Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch

Three types of white perch (Morone americana) vitellogenin (VtgAa, VtgAb, and VtgC) were purified, labeled with digoxigenin (DIG), and subjected to Vtg receptor (Vtgr) binding assays in 96-well plates coated with perch ovarian membrane proteins or to ligand blotting procedures. Binding specificity was evaluated by incubating membrane protein preparations with constant amounts of DIG-Vtg tracer (VtgAa, VtgAb, VtgC, or a mixture of VtgAa and VtgAb [VtgAa/b]) alone or in the presence of unlabeled Vtg ligands. At 250-fold excess molar concentration relative to the tracer, VtgAa and VtgAb were each able to displace only approximately 50% of bound DIG-VtgAa/b, but VtgAa/b could fully displace DIG-VtgAa and DIG-VtgAb under the same conditions. Over a broad range of excess molar ratios, unlabeled VtgAa and VtgAb each displaced their respective DIG-Vtg tracer much more effectively than each did the heterologous tracer (DIG-VtgAb and DIG-VtgAa, respectively). Ligand blotting revealed three forms of Vtgr, a large receptor (>212 kDa) that bound only to VtgAa and two smaller receptors (～ 116 and ～ 110.5 kDa) that bound preferentially to VtgAb. The VtgC did not specifically bind to ovarian membrane proteins in either assay. Collectively, these results indicate the presence of a system of multiple ovarian Vtgrs with disparate binding to the three types of Vtg present in higher-order teleosts (Acanthomorpha). To our knowledge, this is the first report on binding of multiple types of Vtg to multiple forms of Vtgr in any vertebrate.     

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

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

Maturation and fecundity of a stock-enhanced population of striped bass in the Savannah River Estuary, U.S.A

The striped bass Morone saxatilis population in the Savannah River (south-eastern U.S.A.) collapsed in the 1980s, and recent eorts to restore the population have resulted in increased catch-per-unit-eort (CPUE) of striped bass in the Savannah River Estuary (SRE). The abundance of eggs and larvae, however, remain well below historic levels. The primary cause of the population decline was remedied, and environmental conditions seem suitable for striped bass spawning. Regression analysis of data derived from ultrasonic imaging of 31 striped bass resulted in a statistical model that predicted ovary volume well (r²=0.95). The enumeration of oocytes from ovarian tissue samples and the prediction of ovary volume allowed fecundity to be estimated without sacrificing the fish. Oocyte maturation in Savannah River striped bass seemed to progress normally, with oocytes developing to final stages of maturity in larger fish (>750 mm L _T). Additionally, fecundity estimates were comparable to a neighbouring striped bass population. The environmental cues needed to trigger development and release of striped bass oocytes into the SRE appeared to be present. If most of the striped bass females in the SRE are still young (<7 years), the ability to produce large numbers of eggs will be limited. As these young fish mature, egg production probably will increase and the density of striped bass eggs eventually will approach historic levels, provided suitable habitat and water quality are maintained.     

Ultrahistology of oogenesis and vitellogenesis in the spider mite Tetranychus urticae

Histology of the ovary of the spider mite Tetranychus urticae is described light and electron microscopically with special reference to oogenesis and vitellogenesis of this mite. Morphology of the ovary is comparable to the typical sac-like chelicerate ovary with oocytes protruding from the ovarian surface, thus resulting in a grape-like appearance. According to different oogenetic stages, a germ, pre-vitellogenic and vitellogenic region can be observed. Oogonia and primary oocytes characterized by extranuclear material or 'yolk nuclei' are situated in the germ region. Primary oocytes develop into three-nucleated nurse cells situated in the periphery of the pre-vitellogenic region, and into pre-vitellogenic oocytes protruding from the ovarian surface. Growth of oocytes is performed while they are in ovarian pouches by uptake of nurse cell cytoplasm and following extraovarian yolk precursors. Intraoocyte yolk synthesis interpreted from altered cytoplasmic organelles also occurs. Processes taking place during oogenesis and vitellogenesis in T. urticae are compared to published information on yolk synthesis of other animal species.     

Machine learning reveals sex‐specific 17β‐estradiol‐responsive expression patterns in white perch (Morone americana) plasma proteins

With growing abundance and awareness of endocrine disrupting compounds (EDCs) in the environment, there is a need for accurate and reliable detection of EDC exposure. Our objective in the present study was to observe differences within and between the global plasma proteomes of sexually mature male and female white perch (Morone americana) before (Initial Control, IC) and after 17β‐estradiol (E₂) induction. Semiquantitative nanoLC‐MS/MS data were analyzed by machine learning support vector machines (SVMs) and by two‐way ANOVA. By ANOVA, the expression levels of 44, 77, and 57 proteins varied significantly by gender, treatment, and the interaction of gender and treatment, respectively. SVMs perfectly classified male and female perch IC and E₂‐induced plasma samples using the protein expression data. E₂‐induced male and female perch plasma proteomes contained significantly higher levels of the yolk precursors vitellogenin Aa and Ab (VtgAa, VtgAb), as well as latrophilin and seven transmembrane domain‐containing protein 1 (Eltd1) and kininogen 1 (Kng1). This is the first report that Eltd1 and Kng1 may be E₂‐responsive proteins in fishes and therefore may be useful indicators of estrogen induction.     

Use of cellular oncogene probes to identify Morone hybrids.

We tested the ability of cellular oncogene (c-onc) probes to identify F1 hybrids and the lineage of known backcrosses within the fish genus Morone. Total DNA was isolated from five to 14 individuals per North American Morone species (striped bass, white bass, white perch, and yellow bass). The DNA was digested with two restriction enzymes, Eco RI and Hin dIII, Southern blotted, and hybridized to six different c-onc probes including v-abl, v-erb B, c-myc, c-H-ras, c-K-ras, and v-src. We found fixed genotypic differences among the four species for all six probes in single restriction enzyme digests. The heritability of these nuclear DNA genotypes was evaluated in hatchery-produced F1 Morone hybrids (striped bass x white bass and striped bass x white perch) tested with the six informative single probe/restriction enzyme combinations. All F1 individuals exhibited heterozygosity in all diagnostic nuclear DNA fragments, confirming the Mendelian inheritance of these genotypes in these fish. Furthermore, analysis of these nuclear DNA genotypes in hatchery-produced backcrosses of F1 hybrids striped bass x (white bass x striped bass) detected both recombinant and parental genotypes at all six polymorphic c-onc sequences. The lineage of suspected Morone hybrids of unknown descent collected from Lewis Smith Lake, Alabama, and from the Occoquan River, Virginia, was determined using the c-onc probes. Our results suggest that c-onc probes are suitable markers to unequivocally identify F1 hybrids and backcrosses and to quantify introgression in natural populations of fishes. The addition of RFLP analysis of mtDNA provided a complete ancestral history of individual fish.     

Evolution of an MHC class Ia gene fragment in four North American Morone species

A nucleotide sequence analysis of a fragment of a Morone MHC class Ia gene detected high levels of polymorphism in striped bass Morone saxatilis, white perch Morone americana and yellow bass Morone mississippiensis. Extremely low levels of MHC diversity, however, were detected in white bass Morone chrysops, suggesting the possibility of a severe population bottleneck for this species.     

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana cttesbeiana oocytes

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.     

Some aspects of the reproductive biology of perch Perca fluviatilis L. A histological description of the reproductive cycle

Histological changes in the perch ovary during development are described and related to changes in gonad weight and to a macroscopic scale of maturity stages after Kesteven (1960). A single organ formed by the fusion of two primordial ovaries, a central internal oviduct, and an extremely thick chorion surrounding oocytes are characteristic features of the perch ovary. Resting oocytes can be differentiated from developing oocytes which are larger and possess a granulosa layer which permits formation of the chorion and yolk acquisition. In the maturing ovary, oocyte diameter increases rapidly and the chorion, which possesses four layers, the tunica propria, two middle zones forming a fine network, and the zona radiata, also expands. The spent ovary is disorganized with follicles and tunica wall contracted, and a number of residual oocytes may be apparent. There was no evidence of pre-ovulatory degeneration although autolysis of a small number of residual oocytes was observed. The gonadosomatic index was highest for males in September and at a maximum in females immediately prior to spawning. After spawning the index fell rapidly with lowest values recorded in June and July. Variations in the gonadosomatic index are related to developmental stages in females.     

An ultrastructural study of oogenesis in a marine triclad

The ultrastructural features of oocyte differentiation were studied in the marine triclad Cercyra hastata. Oocytes at several stages of maturation, each surrounded by follicle cell projections, are present within each of the two ovaries. A pre-vitellogenic and a vitellogenic stage have been detected in the oogenesis of C. hastata. The pre-vitellogenic stage is mainly characterized by an increase in the nuclear and nucleolar volume and activity, and the appearance and development of cortical granule precursors which are elaborated by the Golgi complex. In early phases of the vitellogenic stage, intense delamination and blebbing of the nuclear envelope occurs which probably contributes to an increase in number of cytoplasmic membranes and to transfer of nuclear material to the cytoplasm. The rough endoplasmic reticulum is extensively developed and often assumes a ‘whorl’ array. Several areas of yolk precursor formation appear in the whorls. Numerous 2–5 μm protein yolk globules are subsequently formed which appear surrounded by a double membrane (cisternae of the smooth endoplasmic reticulum) and become randomly distributed throughout the cytoplasm of mature oocytes. The peripheral ooplasm is occupied by a monolayer of electron-dense cortical granules. Finally, the evolutionary significance of the autosynthetic mechanism of yolk production is discussed.     

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

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

First feeding and subsequent growth performances in piscivorous larvae of Chinese perch Siniperca chuatsi (Perciforms: Sinipercidae)

The success of first feeding is influenced by many factors, such as prey availability, and is critical to subsequent larval growth performances. To test the advantages of prey exposure before complete yolk absorption in piscivorous larvae, feeding incidence and specific growth rate were longitudinally measured in Chinese perch Siniperca chuatsi larvae first fed at different ages. The results showed that Chinese perch larvae were able to capture live piscine prey at 4 DAH (days after hatching), 1 day before complete yolk absorption. The feeding incidence and specific growth rate were higher in larvae first fed at 4 DAH than in larvae first fed at 5 or 6 DAH. These results indicate that prey exposure experience can facilitate the onset of first feeding, and that successful exogenous feeding, even before complete yolk absorption, is important for growth performances of piscivorous fish larvae.     

The histological and histochemical studies on the ovary in relation to vitellogenesis in the dragonfly, Orthetrum chrysis Selys (Libellulidae: Odonata).

The ovaries consist of large number of panoistic ovarioles in the last instar nymph and the adult dragonfly Orthetrum chrysis (Selys). In the nymph the vitellaria are compactly filled with the primary oocytes and the vitellogenesis takes place only in the adult stage. During vitellogenesis oocytes change widely in their shape, size and cytological organisation and their developmental stages can be divided into pre-vitellogenic, early-vitellogenic, vitellogenic, late-vitellogenic and maturation age. PAS-positive material appears first around the germinal vesicle in the early-vitellogenic stage and lateron it migrates towards the periphery. Glycogen appears in the late-vitellogenic stage. DNA is abundantly present in the nuclei of the oocytes during the pre-vitellogenic and completely absent in early-vitellogenic, vitellogenic, late-vitellogenic and maturation stages. It is observed in the nuclei of follicular epithelial cells of all the stages. RNA is abundantly present in cytoplasm of the pre-vitellogenic oocytes but lateron is gradually decreases. During the early-vitellogenic and vitellogenic stages high concentration of RNA in the follicular epithelial cells has been observed. The protein bodies appear first in the interfollicular spaces and towards the periphery of the oocytes just near the enveloping follicular epithelial cells, during the early-vitellogenic stage suggesting the formation of yolk proteins from the haemolymph. In Orthetrum chrysis the sudanophilic bodies appear first in the follicular cells and then lie in the peripheral region of the oocytes suggesting the incorporation of yolk lipid either from the follicular epithelium or from the haemolymph through the follicular epithelium. The phospholipids are synthesised in pre-vitellogenic to the late-vitellogenic stages. In the late-vitellogenic stages the phospholipid granules are present abundantly in the follicular epithelium while in the maturation stage they disappear suggesting their utilisation in the formation of membranes like vitelline and chorion. The neutral fats are present in the form of large number of droplets in the oocytes during the maturation stage.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Vitellogenocytes in the Hepatopancreas of Carcinus maenas and Libinia emarginata (Decapoda brachyura)

Summary

In addition to the ovary, the hepatopanocreas of female decapod crustaceans, Carcinus maenas, and Libinia emarginata is a source of yolk protein. The specific cells in the hepatopancreas that localize vitellogenins on tissue sections are revealed with lipovitellin-specific antiserum. These cells, designated vitellogenocytes, are believed to be responsible for vitellogenin synthesis in the hepatopanocreas. This conclusion is based upon immunolocalization which demonstrates a temporal relationship with vitellogenin synthesis in the hepatopanocreas. Specifically, when the oocytes are most active in vitellogenin uptake, the hepatopanocreas is producing vitellogenins most abundantly. Vitellogenocytes are relatively large and polymorphic, similar to the reserve-inclusion cells that were described by others. Yolk protein was not detected in other cells of the hepatopancreas, male reserve-inclusion cells, or pre-vitellogenic oocytes by the same method of staining. Vitellogenocytes resemble cyanocytes, the source of hemocyanin. Whether the vitellogenocytes and their precursors are related to other populations of hepatocytes, such as cyanocytes, is not known and has not yet been studied.     

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

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

Ecdysteroids regulate yolk protein uptake by Drosophila melanogaster oocytes

Juvenile hormones (JHs) are thought to drive the regulation of yolk protein uptake by ovaries in Drosophila melanogaster. However, the level of JH production in a mutant stock (ap(56f)) is depressed yet the flies are normally vitellogenic. The production of ecdysteroids by these ap(56f) ovaries in vitro is elevated above that of wild-type ovaries. The incubation of wild-type ovaries in the presence of 0.1mM JHB(3) increased ecdysteroid biosynthesis only during the first 18h following eclosion. Female Drosophila melanogaster undergo a pre-vitellogenic reproductive diapause when exposed to low temperature (11 degrees C) and a short-day photoperiod (L12:D12). The rate of ecdysteroid synthesis by the ovaries, but not JH production, increased within 12h of a temperature upshift to 25 degrees C from a basal level of 20+/-1pg/10 pair of ovaries/5h to a sustained level of 150+/-20pg/10 pair/5h. Vitellogenic oocytes were noted in all females within 12h of this temperature upshift. Diapause was also terminated by the injection of 1&mgr;g of 20-hydroxyecdysone into the abdomens of diapausing females as determined by an increase in ovary size, and the appearance of vitellogenic oocytes as compared to controls. These results are consistent with a revised model for the regulation of yolk protein uptake by ovaries in which ecdysteroids, and not JHs, play the prominent role.