Processing Of Vitellogenin Research Articles

Dynamics of composition and morphology in oocytes of Eurasian perch, Perca fluviatilis L., during induced spawning

Histological and chemical composition (water, protein, lipids, fatty acid profile [FA]) analyses of gonads and ovulated eggs in various (I–VII) maturation stages of wild Eurasian perch, Perca fluviatilis L., were carried out on females during induced (hCG 500IUkg−1) spawning under controlled conditions. The results indicate that the process of vitellogenesis is finalized in the first two maturation stages (I and II). An analysis of the gonad composition and the FA profile indicated that the proper process of the final oocyte maturation (FOM; germinal vesicle [GV] migration and coagulation of oil droplets, GV disintegration) does not begin until stage III and ends with ovulation (stage VII). This follows from the fact that in stages I–II, fat was incorporated into the oocytes and n-3 polyunsaturated fatty acids accounted for the highest share of FA built into the oocytes at that time. Additionally, it was found that in the Eurasian perch the oocytes are hydrated just before the ovulation itself, after proteolytic processing of vitellogenin into a homogenous yolk mass or at the end of this process. On the other hand, the hydration process itself proved the least intensive (only 4%) of the processes previously described in the teleosts. On the basis of the obtained results, it is recommended that hormonal stimulation should not be performed during artificial spawning of the Eurasian perch in the first two specified maturation stages since this could possibly adversely affect the quality of the obtained eggs.

Thyroglobulin type-1 domain protease inhibitors exhibit specific expression in the cortical ooplasm of vitellogenic rainbow trout oocytes.

The synthesis, uptake, and processing of yolk proteins remain poorly described aspects of oviparous reproductive development. In this study, we report the identification and characterization of two protease inhibitors in rainbow trout ovary whose expression and distribution are directly associated with yolk protein uptake in vitellogenic oocytes. The first transcript, termed "oocyte protease inhibitor-1" (OPI-1), is predicted to encode a 9.1 kDa, 87 amino acid protein containing a single thyroglobulin type-1 (TY) domain, identifying it as a putative TY domain inhibitor. The second transcript, termed OPI-2, is predicted to encode an 18.3 kDa, 173 amino acid protein with two similar, but not identical, TY domains. Messenger RNA expression of both genes was first detected in ovarian tissues at the onset of vitellogenesis, and persisted throughout the vitellogenic growth phase. We did not detect expression of either gene in previtellogenic ovaries, nor in any somatic tissues examined. Expression of OPI-1 mRNA was significantly reduced in atretic follicles as compared to healthy vitellogenic follicles, suggesting a downregulation of inhibitor expression during oocyte atresia. Western immunoblot analyses of whole yolk from vitellogenic oocytes revealed the presence of two immunoreactive proteins that corresponded to the predicted sizes of OPI-1 and OPI-2. We detected strong crossreactivity of this antiserum with specific vesicles in the cortical ooplasm of vitellogenic oocytes, in regions directly associated with vitellogenin processing. The identification of OPI-1 and OPI-2 provides new evidence for the expression of multiple TY domain protease inhibitors likely involved in the regulation of yolk processing during oocyte growth in salmonids.

Oosorption in the stink bug, Plautia crossotastali: induction and vitellogenin dynamics

Oosorption, resorption of developing oocytes in the ovary, in P. c. stali is characterized by changes in appearance of oocytes from opaque greyish green or orange to transparent, degeneration of yolk granules and disappearance of oocyte contents. Starvation and virginity were indicated to be factors that induce oosorption. SDS PAGE/Western blotting analysis using anti-vitellogenin antiserum detected two major and many minor bands in haemolymph samples. Egg extracts showed a more complicated set of positive bands in the same analysis. Yolk protein, vitellin, therefore, seemed to be formed after complicated processing of vitellogenin following its uptake by the oocytes. In starved, oosorption-induced females, vitellogenin concentration in the haemolymph was lower than that of fed females, and Western blotting failed to detect either oosorption-specific or ovary-specific peptide fragments in haemolymph samples collected from those females. These results suggest that once oosorption was induced vitellogenin/vitellin in oocytes was degraded rapidly and released into the haemolymph in the form of amino acids or small peptides too small to be recognized by the anti-vitellogenin antiserum.

Molecular cloning and expression of ovarian cathepsin D in seabream, Sparus aurata.

In marine fish producing pelagic eggs, the acquirement of buoyancy by the eggs through the hydration process is a key event of reproduction; moreover, the yolk proteolysis, which leads to buoyancy, seems to affect the fertility and survival of the spawned eggs. Recently we demonstrated that cathepsin D is the aspartic protease responsible for this intraoocytic processing of vitellogenin into yolk proteins. In the present study, we isolated, cloned, and sequenced the cDNA encoding cathepsin D and studied expression of the message by Northern blotting and whole-mount in situ hybridization. The full-length seabream cathepsin D cDNA is 1837 base pairs long, encoding a protein of 400 amino acids (aa) consisting of a signal peptide of 19 aa, a prosequence of 44 aa, and a mature peptide of 336 aa. An absolute sequence conservation at the aspartyl residues (+33 and +221) was found, and there are three potential N-glycosylation sites at +70, aa +189, and aa +274. The aa sequence of seabream cathepsin D reveals a high degree of sequence similarity with cathepsin D mRNAs from other organisms (73% sequence homology to mouse and rat, 72% to human and trout, 69% to chicken, 66% to pig, and 65% to Xenopus). The cathepsin D mRNA in floating eggs was present as a single band that was approximately 1.9 kilobases (kb) in size, while in the sinking eggs there were several fast-migrating bands (size range 1.3-0.2 kb). Whole-mount hybridization was used to investigate transcription of cathepsin D in the developing embryo; during the hatching period, cathepsin D mRNA-positive cells were distributed in a wide region between the trunk and the tail, and in the ventral region over the yolk ball. The highest levels of cathepsin D enzymatic activity were found in the sinking eggs and during the hatching period of embryonic development. These data suggest that cathepsin D can be considered a possible marker for egg quality.

Maternal factors and the evolution of developmental mode: evolution of oogenesis in Heliocidaris erythrogramma.

Evolutionary change in developmental mode in sea urchins is closely tied to an increase in maternal provisioning. We examined the oogenic modifications involved in production of a large egg by comparison of oogenesis in congeneric sea urchins with markedly different sized oocytes and divergent modes of development. Heliocidaris tuberculata has small eggs (95 microm diameter) and the ancestral mode of development through feeding larvae, whereas H. erythrogramma has large eggs (430 microm diameter) and highly modified non-feeding lecithotrophic larvae. Production of a large egg in H. erythrogramma involved both conserved and divergent mechanisms. The pattern and level of vitellogenin gene expression is similar in the two species. Vitellogenin processing is also similar with the gonads of both species incorporating yolk protein from coelomic and hemal stores into nutritive cells with subsequent transfer of this protein into yolk granules in the developing vitellogenic oocyte. Immunocytology of the eggs of both Heliocidaris species indicates they incorporate similar levels of yolk protein. However, H. erythrogramma has evolved a highly divergent second phase of oogenesis characterised by massive deposition of non-vitellogenic material including additional maternal protein and lipid. Maternal provisioning in H. erythrogramma exhibits recapitulation of the ancestral vitellogenic program followed by a novel oogenic phase with hypertrophy of the lipogenic program being a major contributor to the increase in egg size.

The Biosynthesis and Processing of Vitellogenin in the Fat Bodies of Females and Males of the Cockroach Leucophaea maderae

The juvenile hormone analog (JHA) methoprene was used to induce the synthesis of the yolk protein precursor vitellogenin (Vg) in adult females and males of the cockroach Leucophaea maderae. The female- and male-produced vitellogenin (VgF and VgM, respectively) contained polypeptides of 112, 95, 92, and 54kDa. Also present in the secreted vitellogenins was a small quantity of a short-lived transitional 155kDa Vg polypeptide, and a variable amount of an 85kDa species. Quantitatively, the VgF and VgM were significantly different in the Vg112 and Vg95 units (VgF>VgM), and in the Vg85 polypeptide (VgF<VgM). In the present study, the biosynthesis of Vg precursors in the fat bodies of females and males was examined using a short radiopulse with 35S-methionine/cysteine and 32P-orthophosphate. The glycosylation of the Vg precursors was examined by digestion with endoglycosidase H and by the inhibition of N-linked glycosylation with tunicamycin. The data showed that in both females and males, the synthesis of the vitellogenin precursor occurred in a stepwise fashion: (1) the co-translational glycosylation of Vg203; (2) the post-translational phosphorylation of Vg203 to form Vg220; (3) the proteolytic processing of Vg220 to form the constituent Vg polypeptides. The 203 and 220kDa Vg precursors of females and males appeared to be similarly glycosylated and phosphorylated. The additional processing of Vg112 to Vg85 was more pronounced in the fat bodies of males than in females, and appears to account for the quantitative difference in the distribution of these polypeptides in VgF and VgM. Finally, the major oligosaccharides of VgF and VgM appear to be those of N-linked mannose residues. The treatment of females and males with tunicamycin indicated that the co-translational glycosylation of Vg precursors was required for the phosphorylation of the Vg precursor, as well as the secretion of Vg from the fat body.

Trace mineral metabolism in the avian embryo

Trace mineral metabolism in the developing avian embryo begins with the formation of the egg and the trace mineral stores contained within it. Vitellogenin, the yolk precursor protein, serves as a trace mineral transporting protein that mediates the transfer of these essential nutrients from stores within the liver of the hen to the ovary and developing oocyte, and hence, to the yolk of the egg. Lipovitellin and phosvitin, derived from intraoocytic proteolytic processing of vitellogenin, are also trace mineral binding proteins that form important storage sites within the granule subfraction of yolk. The mobilization and uptake of egg trace mineral stores is mediated by the extra-embryonic membranes, principally the yolk sac membrane. The yolk sac also serves as a short-term storage site for trace minerals. Because it is an important site of plasma protein synthesis, the yolk sac has the ability to regulate the export of trace minerals to the embryo during development. Within the embryo, specific metaloproteins function in the interorgan transport cellular uptake, and intracellular storage of trace minerals. Thus, embryonic trace mineral homeostasis is established through the coordinated actions of the yolk sac, which mobilizes and exports trace minerals derived from egg stores; the vitelline circulation, which transports them to the embryo; and the liver, which accumulates trace minerals and distributes them to the rest of the tissues of the embryo via the embryonic circulation.

Salt concentration-dependency of vitellogenin processing by cathepsin D in Xenopus laevis.

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.

Vitellogenin and lipovitellin: zinc proteins of Xenopus laevis oocytes.

Xenopus laevis vitellogenin is a plasma protein that contains a total of 5 mol of metal/440 kDa dimer, 2 mol of zinc, and 3 mol of calcium (Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413]. There are no other group IIB or transition metals in the molecule. The zinc atoms are removed instantaneously by 1,10-phenanthroline (OP) (pK 4.8). Once internalized by receptor-mediated endocytosis, vitellogenin is cleaved into multiple polypeptides, i.e., the two lipovitellin subunits (1 and 2) plus phosvitin; these are then stored as microcrystals within yolk platelets. We here show by metal analysis of the individual proteins generated by vitellogenin processing that zinc and calcium occur in different domains of the vitellogenin polypeptide chain. All of the vitellogenin zinc is present in lipovitellin, in amounts equal to 1 mol of zinc/141 kDa. Calcium, in contrast, is detected exclusively in phosvitin which, in addition, contains 3 mol of magnesium/35 kDa, apparently acquired following vitellogenin entry into the oocyte. The zinc in lipovitellin is removed by OP in a concentration-dependent manner with a pK of 4.8, identical to that obtained for vitellogenin, and by exposure to acidic conditions (below pH 5). Following removal of zinc, the two lipovitellin subunits remain associated, suggesting that zinc is not involved in their interaction. On exposure to 1% SDS, lipovitellin does dissociate into 106 and 33 kDa subunits. The presence of stoichiometric quantities of zinc in both vitellogenin and lipovitellin calls for the study of the hitherto unrecognized biochemistry and functions of these proteins in zinc metabolism and development of the frog oocyte and embryo.

Vitellogenesis in the sand worm, Nereis diversicolor

1. 1. Vitellin (V400) was identified in mature oocytes of Nereis diversicolor as a high molecular-weight lipoglycoprotein with a relative M r of 400,000. The yolk protein was purified by a two-step chromatographic procedure, using gel permeation and anion-exchange chromatography. 2. 2. Upon dissociation by SDS, vitellin was shown to release several polypeptide subunits which ranged in relative M r from 140,000 to 14,000. 3. 3. Vitellogenin (Vg) was identified in the coelomic fluid as a high molecular-weight protein with a relative M r of 530,000. This protein is composed of a single polypeptide with a relative M r of 175,000 (YP175). 4. 4. After uptake by the maturing oocyte, Vg is processed into V400 by progressive loss in molecular weight and probable cleavage. 5. 5. Electrophoretic analysis of the oocyte protein content during oocyte growth revealed that the processing of Vg does not occur in the earlier stage of vitellogenesis. Vg accumulates in the oocytes of young females without undergoing any significant structural modification. This suggests that the activation of the vitellogenin processing in nereid oocytes depends upon the developmental stage.

The female- and male-produced vitellogenins of Leucophaea maderae

Vitellogenin synthesis was induced in adult females and males as well as in last-instar nymphs of both sexes of Leucophaea maderae by the juvenile hormone analogue methoprene. Vitellogenic competency in females and males is developed during the last nymphal instar, but maximal vitellogenic competency in females is attained only after emergence. No increase in competency is observed for adult males. Once vitellogenic competency is established, last-instar female nymphs synthesize vitellogenin at a higher rate than either adult or last-instar males. Female- and male-produced vitellogenins are different in their polypeptide compositions. The difference in composition may reflect differences in vitellogenin processing in the female and male fat body. Female- and male-produced vitellogenins are taken up into competent oöcytes at different rates in vivo and in vitro, i.e. the female-produced vitellogenin is preferred over the male-produced vitellogenin.

Vitellogenin synthesis, processing and hormonal regulation in the tick, Ornithodoros parkeri (Acari:Argasidae)

This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri. Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [ 35S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.

In vivo ovarian uptake and processing of vitellogenin in the rainbow trout, Salmo gairdneri

AbstractThe in vivo uptake and processing of vitellogenin (VTG) into developing oocytes of rainbow trout were studied. Oestrogenized trout injected with 32P and 3H‐leucine incorporated these labels into newly synthesized vitellogenin. This dual‐labelled VTG was purified from the serum and injected intraperitoneally into sexually maturing female trout. The 32P. 3H‐VTG was readily taken up from the peritoneal cavity into the blood, without degradation, and thus became available for sequestration by developing oocytes. Ovarian uptake of 32P. 3H‐VTG was very rapid. Eight h after injection, approximately half of the labelled VTG was in the blood, but this figure had fallen to 12% after 48 h, and to 0.2% after 2 weeks. This fall in blood 32P. 32H‐VTG level coincided with increases in the level of both radiolabels in the ovary. Eight hours post‐injection, a significant amount of 32P. 32H‐VTG was found in the ovary homogenate. Most of this material was intact, primarily representing VTG bound to the follicle surface. However, small amounts were contained within the follicles and probably represented newly sequestered VTG which had not yet been degraded into the yolk proteins, lipovitellin and phosvitin. Twenty‐four hours post‐injection, approximately 25% of the injected 32P. 3H‐VTG had been sequestered by the ovary. Although a small proportion of the labels were associated with intact VTG, the majority of the 3H and 32P eluted with lipovitellin and phosvitin, respectively, the yolk proteins derived from VTG. After 2 weeks, nearly 70% of the 32P. 3H‐VTG initially injected had been incorporated into the ovaries, all of which was present as labelled yolk proteins. The rate of uptake of VTG was highest in the fish with the largest follicles, where it approached 120 ng.mm2 follicle surface h−1. The results demonstrate that both the uptake and processing of VTG by trout oocytes are rapid features that are reflected in the rate of ovary growth seen during exogenous vitellogenesis.

Multivesicular bodies play a key role in vitellogenin endocytosis by Xenopus oocytes

A combination of electron microscopic tracers and subcellular fractionation has been used to examine the endocytic pathway of the yolk protein precursor, vitellogenin (VG), in Xenopus oocytes. VG was adsorbed to colloidal gold, and the organelles traversed by newly internalized ligand were examined at various time intervals after endocytosis. VG-Au enters oocytes via coated pits and vesicles and then appears rapidly in tubular endosomes and multivesicular bodies (MVBs). MVBs play a central role in VG processing for storage; the large majority of newly internalized VG enters this compartment, remaining there for up to several hours. Condensation of VG into crystalline bodies begins in MVBs, and continues with growth of the crystals until typical platelets are formed. When oocytes are exposed to high [VG], MVBs containing large amounts of internalized VG are morphologically indistinguishable from the primordial yolk platelets described earlier ( Dumont, 1978). The use of VG-Au particles of two sizes demonstrates that gold particles in early MVBs were generally associated with the limiting membrane of these organelles, while older MVB compartments have gold particles well separated from the limiting membranes, suggesting that dissociation of VG from its receptor occurs in this compartment. Newly internalized ligand preferentially forms a new MVB, rather than fusing and mixing with previously formed MVBs. Progressive yolk protein condensation gradually transforms MVBs into yolk platelets over a period of several hours. Analysis of 125I-VG-Au behavior after sucrose gradient fractionation of oocytes allowed correlation of biochemical compartments with those observed in the electron microscope. MVBs containing yolk in progressive stages of condensation were found at densities from 1.16 up to 1.21 g/cc. The final, rate-limiting step in VG transport is a shift of ligand from light (1.21 g/cc) to heavy (1.23 g/cc) platelet compartments ( Wall and Meleka, 1985). The morphological correlate of this process is movement of VG-Au from small (<3–4 μm diameter) to large (>4 μm diameter) platelets.

Processing of pro-vitellogenin in insect fat body: A role for high-mannose oligosaccharide

Several discrete events were resolved in the processing of vitellogenin in Blattella germanica. Using tunicamycin to inhibit the synthesis of high-mannose oligosaccharide, a high molecular weight pro-vitellogenin peptide (apo-proVG, M r 215,000) was identified in fat body. Dosages of tunicamycin which inhibited glycosylation of vitellogenin by 98% inhibited its synthesis by as much as 59%, yet led to an intracellular accumulation of apo-proVG. Reversibility and dose dependency of these effects on vitellogenin synthesis, glycosylation, proteolytic processing, and secretion were demonstrated. In control insects, glycosylation of apo-proVG yielded a M r 240,000 pro-vitellogenin peptide (proVG). FITC-Concanavalin A bound to purified proVG but not to apo-proVG, thus confirming an absence of high-mannose oligosaccharide in the apo-protein. Following its glycosylation, proVG was processed rapidly in fat body to M r 160,000 (VG160) and M r 102,000 (VG102) peptides which subsequently were secreted into hemolymph. After uptake into developing oocytes, the VG160 peptide was processed further prior to chorionation, yielding subunits of M r 95,000 and 50,000. Uniqueness of the peptides of mature vitellin ( M r 102,000, 95,000, and 50,000) was indicated by comparison of the CNBr fragments of each purified subunit. Staining of CNBr fragments with FITC-Concanavalin A also indicated that high-mannose oligosaccharides are attached at one or more sites within each vitellin subunit. Resolution of the substructure of this insect vitellin and identification of events involved in the processing and secretion of its fat body apo-protein provide a basis for further study of the assembly and transport of vitellogenin, its packaging in eggs, and utilization during embryogenesis.

Proteolytic enzymes and blood digestion in the mosquito, Culex nigripalpus

AbstractThe synthesis of proteolytic enzymes in the fat body and midgut of female Culex nigripalpus was followed. The effects of brain factor(s) and RNA levels in the fat body were correlated with the synthesis of proteolytic enzymes. Trypsinlike activity in the midgut of C. nigripalpus accounted for 80% of total proteolytic activity, whereas chymotrypsinlike activity accounted for 5–7% of total proteolytic activity. Synthesis of porteases in the midgut and fat body reached a peak at 35 h and 22 h after the blood meal, respectively. In the fat body, proteolytic enzyme activity fell to a low level 30 h after the blood meal, but activity in the midgut reached a low level 58 h after the blood meal. The presence of low protease activity in the fat body at the time of peak vitellogenin synthesis indicated that processing of vitellogenin was not done in this tissue.Fat bodies incubated in vitro in the presence of [14C]valine synthesized a [14C]labeled trypsinlike molecule identified as such with antitrypsin antibodies and specific substrate p‐toluene‐sulphonyl‐L‐arginine methylester (TAME) and on disc gel electrophoresis in the presence of dodecyl sulfate. The sizes of the proteins found inside and outside the peritrophic membrane were determined by gel‐chromatography and disc gel electrophoresis in the presence of dodecyl sulfate. The molecular weight (± SEM) of the largest polypeptide that migrated through the peritrophic membrane into the ectoperitrophic space was found to be 23,000 ± 2,000 daltons. Based on these results, a model is proposed to account for blood digestion in the mosquito midgut, along with the role of the peritrophic membrane.