The effects of thymol, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on viability, the Nosema sp. spore load and the expression of vg and sod-1 genes in infected honey bees.

This study was done to investigate the effects of thymol, fumagillin, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on Nosema sp. spore load, the expression of vitellogenin (vg) and superoxide-dismutase-1 (sod-1) genes and mortality of bees infected with N. ceranae. Five healthy colonies were assigned as the negative control, and 25 Nosema sp. infected colonies were assigned to five treatment groups including: the positive control: no additive to sirup; fumagillin 26.4 mg/L, thymol 0.1 g/L, Api-Bioxal 0.64 g/L and Nose-Go 5.0 g/L sirup. The reduction in the number of Nosema sp. spores in fumagillin, thymol, Api-Bioxal and Nose-Go compared to the positive control was 54, 25, 30 and 58%, respectively. Nosema sp. infection in all infected groups increased (p < .05) Escherichia coli population compared to the negative control. Nose-Go had a negative effect on lactobacillus population compared to other substances. Nosema sp. infection decreased vg and sod-1 genes expression in all infected groups compared to the negative control. Fumagillin and Nose-Go increased the expression of vg gene, and Nose-Go and thymol increased the expression of sod-1 gene than the positive control. Nose-Go has the potential to treat nosemosis if the necessary lactobacillus population is provided in the gut.

Vitellogenin 1 is essential for fish reproduction by transporting DHA-containing phosphatidylcholine from liver to ovary.

Vitellogenins (Vtgs) are essential for female reproduction in oviparous animals, yet the exact roles and mechanisms remain unknown. In the present study, we knocked out vtg1, which is the most abundant Vtg in zebrafish, Danio rerio via the CRISPR/Cas 9 technology. We aimed to identify the roles of Vtg1 and related mechanisms in reproduction and development. We found that, the Vtg1-deficient female zebrafish reduced gonadosomatic index, egg production, yolk granules and mature follicles in ovary compared to the wide type (WT). Moreover, the Vtg1-deficient zebrafish diminished hatching rates, cumulative survival rate, swimming capacity and food intake, but increased malformation rate, and delayed swim bladder development during embryo and early-larval phases. The Vtg1-deficiency in female broodstock inhibited docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) transportation from liver to ovary, which lowered DHA-PC content in ovary and offspring during larval stage. However, the Vtg1-deficient zebrafish increased gradually the total DHA-PC content via exogeneous food intake, and the differences in swimming capacity and food intake returned to normal as they matured. Furthermore, supplementing Vtg1-deficient zebrafish with dietary PC and DHA partly ameliorated the impaired female reproductive capacity and larval development during early phases. This study indicates that, DHA and PC carried by Vtg1 are crucial for female fecundity, and affect embryo and larval development through maternal-nutrition effects. This is the first study elucidating the nutrient and physiological functions of Vtg1 and the underlying biochemical mechanisms in fish reproduction and development.

Protective effect of Lactococcus laudensis and Pediococcus parvulus against neuropathy due to amyloid-beta in Caenorhabditis elegans.

In Alzheimer's disease (AD), the amyloid-ß (Aß) protein begins to accumulate in the brain 20 years prior to any dementia symptoms manifestation, in which Aß aggregates in the brain, causing destruction of nerve cells and resulting in memory impairments. Lifestyle and diet appear to inhibit Aß production and amyloid deposition. Therefore, identifying factors that prevent Aß production and administering them before the onset of AD, may be an effective preventive method. Lactic acid bacteria (LAB) exhibit various health effects on the host and are expected to have protective effects on neurological functions via brain-gut correlation. However, the protective effects of LAB against Aß are not well understood. We investigated whether LAB feeding could ameliorate the toxicity of Aß peptide accumulation in transgenic Caenorhabditis elegans expressing the human Aß peptide in neurons or muscle as an AD model. Aß expressed in muscle caused myopathy and worm paralysis, while Aß in neurons disturbed chemotactic activity. Among 14 screened strains, Lactococcus laudensis (LL) and Pediococcus parvulus (PP) prevented the AD worms from losing their chemotaxis behavior and becoming paralyzed by the Aß peptide. Immunostaining and western blotting indicated that Aß peptide was significantly suppressed in worms fed these two strains, and binding of the Aß to vitellogenin was particularly inhibited. Conversely, the mRNA level of the Aß gene did not change between LL- or PP-fed worms and the control. In conclusion, LL and PP alleviate neurotoxicity by inhibiting Aß accumulation; AD model worms can be used to screen efficient LAB for AD prevention.

Reproductive toxicity by exposure to low concentrations of pesticides in Caenorhabditis elegans.

In view of the recurrent applications of pesticides in agricultural producing countries, the increased presence of these substances in the environment raise a demand for the evaluation of adverse effects on non-target organisms. This study assesses the impact of exposure to five pesticides suspected of being endocrine disruptors (atrazine, 2,4-dichlorophenoxyacetic acid, mancozeb, chlorpyrifos and cypermethrin) on the reproductive development of the nematode Caenorhabditis elegans. To this end, nematodes in the L4 larval stage were exposed to different concentrations of pesticides for 24 h and the consequences on brood size, percentage of gravid nematodes, expression of reproductive-related genes and vitellogenin trafficking and endocytosis were measured. Moreover, 17ß-estradiol was used as an estrogenic control for endocrine disrupting compounds throughout the work. The results showed that all the pesticides disturbed to some extent one or more of the evaluated endpoints. Remarkably, we found that atrazine, 2,4-dichlorophenoxyacetic acid and chlorpyrifos produced comparable responses to 17ß-estradiol suggesting that these pesticides may have estrogen-like endocrine disrupting activity. Atrazine and 17ß-estradiol, as well as 2,4-dichlorophenoxyacetic acid and chlorpyrifos to a lesser extent, decreased the brood size, affected vitellogenin trafficking and endocytosis, and changed the expression of several reproductive-related genes. Conversely, mancozeb and cypermethrin had the least impact on the evaluated endpoint. Cypermethrin affected the brood size at the highest concentration tested and mancozeb altered the distribution of vitellogenin only in approximately 10% of the population. However, both products overexpressed hus-1 and vit-2 genes, indicating that an induction of stress could interfere with the normal development of the nematode. In conclusion, our work proved that C. elegans is a useful biological model to identify the effects of estrogen-like endocrine disruptor compounds, and the sublethal endpoints proposed may serve as an important contribution on evaluating environmental pollutants.

In vitro induction of catfish, Clarias batrachus, oocyte maturation by conspecific vitellogenin 1 (CFVg1).

Present study demonstrates that conspecific vitellogenin1 (CFVg1) induces oocyte maturation in the catfish, Clarias batrachus. CFVg1 is able to develop fertilizable eggs in the Clarias batrachus. Therefore, different in vitro oocyte culture experiments were designed to see whether CFVg1 has efficacy of oocyte maturation and its pathway. In in vitro oocyte culture experiment, CFVg1 showed a dose- and time-dependent response and 64% maturation was obtained at the dose level of 10 µg/ml or more. CFVg1 induction of oocyte maturation was confirmed by co-incubating CFVg1 with CFVg1-antiserum (a-CFVg1), which inhibited the CFVg1-induced oocyte maturation. To answer issues lead to the understanding of the mechanism of vitellogenin (Vg) on oocyte maturation, trypsin digested CFVg1 and Indian major carp Cirhinus mrigala Vg HAI (Hydroxy appetite peak I) also showed significant level of maturation. Actinomycin-D and cycloheximide blocked the effect of CFVg1, indicating that CFVg1 acts through transcription and translation. Theophylline, the phosphodiesterase inhibitor, and cAMP also inhibited the stimulatory effect of CFVg1 on oocyte maturation, indicating indirectly that CFVg1-induced oocyte maturation by decreasing the intracellular cAMP possibly by activating the phosphodiesterase enzyme. Trilostane, the 3ß-HSD-blocker, did not inhibit the CFVg1-induced oocyte maturation but wortmannin and Ly294002 two mechanistically different specific inhibitors of PI3 kinase blocked the oocyte maturation. The results thus indicate that oocyte maturation in catfish by Vg may be regulated by two pathways: (1) through decreasing the intraoocyte cAMP level by activating the cAMP-PKA pathway and (2) by cAMP-dependent PI3K/Akt pathway. Therefore, there might be role of vitellogenin itself in initiation of oocyte maturation.

Vitellogenin 2 promotes muscle development and stimulates the browning of white fat.

Eggs are rich in nutrients and contain a lot of protein. Although eggs have proved to accelerate the growth of C2C12 cells, the regulatory and mechanism of fertilized egg yolk extract (FEYE) on skeletal muscle development and fat metabolism remains unclearly. The mice were treated with FEYE by gavage for 24 d, we found that FEYE can inhibit the expression of skeletal muscle atrophy genes such as MSTN and Murf-1, and up-regulate the expression levels of MYOD, MYOG and Irisin. In addition, the treatment of FEYE induced UCP1 and PGC1α high expression in WAT, thereby causing WAT browning reaction. In order to confirm the composition of FEYE, we performed protein full spectrum identification (LC MS/MS) analysis and found the most enriched component is vitellogenin 2 (VTG2). Therefore, we added the recombinant protein VTG2 to C2C12 cells and found that VTG2 promoted the proliferation and differentiation of C2C12 cells. After that, we further proved that VTG2 inhibited the expression of MSTN and improved the expression of MYOD and Irisin. Finally, the dual luciferase test proved that VTG2 directly inhibited the transcriptional activity of MSTN. Our results conclude that FEYE inhibits the expression of MSTN in muscle tissues by delivering VTG2, thereby promoting skeletal muscle development, and can also promote the expression level of FNDC5 in serum. Then, FNDC5 acts on the fat through the serum, stimulating the browning reaction of white adipocytes. Therefore, VTG2 can be used to stop muscle consumption, improve skeletal muscle aging, and prevent obesity.

C-Terminal Fragment of Vitellogenin II, a Potential Yolkin Polypeptide Complex Precursor Protein-Heterologous Expression, Purification, and Immunoregulatory Activity.

The aim of this research was to analyze the heterologous expression, purification, and immunoregulatory activity of recombinant YGP40 (rYGP40), the potential precursor of the yolkin peptide complex. The ygp40 coding sequence was codon optimized, successfully expressed in the E. coli system, and purified from inclusion bodies with a yield of about 1.1 mg/L of culture. This study showed that the protein exhibits immunomodulatory activity, expressed by the stimulation of TNF-α and IL-10 production and nitric oxide induction at a level comparable to that of the natural yolkin peptide complex obtained by other authors from hen egg yolk. At the highest dose of 100 µg/mL, rYGP40 also caused the up-regulation of iNOS expression in murine bone marrow-derived macrophages (BMDM). Moreover, no cytotoxic effects of rYGP40 on the BMDM cell line were observed.

Effects of yolkin on the immune response of mice and its plausible mechanism of action.

Yolkin is a product of proteolytic degradation of vitellogenin, a protein contained in eggs' yolk, with already described procognitive properties. Here, we investigated effects of yolkin on the humoral and cellular immune response in mice, phenotype of cells from lymphoid organs and function of innate immunity cells. In vitro studies included effects of yolkin on mitogen-induced thymocyte proliferation, percentage of CD19 cells in bone marrow cells culture, expression of signaling molecules in Jurkat cells, interleukin 2 receptor (IL-2R) subunits in WEHI 231 cells and susceptibility of these cells to anti-Ig-induced cell death. The results showed that repeatable i.p. injections of yolkin stimulated the humoral immune response to sheep red blood cells (SRBC) irrespective of the time of the treatment. On the other hand, yolkin inhibited contact sensitivity to oxazolone. Treatment of mice with yolkin diminished the percentage of double positive cells and increasing the content of single positive CD4+ and CD8+ cells in the thymus. At the same time an increase of percentage of CD19 + B cells in the spleen and mesenteric lymph nodes was observed. In addition, the protein, given i.p., diminished ex vivo ability to synthesize nitric oxide by resident, peritoneal macrophages, stimulated with lipopolisaccharide (LPS). In vitro studies showed that yolkin increased CD19+ cell content in bone marrow cell population. The protein also enhanced proliferation of thymocytes to concanavalin A and stimulated expression of MAP kinases in Jurkat cells. In WEHI 231 B cell line yolkin caused a loss of IL-2R gamma chain expression, correlated with an increased resistance of these cells to proapoptotic action of anti-Ig antibodies. In conclusion, this is a first demonstration of immunotropic properties of yolkin in in vitro and in vivo tests. The results provide evidence for induction of maturation and stimulatory signals in immature T and B cells by the protein, suggesting its potential role in the development of an embryo's immune system.

Identification and evaluation of vaccine candidate antigens from the poultry red mite (Dermanyssus gallinae).

An aqueous extract of the haematophagous poultry ectoparasite, Dermanyssus gallinae, was subfractionated using anion exchange chromatography. Six of these subfractions were used to immunise hens and the blood from these hens was fed, in vitro, to poultry red mites. Mite mortality following these feeds was indicative of protective antigens in two of the subfractions, with the risks of mites dying being 3.1 and 3.7 times higher than in the control group (P<0.001). A combination of two-dimensional immunoblotting and immunoaffinity chromatography, using IgY from hens immunised with these subfractions, was used in concert with proteomic analyses to identify the strongest immunogenic proteins in each of these subfractions. Ten of the immunoreactive proteins were selected for assessment as vaccine candidates using the following criteria: intensity of immune recognition; likelihood of exposure of the antigen to the antibodies in a blood meal; proposed function and known vaccine potential of orthologous molecules. Recombinant versions of each of these 10 proteins were produced in Escherichia coli and were used to immunise hens. Subsequent in vitro feeding of mites on blood from these birds indicated that immunisation with Deg-SRP-1 (serpin), Deg-VIT-1 (vitellogenin), Deg-HGP-1 (hemelipoglycoprotein) or Deg-PUF-1 (a protein of unknown function) resulted in significantly increased risk of mite death (1.7-2.8times higher than in mites fed blood from control hens immunised with adjuvant only, P<0.001). The potential for using these antigens in a recombinant vaccine is discussed.

Vitellogenin from the silkworm, Bombyx mori: an effective anti-bacterial agent.

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.

Estrogenic potential of benzotriazole on marine medaka (Oryzias melastigma).

This study, for the first time, assessed the reproductive effects of benzotriazoles, widely used industrial chemicals, on marine fish. Marine medakas (Oryzias melastigma) were exposed to 0.01, 0.1, and 1mg/L benzotriazole for periods of four and 35 days. The results that are obtained showed that the expression levels of CYP1A1 were down-regulated in the liver, gills and intestines of both males and females. Vitellogenin (VTG) was highly induced in the liver, gills and intestine of both male and female marine medaka, and CYP19a was up-regulated in the ovaries especially after being exposed for 35 days. Most importantly, the results of the present study suggest that even at environmentally relevant concentrations detected in the aquatic environment, 0.01 mg/L, benzotriazole also caused notable changes in expression levels of VTG, CYP1A1 and CYP19a. More concerns about the toxicity of benzotriazoles on marine animals should be raised.

Atlantic salmon (Salmo salar L.) serum vitellogenin neutralises infectivity of infectious pancreatic necrosis virus (IPNV).

Vitellogenin is a phosphoglycoprotein which represents the main precursor of the egg yolk in teleost fish. This reproductive protein was also demonstrated to play an important role in innate immunity by acting as a pattern recognition molecule capable of binding to bacteria, fungi and enhancing macrophage phagocytosis. The presented results demonstrate that, egg homogenate, ovarian fluid and serum of mature female Atlantic salmon have high neutralising ability for infectious pancreatic necrosis virus (IPNV). Vitellogenin from mature female Atlantic salmon serum, purified by immuno-affinity on a column matrix coated with monoclonal anti-Atlantic salmon vitellogenin antibody, was able to neutralise between 9.1 x 10(4) and 3.09 x 10(5) TCID(50) IPNV mg(-1) of protein. To the author's knowledge, this is the first time that the neutralising activity of vitellogenin on a teleost virus has been demonstrated. The results may explain why IPNV is difficult to detect by culture methods in ovarian fluid and egg homogenates from carrier mature females and suggest a possible means of vertical transmission via the egg.

Vitellogenin is a cidal factor capable of killing bacteria via interaction with lipopolysaccharide and lipoteichoic acid.

Vitellogenin (Vg) has been shown to be involved in host immune defense. However, the underlying mechanism by which Vg functions is largely unknown, and which component in Vg is essential for the execution of its immune role remains lacking. Here, we demonstrate clearly that fish Vg is capable of killing the whole cells of Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus rather than their protoplasts; and that Vg has distinct binding sites specific for lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN), respectively. Of note, the interaction between Vg and bacterial cells via the different binding sites results in distinct effects: the binding of Vg to E. coli via LPS and to S. aureus via LTA is lethal, whereas the binding of Vg to S. aureus via PGN is not. Moreover, Vg exhibits a lectin-like activity because its antibacterial activities can be suppressed by the carbohydrates like d-mannose, N-acetyl-d-glucosamine and d-fucose. Finally, the polypeptide chain integrity and carbohydrate residues of Vg are indispensable for its antibacterial activity, but the lipidation and phosphorylation are not necessary. Taken together, Vg is a bacteriocidal factor capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls rather than attacking their plasma membranes.

Implication of gap junction coupling in amphibian vitellogenin uptake.

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.

In vitro effect of vitellogenin on steroid production by ovarian follicles of greenback flounder Rhombosolea tapirina.

Ovarian follicles from vitellogenic greenback flounder (Rhombosolea tapirina) were incubated in L15 medium alone, or containing human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP) or the steroid precursors testosterone (T), 17-hydroxyprogesterone (17P) and androstenedione (A) in the presence of vitellogenin (Vtg) at 0.1-5.0mg mL(-)(1). Medium concentrations of 17beta-estradiol (E(2)) and T were measured by radioimmunoassay. HCG generally stimulated follicular E(2) but not T production, whereas 17P, A and T stimulated production of E(2), T, and E(2) respectively. Treatment of follicles with dbcAMP inhibited follicular E(2) production, but increased follicular T production at high doses. The effect of low concentrations of Vtg on follicular steroid production was variable; however, higher doses of Vtg significantly suppressed basal, hCG-, dbcAMP- and steroid precursor-stimulated follicular E(2) and T production. The results of this study show that high concentrations of Vtg may suppress follicular steroid production by interfering in the steroidogenic pathway. This suggests that Vtg may regulate its own production by limiting the ovarian production of E(2).

Vitellogenin is a novel player in defense reactions.

The significance of the presence of vitellogenin (Vg), a female-specific protein, in male and juvenile fish is uncertain. Here we show that the Vg purified from the rosy barb Puntius conchonius possesses both antibacterial and hemagglutinating activities in vitro, and the male fish challenged with Escherichia coli synthesize Vg. These suggest that in addition to being involved in yolk protein formation, Vg is also involved in defense reactions, a novel function assigned to Vg.

Single-cell protein diet of a novel recombinant vitellogenin yeast enhances growth and survival of first-feeding tilapia (Oreochromis mossambicus) larvae.

Yeast single-cell protein (SCP) is a high-nutrient feed substitute. This study evaluates the dual applications of a novel recombinant Pichia pastoris SMD1168H (SMD) yeast, expressing a tilapia vitellogenin protein (rVtg), as an SCP diet for Artemia and the first-feeding fish larvae. Instar II Artemia fed rVtg, rVtg precultured in 5% fish oil (rVtg-FO), Saccharomyces cerevisiae (SC), or native SMD had greater lipid contents (P < 0.05) than the freshly hatched. Lipid deposition in the Artemia fed rVtg or rVtg-FO was greater (P < 0.05) than in those fed SMD or SC. Diet-induced accumulation of low levels of docosahexaenoic acid [22:6(n-3)] was detected only in Artemia fed the rVtg-based diets. Tilapia (Oreochromis mossambicus) larvae were fed solely yeast diets singly or in combination (d 3-22), or a staggered regimen of yeast (d 3-12) followed by unenriched or yeast-enriched Artemia (d 13-22). The larvae fed rVtg for 22 d increased in length and weight (P < 0.05), whereas those fed SC or SMD suffered growth suppression and high mortality. Such adverse consequences were ameliorated when 50% of SC was substituted with rVtg. The larvae prefed rVtg followed by a dietary switch to Artemia preenriched for 48 h with rVtg or rVtg-FO were greatest in length, had the highest weight gain, and lived the longest. Besides delivering rVtg protein, essential fatty acids and amino acids, rVtg may have probiotic effects in enhancing larval survival. This study suggests the feasibility of using the rVtg yeast as an Artemia booster and an SCP first feed for larvae.

Sex change in the protandrous black porgy, Acanthopagrus schlegeli: a review in gonadal development, estradiol, estrogen receptor, aromatase activity and gonadotropin.

Black porgy, Acanthopagrus schlegeli Bleeker, a marine protandrous hermaphrodite, is functional male for the first two years of life but begins to sexually change to female after the third year. Testicular tissue and ovarian tissue was separated by connective tissue in the bisexual gonad. This sex pattern provides a very good model to study the endocrine mechanism of sex change in fish. The annual profiles of plasma estradiol, vitellogenin and 11-ketotestosterone concentrations in males were significantly different from those in the three-year-old females. Significantly high levels of plasma estradiol during the prespawning/spawning season and low levels of plasma 11-ketotestosterone during the spawning season were observed in the inversing females. No difference of plasma testosterone levels was observed in males and females. Oral administration of estradiol stimulated high levels of gonadal aromatase activity, plasma gonadotropin II levels and sex change in the two-year-old fish. Exogenous estradiol administered for 5-6 months induced a reversible sex change in one- and two-year-old fish. The sensitive period for estradiol treatment of sex change is from early prespawning to spawning season. Implantation with testosterone for more than a year could not block the natural sex change in three-year-old fish. Exogenous aromatase inhibitors (1,4,6-androstatriene-3,17-dione or fadrozole) suppressed aromatase activity in the brain. Oral administration with aromatase inhibitors for a year further inhibited the natural sex change in three-year-old black porgy and all fish became functional male with spermiation. Estrogen receptor alpha gene in the ovarian tissue of bisexual gonad is significantly less expressed than that in the vitellogenic ovary of female on the basis of reverse-transcription polymerase-chain reaction. There was no difference in the annual profiles of the plasma gonadotropin II levels in the males and natural inversing females. Plasma gonadotropin II levels were significantly higher in estradiol-treated group than those in the control. It is concluded that estradiol, aromatase activity and estrogen receptor in the ovarian tissue play an important role in the natural and controlled sex change in black porgy. The association of gonadotropin and sex change in black porgy is not clear.

Dose-response relationships and pharmacokinetics of vitellogenin in rainbow trout after intravascular administration of 17alpha-ethynylestradiol.

A commonly used endpoint in bioassays testing the estrogenicity of chemicals is the induction of the egg yolk precursor vitellogenin (VTG) in male fish. However, relatively little is known about the kinetics of induction and elimination of VTG in fish exposed to xenoestrogens. In this study, we administered graded intra-arterial doses (0.001, 0.1, 1.0 and 10.0 mg/kg) of 17alpha-ethynylestradiol (EE(2)) to male rainbow trout via a dorsal aortic cannula which allowed repetitive blood sampling from individual fish for up to 48 days after injection. The plasma concentrations of VTG was quantified using an enzyme-linked immunosorbent assay procedure and the simultaneous concentrations of EE(2) were determined by gas chromatography-mass spectrometry. The pattern of VTG induction was similar for all doses of EE(2), with a 12-h lag-time before increase from basal levels (0.006-0.008 microg/ml), then increasing sharply to maximum levels within 7-9 days (C(max)=0.05, 711, 1521 and 2547 microg/ml VTG for the 0.001, 0.1, 1.0 and 10.0 mg/kg doses, respectively). After induction by EE(2), VTG declined mono-exponentially with an elimination half-life of 42-49 h. The half-life of VTG increased to 145 h in the 10 mg/kg treated fish. The pharmacokinetics of EE(2) were distinctly nonlinear with substantial increases in the elimination half-life with increasing dose. The plasma concentration-time profiles of EE(2) were influenced by enterohepatic recirculation that caused multiple or secondary peaks in the profiles. In a separate experiment, the pharmacokinetics of purified VTG was characterized after intra-arterial injection in trout. After direct injection of VTG, plasma levels declined tri-exponentially with an apparent steady-state volume of distribution of 837 ml/kg; total body clearance was 31.1 ml/h per kg, and the elimination half-life was 43.7 h.