Article

Characterization of vitellogenin from White Sturgeon, Acipenser transmontanus

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Abstract

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age.

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... Complete and partial Vg deduced amino acid sequences were obtained from the GenBank database and published papers: boll weevil, Anthonomus grandis (M72980; Trewitt et al. 1992); chicken, Gallus gallus (M18060; van het Schip et al. 1987); frog, Xenopus laevis (Y00354; Gerber-Huber et al. 1987); gypsy moth, Lymantria dispar (L28097; Hiremath et al. 1994); lamprey, Ichthyomyzon unicuspus (M88749; Sharrock et al. 1992); nematode, Caenorhabditis elegans (X56121 and X56213; Spieth et al. 1985Spieth et al. , 1991; mummichog, Fundulus heteroclitus (U07055; LaFleur et al. 1995); sawfly, Athalia rosae (Kageyama et al. 1994); silkworm moth, Bombyx mori (D13160; Yano et al. 1994a); white sturgeon, Acipenser transmontanus (U00455; Bidwell and Carlson 1995); and yellow fever mosquito, Aedes aegypti (U02548; Chen et al. 1994). ...
... Insect Vgs: AgVg, BmVg, AaVg (abbreviations and citations as in Fig. 1). Vertebrate Vgs: AtVg, sturgeon, Acipenser transmontanus (Bidwell and Carlson 1995); XlVg, frog, Xenopus laevis ; GgVg, chicken, Gallus gallus (van het ; FhVg, mummichog, Fundulus heteroclitus (LaFleur et al. 1995); IuVg, lamprey, Ichthyomyzon unicuspus (Sharrock et al. 1992). Nematode Vgs: CeVg5 and CeVg6, nematode, Caenorhabditis elegans (Spieth et al. 1985(Spieth et al. , 1991. ...
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The eggs of most oviparous animals are provisioned with a class of protein called vitellogenin (Vg) which is stored as the major component of yolk. Until recently, deduced amino acid sequences were available only from vertebrate and nematode Vgs, which proved to be homologous. The sequences of several insect Vgs are now known, but early attempts at pairwise alignments with vertebrate and nematode Vgs have been problematic, leading to conflicting conclusions about how closely insect Vgs are related to the others. In this paper we demonstrate that insect Vg sequences can be confidently aligned with one another along their entire lengths and with multiple vertebrate and nematode Vg sequences along most of their spans. Although divergence is high, conservation among insect, vertebrate, and nematode Vg sequences is widespread with a preponderance of glycine, proline, and cysteine residues among strictly conserved amino acids, establishing conclusively that Vgs from the three phyla are homologous. Areas of least-certain alignment are primarily in and around insect and vertebrate polyserine domains which are not homologous. Phylogenetic reconstructions of Vgs based on sequence identities indicate that the insect lineage is the most diverged and that the mammalian serum protein, apolipoprotein B-100, arose from a Vg ancestor after the nematode/vertebrate divergence.
... All of these vitellogenins were expressed in female liver with a level significantly higher than in male liver. As the precursor of a major egg yolk protein, vitellogenin is normally observed to be synthesized in the liver [32,33] then transported to the ovaries where it is sequestered to serve as an energy reserve for the developing embryo. A number of various vitellogenin genes have been identified in different species of fishes [32,33,34,35,36,37,38] and all of these reports show the expression of vitellogenin to be liver specific (or predominant). ...
... As the precursor of a major egg yolk protein, vitellogenin is normally observed to be synthesized in the liver [32,33] then transported to the ovaries where it is sequestered to serve as an energy reserve for the developing embryo. A number of various vitellogenin genes have been identified in different species of fishes [32,33,34,35,36,37,38] and all of these reports show the expression of vitellogenin to be liver specific (or predominant). Since these reports all stem from vitellogenin genes in oviparous fishes, this may hallmark a difference in tactics by viviparous fishes such as Xiphophorus. ...
Article
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Xiphophorus models are important for melanoma, sex determination and differentiation, ovoviviparity and evolution. To gain a global view of the molecular mechanism(s) whereby gene expression may influence sexual dimorphism in Xiphophorus and to develop a database for future studies, we performed a large-scale transcriptome study. The 454-FLX massively parallel DNA sequencing platform was employed to obtain 742,771 and 721,543 reads from 2 normalized cDNA libraries generated from whole adult female and male X. maculatus Jp 163 A, respectively. The reads assembled into 45,538 contigs (here, a "contig" is a set of contiguous sequences), of which, 11,918 shared homology to existing protein sequences. These numbers estimate that the contigs may cover 53% of the total number of Xiphophorus transcriptome. Putative translations were obtained for 11,918 cDNA contigs, of which, 3,049 amino acid sequences contain Pfam domains and 11,064 contigs encode secretory proteins. A total of 3,898 contigs were associated with 2,781 InterPro (IPR) entries and 5,411 contigs with 132 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. There were 10,446 contigs annotated with 69,778 gene ontology (GO) terms and the three corresponding organizing principles. Fifty-four potential sex differentially expressed genes have been identified from these contigs. Eight and nine of these contigs were confirmed by real-time PCR as female and male predominantly expressed genes respectively. Based on annotation results, 34 contigs were predicted to be differentially expressed in male and female and 17 of them were also confirmed by real-time PCR. This is the first report of an annotated overview of the transcriptome of X. maculatus and identification of sex differentially expressed genes. These data will be of interest to researchers using the Xiphophorus model. This work also provides an archive for future studies in molecular mechanisms of sexual dimorphism and evolution, and can be used in comparative studies of other fish.
... One of the largest vitellogenin clones, A376, containing a 2.2 kb and II (fuVgI and fuVgII ) (La Fleur et al., 1995a,b), white sturgeon (Acipenser transmoutanus) Vitellogenin insert was sequenced completely for both strands. For manual sequencing, double-stranded plasmid DNAs (wsVg) (Bidwell and Carlson, 1995), lamprey (Ichthyomyzon unicuspus) Vitellogenin ( lpVg) (Sharrock were used as templates and sequenced with the T7 Sequencing Kit from Pharmacia according to the manu- et al., 1992), and yellow-fever mosquito (Aedes aegypti) Vitellogenin (moVg) (Chen et al., 1994). Two relatively facturer's instructions. ...
... Both the cross-hybridization among different vitellogenin gene nucleotide and amino acid sequences are most similar probes. to those from white sturgeon vitellogenin cDNA (Bidwell and Carlson, 1995). When the zebrafish Vitellogenin3 sequence was aligned with that of white sturgeon vitellogenin (Fig. 1 ), we found that the zebra- 3. Results fish Vitellogenin3 has no phosvitin domain or polyserine region in spite of the high conservation of the lipovitellin By partially sequencing 401 random cDNA clones I and II regions between the two vitellogenin sequences. ...
Article
By analysis of zebrafish EST (expressed sequence tag) clones from an adult cDNA library, we have identified 44 clones, about 11% of the adult EST clones, encoding vitellogenins. These vitellogenin EST clones have been derived from at least seven distinct vitellogenin genes. One of the largest vitellogenin cDNA clones, vg3, and its 5' extended clone isolated by 5' RACE (rapid amplification of cDNA ends)-PCR, have been sequenced completely. The deduced complete sequence includes a predicted mature vitellogenin of 1233 amino acids and a truncated signal peptide of 18 amino acids. Interestingly, the predicted vitellogenin has no polyserine phosvitin domain. The lack of the phosvitin domain was confirmed by isolation and sequencing of the vg3 genomic region. Phylogenetic analysis indicates that the phosvitinless vitellogenin is an intermediate between invertebrate vitellogenins and all known vertebrate vitellogenins, and thus may represent a primitive vertebrate vitellogenin. Like other vitellogenins in vertebrates, the phosvitinless vitellogenin is also synthesized mainly in the liver and weakly in the intestine.
... We found vtgr expression in eel testis, as also reported in mammals (Takahashi et al., 1992) and amphibians (Okabayashi et al., 1996). Low levels of vtgr transcripts have been detected in the testis of a non-teleost actinopterygian, the white sturgeon (Acipenser transmontanus), and in some teleosts, zebrafish (Danio rerio), and tilapia (Oreochromis mossambicus) (Bidwell and Carlson, 1995). It was hypothesized that vtgr may be involved in nutrient transport, uptake of hormones, vitamins, and other biomolecules for white sturgeon spermatocytes (Bidwell and Carlson, 1995). ...
... Low levels of vtgr transcripts have been detected in the testis of a non-teleost actinopterygian, the white sturgeon (Acipenser transmontanus), and in some teleosts, zebrafish (Danio rerio), and tilapia (Oreochromis mossambicus) (Bidwell and Carlson, 1995). It was hypothesized that vtgr may be involved in nutrient transport, uptake of hormones, vitamins, and other biomolecules for white sturgeon spermatocytes (Bidwell and Carlson, 1995). ...
Article
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In teleosts, vitellogenin ( Vtg ) is a phospholipoglycoprotein synthesized by the liver, released into the blood circulation and incorporated into the oocytes via endocytosis mediated by the Vtg receptor ( VTGR ) to form the yolk granules. The VTGR is crucial for oocyte growth in egg-laying animals but is also present in non-oviparous vertebrates, such as human. The VTGR belongs to the low-density lipoprotein receptor superfamily ( LDLR ) and is also named very-low-density lipoprotein receptor ( VLDLR ). In this study, we identified and phylogenetically positioned the VTGR of a basal teleost, the European eel, Anguilla anguilla . We developed quantitative real-time PCR ( qRT-PCR ) and investigated the tissue distribution of vtgr transcripts. We compared by qRT-PCR the ovarian expression levels of vtgr in juvenile yellow eels and pre-pubertal silver eels. We also analyzed the regulation of ovarian vtgr expression throughout vitellogenesis in experimentally matured eels. The Vtg plasma level was measured by homologous ELISA experimental maturation. Our in silico search and phylogenetical analysis revealed a single vtgr in the European eel, orthologous to other vertebrate vtgr . The qRT-PCR studies revealed that vtgr is mainly expressed in the ovary and also detected in various other tissues such as brain, pituitary, gill, fat, heart, and testis, suggesting some extra-ovarian functions of VTGR. We showed that vtgr is expressed in ovaries of juvenile yellow eels with no higher expression in pre-pubertal silver eels nor in experimentally matured eels. This suggests that vtgr transcription already occurs during early pre-vitellogenesis of immature eels and is not further activated in vitellogenic oocytes. European eel Vtg plasma level increased throughout experimental maturation in agreement with previous studies. Taken together, these results suggest that vtgr transcript levels may not be a limiting step for the uptake of Vtg by the oocyte in the European eel.
... At 550 dph in Russian sturgeon it was also detected, but the highest observed expression of vtg was seen in the gonads of intersex individual (Fajkowska et al. 2016). In adolescent white sturgeon (2-year-old), in one study, vtg transcripts were not found at that stage of development, neither in the ovaries nor in the testes (Bidwell & Carlson 1995). In another study, however, vtg expression was identified but the sex of the examined individuals was not stated (Bidwell et al. 1991). ...
... In another study, however, vtg expression was identified but the sex of the examined individuals was not stated (Bidwell et al. 1991). In 4-yearold white sturgeon vtg exhibited sex-differentiating expression with higher expression observed in males (Bidwell & Carlson 1995) while in 1600 dph (4.38-year-old) Russian sturgeon the highest levels were detected in the intersex gonads (Fajkowska et al. 2016). Again in adult shovelnose sturgeon, no differences in vtg expression between males, females or the intersex gonads were seen (Amberg et al. 2010). ...
Article
Sex determination is a complex process, especially in fish where multiple models of sex determination can be identified. The fate of differentiating gonads may depend on species genetics, environmental or behavioural factors. In the case of the sturgeon, sex determination is most likely of genetic origin and is characterized by a ZW female heterogametic system. However, molecular mechanisms of sex development and differentiation in sturgeons are poorly understood. Due to the lack of sexual dimorphism and a long period of maturation combined with invaluable caviar obtained from sturgeons, plenty of research revolving around discovering master sex‐determining gene has been done. Yet, despite numerous studies utilizing multiple approaches and techniques to find key genes involved in sex differentiation, no master sex‐determining genes were identified in sturgeons. Nevertheless, the latest research provided a great deal of data regarding the expression of genes commonly related with sex development and differentiation in vertebrates. Several of these genes were found to be connected with the same processes in sturgeons. This review attempts to summarize research into the expression of genes involved in sex differentiation and development in sturgeons.
... Among sturgeon fishes, Sterlet sturgeon (Acipenser ruthenus Linnaeus, 1758), are considered as a suitable model for studying reproductive physiology of sturgeon (Holcik, 1989), due to quicker sexual maturation, small size and lower maintenance costs compared to the other sturgeons. The molecular changes in oocytes during the oogenesis from the early oogonia to mature oocytes are well characterized in teleosts (Bidwell and Carlson, 1995). Unlike bony fish, little information is available on sturgeon species. ...
... Unlike bony fish, little information is available on sturgeon species. Following these molecular events and comparing them among various species, a framework will be provided for understanding the mechanisms involved in the oocyte maturation in sturgeon (Bidwell and Carlson, 1995). Therefore, studying the ovarian proteome and measuring the blood value of sex steroids and vitellogenin in Sterlet, as a sturgeon, in different stages of sexual maturation can answer many of the questions about the reproductive physiology female sturgeons. ...
Article
One of the challenges of sturgeon aquaculture is that sturgeon takes an extended amount of time to reach sexual maturity. The pattern of the protein expression in relation to the late maturity of sturgeon can help to better understand changes in sexual maturity. 17β-estradiol (E2), testosterone (T) and vitellogenin (Vtg) levels were examined at all stages of sexual maturation in Sterlet sturgeon (Acipenser ruthenus). Two-dimensional gel electrophoresis and mass spectrometry analysis were used to show the pattern of the ovarian proteins. The T levels increased from the previtellogenic to the postvitellogenic stages (P < 0.05) and Vtg showed a decremental pattern in pre- and postvitellogenic, and atresia (not significantly). The analysis showed 900 protein spots, 19 of which were successfully identified and had significant differences between the previtellogenic and the vitellogenic groups (P < 0.05). Among the identified proteins, 40% involved in cell defense (heat shock protein, Glutathione peroxidase, natural killer enhancing factor, peroxiredoxin-2), 30% in transcription and translation (constitutive photomorphogenesis 9 and Ybx2), 20% in metabolism and energy production (triose-phosphate isomerase (TPI)) and 10% in transport (glycolipid transfer protein). In the vitellogenic stage, the proteins were related to metabolism and energy production (TPI, ES1, creatin kinase, enolase, nucleoside diphosphate kinase, 50%), cell defense (thioredoxin and dislophid isomerase, 20%) and transport (fatty acid binding protein, 10%). Our findings show changes in protein expression pattern from cell defense to metabolism during egg development.
... Over the years, many studies resumed the possibility of an extrahepatic origin for VTG protein by using biochemical and molecular approaches (Table 1). Investigations carried out on oviparous fish suggest that estrogenic hormones and estrogen-like compounds are able to induce the expression of the VTG gene in both hepatic and extrahepatic tissues [36][37][38]. It has been demonstrated that in male zebrafish 17a-ethinylestradiol induces VTG expression and synthesis in dose-and time-dependent pattern in skin and eye [39] and estradiol-17b or EDCs are able to determine the VTG synthesis in the heart, cerebral tissue [40], epidermis [41], gills [42,43], white adipose tissue [44], intestine and muscle tissue [38]. ...
... Many studies focused on gonads of male fish experimentally exposed to E2 or estrogen-like substances. In Acipenser transmontanus, VTG transcripts were detected in the testis of the E2-treated samples [36]; a same result was obtained also in zebrafish [38]. In the testis of Oryzias latipes treated with E2 or NP, spermatocytes are able to transcribe the VTG gene [37,45]. ...
Article
In the last years, the hormonal balance is threatened by the interferences of substances with hormone-like action (endocrine disruptor chemicals, EDCs) that may harm animal reproduction. Most EDCs are resistant to environmental degradation and are considered ubiquitous contaminants. EDCs may have synthetic or natural origins. Pesticides used in intensive agriculture contain large amounts of chemicals with estrogenic properties, such as the alkylphenol nonylphenol (NP). Besides, animal feeding operations are important sources of natural estrogen metabolites introduced into the environment through manure application in organic farming. In both cases, EDCs can reach animals, including humans particularly at risk due to their position in the food chain. This is the reason for which it is important to use terrestrial vertebrates as sentinels in soil biomonitoring programmes. Today, the most validated biomarker of estrogenic exposure is the expression in male liver of the vitellogenin (VTG), an estrogen-dependent glycolipophosphoprotein naturally expressed only in the liver of oviparous females during the reproductive season. This report summarizes the data available on the EDC-dependent expression and the synthesis of VTG in male vertebrates, highlighting our latest studies that demonstrate the ability of testis and epididymis of the lacertid Podarcis sicula to synthesize VTG following estrogenic exposure. These findings provide, for the first time, evidence on an extrahepatic expression and synthesis of VTG in a terrestrial vertebrate and lay the groundwork for a new value of the VTG as a biomarker of environmental contamination. In addition, the results open a new scenario on the role of VTG in cells other than oocytes.
... The knowledge of vtg expression in sturgeons is limited. In white sturgeon Acipenser transmontanus Richardson 1837, vtg transcripts were detected in livers of oestrogen-treated sexually differentiated and undifferentiated sturgeons and on a low level in gonads of undifferentiated oestrogen-treated sturgeons, but surprisingly also in testes of differentiated oestrogen-treated and control males (Bidwell & Carlson, 1995). vtg expression was also found in livers of 1 year-old Chinese sturgeon Acipenser sinensis Gray 1835, treated with 4-nonylophenol and 17--oestradiol and all treated sturgeons expressed vtg, while in untreated individuals no expression was found. ...
... The highest vtg expression in 550 dph was observed in intersex gonads but unexpectedly the lowest level was in ovaries. It was previously suggested by Bidwell & Carlson (1995) that expression of vtg in the oestrogen-stimulated undifferentiated gonads may be a nonspecific effect on oestrogen-responsive tissue. The high vtg expression in gonads may therefore be caused by uncharted germinal tissue response to oestrogenic compounds or other non-germinal gonadal tissues response, such as white fat tissue, which is known to express vtg1 gene in zebrafish Danio rerio (Hamilton 1822) (Tingaud-Sequeira et al., 2012), therefore variances in white fat tissue amount in gonadal samples could cause differences in detected vtg levels. ...
Article
Expression of the dmrt1 and vtg genes was described using the real-time PCR (rt-PCR) method from 25 to 1600 days post-hatch (dph) in cultured Russian sturgeon Acipenser gueldenstaedtii. The level of dmrt1 transcription in gonads in subsequent studied periods increased exponentially while vtg expression increased in gonads and livers of A. gueldenstaedtii examined, but in later stages of development. Both dmrt1 and vtg genes showed elevated expression in intersex individuals probably caused by dietary exposure to phyto-oestrogens.
... These AA sequences did not show any alignment with the N-termini of teleost Vtgs. Therefore, internal AA sequences of E110 (E110-Lys1, E110-Lys2, E110-Lys3 and E110-Lys4) were analyzed following the digestion of E110 with Lys-C endopeptidase and the resulting sequences are listed in (Bidwell and Carlson 1995). In addition, the N-termini of E45 and E30 were also aligned with high similarity to parts of the Lv sequence of white sturgeon vtg (Bidwell and Carlson 1995). ...
... Therefore, internal AA sequences of E110 (E110-Lys1, E110-Lys2, E110-Lys3 and E110-Lys4) were analyzed following the digestion of E110 with Lys-C endopeptidase and the resulting sequences are listed in (Bidwell and Carlson 1995). In addition, the N-termini of E45 and E30 were also aligned with high similarity to parts of the Lv sequence of white sturgeon vtg (Bidwell and Carlson 1995). Thus far, based on the above results, Vtg210 appeared to be Vtg polypeptide, while E110 and other minor components (E45 and E30) appeared to consist of Lv. ...
Article
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Elasmobranchs (sharks and rays) exhibit unique reproductive characteristics and, in contrast to the situation in teleosts, very little is known about the identity, structure and physical characteristics of their egg yolk proteins. The aims of this study were to (1) detect and purify the vitellogenin (Vtg; egg yolk precursor) and yolk proteins (YPs) of the cloudy catshark (Scyliorhinus torazame), (2) examine the relationships between Vtg and YPs and (3) characterize and classify the deduced primary structure of the Vtg transcript (vtg). The apparent molecular weights of purified Vtg and putative Vtg-related YPs (lipovitellin: Lv, phosvitin: Pv) were determined by gel filtration and were ~560, >669 and ~58 kDa, respectively. Following SDS-PAGE, these purified products (i.e., Vtg, Lv and Pv) appeared as bands of ~210, ~110 and ~22 kDa, respectively. On Western blots, antisera against purified Vtg, Lv and Pv recognized the ~210 kDa Vtg band. Catshark Pv, in contrast to teleost Pvs, had a very low serine content. The catshark Vtg cDNA sequence (vtg) appeared to contain an open-reading frame consisting of domains encoding Lv, Pv and β'-component (β'-c). A phylogenetic analysis, with a consideration of genome duplication events, placed catshark vtg into the 'vtgAB type.' It is concluded that at least a single major type of Vtg protein, which is transcribed and translated from catshark vtgAB gene, is the precursor of three egg yolk proteins (Lv, Pv and β'-c) in catshark.
... Prior to the uptake of VTG into Chinese sturgeon ovary, massive VTGRs are synthesized and prepared for the selective acceptance and transportation of VTG [50,51]. The highest temporal expression of vtgr in stage II might indicate the active transport of VTG into oocytes through VTGR for ovary development. ...
Article
Ovary development of Chinese sturgeon (Acipenser sinensis) in controlled breeding has been reported to respond to dietary lipid levels. However, the corresponding molecular regulatory mechanism about ovary development of Chinese sturgeon is still unclear. To elucidate the molecular mechanism of vitellogenic deposition and hydrolysis, six key genes, namely, vtgr (vitellogenin receptor), atp6v1c1 (Vacuolar H⁺-ATPase subunit c1), atp6v1h (Vacuolar H⁺-ATPase subunit h), ctsb (cathepsin B), ctsd (cathepsin D) and ctsl (cathepsin L) involved in vitellogenic deposition and hydrolysis of Chinese sturgeon were cloned and characterized, and their spatio-temporal mRNA expression profiles as well as transcriptional responses to dietary lipid level were investigated. The full-length cDNA sequences of these six genes showed similar domain structure to their respective orthologous genes from other vertebrates. Tissue-specific expression patterns of these genes were observed in ovary, liver, muscle, spleen, brain, gill, intestine, heart, stomach and kidney. Ovarian expression level of vtgr was the highest in stage II, and ctsl expression was the highest in stage IV, while the mRNA expressions of other 4 genes were the highest in stage III. The increase of dietary lipid level promoted ovary development and elevated the expressions of vtgr, atp6v1c1, atp6v1h, ctsb and ctsd in the ovary. The results of the present study indicated that these genes are crucial for vitellogenic deposition, and provided a preliminary understanding on the molecular regulation of vitellogenic deposition and hydrolysis during ovary development of Chinese sturgeon.
... However, neither gulf sturgeon nor lake sturgeon purified Vtg protein is commercially available for developing a quantitative assay. Given that sequences of the sturgeon Vtg gene appear well-conserved across phyla (Bidwell and Carlson, 1995), we attempted to use shovelnose sturgeon (Scaphirhynchus platoyrnchus) and white sturgeon (A. transmontanus) reagents (i.e. ...
Article
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This study focused on identifying the sex of lake sturgeon by measuring the sex hormones estradiol and testosterone, and the phosphoprotein vitellogenin (Vtg) in blood plasma by radioimmunoassay and enzyme-linked immunosorbent assay, respectively, and evaluating these techniques as tools in lake sturgeon population management. Surveys of the St Clair River (SCR) lake sturgeon population have characterized it as rebounding by having steady or increasing recruitment since 1997. However, researchers have not been able to effectively determine the sex for most of the sturgeon they capture because few fish caught during surveys are releasing gametes. A total of 115 fish were sampled from May through June in 2004 and 2005 from the SCR, Michigan, USA. Of these, only four females and eight males were verified (i.e. they were releasing gametes at time of capture), resulting in very few fish with which to validate blood hormone and Vtg biomarkers of sex. Fifty-six percent of the fish were assigned a sex designation based on biomarker criteria. Correspondence between actual gonadal sex and biomarker-directed classification was good for the small subset of fish for which gonadal sex was definitively determined. Moreover, application of the steroid values in a predictive sex assignment model developed for white sturgeon misclassified only the same two fish that were misclassified with the steroid and Vtg biomarkers. The experimental results suggest a sex ratio of 1 : 2.7 (F:M), however more conclusive methods are needed to confirm this ratio because so few fish were available for sex validation. Of the 43 males, 14 were within the legal slot limit, 11 were smaller than 1067 mm total length (TL), and 18 were larger than 1270 mm TL. All 15 females were larger than 1270 mm TL, and thus protected by the slot limit criteria. Considering that lake sturgeon are threatened in Michigan, an advantage to using blood plasma assays was that fish were not harmed, and sample collection was quick, simple, and inexpensive. However, because a sufficiently large number of fish could not be validated for gonadal sex due to handling restrictions given the fish’s protected status, assignment of sex is not based on a robust multi-variate model. An immediate alternative may be to use other non-invasive field methods (e.g. ultrasound, fiber-optic endoscope) to provide a more timely classification while establishing well-validated plasma hormone and Vtg-based predictive models for sex assignment of lake sturgeon.
... The zebrafish (Danio rerio) genome contains at least seven vtgs that encode homologous Vtgs with three distinct types mapped onto two different chromosomes (Wang et al., 2005). One vtg transcript has been characterized in the silver lamprey, Ichthyomyzon unicuspies (Sharrock et al., 1992) and the white sturgeon, Acipenser trasmontanus (Bidwell and Carlson, 1995); at least two have been cloned in mummichog (Fundulus heteroclitus) (LaFleur et al., 1995LaFleur et al., , 2005), Japanese medaka (Oryzias latipes) (GenBank http://www.ncbi.nlm.nih.gov/Genbank/index.html accession numbers: AB064320 and AB074891;Tong et al., 2004), Japanese goby, Acanthogobius flavimanus (Ohkubo et al., 2004), tilapia (Oreochromis aureus) (Lee et al., 1994;Lim et al., 2001), and haddock (Melanogrammus aeglefinus) (Reith et al., 2001); and multiple transcripts were demonstrated in mosquitofish (Gambusia affinis) (Sawaguchi et al., 2005a, b). ...
Chapter
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The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules. Oocyte growth, particularly in oviparous species, is characterized by the intense deposition of products; e.g. RNAs, proteins (including growth and transcription factors), lipids, vitamins, and hormones. The deposit and storage occur in teleost fish species in a physiologically arrested cell at the G2/M border in first meiotic prophase (see Chapter 1).Whereas the nucleus remains in the diplotene stage, maternal RNAs are produced endogenously by the oocyte (see Chapters 3 and 6).
... As in other egg-laying vertebrates, the hepatic synthesis of VTG in sturgeon is induced by estrogen (Moberg et al., 1991). The VTG of white sturgeon was previously investigated by Kroll (1990), Bidwell et al. (1991), and Bidwell and Carlson (1995), and the relative concentrations of circulating VTG were estimated by plasma protein phosphorus or total plasma calcium (Doroshov et al., 1997). Plasma VTG during ovarian maturation was measured by ELISA in Acipenser baerii cultured in France (Cuisset et al., 1991) and by radial immunodiffusion in fertile hybrid Huso huso  Acipenser ruthenus cultured in Japan (Fujii et al., 1991). ...
Article
We developed an enzyme immunoassay for plasma vitellogenin (VTG) of white sturgeon Acipenser transmontanus, cultured in California. The assay, based on polyclonal rabbit antibody to purified VTG, was tested for specificity and applied on sturgeon broodstocks (age 4–12 years) reared at three locations. Plasma VTG concentrations were correlated with total plasma calcium and stage of ovarian maturity. Females in the previtellogenic ovarian stage and at the onset of yolk deposition had low VTG concentrations, but plasma VTG and calcium increased after onset of yolk deposition in the oocytes. Total plasma calcium exhibited a linear relationship with plasma VTG. Concentrations of both metabolites in plasma discriminate previtellogenic and vitellogenic females, but fail to discriminate specific stage of ovarian vitellogenesis. The immunoassay for plasma VTG or flame photometry for total plasma calcium can be utilized to segregate vitellogenic females in cultured sturgeon broodstocks.
... This observation suggests that limited proteolysis of the yolk proteins occurs during oocyte maturation in the catfish C. gariepinus. These results are in agreement with the studies on other freshwater benthophil fish, where maturational cleavage/nicking occurs but yolk proteins are not essentially degraded to free amino acids, e.g., rainbow trout, O. mykiss (Tyler 1993;Milla et al. 2006), zebrafish, D. rerio (Dosch et al. 2004), white sturgeon, Acipenser transmontanus (Bidwell and Carlson 1995), hybrid sturgeon, bester (Huso huso x Acipenser ruthenus) (Hiramatsu et al. 2002). ...
Article
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Yolk processing pathways vary in the oocytes of benthophil and pelagophil teleosts. The present study investigated the yolk processing pattern in the oocytes of the fresh water catfish Clarias gariepinus at vitellogenic, maturation, and ovulated stages. This study concludes that during maturation stage, an electrophoretic shift in the major peptide band on Polyacrylamide gel electrophoresis (PAGE) occurs due to a decrease in the size of the yolk protein. The PMF spectrum of corresponding peptides from vitellogenic and ovulated oocytes revealed a difference in the minor ions. A minor difference in the molecular weight of the corresponding peptides occurs due to a difference in their amino acid composition. Maximal activity of the proteases cathepsin D and cathepsin B was observed in the vitellogenic oocytes, thus confirming their role in the processing of yolk. A significant transient increase in the activity of cathepsin B in the mature oocytes also suggests its role in oocyte maturation.
... It is commonly accepted that production of vitellogenins does not occur in males normally, but can be induced upon exposure to estrogenic compounds (Stroemqvist et al. 2010;Hashimoto et al. 2000;Wang et al. 2005;Islinger et al. 2003;Henry et al. 2009;Hiramatsu et al. 2005). However, several studies have also reported low levels of vitellogenin mRNAs or proteins found in presumably unexposed male fish (Bidwell and Carlson 1995;Copeland et al. 1986;Gomez-Requeni et al. 2010). Our data on the detection of peptide evidence for the presence of vitellogenins or their derivatives in the testis of unexposed zebrafish further supports these observations. ...
Article
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The molecular mechanisms controlling sex determination and differentiation in zebrafish (Danio rerio) are largely unknown. A genome-wide analysis may provide comprehensive insights into the processes involved. The mRNA expression in zebrafish gonads has been fairly well studied, but much less data on the corresponding protein expression are available, although the proteins are considered to be more relevant markers of gene function. Because mRNA and protein abundances rarely correlate well, mRNA profiles need to be complemented with the information on protein expression. The work presented here analyzed the proteomes of adult zebrafish gonads by a multidimensional protein identification technology, generating the to-date most populated lists of proteins expressed in mature zebrafish gonads. The acquired proteomics data partially confirmed existing transcriptomics information for several genes, including several novel transcripts. However, disagreements between mRNA and protein abundances were often observed, further stressing the necessity to assess the expression on different levels before drawing conclusions on a certain gene’s expression and function. Several gene groups expressed in a sexually dimorphic way in zebrafish gonads were identified. Their potential importance for gonad development and function is discussed. The data gained in the current study provide a basis for further work on elucidating processes occurring during zebrafish development with use of high-throughput proteomics. Electronic supplementary material The online version of this article (doi:10.1007/s10695-010-9464-x) contains supplementary material, which is available to authorized users.
... The putative N-glycosylation sites are double underlined c Very low levels of wcmmVtg mRNA were also detected in the gill filament and testis of white cloud mountain minnows. The expression of Vtg in the gill filament has not yet been reported in other fish, but the Vtg mRNA was detected in the testis of normal male white sturgeons (Acipenser transmontanus) by Northern blot analysis (Bidwell and Carlson 1995). The roles of wcmmVtg in the gill filament and testis of white cloud mountain minnows remain to be identified. ...
Article
Full-text available
Vitellogenins (Vtgs), the precursors for the yolk proteins, are very important for the embryonic development of teleosts, and have also been studied extensively as biomarkers for environmental estrogenic mimics. The cDNA for a Vtg was isolated from the liver of the female white cloud mountain minnow (Tanichthys albonubes) by 3'- and 5'-RACE methods. It is 4,171 bp in full length, and encodes a putative protein of 1,326 amino acids. This putative Vtg, designated as wcmmVtg, contains complete portions of LVI and PV, but lacks the C-terminal half of LVII and thus belongs to type I vitellogenin. In addition to the liver of the female fish, wcmmVtg was also shown to be expressed in the ovary. During ovarian development, the mRNA expression of wcmmVtg in both the liver and ovary was continuously increased from the previtellogenic to late vitellogenic stages, but then decreased significantly at post-spawning stage. In the male fish, expression of wcmmVtg mRNA was induced in the liver by treatment with E2 (10 and 100 ng/l) for 14 days. These results suggest that the Vtg originated from the ovary of the white cloud mountain minnow may also contribute to the accumulation of yolk proteins during oocyte growth, and that the male white cloud mountain minnow is sensitive to the estrogenic treatment in terms of Vtg mRNA expression, which could also be applied to monitor the environmental estrogenic mimics.
... 45 Mio Jahren voneinander getrennt haben (Nelson 1994). Bezüglich der Konservierung der Aminosäuresequenz verringert sich die Homologie mit abnehmender evolutionärer Verwandtschaft: während ein Alignment mit Euteleostei, wie Fundulus heteroclitus oder der Regenbogenforelle (Mouchel et al. 1996), eine Übereinstimmung von mehr als 50 % identischer Aminosäuren ergibt, liegt die Aminosäuresequenzidentität von M-Vg1.6 zu anderen Vertebraten wie dem Stör (Acipenser transmontanus; Bidwel and Carlson 1995), dem südafrikanischen Krallenfrosch (Xenopus laevis, Gerber-Huber et al. 1987) oder dem amerikanischen Neunauge (Ichthyomyzon unicuspis; Sharrock et al. 1992) nur bei 40 -30 % mit absteigender Ähnlichkeit in Reihenfolge ihrer Aufzählung. Die Identifikation von M-Vg1.6 als 5'-Ende der Vitellogenin-cDNA kann auch durch eine ProDom-Computer-Analyse (Corpet et al. 1998) gestützt werden; M-Vg1.6 weist die typische Lipovitellin 1 (Lv1)-Domäne auf, die in der N-terminalen Region des Proteins zu finden ist. ...
Article
Vitellogenin und Estrogenrezeptoren stehen in der Leber von Fischen unter estrogener Kontrolle und werden in männlichen Individuen nur gering exprimiert. Durch exogene Applikation Estrogen-wirksamer Stoffe wird jedoch auch bei Männchen eine Expression induziert, so dass beide Gene als Marker für estrogene Wirkungen dienen können. Unter dieser Voraussetzung wurde in der vorliegenden Dissertation ein Nachweissystem für die Expression beider Gene in Hepatocytenkulturen aus der Regenbogenforelle entwickelt. In einem ersten Bewertungsansatz wurde das estrogene Potential ausgewählter Xenoestrogene erfasst. Ebenso konnte in einer Stichprobe bei 6 Abwässern aus dem Schweizer Mittelland die Anwendbarkeit der Methode auf komplexe Lösungen nachgewiesen werden. Für eine grundlegende Beurteilung des estrogenen Potentials einer Substanz sind jedoch In vivo-Untersuchungen unerlässlich, da nur die Belastung eines intakten Organismus Aufschluss über Bioakkumulation, metabolische Umsetzung und Exkretion gibt. Zur Etablierung geeigneter Nachweissysteme zur Expression Estrogen-regulierter Gene in den beiden Modellfischarten Medaka und Zebrabärbling wurde zunächst Sequenzinformation über die cDNAs von Vitellogeninen beider Fischarten sowie Estrogenrezeptor des Zebrabärblings ermittelt. Mit Hilfe dieser Daten wurden auf semiquantitativer RT-PCR basierende Nachweissysteme für diese Genprodukte sowie für Estrogenreptor und Choriogenin H des Medakas bzw. ZP2 des Zebrabärblings entwickelt. Anschließend erfolgten vergleichende Studien zur zeitlichen und dosisabhängigen Expression dieser Gene nach Exposition verschiedener Xenoestrogene. Bei der Auswertung der Ergebnisse waren distinkte Unterschiede in der Empfindlichkeit beider Fischarten für die einzelnen Modellsubstanzen festzustellen. Für die Bewertung der Emission endokrin wirksamer Substanzen in das Freiland ergibt sich hieraus die Konsequenz, dass signifikante Artunterschiede in der estrogenen Sensibilität zu berücksichtigen sind.
... The primary structures are now known for several insect Vgs including that of the boll weevil, Anthonomus grandis (Trewitt et al., 1992), the yellow fever mosquito, Aedes aegypti , the silk moth, Bombyx mori , the gypsy moth, Lymantria dispar (Hiremath and Lehtoma, 1997), a sawfly, Athalia rosae (Nose et al., 1997), and a parasitoid wasp, Pimpla nipponica (Nose et al., 1997). Deduced amino acid sequences also have been reported for the Vgs of various species of vertebrates van het Schip et al., 1987;Sharrock et al., 1992;LaFleur et al., 1995;Bidwell and Carlson, 1995) and two species of nematode (Spieth et al., 1985(Spieth et al., , 1991Winter et al., 1996). Although the primary function of Vg is to provide a pool of amino acids for the embryo, it also functions as a carrier of carbohydrates, lipids, phosphates, vitamins, metals, and hormones (Lagueux et al., 1981;Kawooya et al., 1988;Byrne et al., 1989;Niimi et al., 1993;Mac Lachlan et al., 1994;Sunderman et al., 1995). ...
Article
The recent cloning and sequencing of several insect vitellogenins (Vg), the major yolk protein precursor of most oviparous animals, and the mosquito Vg receptor (VgR) has brought the study of insect vitellogenesis to a new plane. Insect Vgs are homologous to nematode and vertebrate Vgs. All but one of the insect Vgs for which we know the primary structure are cleaved into two subunits at a site [(R/K)X(R/K)R or RXXR with an adjacent beta-turn] recognized by subtilisin-like proprotein convertases. In four of the Vgs, the cleavage site is near the N-terminus, but in one insect species, it is near the C-terminus of the Vg precursor. Multiple alignments of these Vg sequences indicate that the variation in cleavage location has not arisen through exon shuffling, but through local modifications of the amino acid sequences. A wasp Vg precursor is not cleaved, apparently because the sequence at the presumed ancestral cleavage site has been mutated from RXRR to LYRR and is no longer recognized by convertases. Some insect Vgs contain polyserine domains which are reminiscent of, but not homologous to, the phosvitin domain in vertebrate Vgs. The sequence of the mosquito VgR revealed that it is a member of the low-density lipoprotein receptor (LDLR) family. Though resembling chicken and frog VgRs, which are also members of the LDLR family, it is twice as big, carrying two clusters of cysteine-rich complement-type (Class A) repeats (implicated in ligand-binding) instead of one like vertebrate VgRs and LDLRs. It is very similar in sequence and domain arrangement to the Drosophila yolk protein receptor (YPR), despite a non-vitellogenin ligand for the latter. Though vertebrate VgRs, insect VgR/YPRs, and LDLR-related proteins/megalins all accommodate one cluster of eight Class A repeats, fingerprint analysis of the repeats in these clusters indicate they are not directly homologous with one another, but have undergone differing histories of duplications, deletions, and exon shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.
... The rapid growth of the oocyte prior to ovulation in most oviparous vertebrates is mainly due to the incorporation of the yolk protein precursor vitellogenin (Wallace 1985). Vitellogenin is synthesized by the liver (Chen 1983, Bidwell & Carlson 1995) and transported to the ovaries where it is sequestered to serve as an energy reserve for the developing embryo. Another feature of oocyte development is the formation of an extracellular eggshell, or vitelline envelope (Dumont & Brummet 1985). ...
Article
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Fish oogenesis represents pleiotropic cytodifferentiative programs including hepatic synthesis of the molecular components for both the eggshell and the oocytic energy deposits. Both hepatic processes are directly controlled by plasma levels of estradiol (E2), and injected E2 induces both biogenetic processes in prepubertal fish of both sexes. This work compares the temporal pattern of E2-induced biosynthesis of zona radiata proteins (zr-proteins) and vitellogenin in Atlantic salmon (Salmo salar L.) in vivo and in vitro. We monitored the presence of plasma zr-proteins and vitellogenin, using homologous polyclonal antiserum to zr-proteins and a monoclonal antibody to vitellogenin. Zr-proteins were induced by all E2 concentrations (0.001-1.1 mg/kg body weight (bw)) within one week of exposure while vitellogenin was not induced until two weeks post-injection and then only in plasma from fish injected with high E2 concentrations (0.4 mg or 1.1 mg/kg bw). After E2 treatment, hepatocytes isolated from male fish synthesized zr-proteins and vitellogenin in vitro. However, zr-proteins were secreted into the medium two days before vitellogenin, as measured by ELISA. The data indicate a preferential induction of zr-proteins compared with vitellogenin, both with regard to E2 sensitivity and response time to E2 treatment. These findings suggest an obligate sequence in salmon oogenesis. During sexual maturation low E2 levels at first induce only zonagenesis, while increasing levels of E2 subsequently induce both zonagenesis and vitellogenesis. In nature, the interval between zonagenesis and vitellogenesis may, therefore, be considerable. The data suggest new control mechanisms in fish oogenesis.
... This variation could also be related to the energy cost that is needed for the formulation of the gonads. The vitellogenin is synthesized by the liver (Chen, 1983; Bidwell and Carlson, 1995) and transported to the ovaries by blood circulation (Ng and Idler, 1983) where it is sequestered to serve as an energy reserve for the developing embryo (Celius and Walther, 1998). Based on the gonadosomatic index values, AristotleÕs catfish is a spring spawner (April–June). ...
Article
The life history characteristics of Aristotle’s catfish, Silurus aristotelis (Agassiz 1856) were studied in Lake Pamvotis (northwestern Greece). Samples were collected on a monthly basis using gillnets, trammel-nets and traps. Total lengths ranged from 11.1 to 36.7 cm. Sex ratio was biased toward females (F : M = 1.8 : 1) and was statistically different from unity (χ2 = 46.94, P < 0.001). Spawning is from April to June. The relationship between total length and total weight showed positive allometric growth for males (TW = 0.0035 × TL3.21, r2 = 0.93, n = 198, P < 0.001) and females (TW = 0.0066 × TL3.02, r2 = 0.95, n = 363, P < 0.001). Age was determined on the annual growth marks formed on the spine of the pectoral fin. Based on cross-section readings of the spine, lifespan of the Aristotle’s catfish was 5 years. Age classes 1 and 2 dominated the catches (39.1 and 40.0% of the total sample, respectively). Back-calculated lengths at age showed a rapid increase in fish size during the first year of life, reaching 61.1% of maximum attainable length, and a declining growth rate thereafter. Growth parameters were calculated as L∞ = 36.12 cm, K = 0.37 year−1, t0 = −0.76 year based on the observed lengths at age and as L∞ = 28.19 cm, K = 0.53 year−1, t0 = −0.62 year based on the back-calculated lengths at age. It seems that some of the life history traits (longevity, growth pattern, reproductive period) are influenced significantly by adverse effects of pollution and eutrophication on the lacustrine ecosystem.
... The growth phase consists of two different processes, zonagenesis, the synthesis of eggshell and vitellogenesis, the synthesis of yolk material (vitellogenin, (VTG)). Both proteins are physiologically induced by estrogens, synthesized in the liver and transported to the ovary during oogenesis in several fish species [3][4][5][6][7]. The ZR of a number of fish species consists of 2 to 4 protein monomers [4,5,8]. ...
Article
Estrogen receptor-mediated induction of zona radiata (ZR) and vitellogenin (VTG) mRNA and protein in rainbow trout (Oncorhynchus mykiss) was compared to assess their utility as biomarkers for exposure to estrogenic compounds. Partial sequences of rainbow trout ZR and beta-actin were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers based on conserved regions across a number of species. A 549 bp fragment of the rainbow trout ZR-gene showed a high degree of amino acid sequence identity to that of salmon (77%), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp beta-actin fragment was approximately 100% identical to sequences from several species. Real-time PCR was used to quantify the induction of ZR-gene and VTG in rainbow trout liver after in vivo exposure to estradiol-17 beta (E(2)) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or alpha-zearalenol (alpha-ZEA) (0.1, 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) showed that ZR and VTG were induced in both the liver and the plasma after a single injection of E(2) or alpha-ZEA. ZR was more responsive to low levels of E(2) and alpha-ZEA than VTG, and real-time PCR was shown to be more sensitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensitive biomarker for assessing estrogenic activity.
... The zebrafish (Danio rerio) genome contains at least seven vtgs that encode homologous Vtgs with three distinct types mapped onto two different chromosomes . One vtg transcript has been characterized in the silver lamprey, Ichthyomyzon unicuspies (Sharrock et al., 1992) and the white sturgeon, Acipenser trasmontanus (Bidwell and Carlson, 1995); at least two have been cloned in mummichog (Fundulus heteroclitus) (LaFleur et al., 1995(LaFleur et al., , 2005, Japanese medaka (Oryzias latipes) (GenBank http://www.ncbi.nlm.nih.gov/Genbank/index.html accession numbers: AB064320 and AB074891; Tong et al., 2004), Japanese goby, Acanthogobius flavimanus (Ohkubo et al., 2004), tilapia (Oreochromis aureus) (Lee et al., 1994;Lim et al., 2001), and haddock (Melanogrammus aeglefinus) (Reith et al., 2001); and multiple transcripts were demonstrated in mosquitofish (Gambusia affinis) (Sawaguchi et al., 2005a, b). ...
Book
This book addresses the growing needs in deciphering the biological processes associated with fish reproduction, in view of the growth of aquaculture and the dwindling natural stocks of commercially important fish. It presents a comprehensive overview on egg production in fish, from the standpoint of the oocyte. With this view in mind, the book includes chapters on oocyte development (oogenesis), hormonal regulation and hormone receptors, formation of the egg envelopes, growth, accumulation of nutrients and maternal transcripts, maturation, hydration, ovulation and fertilization. A special emphasis is placed on using state-of-the-art tools including electron microscopy for discerning the ultra-structure of the follicle and genomic/proteomic tools to fully understand biological basis of fish reproduction. Studies on promoting oocyte maturation, ovulation and spawning in farmed fish and preservation of fish oocytes at low temperatures are also included. The book will appeal to University lecturers, students, research scientists and those associated with culture of fish in freshwater or marine aquaculture.
... Therefore, the occurrence of vitellogenin protein in blood plasma and some perinucleolar oocytes, both observed in this study, can be attributed to the onset of the early vitellogenic phase. On the other hand, the presence of vitellogenin protein in some Sertoli cells in this study may be the cause of the previously detected unspecific vitellogenin expression in the sturgeon testes [31,34]. However, the source of the low unspecific vitellogenin expression in the ovaries of the 1600th dph females, reported by Fajkowska et al. [31], is still unknown. ...
Article
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The aim of the study was to raise an issue concerning gonadal impairments in sturgeon reared in recirculating aquatic systems (RAS). In the present study, an in-depth histological evaluation in terms of gonadal pathologies was performed on over-4-year-old (1600 days post-hatching) Russian sturgeon (Acipenser gueldenstaedtii) reared under indoor RAS. A female-biased sex ratio, intersex occurrence, ovarian fat overgrowth, T-cell infiltration and follicle atresia were the most commonly observed disorders in the analyzed gonads. The combined processes of oocyte autophagy and follicular cell apoptosis were engaged in follicular atresia; however, atretic follicles showed a varied morphology, whereas oogonia and oocytes in the early stages of meiosis, as well as spermatogonia, underwent degeneration by apoptosis. The most severe pathology was observed in females with abundant intra-ovarian fat deposition. The extremely fatty ovaries were noted to lose the majority of ovarian follicles, which directly leads to fish sterility. The identified impairments might be related to estrogenic endocrine disruption, as feminization and unspecific vitellogenin synthesis were detected, although the sources of the observed pathologies can be diverse. Therefore, the presented research lays the groundwork for further studies on reproductive disorders in this prized and endangered fish species.
... Molecular characterization of the cDNA of the gene encoding the VTG has received some prior study in sturgeon. The VTG of white sturgeon Acipenser trasmontanus was previously investigated by Kroll (1990), and also one VTG transcript has been characterized in the same species by Bidwell et al. (1991) and Bidwell and Carlson (1995). VTG mRNA expression was used as a marker in juvenile Chinese sturgeon Acipenser sinensis treated with 17b-estradiol and 4-nonylphenol (Zhang et al. 2005). ...
... In our previous work, Fourier transform infrared (FT-IR) spectroscopy (4000-400 cm -1 ) was employed to investigate the biochemical composition of sturgeon plasma [6] and the spectral features could be partially related to sturgeon reproductive maturity at different stages [7]. Other research indicates that sex steroids and vitellogenin (VTG) in sturgeon blood plasma serve as suitable biomarkers for predicting sturgeon maturity [8][9][10][11]. Spectra reflect important chemical information in plasma, including proteins, lipids, polysaccharides, nucleic acids, and constituents important for fish reproduction, such as vitellogenin (VTG) and sex steroids. The raw FT-IR spectral features of white sturgeon plasma were characterized in earlier studies [6,7]. ...
... Molecular characterization of the cDNA of the gene encoding the VTG has received some prior study in sturgeon. The VTG of white sturgeon Acipenser trasmontanus was previously investigated by Kroll (1990), and also one VTG transcript has been characterized in the same species by Bidwell et al. (1991) and Bidwell and Carlson (1995). VTG mRNA expression was used as a marker in juvenile Chinese sturgeon Acipenser sinensis treated with 17b-estradiol and 4-nonylphenol (Zhang et al. 2005). ...
Article
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Plasma chemistry, lipid metabolism and vitellogenin gene expression of captive Sterlet sturgeon Acipenser ruthenus were studied in different maturity stages. A total of 32 fish were sampled, and maturity stages were identified on the basis of histological criteria and direct observation. Females were classified to four groups: previtellogenic, vitellogenic, post-vitellogenic, and atresia. Blood, gonad and liver tissue samples were taken through non-lethal biopsy. Our results showed that plasma levels of glucose, cholesterol, triacylglycerol, high-density lipoprotein, low-density lipoprotein, very low-density lipoprotein, calcium, phosphorus, alkaline phosphatase activity, albumin and total protein increased during ovarian development and were highest at post-vitellogenic stage. The lowest amounts in atresia stage demonstrate that lipid and energy imbalance was related to reabsorption and digestion of the yolk. These results suggested that the VLDL was the main plasma lipoprotein component of Sterlet. We determined that lipoprotein lipase and hepatic lipase activity increased during vitellogenesis process which suggested the role of lipase enzymes in regulating blood lipid metabolism. RT-PCR analysis indicates that Vitellogenin (VTG) mRNA could be detected both in livers and ovaries of female Sterlet. Throughout the study, the expression level of VTG gene showed an increase both in ovaries and in livers reaching its peak at late vitellogenesis stage. This strongly indicated a relation between VTG mRNA and ovarian development.
Article
Yolk platelets, one of the main food stores in the embryonic development, are composed of proteins. However, little is known about the identity of proteins utilized at certain stages of embryogenesis. In this study, we followed the fates of embryonic storage proteins by using an anti‐polyubiquitin monoclonal antibody (mAB) as a probe. The mAb recognized the major storage proteins of Dros op hila, Xenopus and chicken eggs. In the Drosophila embryo, the mAb‐reactive 45‐kDa protein was not used until stage 11 but was used up at stage 16 when the embryo completed segmentation. In the Xenopus embryo, the mAb‐reactive 111 kDa protein was mostly utilized between stages 42 and 45 implying that the protein might be an energy source used just prior to feeding on food. By N‐terminal sequencing the storage protein of Xenopus embryo was identified as a lipovitellin 1. This study confirms that storage proteins are used almost simultaneously at certain stages of embryogenesis and that vitellogenin 1 is the last storage protein in Xenopus embryogenesis.
Article
A 349-residue recombinant polypeptide of Dermatophagoides farinae, Mag 3, has been shown to represent part of a larger 177-kD (M-177) allergen with very high IgE-binding activity. Cloning and sequencing of cDNA from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei was used to characterise the polypeptide containing the Mag 3 sequence. cDNA clones containing the complete sequence of the E. maynei homologue of the M-177 allergen were isolated and analysed. The translation contained not only an amino acid sequence with 90% identity to the 349-residue Mag-3 fragment but also a further sequence with 90% identity to another IgE-binding recombinant D. farinae polypeptide designated Mag 1. The complete sequence encoded a mature polypeptide of 1,650 residues and a molecular mass of 189.5 kD. cDNA clones from D. pteronyssinus also encoded sequences equivalent to the Mag 1 and 3 polypeptides. The M-177 sequence showed strong similarity to the lipid transport apolipophorins found in insect lipophorins. cDNA sequence data show that the D. pteronyssinus and E. maynei homologues of the M-177 high-molecular-weight D. farinae allergen contain sequences equivalent to both the Mag 1 and Mag 3 recombinant IgE-binding fragments. The N-terminal sequence of the full-length 1,650 amino-acid allergen showed strong similarity to the insect apolipophorins which are poorly soluble in aqueous extracts and exist in the lipid transport particles in haemolymph. It is proposed that presentation in lipid particles could be a factor which enhances the immunogenicity of this group of allergens.
Article
Vitellogenesis is an important part of reproductive process in crustaceans, and the process is characterized by the synthesis and accumulation of yolk protein in the developing oocytes. The yolk proteins in crustaceans mainly consist of vitellogenin (Vg) and vitellin (Vn), which are respectively present in extra-oocyte tissues and intra-oocytes. The site and the process of yolk protein synthesis in crustaceans are still controversial. The synthesis site of Vg in a crustacean species, Macrobrachium rosenbergii, is determined by immunological and immunohistochemical techniques, and molecular cloning of a cDNA encoding the primary structure of Vn in this study. The hepatopancrease is clearly shown to be the synthesis site of Vg in this species. The length of Vg mRNA was estimated as about 6 kb from Northern blotting analysis. The partial primary structure of Vg gene is presented, and the post-translational processing are further discussed. For the first time, the partial primary structure of Vg gene and the synthesis site of Vg approached by molecular cloning in crustaceans are presented.
Article
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Mounting evidence confirms that hepatic biosynthetic processes are essential for female sexual maturation in fish, which is directly controlled by estrogens. These oogenetic events (zonagenesis and vitellogenesis) are induced in both sexes by estrogens. In this paper, we report the induction of zona radiata (zr) proteins and vitellogenin in primary hepatocytes from Atlantic salmon (Salmo salar L.) exposed to xenoestrogens and mycotoxins. Cells were treated with doses of 1, 5, and 10 microM 4-nonylphenol (4-NP), o, p'-DDT, lindane ([gamma]-HCH), and bisphenol A (BPA), which all induced zr proteins and vitellogenin in an approximate dose-dependent manner. Hepatocytes were also treated with combinations of xenoestrogens at 1 or 2 microM, resulting in elevated levels of both zr proteins and vitellogenin, compared to single treatment. The estrogenic activity of the mycotoxin zearalenone (ZEA) and its metabolites [alpha]-ZEA) and ss-zearalenol (ss-ZEA)], with regard to zonagenesis and vitellogenesis, was assessed in this assay system. Mycotoxins were used at concentrations of 10, 100, or 1,000 nM. All induced zr proteins and vitellogenin, with [alpha]-ZEA being the strongest inducer. When cells were treated with xenoestrogens or mycotoxins in combination with an estrogen receptor inhibitor (ICI 182,780), the induction of both zr proteins and vitellogenin was inhibited in all cases. Thus, the reported estrogen effects are bonafide estrogen responses. Zona radiata proteins were more responsive than vitellogenin to both xenoestrogens and mycotoxins. The versatility and sensitivity of the hepatocyte assay demonstrates that biosynthesis of zr proteins provides a new supplementary method for estimating xenoestrogenicity and mycotoxin action.
Article
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Molecular cloning of cDNAs encoding an egg yolk precursor, vitellogenin (Vtg), was performed using the liver of estrogen-treated Amur sturgeon. Three kinds of vtg cDNA were cloned and temporarily named as vtg1, vtg2 and vtg3. These vtg cDNAs were obtained as contiguous sequences; each of vtg1, vtg2 and vtg3 sequences contained a complete open reading frame (5,307, 5,247 and 5,319 bp, respectively), each encoding 1,769, 1,749 and 1,773 aa residues, respectively. Similarity among amino acid sequences deduced from the three vtg cDNAs were relatively low, ranging from 64.3% to 47.9%. Three Vtgs appeared to be a complete-type, consisting of all representative yolk protein domains. Phylogenetic analysis revealed that Amur sturgeon Vtg1 formed a clade together with a published white sturgeon Vtg, while Amur sturgeon Vtg2 and Vtg3 formed another distinct clade. The results of phylogenetic analysis confirmed all three Vtgs belong to VtgAB type; vtg1/Vtg1, vtg2/Vtg2 and vtg3/Vtg3 were hereby designated as vtgAB1/VtgAB1, vtgAB2a/VtgAB2a and vtgAB2b/VtgAB2b, respectively. This study provided a basis to understand multiplicity in sturgeon vtg/Vtg and set a stage for their future application as reproductive biomarkers in sturgeon aquaculture.
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A wide range of organic contaminant compounds prevalent in the aquatic environment has been shown to exhibit hormone-disrupting activity. The actual potency of such compounds are low compared with endogenous hormones, such as 17beta-estradiol, but may still produce detrimental biological effects. Induced hormone levels are routinely measured using commercial testing kits, though these fail to relate to actual effects. Field and laboratory studies on the biological effects of environmental estrogens have, in the past, largely relied on assays of vitellogenin (vtg) induction in male fish, reduced growth in testes formation, and intersex incidence. Here, we critically review the current and potential application of molecular techniques in assessing the adverse biological reproductive effects of endocrine-disrupting chemicals in aquatic organisms. The role of fish ( estrogen, androgen, and progestogen) hormone receptors and invertebrate ( ecdysone) hormone receptor, egg production ( vtg and chorion) proteins, steroid biosynthesis enzymes ( aromatase, sulfotransferase, and hydroxysteroid dehydrogenase), DNA damage, apoptosis, and their potential development as biomarkers are discussed in turn. In each case, the sequences characterized are presented and homologies across species are highlighted. Molecular methods of gauging vtg and zona radiata (ZR) expression and protein concentrations have included immunoassay and reverse transcription polymerase chain reaction (RT-PCR). Suggestions for the isolation for key gene expression products ( aromatase, ZR, and vtg, for instance), from a wider range of fish species using degenerate primers, are given. Endocrine disruption in invertebrates has received less attention compared with fish, partly because the knowledge regarding invertebrate endocrinology is limited. Here we review and suggest alternate isolation strategies for key players in the imposex induction process: vitellin (Vn), aromatase, and Ala-Pro-Gly-Trp ( APGW) amide neurohormone. Current molecular-level techniques rely on ligand-binding assays, enzyme-linked immunosorbent assay ( ELISA), and, more recently, gene expression. In the future, more reliance will be placed on the development of gene expression assays using reporter systems combined with cross-species PCR-based or polyclonal antibody-based assays. We discuss the use of recombinant receptors as a means of primary screening of environmental samples for estrogenicity and antiestrogenicity, which avoids species and seasonal variation in receptor response to ligand binding, a recognized problem of earlier bioassays. Most exciting is the potential that microarray and proteomics approaches have to offer. Such techniques are now used routinely in medical research to identify specific genes and proteins affected by treatment with endocrine disruptors, including estradiol. The technique has yet to be used to screen aquatic organisms, but it has the potential to implicate previously unsuspected estradiol-sensitive genes that may later become molecular markers of endocrine disruption.
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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.
Article
The localisation of estrogen receptors (ERalpha and ERbeta) and vitellogenin (VTG) transcripts were examined in the liver and testis in male rainbowfish exposed to 17beta-estradiol (E2; 0, 50 and 500 ng/L) via the water for up to 7 days. The ER transcripts were localised within the perinuclear region of the hepatocytes and were up-regulated with E2 exposure. A parallel induction of liver VTG transcripts and protein was observed within 24h, followed by a time-dependent increase in VTG protein. In the testis, both ERs were up-regulated in the germ and epithelial cells, while VTG protein was detected in the cellular space surrounding the spermatids and in association with the connective tissue of the sperm tubules. These results indicate that the ERs are positively auto-regulated in the liver and testis of male rainbowfish. The cellular localisation of VTG within the testis may suggest implication in the mediation of adverse effects of endocrine disrupting chemicals such as testicular growth inhibition, testis-ova and sex reversal.
Article
Fourier transform infrared spectroscopy (FT-IR, 4000-400 cm(-1)) was applied to blood plasma of farmed white sturgeon (N = 40) to differentiate and predict the stages of ovarian maturity. Spectral features of sex steroids (approximately 3000 cm(-1)) and vitellogenin (approximately 1080 cm(-1)) were identified. Clear segregation of maturity stages (previtellogenesis, vitellogenesis, postvitellogenesis, and follicular atresia) was achieved using principal component analysis (PCA). Progression of oocyte development in the late phase of vitellogenesis was also monitored using PCA based on changes in plasma concentrations of sex steroid and lipid content. The observed oocyte polarization index (PI, a measure of nuclear migration) was correlated with changes in plasma sex steroid levels revealed by FT-IR PCA results. A partial least squares (PLS) model predicted PI values within the range 0.12-0.40 (R = 0.95, SEP = 2.18%) from differences in spectral features. These results suggest that FT-IR may be a good tool for assessing ovarian maturity in farmed sturgeon and will reduce the need for the invasive ovarian biopsy required for PI determination.
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Less is known about the reproductive endocrinology of sturgeons compared to modern teleosts. However, tools to assess the reproductive endocrinology and effects of environmental factors on reproduction do exist. This review utilizes case studies to describe the parameters involved in environmental endocrinology and the management and recovery efforts for the phylogenetically ancient sturgeon and paddlefish (Clade Chondrostei). Specifically, we discuss the use of environmental endocrinology to determine sex and stage of maturity and identify oviposition on spawning grounds, the importance of understanding endocrine disruption pathways, the challenges and benefits of assessing stress in wild populations of sturgeon, and three major physiological events in the reproductive development of farmed sturgeon understanding of which appears to be crucial for improving sturgeon aquaculture.
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Indigenous aquatic population such as fish could be used as a successful test species for evaluating the ecological effects in aquatic environment. In the present study, vitellogenin (Vtg) from slender bitterling (Acheilognathus yamatsutae), an indigenous aquatic species in Korea, was cloned and sequenced to determine if the Vtg gene possesses an important characteristic so as to act as a sensitive biomarker for estrogenic endocrine disrupting chemicals (EEDCs). The sbVtg cDNA is 5010 bp in length, containing a 4653 bp open reading frame, which encodes 1550 amino acid residues. The sbVtg cDNA was divided into lipovitellin heavy chain (LvH), phosvitin (Pv), lipovitellin light chain (LvL) as well as a beta'-component (beta'-c) domain, and belongs to VtgAo2. SbVtg has conserved important sequences for Vtg functions such as signal peptide, VtgR-binding region, and disulfide bond formation, all of which are consistent with those of other teleosts. In addition, the male slender bitterling aqueous exposed to 17 alpha-ethinylestradiol (EE2, 12.5, 25, and 50 ng/L) produced a statistically significant and concentration-dependent increase in hepatic Vtg mRNA expression, which showed a similar pattern to biliary estrogenic activity, measured by ERE-reporter gene assay. Thus, this study clearly indicates that the induction of Vtg in slender bitterling might be a suitable biomarker in toxicological research of EEDCs.
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Males and females of cultured white sturgeon, Acipenser transmontanus, mature at an average age of 4 and 8 years, respectively. However, the onset of ovarian vitellogenesis and puberty are highly asynchronous in the female stock. Gonadal cycles are annual in males and biennial in females, and gametogenesis is influenced by season. Neuroendocrine regulation of reproduction appears to involve a dual gonadotropin system controlling gonadal development and spawning. Labile puberty and sex-specific duration of the gonadal cycle are distinct characteristics of cultured and wild sturgeon. Photoperiod and temperature play a significant role in environmental regulation of the reproductive cycle, but further studies are necessary to elucidate the roles of endogenous and environmental factors in sturgeon reproduction which is critically important for both aquaculture and conservation of endangered wild stocks.
Article
In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo marmorata exposed to 17β-estradiol (E(2)). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E(2), synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E(2) treatment are discussed.
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Vitellogenin (Vg), a major precursor to egg yolk proteins, was purified from plasma of an estradiol-treated female tuatara (Sphenodon punctatus) by MgCl2–EDTA precipitation and DEAE-cellulose chromatography. The amino acid composition of tuatara Vg is similar to that of other vertebrate Vgs and contains a large proportion of serine (13.7 mol/100 mol of total amino acid). The amino acid sequences of the N-terminus of mature Vg (33 residues) and of several trypsin- and CNBr-generated peptides were determined. Six peptide sequences obtained from tuatara Vg could be aligned with Vg sequences from other vertebrates. Reduced and non-reduced forms of tuatara Vg have the same apparent molecular mass (∼218 kDa) when resolved by SDS-PAGE, indicating that inter-chain disulfide bonds are not a feature of the molecule in this species. Western blot analysis with anti-tuatara Vg antiserum indicated that at least some epitopes are shared among Vgs of turtle, alligator and tuatara.
Article
Summary The objective of the study was to assess the changes of vitellogenin (Vg) during the course of the reproductive cycle in Amur sturgeon (Acipenser schrenckii). Vg was purified from the serum of vitellogenic female Amur sturgeon by distilled water and gel filtration. Vg had an apparent molecular mass of 410 kDa and appeared as one band corresponding to 205 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against amur sturgeon lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. Lv was purified from egg extracts by ammonium sulfate solution and gel filtration. Vg was confirmed to be a lipoglycophosphoprotein by staining with Red oil, Molecular Proes’ Pro-Q® Emerald 300 Glycoprotein Gel stain and Pro-Q® Diamond Phosphoprotein Gel Stain. Vg induction was detected after injection of E2 at a concentration of at least 0.5 mg kg−1, and the plasma Vg concentration was increased by injection of the animals to 0.5 mg kg−1 for 5 day late. Immature sturgeons at 1015 g weight do not naturally synthesize Vg, but strongly responded to exogenously added E2. In addition, if they were not stimulated with exogenous E2, the youngest sturgeons did not show any detectable amount of Vg in their plasma, suggesting that Amur sturgeons with a body mass range of 903–1015 g could be used for the induction test, irrespective of their sex. The Enzyme-linked immuno-sorbant assay for sturgeon vg was developed to quantify serum Vg, using purified sturgeon Vg and anti-Vg. The measurable range was from 16.25 to 1000 ng ml−1. The dilution curve in ELISA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Vg levels varied throughout natural vitellogenesis from 0 μg ml−1 (1–3 years old) to approximately 200 μg ml−1 (7–8 years old). We observed an early transitory peak of serum Vg levels 200 μg ml−1 (for 10 months) at the time of early vitellogenesis and high Vg levels 218 μg ml−1 (for 3 months) in spring period before ovulation. It appears that the duration of vitellogenesis in Amur sturgeon is lasting more than 2 years.
Chapter
Males and females of cultured white sturgeon, Acipenser transmontanus, mature at an average age of 4 and 8 years, respectively. However, the onset of ovarian vitellogenesis and puberty are highly asynchronous in the female stock. Gonadal cycles are annual in males and biennial in females, and gametogenesis is influenced by season. Neuroendocrine regulation of reproduction appears to involve a dual gonadotropin system controlling gonadal development and spawning. Labile puberty and sex-specific duration of the gonadal cycle are distinct characteristics of cultured and wild sturgeon. Photoperiod and temperature play a significant role in environmental regulation of the reproductive cycle, but further studies are necessary to elucidate the roles of endogenousand environmental factors in sturgeon reproduction which is critically important for both aquaculture and conservationof endangered wildstocks.
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The coelacanth Latimeria menadoensis, a living fossil, occupies a key phylogenetic position to explore the changes that have affected the genomes of the aquatic vertebrates that colonized dry land. This is the first study to isolate and analyze L. menadoensis mRNA. Three different vitellogenin transcripts were identified and their inferred amino acid sequences compared to those of other known vertebrates. The phylogenetic data suggest that the evolutionary history of this gene family in coelacanths was characterized by a different duplication event than those which occurred in teleosts, amniotes, and amphibia. Comparison of the three sequences highlighted differences in functional sites. Moreover, despite the presence of conserved sites compared with the other oviparous vertebrates, some sites were seen to have changed, others to be similar only to those of teleosts, and others still to resemble only to those of tetrapods.
Article
Numerous studies about sturgeon reproductive physiology over the past twenty years have helped researchers to understand the relationship between how sexual maturation in female sturgeon may affect the quality and yield of caviar. Here, works about white sturgeon (Acipenser transmontanus) female reproductive physiology relating to egg harvest and roe quality are summarized.
Article
Morphological and biochemical changes in white sturgeon (N=11) plasma and ovarian follicles were monitored during late vitellogenesis and follicular atresia using Fourier Transform Infrared Spectroscopy (FT-IR, 4000–400cm−1), with the objective to predict ovarian regression. Spectral features of plasma were correlated with ovarian follicular atresia based upon changes in spectra of sex steroids and vitellogenin. Principal Component Analysis could differentiate ovarian maturity during vitellogenesis and onset and progression of ovarian regression. It was possible to predict the ovarian conditions (late vitellogenesis vs. early atresia) about 70% of the time using Soft Independent Modeling of Class Analogy models. We conclude that FT-IR spectroscopy provides a useful tool for predicting ovarian maturity and optimal time of roe harvest.
Article
We have applied a method for quantifying relative levels of messenger RNA (mRNA) transcription to assess chemically induced gene expression in fathead minnows (Pimephales promelas). Synthetic oligonucleotides designed for the fathead minnow vitellogenin gene transcription product were used in a reverse transcription polymerase chain reaction (RT-PCR) protocol. This sensitive and rapid strategy detected vitellogenin gene transcription in livers of male fathead minnows exposed to concentrations as low as 2 ng/L of the endocrine-disrupting compound 17-α-ethynylestradiol for 24 h. Surprisingly, vitellogenin transcription products also were detected in gill tissue and in 48-h-old posthatch fathead minnow larvae. Relative levels of vitellogenin gene induction among individuals were quantified in a single-step reaction (PCR multiplex) with 18S rRNA universal primers and Competimerst concurrently with fathead minnow vitellogenin oligonucleotides. This quantitative approach will markedly enhance detection of the first cellular event of estrogenic exposure to aquatic ecosystems in both field and laboratory systems. Use of the model provides sensitivity of detection at a concentration below those that cause mortality or visible signs of stress in fish or other aquatic organisms. The model may also provide an in vivo screening method for estrogenlike endocrine-disrupting compounds.
Article
Vitellogenins are serum proteins that are considered major protein components of the yolk of an egg. Vitellogenins are related to other serum proteins that bind lipid in vertebrates, such as apolipoprotein B (apo B). On the other hand, the yolk proteins are low-molecular weight molecules present in the eggs of cyclorrhaphan Diptera. Using several phylogenetic and statistical approaches, the chapter confirms the relationship between the cyclorrhaphan yolk proteins and the lipases, between the vitellogenins and the apo B family. It exhibits that the lipophorins are also significantly similar to this family. The existence of a conserved region in the deduced amino-acid sequences of vitellogenin genes of three insects— namely, mosquito, boll weevil, and silkworm is also reported. This region is conserved in other vitellogenin genes and contains a region identified as α-helix-rich in the lamprey and that the yolk protein sequences align with this conserved region. The chapter presents an evidence that can indicate either a distant relationship between yolk protein genes and vitellogenin genes or that they have evolved convergently owing to ligand binding constraints.
Article
Many studies link pulp and paper mill effluent (PPME) exposure to adverse effects in fish populations present in the mill receiving environments. These impacts are often characteristic of endocrine disruption and may include impaired reproduction, development and survival. While these physiological endpoints are well-characterized, the molecular mechanisms causing them are not yet understood. To investigate changes in gene transcription induced by exposure to a PPME at several stages of treatment, male and female fathead minnows (FHMs) were exposed for 6 days to 25% (v/v) secondary (biologically) treated kraft effluent (TK) or 100% (v/v) combined mill outfall (CMO) from a mill producing both kraft pulp and newsprint. The gene expression changes in the livers of these fish were analyzed using a 22K oligonucleotide microarray. Exposure to TK or CMO resulted in significant changes in the expression levels of 105 and 238 targets in male FHMs and 296 and 133 targets in females, respectively. Targets were then functionally analyzed using gene ontology tools to identify the biological processes in fish hepatocytes that were affected by exposure to PPME after its secondary treatment. Proteolysis was affected in female FHMs exposed to both TK and CMO. In male FHMs, no processes were affected by TK exposure, while sterol, isoprenoid, steroid and cholesterol biosynthesis and electron transport were up-regulated by CMO exposure. The results presented in this study indicate that short-term exposure to PPMEs affects the expression of reproduction-related genes in the livers of both male and female FHMs, and that secondary treatment of PPMEs may not neutralize all of their metabolic effects in fish. Gene ontology analysis of microarray data may enable identification of biological processes altered by toxicant exposure and thus provide an additional tool for monitoring the impact of PPMEs on fish populations.
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1.1. A chromatographic procedure for isolating intact, highly purified vitellogenin from the plasma of the goldfish, Carassius auratus, is described.2.2. Vitellogenin is normally present in the plasma of females during the breeding season, but is not found in males; however, estradiol-17β induces the appearance of vitellogenin in male plasma.3.3. Native (dimeric_ goldfish vitellogenin has a molecular weight of about 380,000 and contains 15.4% protein-nitrogen and 0.79% protein-phosphorus; amino acid profiles were compared for vitellogenins from goldfish, Salmo gairdneri and Xenopus laevis.4.4. At least three polypeptides, ranging in molecular weight from 140,000 to 147,000, were identified by sodium dodecyl sulfate-gel electrophoresis of vitellogenin; native (dimeric) vitellogen is comprised of two such polypeptides together with approx 20% lipid.5.5. Native goldfish yolk protein consists of a lipovitellin and a phosvitin fraction; the former yields one class of at least two large polypeptides () and another class of up to four small polypeptides () on sodium dodecyl sulfate-gel electrophoresis, while the phosvitin fraction yields at least two polypeptides () on Sephadex-gel filtration.6.6. In this teleostean species, multiple vitellogenin polypeptides are apparently processed into multiple yolk protein polypeptides within the growing oocyte.
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One of the most obvious characteristics of the egg cells of oviparous animals is their large size resulting to a major extent from the deposition of nutritional reserves, mainly constituted of yolk proteins. In general, these are derived from a precursor called vitellogenin, which undergoes posttranslational modifications during secretion and during transport into and storage within the oocytes. Comparative analysis of the structural organization of the vitellogenin gene and of its product in different species shows that the vitellogenin gene is very ancient and that in vertebrates the gene may have more resemblance to the earliest gene than in invertebrates.
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Experimental results on fast-twitch muscle of rainbow trout following exercise and during subsequent recovery lead us to a reinterpretation for the function of the components of the purine nucleotide cycle (PNC). Exhaustive exercise depletes tissue ATP by more than 90% and results in a stoichiometric gain in IMP and ammonium ions. Simultaneously, white-muscle aspartate decreases by half, but its maximum contribution can account for less than 2% of the accumulated ammonium. Of the three enzymes of the purine nucleotide cycle, AMP deaminase, adenylosuccinate synthetase and adenylosuccinate lyase, only AMP deaminase is functional during exhaustive exercise. During the slow (>15 hour) recovery, AMP deaminase is effectively shut off, while the other two enzymes replenish the adenylate pool. At all times, a tight inverse correlation exists between ATP and IMP concentrations. Tissue ammonium and malate supply the required aspartate. Theoretical treatment with speciai attention to proton dynamics in a potentially anaerobic tissue also leads to the conclusion that rather than constituting a true cycle, distinct parts of the PNC are temporally segregated. We hypothesize that during periods of high energy demand, exclusively AMP deaminase is activated as a means (1) to push the myokinase reaction toward ATP synthesis, (2) to supply allosteric effectors, and (3) to remove some of the accumulating protons through the formation of ammonium, all at the expense of the adenylate pool. The process leading to its replenishment, which involves the production of two protons and the consumption of a high-energy phosphate, can be active during aerobic recovery only. Full PNC activity during anaerobic muscle work would outweigh any metabolic advantages of the AMP deaminase reaction, and severely aggravate the problem of acid-base regulation.
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Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane.
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A major event during the growth period of oocytes in nonmammalian animals is the accumulation of yolk. The genes coding for the yolk protein precursor, known as vitallogenin, are well characterized in a few vertebrate and invertebrate species. Studies on the evolution of these genes and on the regulatory mechanisms involved in their time, tissue- and hormone-specific expression are presented and discussed in this review.
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Vitellogenin, an ancient animal protein, is the major yolk protein of eggs, where it is used as a food source during embryogenesis. Here it is shown that vitellogenins, including those from the invertebrates Caenorhabditis elegans and Drosophila melanogaster, contain domains that are homologous with parts of apolipoprotein B-100 (apoB-100) of human low-density lipoprotein and human lipoprotein lipase. As vitellogenins are likely to have been used by invertebrates during embryogenesis well before the circulation of lipids appeared in vertebrates, it is suggested that copies of a precursor gene, serving a function similar to vitellogenin, were modified to code for part of apoB-100 and lipoprotein lipase in vertebrates. In addition to providing a link between invertebrates and vertebrates for proteins involved in lipid transport, these homologies suggest new functions for vitellogenin other than being a yolk food for the developing embryo.
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Expression of vitellogenin, the yolk protein precursor, is strictly regulated during development. In previous studies on a variety of organisms, vitellogenin gene expression has been shown to be restricted to one or two tissues in adult female animals. In this report we show that, in contrast, sea urchin vitellogenin is synthesized in both females and males. To identify sea urchin vitellogenin, we raised antibodies specific for the major yolk protein. We show here that a 155-kDa polypeptide, immunoprecipitable by the antibody to the major yolk protein, is synthesized in the intestines of female and male sea urchins and also in ovaries and testes. This 155-kDa polypeptide is converted to a 195-kDa vitellogenin in each of these tissues; further modification to yield the 180-kDa major yolk protein occurs only in the ovary. We have also identified a vitellogenin cDNA clone and used it to study vitellogenin mRNA production. An abundant 5.1-kilobase mRNA was found in the tissues containing vitellogenin. Our results suggest that vitellogenin may serve the following two functions in sea urchins: its classical role as a yolk protein precursor and an unidentified function required by adults of both sexes.
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In Xenopus laevis four estrogen-responsive genes are expressed simultaneously to produce vitellogenin, the precursor of the yolk proteins. One of these four genes, the gene A2, was sequenced completely, as well as cDNAs representing 75% of the coding region of the gene. From this data the exon - intron structure of the gene was established, revealing 35 exons that give a transcript of 5,619 bp without the poly A-tail. This A2 transcript encodes a vitellogenin of 1,807 amino acids, whose structure is discussed with respect to its function. At the nucleic acid as well as at the protein level no extensive homologies with any sequences other than vitellogenin were observed. Comparison of the amino acid sequence of the vitellogenin A2 molecule with biochemical data obtained from the different yolk proteins allowed us to localize the cleavage products on the vitellogenin precursor as follows : NH2- lipovitellin I - phosvitin (or phos-vette II - phosvette I) - lipovitellin II - COOH.
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The structural organization and the coding nucleotide sequence of the Xenopus laevis A2 and the chicken major vitellogenin genes have been compared. Both genes show the same exon-intron organization. However, the degree of homology between the nucleotide and derived amino acid sequences varies extensively along the genes. Several of the 35 exons are quite similar, and a unique cysteine motif in the lipovitellin II domain is conserved between the two genes. In contrast, one internal region is quite divergent. Part of this region encodes phosvitin, which appears to have evolved rapidly by both point mutations and duplications of serines or short other amino acid stretches. On the basis of these observations, we discuss the possible mechanism of evolution of phosvitin in vertebrates.
Article
Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.
Article
This chapter discusses the yolk formation of teleost fishes. The process of vitellogenesis in teleosts has been shown to be similar to that operating in other oviparous vertebrates. The contribution of autosynthetic processes (endogenous vitellogenesis) to the yolk mass in the teleost ovary, relative to exogenous yolk acquired by incorporation of vitellogenin, has not been estimated. Exogenous vitellogenesis can be considered to consist of two phases. The first phase involves the induction of hepatic vitellogenin production under stimulation of ovarian estrogen. During the second phase vitellogenin is taken up from the blood stream and incorporated into ovarian yolk proteins. In salmonids maturational gonadotropin occurs at high levels in plasma around spawning time but is near the lower limit of the radioimmunoassay during the phase of active incorporation of vitellogenin. There appears to be a small increase in maturational gonadotropin coincident with an increase in estradiol in trout plasma early in vitellogenesis, and antibody to maturational gonadotropin inhibits ovarian growth immediately prior to the rapid and massive increase in the ovarian weight. Therefore, there appears to be a low level of maturational hormone present when the fish resumes ovarian development after spawning, but it is sufficient to establish vitellogenin production by the liver.
Chapter
This chapter describes the different aspects of vitellogenesis and oocyte assembly. The major components of fish oocytes are derived from the blood-borne high-molecular-weight compound, vitellogenin, which is synthesized in the liver of oviparous vertebrates. The classification of vitellogenin as a phospholipoglycoprotein indicates the crucial functional groups that are carried on the protein backbone of the molecule—namely, lipids, some carbohydrates, and phosphate groups. In the rainbow trout, estrone administration leads to the induction of vitellogenin synthesis in the liver and its release into the bloodstream, but estrone displays only 5%–12% of the potency of estradiol. The biochemical information concerning vitellogenin clearly indicates that a great deal of posttranslational modification must occur in the liver cell to reach the finished product seen in the serum. An integral part of estradiol action is the observed hypercalcemia in vitellogenic fish, which can largely be ascribed to the calcium-binding properties of phosphorylated and highly charged, components of the native vitellogenin molecule.
Article
Vitellogenin variation was studied in 11 species of Perciformes encompassing the families Cichlidae, Serranidae, Lutjanidae, Centropomidae, and Eleotrididae. Despite the enormous morphological and ecological diversity that characterizes this order of teleosts, northern blot analysis revealed that the Oreochromis aureus vitellogenin gene probe will recognize estradiol-induced vitellogenin messenger RNA of distant percoid relatives under stringent conditions of hybridization, suggesting that the vitellogenin gene is well conserved at least within the suborder Percoidei. Multiple vitellogenin gene transcripts were detected in the Cichlidae and these are correlated with the finding of multiple vitellogenin protein monomers in this family. In other families of non-cichlid percoids, only one vitellogenin transcript, and, correspondingly, a single vitellogenin monomer was observed. No cross hybridization was, however, observed with a representative of the suborder Gobioidei. The pattern of protein bands seen in SDS-PAGE also differed. The main features of vitellogenin protein diversity in the Perciformes are interfamily variability in size and subunit number. © 1992 Wiley-Liss, Inc.
Article
Caenorhabditis elegans vitellogenins are encoded by a family of six genes, one of which,vit-5, has been previously sequenced and shown to be surprisingly closely related to the vertebrate vitellogenin genes. Here we report an alignment of the amino acid sequences of vitellogenins from frog and chicken with those from threeC. elegans genes:vit-5 and two newly sequenced genes,vit-2 andvit-6. The four introns ofvit-6 are all in different places from the four introns ofvit-5, but three of these eight positions are identical or close to intron locations in the vertebrate vitellogenin genes. The encoded polypeptides have diverged from one another sufficiently to allow us to draw some conclusions about conserved positions. Many cysteine residues have been conserved, suggesting that vitellogenin structure has been maintained over a long evolutionary distance and is dependent upon disulfide bonds. In addition, a 20-residue segment shows conservation between the vertebrate and the nematode vitellogenins. This sequence may play a highly conserved role in vitellogenesis, such as specific recognition by oocytes. On the whole, however, selection may be acting more strongly on amino acid composition and codon usage than on amino acid sequence, as might be expected for abundant storage proteins: The amino acid compositions ofvit-2, vit-5, andvit-6 products are remarkably similar, despite the fact that the sequence of thevit-2 protein is only 22% and 50% identical to the sequences ofvit-6 andvit-5 proteins, respectively.
Article
Using a primary culture of trout hepatocytes we compare the kinetics of accumulation of vitellogenin and its messenger RNA after estrogen administration. We found that the cells were more sensitive to estradiol than to other estrogens. The lowest effective concentration of estradiol was 10−9M. At 10−6M the androgens have no effect. Comparison of the primary and secondary stimulation with E2 shows that the initial rate of accumulation of vitellogenin is very much higher in the secondary stimulation. Over a time course of primary stimulation we show that after estradiol withdrawal the rate of accumulation of vitellogenin mRNA in the secondary is a function of time of the first stimulation.
Article
A simple method for generating cDNA libraries from submicrogram quantities of mRNA is describe. It combines classical first-stand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis [Okayama, H., and Berg, P., Mol. Cell. Biol. 2 (1982) 161–170]. Neiher the elaborate vector-primer system nor the classical hairpin loop cleavage by S1 nuclease are used. cDNA thus made can be tailed and cloned without further purification or sizing. Cloning efficiencies can be as high as 106 recombinants generated per μg mRNA, a considerable improvement over earlier methods. Using the fully sequenced1300 nucleotide-long bovine preproenkephalin mRNA, we have established by sequencing that the method yields faithful full-length transcripts. This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.
1.1. Two days after stimulation of male Oreochromis aureus with estradiol at 4 μg/g body weight, two vitellogenins of 300,000 and 500,000 daltons were enhanced with a simulataneous drop in albumin.2.2. Anti-vitellogenin antibodies were highly species-specific. No cross-reaction was observed with distant relatives from family Cyprinidae (Carp and goldfish), nor with vitellogenin of fish from within the same family (Pterophyllum spp.; angel fish).3.3. Of particular interest is the detection of these vitellogenins, albeit in low quantities, in the plasma and gonads of unstimulated male fish. Th gonadal vitellogenins are also inducible by exogenous estradiol.
Article
The Evolution of Vertebrate Design is a solid introduction to vertebrate evolution, paleontology, vertebrate biology, and functional, comparative anatomy. Its lucid style also makes it ideal for general readers intrigued by fossil history. Clearly drawn diagrams illustrate biomechanical explanations of the evolution of fins, jaws, joints, and body shapes among vertebrates. A glossary of terms is included. "A luminous text is matched by lucid drawings rationally placed. . . . A great teaching monograph, the book will charm lay readers of fossil history. For virtually every college & public collection."—Scitech Book News
Article
A procedure for extracting plasmid DNA from bacterial cells 1s described. The method 1s simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.
Article
A specific cell surface receptor mediates the endocytosis of the yolk protein vitellogenin (VTG), a lipoglycoprotein, into growing oocytes of the insect Locusta migratoria. The ability of the VTG receptor to recognize VTG was analyzed in binding tests after modification by five lysine-specific and two other reagents. Progressive chemical modification of the lysyl and arginyl residues resulted in reduction or loss of the derivatized VTG to compete for binding to the VTG receptor with unmodified VTG. Although the precise role of the lysine residues in receptor binding remains to be defined we conclude that they are involved in expression of a recognition site interacting with the binding domain of the VTG receptor. Sulfhydryl groups are not involved in the conformation of the recognition site or binding ability of VTG.
Article
The amino acid sequence of lamprey vitellogenin has been predicted from the nucleotide sequence of cloned cDNA. The sites of proteolytic cleavage that produce the lipovitellin complex from the vitellogenin have been located by comparing the N-terminal sequences of two lamprey lipovitellin polypeptides with the predicted sequence. These results also confirm that the vitellogenin sequence derived here corresponds to the lipovitellin complex for which the crystal structure has been solved previously. Predictions of secondary structure indicate that the region most likely to correspond to the large alpha-helical domain of the crystallographic model consists of vitellogenin residues 300 to 600. Similar to the lipovitellins of Xenopus laevis, lamprey lipovitellin appears to lack approximately 200 C-terminal residues that are present in vitellogenin. However, the lamprey lipovitellin differs from those of Xenopus and chicken in two respects. First, most of the serine-rich domain that is present as the phosvitin polypeptide in the lipovitellins of the higher vertebrates appears to be lost in the maturation of lamprey vitellogenin to lipovitellin. Second, the domains that constitute the large lipovitellin-1 polypeptide in Xenopus and chicken are present in two polypeptides in lamprey, owing to an additional proteolytic processing event.
Article
Vitellogenesis was induced in white sturgeon by administration of estrogen through silastic implants. Vitellogenin mRNA was identified by agarose gel electrophoresis and cell-free translation. A highly abundant 5.7-kb mRNA was induced in the liver of estrogen-treated sturgeon. Cell-free translation of poly(A)+ mRNA showed the induction of two high-molecular-weight proteins of 180 and 120 kDa. These two proteins, encoded by the 5.7-kb mRNA(s), were immunoprecipitated by antiserum to serum from vitellogenic sturgeon. Immunoprecipitations also showed the presence of four other serum proteins synthesized by the liver of estrogen-treated sturgeon. The induction of vitellogenesis by estrogen in sturgeon, which are a primitive teleost, was found to be similar to induction of vitellogenesis in amphibians, avians, and other teleosts. Estrogen treatment induced a highly abundant vitellogenin mRNA as well as several mRNAs for other serum proteins. However, the presence of two distinct vitellogenin monomers in the cell-free translation assay was significantly different from the results in other species.
Article
cDNA synthesised from liver Poly A+ RNA of estradiol-stimulated male Oreochromis aureus was ligated to PTZ-19R and transformed into E. coli. TG-1 to yield 6 x 10(4) clones/ug DNA, of which 6% was vitellogenin positive. Restriction maps indicate 4 possible subgroups of vitellogenin cDNA, with homology, observed by Southern hybridisation, at a fragment flanked by Pst 1 sites. When the amino acid sequence derived from the DNA sequence of pOA Vg 62 was aligned with that of S. gairdneri, X. laevis, C. elegans and D. melanogaster, homologies of 63%, 22%, 21% and 15%, respectively, were obtained. Estradiol induced a vitellogenin gene transcript of 6500 nucleotides at 6 h and reached a maximum at 72 h after stimulation.
Article
Estrogen has a marked effect on the expression of vitellogenin, the egg yolk precursor protein in the liver of egg-laying vertebrates. cDNA clones specific for trout vitellogenin mRNA have been used to study the expression of the vitellogenin genes in rainbow trout (Salmo gairdneri). The steady-state levels of vitellogenin mRNA in the liver of male rainbow trout were measured during primary and secondary stimulation with estradiol. The kinetics of induction in trout appear to be very similar to those seen in Xenopus and chicken in that a lag of approximately 2 days is observed in the accumulation of serum vitellogenin during primary induction. This lag is not observed during the secondary stimulation. The primary induction of vitellogenin mRNA in trout liver, using a single injection of estradiol (3 mg/kg body wt) results in a short-lived rise, reaching a maximum level of 260 ppm total RNA on Day 2. Using silastic implants of estradiol to induce a primary response produces a large increase in the steady-state level of vitellogenin mRNA which reaches a maximum of 2750 ppm total RNA on Day 10. During secondary stimulation, using silastic tubing the maximum level reached was 1200 ppm of the total RNA on Day 7, approximately half the level seen in the primary induction using the silastic implants. The difference in these two levels is due to an increase in the steady-state levels of rRNA, which appear to increase between Days 10 and 21 after the primary stimulation. These results demonstrate that the induction of vitellogenesis in the trout by estradiol involves changes in the steady-state levels of a number of different mRNA and rRNA sequences and resembles that seen in Xenopus and chicken.
Article
Apolipoprotein (apo) B is a major protein component of plasma very low-density and low-density lipoproteins (VLDL and LDL, respectively) and serves as a recognition signal for the cellular binding and internalization of LDL by the apoB/E receptor. In contrast to the situation in mammals, avian apoB is also a component of specialized VLDL particles that are produced by the liver in response to estrogen. These particles transport cholesterol and triglyceride from the liver to the ovary for deposition in egg yolk. We report here the identification and characterization of cDNA clones for chicken apoB and their use in examining the tissue distribution and hormonal regulation of chicken apoB mRNA. The cDNA clones were identified by immunological screening of a phage lambda gt11 library constructed with hen liver mRNA and their identity was supported by sequence comparisons with mammalian apoB. The chicken apoB mRNA is approximately the same size as mammalian apoB mRNA (14 kb), and, as occurs in mammals, is present at high levels in liver and small intestine. Unlike mammals, the chicken apoB mRNA is also found at high levels in the kidney, consistent with previous protein biosynthetic studies. A DNA-excess solution-hybridization assay was used to quantitate apoB mRNA in these tissues and to examine its hormonal regulation. In control roosters the liver and kidney contained 65% and 10%, respectively, as much apoB mRNA as the small intestine. Within 24 h after estradiol administration, apoB mRNA was increased five- to seven-fold in liver but was unchanged in intestine and kidney. The increase in apoB mRNA content and the kinetics of induction parallel hepatic apoB synthesis, indicating that estrogen regulates apoB production through changes in the cellular abundance of apoB mRNA. The apoB mRNA increased rapidly following hormone treatment while the mRNA for another VLDL protein (apoII) showed a lag or slow phase of several hours before significant mRNA accumulation occurred. These data indicate that the liver can respond immediately to estrogen to increase apoB mRNA accumulation, while apoII mRNA accumulation appears to involve additional events or signals which occur slowly and are specific to this gene.
Article
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.
Article
A simple and rapid method for preparing plasmids for restriction enzyme analysis has been developed. Bacteria are boiled for 15–40 s and an insoluble clot of genomic DNA and debris is removed by low-speed centrifugation. Plasmids are recovered from the supernatant by isopropanol precipitation and can be resuspended in buffer and immediately restricted. A 5-ml bacterial culture yields enough plasmids for many restriction enzyme digestions, but a single colony on a petri dish or a 0.5-ml miniculture will suffice for a few experiments. In addition, the procedure can be readily adapted for the preparation of plasmids from liter cultures with quantitative yields.
Article
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
Article
Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.
Article
The synthesis of vitellogenin, the egg-yolk precursor, was induced by estrogenic steroids (estrone, estradiol, and estriol) but not by testosterone, progesterone, or cortisol; estrogen treatment alone did not lead to formation of yolky oocytes in the hypophysectomized catfish. Amplification of vitellogenin synthesis was observed following three spaced injections of estradiol as primary, secondary, and tertiary responses in intact males or hypophysectomized females. The pattern of 32P incorporation indicated that the label was first incorporated into the phosphoprotein fraction of the liver of estrogenized males or females and thereafter labeled vitellogenin appeared in blood. Thus, in the catfish, liver is the site of estrogen-induced synthesis of vitellogenin.
Vitellogenin gene expression in primary culture of male rainbow trout hepato-cytes Goldfish Carassius auratus vitellogenin: induction, isolation, properties and relationship to yolk proteins
  • Le C Vaillant
  • C Guellec
  • F Pakdel
  • Valotaire
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  • Wiley Hs
  • G Delahunty
  • Wallace
Vaillant C, Le Guellec C, Pakdel F, Valotaire Y (1988) Vitellogenin gene expression in primary culture of male rainbow trout hepato-cytes. Gen Comp Endocrinol 70:284-290 de Vlaming VL, Wiley HS, Delahunty G, Wallace RA (1980) Goldfish Carassius auratus vitellogenin: induction, isolation, properties and relationship to yolk proteins. Corn Biochem Physiol 67B:613-623
Vitellogenin motifs conserved in nematodes and vertebrates A rapid procedure for creating nested sets of deletions using mini-prep plasmid DNA samples
  • J Speith
  • M Nittleton
  • Lea E K Zucher-Aprison
Speith J, Nittleton M, Zucher-Aprison E, Lea K, Blumenthal T (1991) Vitellogenin motifs conserved in nematodes and vertebrates. J Mol Evol 32:429-438 Steggles AW (1989) A rapid procedure for creating nested sets of deletions using mini-prep plasmid DNA samples. Biotechniques 7:241-242
A simple and efficient method for gen-erating cDNA libraries Vitellogenin gene expression in male rainbow trout Salmo gaird-neri
  • U Gubler
  • Le Hoffman
  • K Guellec
  • K Lawless
  • Kress Y M Valotaire
  • Tenniswood
Gubler U, Hoffman BJ (1983) A simple and efficient method for gen-erating cDNA libraries. Gene 25:263-269 Le Guellec K, Lawless K, Valotaire Y, Kress M, Tenniswood M (1988) Vitellogenin gene expression in male rainbow trout Salmo gaird-neri. Gen Comp Endocrinol 71:359-371
Role of lysine and arginine residues of vitellogenin in high affinity binding to vitellogenin receptors in locust oocyte membranes Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein
  • Roehrkasten A Ferenz
  • Hj F J Samallo
  • Ophius J M Mojet
  • M Gruber
  • Ab
Roehrkasten A, Ferenz HJ (1992) Role of lysine and arginine residues of vitellogenin in high affinity binding to vitellogenin receptors in locust oocyte membranes. Biochim Biophys Acta 1133:160-166 Schip van bet F, Samallo J, Broos J, Ophius J, Mojet M, Gruber M, Ab G (1987) Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein. J Mol Biol 196:245-260