Vg Levels Research Articles

Molecular characterization and expression pattern of Spodoptera litura (Lepidoptera: Noctuidae) vitellogenin, and its response to lead stress

Vitellogenin (Vg) cDNA from Spodoptera litura Fabricius was cloned and sequenced. The open reading frame (ORF) of Vg cDNA was 5247 nucleotides in length (GenBank Accession no. EU095334), which encoded for a protein of 1748 amino acids. S. litura Vg comprised three conserved regions (Vitellogenin-N domain, DUF1943 and von Willebrand factor type D domain (VWD)), a 17 amino-acid signal peptide and a RXXR cleavage signal (RTIR). The highly conserved GL/ICG motif, the DGXR motif and cysteine residues were found in the C-terminus of the Vg. Vg mRNA was found specifically in the female fat body. Vg expression was first transcribed in 6th day female pupae and levels increased with insect development. The maximum level of Vg mRNA appeared in 24-h-old adults. When S. litura larvae were exposed to lead (Pb) (25–200 mg Pb/kg), there was a significant inhibition in Vg of female adults. The start of Vg expression was advanced ahead by Pb, from 6th day pupae to 3rd day or 4th day pupae. Low levels of Vg in male adults were also induced by low concentrations of Pb (12.5 and 25 mg Pb/kg). These data show that Pb stress elicits an important Vg response in S. litura.

Varietal role in the management of the larger grain borer, Prostephanus truncatus (horn) in stored maize

The study determined the amount of haemolymph vitellogenin (Vg) of the Larger Grain Borer (LGB), Prostephanus truncatus (Horn) vitellogenic females reared on different maize varieties. The varieties were ZM 521, ZM 421, ECAVL1-DLN, WEEVIL A, LOCAL 1 and LOCAL 2. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS - PAGE) was conducted on the insect haemolymph. F1 LGB adults from each variety were used to determine Indices of Susceptibility (IS) of the varieties to the insect pest attack. Percentages of Vg from female insects and IS were in the order ZM 521>ZM 421> ECAVL1-DLN>WEEVIL A >LOCAL 1>LOCAL 2. Weight losses of the varieties after F1 emergence were recorded. Findings showed that the IS and the weight losses were highest in ZM 521 and lowest in LOCAL 2. ANOVA indicated significant differences in the IS, weight loses and Vg levels of insects among the varieties. IS and Vg were positively correlated. Varying Vg levels reflect reproductive capacity of P. truncatus and therefore used to identify resistant maize grains. It is concluded that resistant varieties play a great role in stored maize pest management by reducing LGB populations below economic injury levels (EIL). Tanzania Journal of Science Vol. 32 (2) 2006: pp. 13-22

Effects of o,p'-DDE, heptachlor, and 17β-estradiol on vitellogenin gene expression and the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus

Effects of two endocrine disruptors, o,p'-DDE and heptachlor, and 17β-estradiol (E 2) on vitellogenin (Vg) and the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis were examined in male tilapia. In the first experiment, fish were given 5 weekly injections of either E 2, o,p'-DDE or heptachlor (5 μg/g). E 2 treatment increased plasma Vg and hepatic expression of three Vg genes (Vgs A, B, and C) and estrogen receptor α (ERα), while reducing plasma levels of IGF-I and suppressing the expression of IGF-I, the GH receptor (GHR2) and the putative somatolactin receptor (GHR1). Neither pesticide greatly affected the other parameters examined, except for a significant reduction in expression of GHR2 and increased plasma IGF-I. In the second experiment, fish were given a single injection of o,p'-DDE or heptachlor (100 μg/g), or E 2 (5 μg/g) and sacrificed 5 days post-injection. Treatment with E 2 stimulated expression of all three Vg genes. Both o,p'-DDE and heptachlor increased expression of VgB, whereas only o,p'-DDE increased VgA expression. There was no effect of o,p'-DDE or heptachlor on VgC expression or plasma Vg levels. Treatment with o,p'-DDE and heptachlor as well as E 2 increased ERα and ERβ transcript levels. Similarly, both pesticides increased GHR1 and IGF-I expression, whereas no significant effect of E 2 was observed on GHR1, GHR2 or IGF-I expression. These results indicate that o,p'-DDE and heptachlor have varying temporal and dose effects on modulation of Vg and the GH/IGF-I axis that are distinct from E 2.

Effects of starvation on the vitellogenesis, ovarian development and fecundity in the ectoparasitoid, Nasonia vitripennis (Hymenoptera: Pteromalidae)

AbstractA female‐specific protein, vitellogenin (Vg), and its corresponding egg vitellin (Vt) are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions (sodium dodecyl sulfate—polyacrylamide gel electrophoresis). An indirect sandwich enzyme‐linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.

Changes in the levels of serotonin and dopamine in the central nervous system and ovary, and their possible roles in the ovarian development in the giant freshwater prawn, Macrobrachium rosenbergii

Serotonin or 5-hydroxytryptamine (5-HT) and dopamine (DA) are the two key neurotransmitters that control gonadal development in decapod crustaceans. This study investigated changes in the levels of 5-HT and DA in the CNS and ovary during different phases of the ovarian cycle of the freshwater prawn, Macrobrachium rosenbergii. The levels of 5-HT and DA were quantified by using High Performance Liquid Chromatography with electrochemical detection (HPLC-ECD). Moreover, changes of vitellogenin (Vg) concentrations in the hemolymph after treatment with 5-HT and DA (at doses of 2.5 × 10 −6 and 2.5 × 10 −7 mol per prawn) were also examined. 5-HT exhibited a gradual increase in concentration in the brain and thoracic ganglia from ovarian stage I (0.12 ± 0.01 nmol/mg, 0.22 ± 0.01 nmol/mg, respectively) to reach a maximum (0.66 ± 0.03 nmol/mg, 1.48 ± 0.03 nmol/mg, respectively) at ovarian stage IV. In contrast, DA in the brain and thoracic ganglia showed the highest concentrations at ovarian stage II (0.20 ± 0.01 nmol/mg, 1.27 ± 0.06 nmol/mg, respectively) and then decreased to the lowest concentrations (0.06 ± 0.01 nmol/mg, 0.28 ± 0.04 nmol/mg, respectively) at ovarian stage IV. The ovarian concentration of 5-HT was 0.53 ± 0.11 nmol/mg at ovarian stage I and gradually increased to 1.63 ± 0.16 nmol/mg at ovarian stage IV. In contrast, the concentration of DA was highest at ovarian stage I (29.05 ± 1.31 nmol/mg), and lowest at the ovarian stage IV (11.43 ± 0.74 nmol/mg). Injecting 5-HT into prawns significantly increased Vg concentration in the hemolymph at ovarian stage IV compared to control groups, and injecting DA into prawns had the opposite effect. The inverse relationship between 5-HT and DA levels in neural ganglia and ovaries, and their opposing effects on hemolymph Vg levels suggest that these two transmitters play opposite regulatory roles in controlling ovarian maturation and oocyte development in this species.

Molecular cloning and developmental expression of the vitellogenin gene in the endoparasitoid, Pteromalus puparum

A cDNA of the vitellogenin (Vg) protein gene was isolated from the endoparasitoid Pteromalus puparum and characterized. The putative coding sequence was found to be 5634 bp long, encoding 1803 amino acids in a single open reading frame. The chemically determined N-terminal amino acid sequence of vitellin completely matched the deduced amino acid sequence that follows a putative signal peptide of 17 amino acid residues. The Vg mRNA was detected in the fat body of late female pupae, whereas the ovary and male fat body lacked the Vg transcript. The Vg mRNA level in the fat body depended significantly on the developmental stage, reaching the highest level 0 h after eclosion. The haemolymph Vg titre appeared 24 h after the appearance of Vg transcript. A positive correlation between the titre and transcript level of Vg in individual female wasps was found.

From Hepatopancreas to Ovary: Molecular Characterization of a Shrimp Vitellogenin Receptor Involved in the Processing of Vitellogenin1

We report the first cloning and characterization of cDNA encoding a putative vitellogenin (Vg) receptor (VgR) from the shrimp, Penaeus monodon. The shrimp VgR cDNA is 6.8 kb; the deduced protein has 1943 amino acids with a molecular weight of 211 kDa. VgR is ovary specific and consists of conserved cysteine-rich domains, epidermal growth factor-like domains, and YWTD motifs similar to the low-density lipoprotein, very low-density lipoprotein, and VgR of insects and vertebrates. VgR expression level in the ovary is low during early vitellogenesis and increases to maximum levels in females with a gonadosomatic index of 3-4, presumably when needed for receptor-mediated endocytosis during the rapid phase of extraovarian Vg production by the hepatopancreas. A peptide from the C-terminal end of VgR was synthesized for antibody production. Anti-VgR antibody recognized an ovarian membrane protein, and the level of this protein was high when extraovarian production of Vg reached peak levels. By immunohistochemical analysis, VgR was detected strongly in the membranes of larger oocytes. VgR expression was knocked down after the shrimp were injected with VgR double-stranded RNA, leading to a decrease in VgR protein content in the ovary, but an increase in the hemolymph level of Vg. This study represents the first report of the functional analysis of a putative VgR in a crustacean.

Effects of 4-nonylphenol exposure in mussels ( Mytilus galloprovincialis) and crabs ( Carcinus aestuarii) with particular emphasis on vitellogenin induction

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0–0.2 mg NP l −1), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC 50 value was previously determined. Then, in sublethally exposed (0–1.0 mg NP l −1) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 μg g −1 d.w. in mussels, and from 3.6 to 37 μg g −1 d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.

Vitellogenesis in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae), a vector of Chagas’ disease

Oocyte extracts of anautogenous Dipetalogaster maxima were chromatographed on an ion-exchange column in order to purify vitellin (Vt), the main insect yolk protein precursor. Purified Vt (Mr ∼443kDa) was composed of four subunits with approximate molecular weights of 174, 170, 50, and 44kDa. Polyclonal anti-Vt antibody, which cross-reacted equally with fat body extracts and hemolymph vitellogenin (Vg), was used to measure the kinetics of Vg expression in the fat body and the levels in hemolymph. In addition, morphological and immunohistochemical changes that took place in the ovary during vitellogenesis were analyzed. The study was performed between 2 and 8 days post-ecdysis and between 2 and 25 days post-blood feeding. During the post-ecdysis period, D. maxima showed decreased synthesis of Vg and concomitantly, low levels of Vg in hemolymph (4.5×10−3μg/μl at day 4). After a blood meal, Vg synthesis in the fat body and its levels in hemolymph increased significantly, reaching an average of 19.5μg/μl at day 20. The biochemical changes observed in the fat body and hemolymph were consistent with the histological and immunohistochemical finds. These studies showed noticeable remodeling of tissue after blood feeding.

Control ofDrosophilawing growth by thevestigialquadrant enhancer

Following segregation of the Drosophila wing imaginal disc into dorsal (D) and ventral (V) compartments, the wing primordium is specified by activity of the selector gene vestigial (vg). In the accompanying paper, we present evidence that vg expression is itself driven by three distinct inputs: (1) short-range DSL (Delta/Serrate/LAG-2)-Notch signaling across the D-V compartment boundary; (2) long-range Wg signaling from cells abutting the D-V compartment boundary; and (3) a short-range signal sent by vg-expressing cells that entrains neighboring cells to upregulate vg in response to Wg. Furthermore, we showed that these inputs define a feed-forward mechanism of vg autoregulation that initiates in D-V border cells and propagates from cell to cell by reiterative cycles of vg upregulation. Here, we provide evidence that this feed-forward mechanism is required for normal wing growth and is mediated by two distinct enhancers in the vg gene. The first is a newly defined ;priming' enhancer (PE), that provides cryptic, low levels of Vg in most or all cells of the wing disc. The second is the previously defined quadrant enhancer (QE), which we show is activated by the combined action of Wg and the short-range vg-dependent entraining signal, but only if the responding cells are already primed by low-level Vg activity. Thus, entrainment and priming constitute distinct signaling and responding events in the Wg-dependent feed-forward circuit of vg autoregulation mediated by the QE. We posit that Wg controls the expansion of the wing primordium following D-V segregation by fueling this autoregulatory mechanism.

Circannual variation in plasma vitellogenin and gonadotropin II levels in relation to annual ovarian cycle in female mrigal, Cirrhinus mrigala

Two forms (HA I and HA II) of vitellogenin (Vg), the yolk-precursor protein, were purified from the plasma of estradiol-17β (E 2)-treated Indian major carp, mrigal ( Cirrhinus mrigala), by gel filtration on Ultrogel AcA 34 followed by adsorption chromatography on Hydroxylapatite (HA)-Ultrogel. Native HA I and HA II had molecular weights of 500 kDa and 550 kDa, respectively. The apparent masses of purified HA I and HA II after SDS-PAGE under reducing condition were 75 kDa and 85 kDa, respectively. HA I was found to be lipid rich whereas HA II was phosphorous rich. The co-purified Vg containing both HA I and HA II was used to raise polyclonal antisera (a-Vg) and its specificity was assessed by Western blot analysis. In a double immunodiffusion test, the plasma of vitellogenic females and E 2-treated males as well as crude egg yolk protein (CYP), cross-reacted with a-Vg giving two precipitin lines in each case, thereby indicating the presence of two forms of Vg. HA I and HA II each gave single precipitin lines. No cross-reaction was observed with control male plasma. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using a-Vg and HA I. The detection limit of the assay was 6.25 ng/ml, and the intra- and inter-assay variations were 4.48 and 7.57%, respectively. Displacement curves parallel to the standard (HA I) were obtained with plasma samples from vitellogenic female and E 2-treated male mrigal. The assay was validated by estimating Vg in plasma samples from adult female mrigal captured in the field throughout the year and compared with ovarian development. Annual profiles of plasma Vg and gonadotropin (GTH II: estimated by common carp GTH II ELISA) levels presented a good correlation with gonadosomatic index (GSI) and mean number of vitellogenic oocytes during different reproductive phases, i.e., preparatory (Feb–Apr), pre-spawning (May–Jun), spawning (Jul–Aug) and post-spawning (Sep–Jan).

Vitellin of Pteromalus puparum (Hymenoptera: Pteromalidae), a pupal endoparasitoid of Pieris rapae (Lepidoptera: Pieridae): Biochemical characterization, temporal patterns of production and degradation

Vitellin (Vt) and vitellogenin (Vg) profiles were analyzed in Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. Non-denaturing and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analyses indicated that both native Vt and Vg were likely 370 kDa in size, consisting of two subunits of approximate 206 and 165 kDa. An indirect double antibody enzyme-linked immunosorbent assay (ELISA) for monitoring hemolymph Vg and ovarian Vt levels was developed using a monoclonal antibody and a polyclonal antibody made specially against P. puparum Vt. The synthesis and uptake of Vg in this wasp was initiated immediately after adult eclosion. The hemolymph Vg and ovarian Vt reached their highest level of 0.58 and 4.51 μg per female 24 and 48 h after adult eclosion, respectively. Both Vg synthesis and uptake were in parallel with ovarian development. The Vt levels in the developing embryos decreased progressively except 12 h after parasitism. Meanwhile, nine new polypeptides with sizes ranging from 59.2 to 151 kDa, possibly resulting from the limited proteolysis of Vt originally accumulated in newly laid eggs, were detected de-novo during embryonic development using Western blotting with the monoclonal antibody against Vt. These studies provide the basis for future investigation into endocrinal regulations of vitellogenesis and understanding the reproductive strategy in this wasp.

Effects of 4-n-Nonylphenol and Tamoxifen on Salmon Gonadotropin-Releasing Hormone, Estrogen Receptor, and Vitellogenin Gene Expression in Juvenile Rainbow Trout

Alkylphenols such as nonylphenol (NP) are one of a wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of hepatic vitellogenin (Vg) gene expression is widely used as a biomarker for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the brain. To evaluate brain and liver effects of 4-n-nonylphenol (4-NP), juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne 4-NP or 17beta-estradiol (E(2)). Changes in mRNA levels of salmon gonadotropin-releasing hormone (sGnRH) and estrogen receptor alpha (ERalpha) isoforms in the brain and ERalpha isoforms and Vg in the liver were measured after 3 and 6 days of exposure, with the help of a relative RT-PCR-based quantification method. Fish were treated with increasing doses of 4-NP ranging from 0.01 to 10 microM (2.2 microg/l to 2.2 mg/l), and results were compared to the effect of E(2) or tamoxifen, a specific ER modulator. In liver, E(2) and the highest doses of 4-NP increased Vg and ERalpha long-isoform mRNA levels within 3 or 6 days of exposure, but 4-NP did not have any effect on ERalpha short-isoform transcription level. In the brain, 4-NP reduced sGnRH2 gene expression in a dose-dependent manner, but did not modify sGnRH1 or ERalpha mRNA levels.

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.

Characterization of vitellin from the ovaries of the banana shrimp Litopenaeus merguiensis

Vitellin (Vt) was purified from ovary extracts of mature females of the banana shrimp Litopenaeus merguiensis using DEAE-Sephacel and Superdex 200 columns. Native Vt had an apparent molecular mass of 398 kDa as determined by native PAGE and by gel filtration chromatography. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of two major subunits of 87 and 78 kDa, although some faint bands were also detected. The N-terminal 10 amino acids sequence of the 78 kDa subunit is identical to that of Litopenaeus vannamei Vt and very similar to that of Litopenaeus japonicus vitellogenin (Vg) as well as Litopenaeus semisulcatus Vt, with an identity of 89%. Anti-Vt polyclonal antibody raised against purified Vt shows a high specificity with only ovarian Vt and hemolymph Vg of vitellogenic shrimps in double immunodiffusion and Western blot assays. Vg and Vt concentrations in hemolymph, hepatopancreas and ovaries were measured by ELISA. Vg concentrations increased in the hemolymph in the early stages of ovarian development and declined in the maturation stages. As there were undetectable concentrations of Vg in the hepatopancreas while an elevation of Vg levels occurred in the hemolymph, during the time that Vt was accumulating in the ovaries during oogenesis, this would suggest that the contribution of Vg synthesized by the hepatopancreas only might be not sufficient for adequate development of the oocytes in the banana shrimp L. merguiensis during vitellogenesis.

Disparate effects of constant and annually-cycling daylength and water temperature on reproductive maturation of striped bass ( Morone saxatilis)

Adult striped bass ( Morone saxatilis) were exposed to various combinations of constant or anually-cycling daylength and water temperature. Constant conditions (15 h days, 18 °C) were those normally experienced at spawning and cycling conditions simulated natural changes at Chesapeake Bay latitude. Females exposed to constant long (15 h) days and cycling water temperature (TEMPERATURE group) had blood plasma levels of sex steroids (testosterone [T] and estradiol-17β [E 2]) and vitellogenin (Vg), and profiles of oocyte growth, that were nearly identical to those of females held under a natural photothermal cycle (CONTROL group). Several fish from these two groups were induced to spawn fertile eggs. Females constantly exposed to warm water (18 °C), with or without a natural photoperiod cycle (PHOTOPERIOD and STATIC groups, respectively), had diminished circulating levels of gonadal steroid hormones and Vg, impaired deposition of yolk granules in their ooplasm, and decreased oocyte growth, and they underwent premature ovarian atresia. Males exposed to cycling water temperature (CONTROL and TEMPERATURE groups) spermiated synchronously during the natural breeding season, at which time they also had had high plasma androgen (T and 11-ketotestosterone [11-KT]) levels. The timing of spermiation was highly asynchronous among males in groups of fish held constantly at 18 °C (STATIC and PHOTOPERIOD groups) and this asynchrony was associated with diminished plasma androgen levels. Termination of spermiation by males exposed to cycling water temperature coincided with a sharp decline in levels of plasma androgens about a month after water temperature rose above 18 °C. In contrast, most males held constantly at 18 °C sustained intermediate levels of plasma androgens and spermiated until the end of the study in late July. The annual cycle of water temperature clearly plays a prominent role in the initiation, maintenance, and termination of the striped bass reproductive cycle. In females, a decrease in water temperature below values experienced at spawning appears to be required for vitellogenesis and oocyte growth to proceed normally. Constant exposure of males to spawning temperature disrupts synchronous spermiation but also delays testicular regression, which may be useful for spawning fish after the natural reproductive season.

Purification of two lipovitellins and development of immunoassays for two forms of their precursors (vitellogenins) in medaka ( Oryzias latipes)

Two distinct yolk proteins (YP1 and YP2) were purified from the ovary of medaka, and specific antisera against YPs were generated to characterize YPs and reveal their relation to two vitellogenins (Vg1 and Vg2). The molecular masses of purified YP1 and YP2 on gel filtration were 270 and 380 kDa, respectively. YPs were confirmed to be lipoproteins by staining with Sudan black. Amino acid compositions of YP1 and YP2 were similar to those of Vg1 and Vg2, respectively. In double immunodiffusion using anti-Vg1, a precipitin line of YP1 formed a spur against the Vg1 line. YP2 and Vg2 were reacted with anti-Vg2, and a precipitin line of YP2 formed a fuse against the Vg2 line. These biochemical and immunological analyses of purified YPs revealed that YP1 is lipovitellin 1 (Lv1) derived from Vg1 and YP2 is lipovitellin 2 (Lv2) derived from Vg2. Using specific antibodies against Lvs and Vgs, specific, high sensitivity chemiluminescent immunoassays (CLIAs) for two Vgs were developed to reveal basal Vg levels and response to exogenous estradiol-17β (E 2). The measurable range of both CLIAs was from 0.975 to 1000 ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (≦0.57%). When immature fish were immersed in water containing E 2 for 1 h, both Vgs were induced by 0.5 μg/L of E 2 at 24 h after treatment. Vg1 increased in a concentration-dependant manner up to 100 μg/L E 2, while Vg2 reached a plateau at 10 μg/L of E 2. The ratio of Vg1:Vg2 in E 2-treated fish changed in a concentration-dependent manner from 1.5:1 to 8.5:1. The results from E 2-treatment suggest that differential regulation may control the expression of medaka Vgs.

Annual changes in serum levels of two choriogenins and vitellogenin in masu salmon, Oncorhynchus masou

Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17β (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low (∼0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61 ± 0.08, 0.98 ± 0.18 and 10.93 ± 3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth.

Starvation affects vitellogenin production but not vitellogenin mRNA levels in the lubber grasshopper, Romalea microptera

The interaction of juvenile hormone (JH) and nutrition was studied during the oviposition cycle of the Eastern Lubber grasshopper ( Romalea microptera). Starvation of females early or in the middle of the cycle inhibited oocyte growth. Starvation for 4 days also reduced hemolymph levels of JH III and vitellogenesis (Vg) to 25% and 15%, respectively, of the levels in fed animals. Likewise, Vg production by fat body fragments incubated in vitro was reduced to 2% of the levels in fed animals and total protein synthesis was reduced to 25%, suggesting that starvation had a stronger effect on Vg synthesis than on protein synthesis. These effects were reversed when starved animals were fed again. However, fat body levels of Vg-mRNA were similar in fed and starved animals, indicating that starvation did not affect transcript levels. We tested whether the decline in JH levels mediated the other starvation effects by infusing animals with JH III or vehicle for 2 days at the onset of starvation. Infusion of JH elevated JH and Vg-mRNA levels 670% and 103%, respectively, above the levels in vehicle-infused animals. However, Vg production and hemolymph levels of Vg were similar to the levels in vehicle-infused animals. These data suggest that JH alone is insufficient to stimulate Vg production.

Effects of Nonylphenol and Triclosan on Production of Plasma Vitellogenin and Testosterone in Male South African Clawed Frogs (Xenopus laevis)

We investigated the effects of nonylphenol (NP) and triclosan (TCS) on production of vitellogenin (Vg), testosterone (T), and hepatic cytochrome P450 1A and 2B activities in male South African clawed frogs (Xenopus laevis). In a 14-d waterborne exposure test, no significant differences in the level of plasma Vg synthesis in male frogs were observed among the control, 10, 50, and 100 microg/l NP and 20, 100, and 200 microg/l TCS treatment groups. Intraperitoneal injection of male frogs with 2, 20, and 200 microg/g body weight NP resulted in no significant differences in plasma Vg levels among the control and all treatment groups. However, the levels of plasma Vg in all TCS treatment groups (intraperitoneal injection of 4, 40, and 400 microg/g body weight) were lower than that in the solvent control group, and male frogs injected with high doses of NP or TCS had lower T levels than the control group. No significant differences in hepatic cytochrome P450 1A and 2B activities were observed among the all treatment groups. Male frogs injected with 20 microg/g body weight of estradiol-17beta had significantly higher plasma Vg levels than the control group. These results suggest that profiles of plasma Vg and T production in male Xenopus laevis could be useful biomarkers for detecting hormonally active agents.