Vitelline Envelope Proteins Research Articles

Seasonal Variations of Vitelline Envelope Proteins, Vitellogenin, and Sex Steroids in Male and Female Eelpout (Zoarces viviparus)

The seasonal variations of vitelline envelope proteins, vitellogenin (VTG), and reproductive steroids were investigated in feral male and female eelpout, Zoarces viviparus. 17β-Estradiol was present in both sexes with a peak in prespawning fish of 2.6 ng/ml in males and 2.7 ng/ml in females. 11-Ketotestosterone peaked in June at 4.2 and 0.47 ng/ml in males and females, respectively. A surge of testosterone was seen in both sexes in August, just prior to spawning. All steroid levels were low during early pregnancy. The vitelline envelope of the eelpout is composed of two major and one minor protein with molecular weights of 50, 55, and 44 kDa, respectively. An antiserum raised against solubilized vitelline envelope from turbot (Scophthalmus maximus) cross-reacted strongly with the 50-kDa protein from the isolated vitelline envelope and a similar-sized protein in female plasma and plasma from estrogenized males. Interestingly, the 50-kDa protein was also present at low levels in males as demonstrated by ELISA and Western blotting. In males, the 50-kDa protein did not follow the seasonal changes in 17β-estradiol, but instead showed an almost perfect negative correlation with water temperature. VTG was present in female plasma as shown by Western blotting, but VTG was not detectable in male plasma despite relatively high endogenous estrogen levels. This suggests that the VTG induction by estradiol may be modulated by other factors in the eelpout.

Development of Time-Resolved Fluoroimmunoassay for Vitelline Envelope Protein Precursor, Choriogenin, in Marbled Sole Pleuronectes yokohamae

Development of Time-Resolved Fluoroimmunoassay for Vitelline Envelope Protein Precursor, Choriogenin, in Marbled Sole Pleuronectes yokohamae

Properties of the hatching enzyme from Xenopus laevis.

Using an anti-(glutathione S-transferase-UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 micromol.L-1 at 30 degrees C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N-alpha-tosyl-L-lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific 'HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease.

Multiple responses in gene expression in fish treated with estrogen

During the last decade there has been a significant body of research conducted on environmental estrogens. These include industrial, agricultural and pest-control chemicals that bind to the estrogen receptor and induce biological changes during development or reproduction. Most of these changes are probably due to modified gene expression, since estrogen receptors function at this level. We have mapped qualitative gene expression responses (by differential display reverse transcriptase polymerase chain reaction, DD) in adult male sheepshead minnows (Cyprinidon variegatus) receiving high dose injections (5 mg/kg), or constant flow-through aquatic exposures to environmentally relevant concentrations (100 ng/l) of estradiol-17β, and found them nearly identical. We have observed both up-regulation and down-regulation of transcripts, which fit into known responses to estradiol. Among the genes up-regulated are vitellogenin and several vitelline envelope proteins indicating that genes for proteins involved in egg development and maturation are susceptible to environmental estrogen exposure. While physiological changes caused by estradiol treatment are not totally explained by changes at the mRNA level, those changes can nevertheless be used as fingerprints to characterize an in vivo estrogenic response.

Expression of Three Vitelline Envelope Protein Genes in Arctic Char

Several studies have shown effects of estrogenic substances on endocrine and reproductive systems in wildlife. Measurement of plasma vitellogenin (VTG) is a commonly used method to determine exposure to estrogenic substances in fish. There is, however, a growing need for additional sensitive and accurate methods to detect estrogenic substances in vivo. The vitelline envelope proteins (VEPs) have been suggested, in other studies, as suitable biomarkers for estrogenic substances. The present study investigates the induction of VEPs in juvenile Arctic char (Salvelinus alpinus). The results demonstrate that VEP mRNA exhibits earlier induction than estrogen receptor mRNA or VTG mRNA following injection of juvenile Arctic char with a single dose of 17β-estradiol (E2; 10 mg/kg bw). These results indicate that the VEPs have a higher sensitivity for E2 than VTG. However, an early and sex-independent expression of VEPβ in estrogen-unchallenged juvenile Arctic char was observed. These findings suggests that the regulatory mechanisms of VEPs might be more complex than previously thought, which in turn may have implications for the usage of VEPs as biomarkers for xenoestrogen exposure.

Cloning of rainbow trout egg envelope proteins: members of a unique group of structural proteins.

All vertebrate eggs are surrounded by an extracellular envelope that protects the egg and is vital for a successful fertilization. The terminology and functions of the egg envelope vary in different vertebrate groups, but the envelope itself is consistently composed of a few major proteins that are deposited around the oocyte during oocyte growth. Here, we describe the deduced amino acid sequences and tissue expression patterns of the three major egg envelope proteins for rainbow trout (Oncorhynchus mykiss). All three vitelline envelope proteins (VEPs) are expressed in the livers of both male and female fish, with higher expression in females. In addition, VEPgamma mRNA is also detected in the female gonads. To our knowledge, this is the first time that expression of a VEP protein gene has been demonstrated to occur in more than one organ. Sequence comparison reveals that all three VEP proteins share distinct homology with their amphibian, avian, and mammalian counterparts. Whereas mammalian zona pellucida protein 3 isoforms contain two conserved serines needed for sperm binding, these are not conserved in teleost species, in which sperm entry is restricted to the micropyle. Besides the difference in VEPgamma sperm-binding function, the high sequence homology suggests that the egg envelope proteins from these distinct vertebrate groups share a common ancestry and form a unique group of structural proteins.

Induction of Gene Expression in Sheepshead Minnows (Cyprinodon variegatus) Treated with 17β-Estradiol, Diethylstilbestrol, or Ethinylestradiol: The Use of mRNA Fingerprints as an Indicator of Gene Regulation

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variegatus) exposed in vivo to the natural and pharmaceutical estrogens 17β-estradiol, ethinylestradiol, and diethylstilbestrol. This method used differential display reverse transcriptase polymerase chain reaction assays to compare the expression of individual mRNAs from control and estrogen-exposed fish. Forty-eight differentially expressed cDNAs were isolated by this method, including cDNAs for vitelline envelope proteins and vitellogenin. The mRNA expression patterns for fish injected with a pharmacological dose of estradiol (5 mg/kg) were identical to those obtained in fish receiving constant aqueous exposure to 212 ng estradiol/liter. Further, the cDNA “fingerprint” pattern observed in the estradiol-treated fish also matched that obtained in fish receiving continuous-flow aqueous exposures to 192 ng ethinyl estradiol/liter and a nominal concentration of 200 ng diethylstilbestrol/liter. The results demonstrate a characteristic expression pattern for genes upregulated by exposure to a variety of natural and anthropogenic estrogens and suggest this approach may be valuable to examine the potential effects of environmental contaminants on other endocrine-mediated pathways of reproduction, growth, and development.

Molecular components related to egg viability in the gilthead sea bream, Sparus aurata.

In pelagic egg spawners, the production of large numbers of sinking eggs, unable to develop into embryos, represents one of the major limiting factors in controlled reproduction. The aim of this study is to elucidate the molecular differences between floating and nonfloating eggs at cytoplasmic and nuclear level. Comparison of analyses between floating and nonfloating sea bream Sparus aurata eggs evidenced differences in vitelline envelope protein components, such differences being probably related with the hydration process but not with fertilization as supported by the assessment of DNA that doubled after in vitro insemination. These data clearly indicated that the absence of embryo development in nonfloating eggs is not due to lack of fertilization. The cytoplasmic composition was also different, the number of protein components being higher in floating eggs, and these extra components may generate the appropriate osmotic pressure at the base of the hydration process. Some lysosomal enzymes, such as cathepsin D and L both involved in yolk proteolysis, in virgin nonfloating eggs were significantly higher with respect to floating ones; the levels of these two enzymes significantly increased in the latter after fertilization. On the contrary, in nonfloating eggs cathepsin L significantly decreased after fertilization. These changes may be related with a series of metabolic processes vital for the production of viable offspring. The capacity of egg transcription and the protein synthesis in these two types of eggs, indicated by the RNA/DNA and RNA/protein ratios, evidenced that the status of cell transcription rate and protein synthesis capacity is significantly higher in floating eggs. This, in turn, suggested that the lack of embryo development may be due to low levels of proteins involved in cell cycle regulation.

Estrogen-mediated early life stage mortality in zebrafish ( Danio rerio)

Estrogenic substances have been shown to induce estrogen receptor (ER) regulated genes such as vitellogenin and vitelline envelope proteins. Recently we have shown that estrogen is embryo toxic to fish. In the present study specific polychlorinated biphenyl (PCB) congeners were examined for their effect on early life stage mortality in zebrafish ( Danio rerio). A set of eight PCBs, two hydroxylated PCBs and 17β-estradiol were tested. The PCBs were selected based on multivariate physicochemical characterization of all 154 tetra- through hepta-chlorinated PCBs. Three of the PCBs caused significant embryo mortality. Of these PCBs two more were found to also induce ER transcription. Determination of ER mRNA abundance in developing zebrafish embryos showed that the gene was upregulated at mid-blastula transition and returned to low levels prior to organogenesis. Together these results indicate that estrogenic compounds may interfere with development by interacting with ER.

Serum levels of precursors to vitelline envelope proteins (choriogenins) in Sakhalin taimen after treatment with oestrogen and during oocyte growth

In Sakhalin taimen Hucho perryi two choriogenins have been identified in serum, designated high- and low-molecular-weight vitelline envelope-related protein (hVERP and lVERP). Two immunoassays were developed using specific antibodies; single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), for measurement of the precursors to vitelline envelope proteins. ELISA measures hVERP and lVERP in serum at concentrations as low as 16 and 47 ng ml−1, respectively. Using these immunoassays, changes in serum levels of VERP were measured after treatment with E2 and during oocyte growth. When immature 2-year-old taimen were injected with 2 mg E2 per kg body weight, both VERPs were induced in serum within 24 h. In the course of the first ovulation, serum VERPs levels increased in September (8 months before ovulation), reached maximum in January, and decreased sharply prior to ovulation in May. These changes generally paralleled that of vitellogenin except for the drastic decrease of VERPs in May.

Serum levels of precursors to vitelline envelope proteins (choriogenins) in Sakhalin taimen after treatment with oestrogen and during oocyte growth

In Sakhalin taimen Hucho perryi two choriogenins have been identified in serum, designated high- and low-molecular-weight vitelline envelope-related protein (hVERP and lVERP). Two immunoassays were developed using specific antibodies; single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), for measurement of the precursors to vitelline envelope proteins. ELISA measures hVERP and lVERP in serum at concentrations as low as 16 and 47 ng ml−1, respectively. Using these immunoassays, changes in serum levels of VERP were measured after treatment with E2 and during oocyte growth. When immature 2-year-old taimen were injected with 2 mg E2 per kg body weight, both VERPs were induced in serum within 24 h. In the course of the first ovulation, serum VERPs levels increased in September (8 months before ovulation), reached maximum in January, and decreased sharply prior to ovulation in May. These changes generally paralleled that of vitellogenin except for the drastic decrease of VERPs in May.

BIOCHEMICAL AND IMMUNOCYTOCHEMICAL ANALYSIS OF VITELLOGENESIS IN THE OLIVE FRUIT FLY DACUS (BACTROCERA) OLEAE (DIPTERA: TEPHRITIDAE)

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the speciesDacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter-follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin-containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.

Purification of the precursors to vitelline envelope proteins from serum of Sakhalin Taimen, Hucho perryi

High and low molecular weight vitelline envelope-related proteins (hVERP and lVERP) were purified from serum of vitellogenic female Sakhalin taimen (Hucho perryi) by a combination of ion-exchange, hydroxylapatite and gel filtration chromatography. The molecular weight of hVERP was estimated to be 83 kDa by gel filtration, and 48 kDa and 54 kDa in SDS-PAGE under non-reduced and reduced conditions, respectively. The molecular weight of lVERP was 56 kDa by gel filtration, and 42 kDa and 46 kDa on SDS-PAGE (non-reduced and reduced, respectively). Amino acid composition of hVERP was characterized by high content of proline (15.9%) and glutamic acid (13.8%). The lVERP had high contents of glutamic acid (10.8%) and aspartic acid (10.5%). Specific antibodies against hVERP and against lVERP were prepared by immunizing rabbits. The antiserum to hVERP stained bands corresponding to 98 kDa and 48 kDa of vitelline envelope (VE) in SDS-PAGE without reduction, whereas the antiserum to lVERP immunostained 98 kDa and 42 kDa bands. Both specific antibodies recognized the vitelline envelope of vitellogenic oocytes immunocytochemically. Thus, hVERP and lVERP are precursors to vitelline envelope proteins in this species. J. Exp. Zool. 282:385–395, 1998. © 1998 Wiley- Liss, Inc.

Purification of the precursors to vitelline envelope proteins from serum of Sakhalin Taimen,Hucho perryi

High and low molecular weight vitelline envelope-related proteins (hVERP and lVERP) were purified from serum of vitellogenic female Sakhalin taimen (Hucho perryi) by a combination of ion-exchange, hydroxylapatite and gel filtration chromatography. The molecular weight of hVERP was estimated to be 83 kDa by gel filtration, and 48 kDa and 54 kDa in SDS-PAGE under non-reduced and reduced conditions, respectively. The molecular weight of lVERP was 56 kDa by gel filtration, and 42 kDa and 46 kDa on SDS-PAGE (non-reduced and reduced, respectively). Amino acid composition of hVERP was characterized by high content of proline (15.9%) and glutamic acid (13.8%). The lVERP had high contents of glutamic acid (10.8%) and aspartic acid (10.5%). Specific antibodies against hVERP and against lVERP were prepared by immunizing rabbits. The antiserum to hVERP stained bands corresponding to 98 kDa and 48 kDa of vitelline envelope (VE) in SDS-PAGE without reduction, whereas the antiserum to lVERP immunostained 98 kDa and 42 kDa bands. Both specific antibodies recognized the vitelline envelope of vitellogenic oocytes immunocytochemically. Thus, hVERP and lVERP are precursors to vitelline envelope proteins in this species.

Immunochemical detection of precursor proteins of yolk and vitelline envelope, and their annual changes in the blood of Diodon holocanthus

Two distinct groups of female‐specific proteins, vitellogenin (VTG) and vitelline envelope proteins (VEP), were detected in the blood of the porcupine fish Diodon holocanthus, and annual changes in concentration were measured immunochemically. Using antisera against yolk proteins (ab.a‐E) and VEP (ab. a‐VEP), VTG and VEP could be detected in the blood of maturing female fish and oestradiol‐17β (E2)‐treated fish. Neither protein was detected in the blood of male fish. Immunohistochemistry showed that yolk globules and the vitelline envelope enclosing developing oocytes stained with ab.a‐E. The vitelline envelope was stained specifically with ab.a‐VEP. Hepatocytes from the E2‐treated fish had immunoreactivity with both antisera. Thus, VTG and VEP appear to be synthesized in the liver by direct stimulation of E2, released into the circulation, and incorporated into respective target sites. VTG and VEP in female serum maintained high levels from April until June, suggesting that yolk accumulation, as well as vitelline envelope formation, are occurring actively during these months. Unlike VTG, small amounts of VEP were detected between December and March, suggesting that vitelline envelope formation precedes yolk accumulation and that a slightly different hormonal regulation exists in the synthesis of both proteins in the liver during the early phase of oogenesis.

Gamete interactions in Xenopus laevis: identification of sperm binding glycoproteins in the egg vitelline envelope.

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.

Amino acid composition and endocrine control of vitelline envelope proteins in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata).

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17 beta. As males of both species synthesize these proteins after treatment with estradiol-17 beta, the origin is not restricted to the ovaries. Vitellogenin of both European sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa.

Induction of Vitelline Envelope Proteins by Estradiol-17β in 10 Teleost Species

Induction of vitelline envelope proteins by estradiol-17β was investigated in 14 teleost species from five systematic groups to assess whether estradiol-17β controls the synthesis of vitelline envelope proteins in teleost fish. Vitelline envelope proteins were detected in plasma from fish treated with estradiol-17β using three different antisera directed against vitelline envelope proteins from three teleosts. Induction of vitelline envelope proteins was demonstrated in 10 species. In 6 of these species males were available and used to demonstrate that the synthesis of vitelline envelope proteins is not restricted to the ovaries. The immunoreactivity of the proteins varied considerably within and between species. It is suggested that the synthesis of vitelline envelope proteins is controlled by estradiol-17β, in the majority of teleost species.

Isolation, Partial Characterization, Induction, and the Occurrence in Plasma of the Major Vitelline Envelope Proteins in the Atlantic Halibut (Hippoglossus hippoglossus) during Sexual Maturation

The vitelline envelope of Atlantic halibut (Hippoglossus hippoglossus) is composed mainly of two major and two minor proteins. Estradiol-17ß induces the two major vitelline envelope proteins in halibut of both sexes. These proteins were also found in plasma of vitellogenic females. The origin of the two major vitelline envelope proteins is not restricted to the ovary, as male halibut synthesize these proteins after treatment with estradiol-17ß. Individual female halibut were followed and plasma sampled from May to March. Plasma levels of estradiol-17ß increased in October, peaked in early February, and declined until the last ovulation in March. In October, the two major vitelline envelope proteins were first detected in plasma and the amount remained elevated until early March. Prior to the last ovulation, the amount decreased. Plasma vitellogenin varied in a similar way. The results provide strong evidence that the physiological regulation of the synthesis of the major vitelline envelope proteins in teleosts is controlled by the female sex steroid estradiol-17ß.

Isolation, immunochemical detection, and observations of the instability of vitellogenin from four teleosts

AbstractVitellogenin was purified from plasma of estradiol‐17β‐treated cod (Gadus morhua) rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus), and wolffish (Anarhichas lupus) by precipitation with EDTA:Mg2+, distilled water, and high‐performance ion‐exchange chromatography. Vitellogenin of high purity was obtained by precipitation followed by chromatography, as evaluated by an homologous antiserum developed for each species. The instability of vitellogenin demanded consistent low temperature and the use of protease inhibitor before blood sampling. When the necessary precautions were taken, vitellogenin from rainbow trout, turbot, and wolffish eluted as one regular peak during chromatography. Cod vitellogenin eluted as two peaks and these demonstrated identical migration patterns on SDS‐PAGE. The observed differences in stability between the four species suggest that isolation procedures should be modified according to the requirements for each species. Electrophoresis of plasma from treated fish revealed the presence of several smaller proteins, with a molecular mass around 50 kDa, that were considered to be vitelline envelope proteins. Other minor plasma proteins were immunoreactive to antisera, directed against vitellogenin and therefore judged to be fragments of degraded vitellogenin. © 1993 Wiley‐Liss, Inc.