitro analysis of lipovitellin proteolysis indicated that the enzyme involved is a Pefabloc SC-sensitive serine protease. These results demonstrate, for the first time, that a cathepsin D-like protease and serine proteases play key roles in yolk formation and degradation, respectively, in salmonid fishes.     

Lipovitellin constitutes the protein backbone of glycoproteins involved in sperm-egg interaction in the amphibian Discoglossus pictus

Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120‐kDa band released following gel‐into‐gel SDS–PAGE of both glycoproteins share the same N‐terminal amino acid sequence, which itself is similar to the N‐termini of Xenopus liver‐synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120‐kDa band is part of both gps 200 and 270/260. A 117‐kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC‐MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120‐kDa band and is responsible for the formation of the 200–270‐kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti‐vitellogenin antibodies label only the surface of the dimple. Mol. Reprod. Dev. 78:161–171, 2011. © 2011 Wiley‐Liss, Inc.