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.     

Bafilomycin A1 inhibits proteolytic cleavage and hydration but not yolk crystal disassembly or meiosis during maturation of sea bass oocytes

Oocytes of the black sea bass, Centropristes striata, were enlarged in volume more than three-fold over a 24-hr period during oocyte maturation, both in vivo and in vitro. At the same time, the opaque oocytes clarified while the crystalline yolk inclusions lost their ordered structure, fused with one another, and formed a continuous electron-lucent mass. The oocyte size increase was due almost entirely to water uptake, which was accompanied by the accumulation of Na+, K+, and free amino acids (FAAs). The absolute amounts of each of these small molecular weight osmotic effectors increased 2x, 4x, and over 10x, respectively, indicating that the generation of FAAs is the major cause of water uptake during maturation. Amino acid analyses indicated that the amounts of all amino acids except taurine increased, so that selective amino acids were not produced during maturation. The increase in FAAs was accompanied by the loss of certain high-molecular-weight yolk proteins and the generation of many smaller peptides. Oocytes stimulated to undergo maturation in the presence of bafilomycin A1, a specific inhibitor of the vacuolar ATPase-dependent proton pump, clarified and underwent maturation but did not increase significantly in size. Cytological examination revealed that yolk crystals fused and became homogeneous but maintained their electron density. No evidence of proteolysis was found in bafilomycin A1-treated oocytes and the generation of FAAs together with hydration was inhibited in a dose-dependent manner (I50 = 3 nM bafilomycin A1). Taken together, we postulate that the pronounced oocyte hydration in marine teleosts that spawn pelagic (floating) eggs is accomplished by a two-step process whereby (i) K+ influx promotes yolk crystal disassembly and yolk sphere fusion and (ii) acidification of the yolk spheres activates yolk proteolysis and concomitant hydration. Bafilomycin A1 inhibits only the second step so that many of the events of oocyte maturation, including germinal vesicle breakdown, occur in its presence but oocyte hydration is suppressed.     

The Influence of Yolk Protein Proteolysis on Hydration in the Oocytes of Fundulus heteroclitus

Growth in oocytes of many marine teleosts can be attributed to a combination of yolk accumulation during the vitellogenic phase of development and water uptake during meiotic maturation. In the salt marsh fish, Fundulus heteroclitus , hydration associated with maturation gives rise to a greater than two-fold increase in oocyte volume. It has been proposed that a concurrent proteolysis of specific yolk proteins may be the mechanism driving this water uptake. To test this hypothesis, we used various in vitro culture techniques to block or significantly reduce oocyte hydration while allowing meiotic maturation to continue, then examined yolk proteins by SDS-polyacrylamide gel electrophoresis. We were able to dissociate yolk proteolysis from both hydration and nuclear maturation stimulated by a maturation-inducing steroid, 17α-hydroxy- 20β-dihydroprogesterone. It therefore appears that the proteolysis of specific yolk proteins observed in maturing oocytes of marine teleosts is an independent developmental event, and is not directly involved in the hydration mechanism.     

Changes in teleost yolk proteins during oocyte maturation: correlation of yolk proteolysis with oocyte hydration

Yolk proteins of prematuration occytes and postmaturation eggs were compared by SDS gel electrophoresis in several teleosts, including freshwater species that produce demersal eggs, estuarine and marine species with demersal eggs, and marine species with pelagic eggs. In certain teleosts distinct changes in yolk protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins at this time; proteolysis is most pronounced in marine fishes with pelagic eggs. In many teleosts the oocyte swells by hydration during maturation; this hydration is also most pronounced in marine fishes with pelagic eggs. The extent of yolk proteolysis is well correlated with the extent of oocyte hydration during maturation.     

Yolk proteolysis and aquaporin-1o play essential roles to regulate fish oocyte hydration during meiosis resumption

In marine fish, meiosis resumption is associated with a remarkable hydration of the oocyte, which contributes to the survival and dispersal of eggs and early embryos in the ocean. The accumulation of ions and the increase in free amino acids generated from the cleavage of yolk proteins (YPs) provide the osmotic mechanism for water influx into the oocyte, in which is involved the recently identified, fish specific aquaporin-1o (AQP1o). However, the timing when these processes occur during oocyte maturation, and the regulatory pathways involved, remain unknown. Here, we show that gilthead sea bream AQP1o (SaAQP1o) is synthesized at early vitellogenesis and transported towards the oocyte cortex throughout oocyte growth. During oocyte maturation, shortly after germinal vesicle breakdown and before complete hydrolysis of YPs and maximum K(+) accumulation is reached, SaAQP1o is further translocated into the oocyte plasma membrane. Inhibitors of yolk proteolysis and SaAQP1o water permeability reduce sea bream oocyte hydration that normally accompanies meiotic maturation in vitro by 80% and 20%, respectively. Thus, yolk hydrolysis appears to play a major role to create the osmotic driving force, while SaAQP1o possibly facilitates water influx into the oocyte. These results provide further evidence for the role of AQP1o mediating water uptake into fish oocytes, and support a novel model of fish oocyte hydration, whereby the accumulation of osmotic effectors and AQP1o intracellular trafficking are two highly regulated mechanisms.     

Mutant yolk proteins lead to female sterility in Drosophila.

Specific mutations in the yolk protein genes, yp1 and yp2, of Drosophila melanogaster cause the yolk proteins (YPs) they encode to precipitate, ultimately resulting in female sterility. YPs of the yp1 mutant fs(1)1163 are secreted normally but then precipitate as globules and occasionally as crystalline fibers in the subbasement membrane space of the fat body (Butterworth et al., 1991, J. Cell Biol. 112, 727-737). The present ultrastructural and immunological studies of the fat body of the yp2 mutant fs(1)K313 show that YP also precipitates as globules in the same tissue compartment. The globules are also incapable of passing into the hemolymph but they are morphologically distinct from those of fs(1)1163. Similar analyses were performed on developing oocytes in wild type and both mutant strains. YP-containing aggregates, ultrastructurally similar to those in the fat body of each respective mutant, were found in the space between the plasmalemma and the vitelline membrane and embedded within the membrane itself. The evidence suggests that the precipitates interfere with the correct assembly of the eggshell membranes, leading to the sterile phenotype. Immunogold studies demonstrate that newly synthesized YPs in the normal and mutant strains share secretory vesicles with putative, vitelline membrane proteins and that the translocation of follicle cell YP is not through the membrane along the interfollicular spaces but directly through the plasmalemma facing the oocyte. Further the YP precipitates in the mutants permit visualization of the polarity of exocytosis of YP from the follicle cells.     

Evidence for a role of Na+,K(+)-ATPase in the hydration of Atlantic croaker and spotted seatrout oocytes during final maturation.

During final maturation the oocytes of many marine teleosts swell four to five times their original size due to uptake of water. The involvement of active inorganic ion transport and Na+,K(+)-ATPase in oocyte hydration in Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus), marine teleosts which spawn pelagic eggs, was investigated by examining changes in the inorganic ion content of ovarian follicles containing mainly oocytes, by performing in vitro incubations of the follicles with ion channel blockers, and by assaying membrane preparations of ovaries containing hydrating and non-hydrating oocytes for Na+,K(+)-ATPase activity and content. There were marked increases in the contents of K+, Mg++, and Ca++, but not Na+, in oocytes of M. undulatus and C. nebulosus during hydration. Incubation of follicle-enclosed oocytes in K(+)-free medium or with ouabain or amiloride, inhibitors of Na+,K(+)-ATPase and Na+ channels, respectively, blocked gonadotropin-induced oocyte hydration in M. undulatus. In addition, Na+,K(+)-ATPase activity increased threefold and the concentration of the enzyme increased 50% in ovarian tissue during oocyte hydration. These results strongly suggest a major role for active ion regulation by a ouabain-sensitive Na+,K(+)-ATPase system in oocyte hydration in two species of sciaenid fishes.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Oogenesis of microlecithal oocytes in the viviparous teleost Heterandria formosa

Viviparous teleosts exhibit two patterns of embryonic nutrition: lecithotrophy (when nutrients are derived from yolk that is deposited in the oocyte during oogenesis) and matrotrophy (when nutrients are derived from the maternal blood stream during gestation). Nutrients contained in oocytes of matrotrophic species are not sufficient to support embryonic development until term. The smallest oocytes formed among the viviparous poeciliid fish occur in the least killifish, Heterandria formosa, these having diameters of only 400 μm. Accordingly, H. formosa presents the highest level of matrotrophy among poeciliids. This study provides histological details occurring during development of its microlecithal oocytes. Five stages occur during oogenesis: oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis), secondary growth (vitellogenesis), and oocyte maturation. H. formosa, as in all viviparous poeciliids, has intrafollicular fertilization and gestation. Therefore, there is no ovulation stage. The full‐grown oocyte of H. formosa contains a large oil globule, which occupies most of the cell volume. The oocyte periphery contains the germinal vesicle, and ooplasm that includes cortical alveoli, small oil droplets and only a few yolk globules. The follicular cell layer is initially composed of a single layer of squamous cells during early previtellogenesis, but these become columnar during early vitellogenesis. They are pseudostratified during late vitellogenesis and reduce their height becoming almost squamous in full‐grown oocytes. The microlecithal oocytes of H. formosa represent an extreme in fish oogenesis typified by scarce yolk deposition, a characteristic directly related to matrotrophy. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.     

Multiple vitellogenin-derived yolk proteins in gray mullet (Mugil cephalus): disparate proteolytic patterns associated with ovarian follicle maturation

Disparate proteolytic patterns of yolk proteins, derived from three types of vitellogenin (VgA, VgB, and VgC), were observed in gray mullet. Immuno-biochemical analyses of extracts obtained from vitellogenic ovaries (VO) and ovulated eggs (OE) confirmed that a large proportion of VgA-derived lipovitellin (LvA) was degraded into free amino acids (FAAs) during ovarian follicle maturation. The maturation-associated alteration of VgB-derived Lv (LvB) involved only limited proteolysis; the heavy and light LvB chains were dissociated into at least three and one polypeptide fragments, respectively. The native mass of VgC-derived Lv (LvC) exhibited little difference between VO and OE, although it was apparent that the LvC was 'nicked' during maturation, resulting in the appearance of several bands in OE. Similar analyses confirmed that VgA-derived beta'-component (beta'-cA) and VgB-derived beta'-c (beta'-cB) decreased during maturation in both quantity and native mass, while phosvitin derived from either VgA (PvA) or VgB (PvB) appeared to be degraded into FAAs. The pattern of maturation-associated proteolysis of mullet yolk proteins is similar to that reported for other marine teleosts spawning pelagic eggs. However, the depository ratio of the three distinct types of Lv in the mullet VO appeared to be different from that estimated for another marine pelagophil, the barfin flounder. These results support a recent paradigm regarding the significance of Vg multiplicity upon successive physiological events in this group of fishes including the hydration of maturing oocytes, the acquisition of proper egg buoyancy, and the generation of requisite nutrient stocks for each stage of embryogenesis and larval development.     

Biosynthesis of Drosophila yolk polypeptides

The three yolk polypeptides (YPs) of Drosophila are synthesized and secreted by female fat body and ovarian follicle cells, sequestered by pinocytosis into oocytes, and finally deposited into yolk granules. The biosynthesis of the YPs was studied using two-dimensional gels. Labeling the YPs with [³⁵S]-cysteine, an amino acid found only near the amino terminus of YP1 and YP2, showed that an amino terminal peptide is removed from YP1 and YP2 shortly after or during translation. Intermediates in YP biosynthesis corresponding in electrophoretic mobility to pancreatic membrane-processed primary translation products were also detected in a 5-min pulse label with [³⁵S]-methionine. Genetic variants that alter YP structure were used to identify which YP precursor comes from which Yp gene. Pulse labeling with [³⁵S]-methionine revealed that all three YPs becomes more negatively charged, that YP1 and YP2 become heterogeneously charged, and that YP1 gains in apparent molecular weight within 15 min after translation. Injecting female flies with radioiabeled sugars or orthophosphate revealed that the YPs are glycosylated and phosphorylated. Treating hemolymph proteins with phosphatase showed that phosphorylation is responsible for much of the change in charge and increase in molecular weight of the maturing YPs. These experiments with wild-type flies provide a basis for the analysis of mutations at the Yp genes which alter the structure of individual YPs.     

Yolk protein endocytosis by oocytes in Drosophila melanogaster: immunofluorescent localization of clathrin, adaptin and the yolk protein receptor

The process of yolk protein (YP) uptake by developing oocytes in Drosophila melanogaster has been investigated by immunofluorescent localization of the endocytosis proteins, clathrin, alpha-adaptin and the putative yolk protein receptor (YP receptor). Data suggests that YPs from the follicle cells are trafficked into the oocyte during early stages of vitellogenesis, and that hemolymph YPs are sequestered by nurse cells adjacent to the developing oocyte during late stages of vitellogenesis. Yolk proteins were immunolocalized to both follicle cells and nurse cells during these processes. Diapausing female Drosophila melanogaster undergo a pre-vitellogenic arrest of ovarian development associated with the absence of ovarian alpha-adaptin, clathrin and putative YP receptor. Diapause termination by transfer of whole animals from 11 degrees C to 25 degrees C, or by 20-hydroxyecdysone injection, results in the appearance of immunopositive material in the nurse cells for all three proteins between 12 h and 16 h post upshift and within four days of injection. Immunopositive material was not noted in the follicle cells during diapause termination. In vitro warming of diapausing ovaries, or incubation in the presence of 1 &mgr;M 20-hydroxyecdysone failed to initiate early vitellogenic development suggesting that diapause termination requires factor(s) external to the ovary. Western blotting analysis of extracts of 24 h post-eclosion wild type and ap(56f) females identified putative yolk protein receptor with a molecular weight of 208 kDa and clathrin with a molecular weight of 178 kDa.     

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.     

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.     

Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis

The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.     

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.     

Cyto-histological examination of post-vitellogenesis and final oocyte maturation in captive-reared striped bass

The ovarian development of captive-reared, striped bass Morone saxatilis was examined during a 10-week period encompassing the spawning season. Vitellogenic oocytes in March had a mean diameter of 838 ± 18 μm and did not grow significantly thereafter. Except from one non-hormone-treated fish, all females failed to undergo final oocyte maturation (FOM) and their ovaries became atretic with the onset of high spring temperatures. A clearing fixative was found useful in identifying early stages of atresia, evident by the absence of the germinal vesicle (GV). Final oocyte maturation of fish treated with gonadotropin-releasing hormone agonist (GnRHa) consisted of two phases. Early FOM lasted from 1 to 3 weeks, and was associated with lipid-droplet coalescence, and displacement of the GV and yolk globules to the peripheral cytoplasm. Late FOM lasted <24h, and consisted of yolk-globule coalescence and GV breakdown (GVBD). Ovulated eggs had completely coalesced lipid and yolk masses, with cortical alveoli lined against the cell wall. Both phases of FOM were associated with significant increases in oocyte diameter. Striped bass oocytes showed important morphological differences compared to oocytes of other members of the Moronidae family, in terms of percentage lipid content, chorion thickness and degree of hydration after ovulation.     

The maturational disassembly and differential proteolysis of paralogous vitellogenins in a marine pelagophil teleost: a conserved mechanism of oocyte hydration

A structural analysis of the differential proteolysis of vitellogenin (Vtg)-derived yolk proteins in the maturing oocytes of a marine teleost that spawns very large pelagic eggs is presented. Two full-length hepatic cDNAs (hhvtgAa and hhvtgAb) encoding paralogous vitellogenins (HhvtgAa and HhvtgAb) were cloned from nonestrogenized Atlantic halibut, and the N-termini of their subdomain structures were mapped to the oocyte and egg yolk proteins (Yps). The maturational oocyte Yp degradation products were further mapped to the free amino acid (FAA) pool in the ovulated egg. The deduced amino acid sequences conformed to the linear NH(2)-(LvH-Pv-LvL-beta'-CT)-COO(-) structure of complete teleost Vtgs. However, the Yps did not match the expected cleavage products of complete Vtgs. Specifically, the phosvitin subdomain of the HhvtgAa paralogue remains covalently attached to the lipovitellin light chain, while the phosvitin subdomain of the HhvtgAb paralogue remains covalently attached to a C-terminal fragment of the lipovitellin heavy chain (LvH). During oocyte hydration, the LvH of the HhvtgAa paralogue is disassembled and extensively degraded to FAA. In the HhvtgAb paralogue, the LvH is nicked in the C-sheet in a manner similar to that seen in lamprey and other teleosts. A small part of the C-terminal end of the LvH-Ab undergoes proteolysis to FAA, together with the phosvitin, beta' component, and much ( approximately 65%) of the lipovitellin light chain (LvL-Ab). The independently measured FAA pool in the ovulated egg corroborates that calculated from differential proteolysis of the Yps. Based on the 3:1 (HhvtgAb:HhvtgAa) Yp expression ratio, each paralogue contributes approximately equal amounts of FAA to the organic osmolyte pool of the hydrating oocyte during maturation.