Vitellogenin Gene Expression Research Articles

Physiology of reproductive worker honey bees (Apis mellifera): insights for the development of the worker caste.

Reproductive and behavioural specialisations characterise advanced social insect societies. Typically, the honey bee (Apis mellifera) shows a pronounced reproductive division of labour between worker and queen castes, and a clear division of colony roles among workers. In a queenless condition, however, both of these aspects of social organisation break down. Queenless workers reproduce, forage and maintain their colony operating in a manner similar to communal bees, rather than as an advanced eusocial group. This plasticity in social organisation provides a natural experiment for exploring physiological mechanisms of division of labour. We measured brain biogenic amine (BA) levels and abdominal fat body vitellogenin gene expression levels of workers in queenright and queenless colonies. Age, ovary activation and social environment influenced brain BA levels in honey bees. BA levels were most influenced by ovary activation state in queenless bees. Vitellogenin expression levels were higher in queenless workers than queenright workers, but in both colony environments vitellogenin expression was lower in foragers than non-foragers. We propose this plasticity in the interacting signalling systems that influence both reproductive and behavioural development allows queenless workers to deviate significantly from the typical worker bee reaction norm and develop as reproductively active behavioural generalists.

The mechanism of regulation of ovarian maturation by red pigment concentrating hormone in the mud crab Scylla paramamosain

In this study a full-length cDNA (Sp-RPCH) was cloned from the eyestalk ganglia of the mud crab Scylla paramamosain. Sp-RPCH is 660 base pairs in length and its open reading frame encodes a precursor that is predicted to be processed into a 25-residue signal peptide, a mature red pigment concentrating hormone (RPCH, an octapeptide), and a 75-residue precursor-related peptide. Phylogenetic analysis indicates that it clusters with other crustacean RPCHs and belongs to the adipokinetic hormone/RPCH peptide superfamily. Sp-RPCH gene expression was detected, using an end-point polymerase chain reaction (PCR), not only in the eyestalk ganglia but also in the brain and thoracic ganglia. Quantified using a real-time PCR, Sp-RPCH gene expression levels in the three tissues fluctuated along a cycle of ovarian maturation, with the levels progressively increased from stages I to IV, after which the expression levels decreased (although they remained significantly higher than stage I levels) when the ovary reached the mature stage (stage V). It was demonstrated using a patch clamp analysis that synthetic RPCH was able to evoke a Ca2+ current in dissociated brain neurons and synthetic RPCH significantly increased the mean oocyte diameter of the ovarian tissues co-cultured with the eyestalk ganglia, brain, or thoracic ganglia; the stimulatory effect of RPCH was absent when the nervous tissues were not included in the ovarian incubation. Animals administrated with RPCH had significantly higher levels of gonad-somatic index, hepatopancreas-somatic index, and vitellogenin gene expression, when compared to control animals receiving a saline injection. The combined results clearly show that RPCH is involved in ovarian maturation in the mud crab; the stimulatory effects of RPCH are likely mediated by its actions on the release from the nervous tissues of factor(s) that directly regulate vitellogenesis in the ovary and hepatopancreas.

Interactive effects of chronic waterborne copper and cadmium exposure on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas)

The present study was carried out to examine the interactive effects of chronic waterborne copper (Cu) and cadmium (Cd) on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas). Trios (1 male: 2 female; n=5) of fish were exposed for 21days to: (i) control (no added Cu or Cd), (ii) waterborne Cu (75μg/L), (iii) waterborne Cd (5μg/L), and (iv) Cu and Cd mixture (75 and 5μg/L, respectively). Reproductive output (cumulative egg production) was significantly reduced by Cu but not by Cd. Interestingly however, no spawning occurred in fish exposed to the mixture of waterborne Cu and Cd. In general, both Cu and Cd accumulation in target tissues (gill, liver, gonad and carcass) increased significantly in fish exposed to Cu and Cd mixture, and no interaction between Cu and Cd accumulation was observed in any tissues, except in the liver where Cu accumulation was significantly reduced by Cd. The expression of female hepatic estrogen receptor genes (ER-α and ER-β) was most significantly elevated in fish exposed to Cu and Cd mixture, whereas vitellogenin gene expression was reduced maximally in the same exposure. Similarly, the hepatic expression of the metallothionein gene was most significantly upregulated in fish exposed to Cu and Cd mixture. Moreover, the circulating estradiol level in females was significantly decreased only during the co-exposure of waterborne Cu and Cd. Overall, the present study indicates that the interaction of chronic waterborne Cu and Cd exposure may elicit greater than additive effect on reproductive output in fish.

Estrogen-responsive genes encoding egg yolk proteins vitellogenin and apolipoprotein II in chicken are differentially regulated by selective estrogen receptor modulators

In a hen, large quantities of the egg yolk proteins, apolipoprotein II (apo-II) and vitellogenin (VG), are expressed in the liver and transported to the oviduct during egg production. Estrogenic stimulation of the hepatic expression of apo-II and VG is due to both transcriptional increase and mRNA stabilization. The nucleolytic degradation of apo-II messenger RNA (mRNA) is prevented by estrogen-regulated mRNA-stabilizing factor (E-RmRNASF). Gene-specific effects of a select panel of selective estrogen receptor modulators (SERMs) on the hepatic expression of the estrogen-responsive genes encoding apo-II, VG, and E-RmRNASF in the chicken liver were investigated. In the present study, 6-week-old roosters were treated with the vehicle, estrogen, the SERMs genistein, resveratrol, tamoxifen, pterostilbene, raloxifene, catechin, and clomiphene or a combination of estrogen and a 200-fold excess of each of the SERMs. Results from mRNA stabilization studies conducted to investigate the stimulation of expression of E-RmRNASF in the liver by these agents showed that the expression of E-RmRNASF in the liver was stimulated by estrogen and the SERMs genistein, resveratrol, tamoxifen, pterostilbene, and catechin but not by the vehicle, clomiphene or raloxifene. The expression of apo-II and VG from the aforementioned treatments was determined by Northern blot analysis, RNase protection assays, and Western blot analysis. The transcription and protein expression of both apo-II and VG genes were seen in response to treatment with estrogen but not with the SERMs or combinations of estrogen and each of the SERMs. The SERMs that stimulated the expression of E-RmRNASF antagonized the stimulation of the expression of both apo-II and VG by estrogen, demonstrating a gene-specific, selective regulation of the aforementioned genes in the chicken liver by the SERMs. The above panel of SERMs may likely have adverse effects on egg production.

Tamoxifen persistently disrupts the humoral adaptive immune response of gilthead seabream (Sparus aurata L.)

There is increasing concern about the possible effect of pharmaceutical compounds may have on the fish immune system. Bath exposition of 17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives, altered the immune response of the gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. Tamoxifen (Tmx) is a selective estrogen-receptor modulator used in hormone replacement therapy, the effects of which are unknown in fish immunity. This study aims to investigate the effects of dietary administration of EE2 (5 μg/g food) and Tmx (100 μg/g food) on the immune response of gilthead seabream, and the capacity of the immune system to recover its functionality after a recovery period. The results show for the first time the reversibility of the effect of EE2 and Tmx on the fish immune response. Tmx promoted a transient alteration in hepatic vitellogenin gene expression of a different magnitude to that produced by EE2. Both, EE2 and Tmx inhibited the induction of interleukin-1β gene expression while reversed the inhibition of ROI production in leukocytes following vaccination. However, none of these effects were observed after ceasing EE2 and Tmx exposure. EE2 and Tmx stimulated the antibody response of vaccinated fish although Tmx, but not EE2, altered the antibody response and modulated the percentage of IgM+ B lymphocytes of vaccinated fish during the recovery phase. Taken together, our results suggest that EE2 and Tmx might alter the capacity of fish to appropriately respond to infection and show that Tmx has a long-lasting effect on humoral adaptive immunity.

Silencing a sugar transporter gene reduces growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The brown planthopper (BPH), Nilaparvata lugens, sugar transporter gene 6 (Nlst6) is a facilitative glucose/fructose transporter (often called a passive carrier) expressed in midgut that mediates sugar transport from the midgut lumen to hemolymph. The influence of down regulating expression of sugar transporter genes on insect growth, development, and fecundity is unknown. Nonetheless, it is reasonable to suspect that transporter-mediated uptake of dietary sugar is essential to the biology of phloem-feeding insects. Based on this reasoning, we posed the hypothesis that silencing, or reducing expression, of a BPH sugar transporter gene would be deleterious to the insects. To test our hypothesis, we examined the effects of Nlst6 knockdown on BPH biology. Reducing expression of Nlst6 led to profound effects on BPHs. It significantly prolonged the pre-oviposition period, shortened the oviposition period, decreased the number of eggs deposited and reduced body weight, compared to controls. Nlst6 knockdown also significantly decreased fat body and ovarian (particularly vitellogenin) protein content as well as vitellogenin gene expression. Experimental BPHs accumulated less fat body glucose compared to controls. We infer that Nlst6 acts in BPH growth and fecundity, and has potential as a novel target gene for control of phloem-feeding pest insects.

Assessment of biomarkers for contaminants of emerging concern on aquatic organisms downstream of a municipal wastewater discharge

Contaminants of emerging concern (CECs), including pharmaceuticals, personal care products and estrogens, are detected in wastewater treatment plant (WWTP) discharges. However, analytical monitoring of wastewater and surface water does not indicate whether CECs are affecting the organisms downstream. In this study, fathead minnows (Pimephales promelas) and freshwater mussels Pyganodon grandis Say, 1829 (synonym: Anodonta grandis Say, 1829) were caged for 4weeks in the North Saskatchewan River, upstream and downstream of the discharge from the WWTP that serves the Edmonton, AB, Canada. Passive samplers deployed indicated that concentrations of pharmaceuticals, personal care products, an estrogen (estrone) and an androgen (androstenedione) were elevated at sites downstream of the WWTP discharge. Several biomarkers of exposure were significantly altered in the tissues of caged fathead minnows and freshwater mussels relative to the upstream reference sites. Biomarkers altered in fish included induction of CYP3A metabolism, an increase in vitellogenin (Vtg) gene expression in male minnows, elevated ratios of oxidized to total glutathione (i.e. GSSG/TGSH), and an increase in the activity of antioxidant enzymes (i.e. glutathione reductase, glutathione-S-transferase). In mussels, there were no significant changes in biomarkers of oxidative stress and the levels of Vtg-like proteins were reduced, not elevated, indicating a generalized stress response. Immune function was altered in mussels, as indicated by elevated lysosomal activity per hemocyte in P. grandis caged closest to the wastewater discharge. This immune response may be due to exposure to bacterial pathogens in the wastewater. Multivariate analysis indicated a response to the CECs Carbamazepine (CBZ) and Trimethoprim (TPM). Overall, these data indicate that there is a 1km zone of impact for aquatic organisms downstream of WWTP discharge. However, multiple stressors in municipal wastewater make measurement and interpretation of impact of CECs difficult since water temperature, conductivity and bacteria are also inducing biomarker responses in both fish and mussels.

High juvenile hormone titre and abdominal activation of JH signalling may induce reproduction of termite neotenics.

Termite castes are a key example of polyphenism, in which reproductive division of labour is clearly seen in colonies. The reproductive castes in termites include primary and neotenic reproductives; primary reproductives found a new colony whereas neotenics succeed them in the reproductive role when the primary reproductives die or become senescent. Neotenics usually differentiate from nymphs or workers by developing functional gonads while retaining juvenile characteristics; however, the developmental mechanism during neotenic differentiation remains poorly understood. Juvenile hormone (JH) mediates a number of aspects of developmental regulation in caste differentiation in termites. In the present study we quantified JH titres in neotenic reproductives of Reticulitermes speratus, and compared these with other developmental stages. In addition, expression changes in JH signalling gene homologues (Methoprene-tolerant [Met], Krüppel-homolog1, Broad-Complex) in the head, thorax and abdomen were investigated during neotenic differentiation. Finally, we examined the function of Met in reproduction of neotenics by RNA interference (RNAi). Our results showed that the JH titres of neotenics were significantly higher than those of nymphs and workers. JH signalling genes were highly expressed in neotenic abdomens, compared with those in workers and nymphs. Met RNAi resulted in the inhibition of vitellogenin gene expression in newly moulted neotenics. These results suggest that the fertility of neotenics might be controlled by a large increase of JH titres and body-part-specific activation of JH signalling pathways.

17α-Ethinylestradiol can disrupt hemoglobin catabolism in amphibians

Different chemical substances, which enter the environment due to anthropogenic influences, can affect the endocrine system and influence development and physiology of aquatic animals. One of these endocrine disrupting chemicals is the synthetic estrogen, 17α-ethinylestradiol (EE2), which is a main component of various oral contraceptives and demonstrably affects many different aquatic vertebrates at extremely low concentrations by feminization phenomena. The aim of the present study was to investigate whether a four week exposure to three different concentrations of EE2 (0.3ng/L, 29.6ng/L and 2960ng/L) affects the catabolism of hemoglobin of the amphibian Xenopus laevis. The results of this study demonstrate for the first time that beside an increase of the hepatic vitellogenin gene expression, exposure to EE2 also decreases the gene expression of the hepatic heme oxygenase 1 and 2 (HO1, HO2), degrading heme of different heme proteins to biliverdin, as well as of the biliverdin reductase A (BLVRA), which converts biliverdin to bilirubin. The results further suggest that EE2 already at the environmentally relevant concentration of (29.6ng/L) can disrupt hemoglobin catabolism, indicated by decreased gene expression of HO2, which becomes evident at the highest EE2 concentration that led to a severe increase of biliverdin in plasma.

Differential regulation of hepatopancreatic vitellogenin (VTG) gene expression by two putative molt-inhibiting hormones (MIH1/2) in Pacific white shrimp (Litopenaeus vannamei)

Molt-inhibiting hormone (MIH), a peptide member of the crustacean hyperglycemic hormone (CHH) family, is commonly considered as a negative regulator during the molt cycle in crustaceans. Phylogenetic analysis of CHH family peptides in penaeidae shrimps suggested that there is no significant differentiation between MIH and vitellogenesis-inhibiting hormone (VIH, another peptide member of CHH family), by far the most potent negative regulator of crustacean vitellogenesis known. Thus, MIH may also play a role in regulating vitellogenesis. In this study, two previously reported putative MIHs (LivMIH1 and LivMIH2) in the Pacific white shrimp (Litopenaeus vannamei) were expressed in Escherichia coli, purified by immobilized metal ion affinity chromatography (IMAC) and further confirmed by western blot. Regulation of vitellogenin (VTG) mRNA expression by recombinant LivMIH1 and LivMIH2 challenge was performed by both in vitro hepatopancreatic primary cells culture and in vivo injection approaches. In in vitro primary culture of shrimp hepatopancreatic cells, only LivMIH2 but not LivMIH1 administration could improve the mRNA expression of VTG. In in vivo injection experiments, similarly, only LivMIH2 but not LivMIH1 could stimulate hepatopancreatic VTG gene expression and induce ovary maturation. Our study may provide evidence for one isoform of MIH (MIH2 in L. vannamei) may serve as one of the mediators of the physiological progress of molting and vitellogenesis. Our study may also give new insight in CHH family peptides regulating reproduction in crustaceans, in particular penaeidae shrimps.

Differential response of two somatolactin genes to zinc or estrogen in pituitary of Cyprinus carpio

Environmental changes affect gene expression that we addressed in the pituitary, a central regulatory organ at the interface between the central nervous system and the endocrine system. With the aim to reveal effects of changes in the aquatic environment on the expression of hypothalamo-hypophyseal factors, we characterized somatolactin (SL) in Cyprinus carpio. SL, a fish specific pituitary hormone belonging to the prolactin (PRL) superfamily, is involved in background adaptation, osmoregulation, reproduction and fatty acid metabolism. Two sl genes, α and β, were discovered in carp and transcripts of both were detected in pituitaries. Clearly, expression of slα and slβ was modulated significantly in pituitary of male adult carp in response to treatment with ZnCl2 (Zn), but only slβ responded to 17β-estrogen (E2), relative to control carp as shown by RT-qPCR analyses. Furthermore, the amount of mRNA of related factors was assessed revealing variable effects on prl, growth hormone (gh), and factors involved in sl regulation: the pituitary transcription factor pit1 and hypothalamic pituitary adenylase cyclase activating peptide (pacap). In parallel, the physiological response of the experimental animals to Zn or E2 was confirmed by showing a significant increase of metallothionein (mt) or vitellogenin (vg) gene expression in liver, classical sentinels for exposure to heavy metal or estrogens.These data suggest that the sl genes seem to be involved in the response to Zn, as well as to estrogen, and could contribute to evaluate biological relevant changes in the aquatic environment.

Sensitization of vitellogenin gene expression by low doses of octylphenol is mediated by estrogen receptor autoregulation in the Bombina orientalis (Boulenger) male liver

This study aimed to elucidate the mechanisms by which alkylphenols disrupt endocrine function in wild amphibians in Korea. To this end, the effects of 4-tert-octylphenol (OP), 17β-estradiol (E2), and estrogen receptor (ER) agonists on the expression profiles of vitellogenin (VTG) and ERs were examined in livers obtained from male Bombina orientalis toads. A single injection of E2 (10μg/kg; 0.03μmol/kg) induced transcription of VTG mRNA at 2 days post injection; however, injection of either the ERα-selective agonist propyl-(1H)-pyrazole-1,3,5-triyl-trisphenol (PPT, 50μg/kg; 0.12μmol/kg) or the ERβ-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN, 50μg/kg; 0.20μmol/kg) did not affect the expression of VTG. This finding suggests that both ERα and ERβ are required to induce transcription of VTG in the male B. orientalis liver. Interestingly, E2, PPT, and DPN induced transcription of ERα, which was also reflected on the protein level; however, these alkylphenols did not affect ERβ transcription. Similarly, VTG transcription was induced by a single injection of 1–100mg/kg (0.04–484.66μmol/kg) OP, while 0.1mg/kg (0.48μmol/kg) OP had no effect on VTG transcription. This result suggests that the lowest observable effect concentration (LOEC) of OP for induction of VTG transcription in the male liver is 1mg/kg (4.84μmol/kg). Furthermore, treatment with E2 (10μg/kg; 0.03μmol/kg) or OP (1mg/kg; 4.84μmol/kg) significantly upregulated ERα transcription, and a 10mg/kg (48.46μmol/kg) dose of OP significantly upregulated ERβ transcription. The ER antagonist ICI 182,780 decreased the basal levels of ERα and ERβ mRNA, and also prevented E2-mediated and OP-mediated induction of VTG, ERα, and ERβ transcription. A second injection of 0.1mg/kg (0.48μmol/kg) OP after a two-day interval significantly upregulated the transcription of VTG and ERα, but not of ERβ. These results suggest that sensitization of VTG transcription by repeated exposure to OP is mediated by the induction of ERα. Different combinations of alkylphenols that are ubiquitous in the freshwater system in Korea could potentially exert a synergistic effect on endocrine disruption. Thus, chronic exposure to alkylphenols, even at their NOECs, could still disrupt endocrine function in B. orientalis.

Nonmonotonic response of vitellogenin and estrogen receptor α gene expression after octylphenol exposure of Cichlasoma dimerus (Perciformes, Cichlidae)

In oviparous vertebrates, vitellogenin (VTG) is mainly produced by the liver in response to estrogen (E2) and its synthesis is traditionally coupled to estrogen receptor alpha induction. Even though VTG is a female-specific protein, chemicals that mimic natural estrogens, known as xenoestrogens, can activate its expression in males causing endocrine disruption to wildlife and humans. Alkylphenols such as nonylphenol (NP) and octylphenol (OP) are industrial additives used in the manufacture of a wide variety of plastics and detergents, and can disrupt endocrine functions in exposed animals. For more than a decade, the freshwater cichlid fish Cichlasoma dimerus has been used for ecotoxicological studies in our laboratory. We recently found an up-regulation of VTG gene expression in livers of male fish exposed to OP, from a silent state to values similar to those of E2-induced fish. To better understand the underlying mechanisms behind the action of xenoestrogens, the aim of this study was to analyze the dose–response relationship of C. dimerus VTG and estrogen receptors (ERs) gene expression after waterborne exposure to 0.15, 1.5, 15, and 150μg/L OP for up to 1 month (0, 1, 3, 7, 14, 21, and 28 days). At the end of the experiment, histological features of exposed fish included active hepatocytes with basophilic cytoplasm and high eosinophilic content in their vascular system due to augmented expression of VTG. In testis, high preponderance of sperm was found in fish exposed to 150μg/L OP. A classic dose–response down-regulation of the expression of Na+/K+-ATPase, a “non-gender specific gene” used for comparison, was found with increasing OP concentrations. No VTG and very low levels of ERα were detected in control male livers, but an up-regulation of both genes was found in males exposed to 0.15 or 150μg/L OP. Moreover, VTG transcripts were significant as early as day 3 or day 1 of exposure to these OP concentrations, respectively. Nearly no response was detected in 1.5 and 15μg/L OP exposed-fish. Data was curve-fitted evidencing a nonmonotonic dose–response curve. Interestingly, ERβ2 mRNA expression was augmented above baseline levels only when males were exposed to the lowest OP concentration. We speculate that genomic control of vitellogenesis is under control of multiple steroid receptors with different affinities for ligands. ERβ isoform, only up-regulated with very low concentrations of ligand, would act as a sensors of OP (or E2) to induce ERα and VTG. With high OP concentrations, the expression of ERα isoform is promptly augmented, with the concomitant VTG transactivation.

Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together with cryopreserved trout hepatocytes for screening estrogenic chemicals, resulting in a reduction of the time required to perform the assay and enabling greater access to the model system through the approach of cryopreservation.

Complex effects of two presumably antagonistic endocrine disrupting compounds on the goldfish Carassius aumtus: A comprehensive study with multiple toxicological endpoints

We studied the effects of endocrine disrupting compounds nonylphenol (NP) and letrozole (LE) on the male goldfish Carassius aumtus. Exposure to NP (20μgl−1) alone caused a significant up-regulation in the expression of aromatase, estrogen receptors and vitellogenin (VTG) genes, an increase in hepatic and plasma VTG concentration, but no obvious testicular impairment. Exposure to LE (1mgkg−1) alone resulted in a significant decline in aromatase activity, reduced levels of plasma 17β-estradiol (E2), and enhanced sperm maturation. Co-exposure with LE (1mgkg−1) could only partially affect some of the estrogenic effects caused by NP (20μgl−1) (i.e. expression of hepatic and brain estrogen receptor genes, hepatic VTG concentration), but inhibit other estrogenic effects (i.e. brain and testicular aromatase activity, plasma E2). In addition, co-exposure resulted in impairment of liver mitochondria (i.e. detachment of ridges from the membrane, and uneven distribution of the cytoplasm with clusters of glycogen granules), but did not cause significant damage to the testes (i.e. the morphology, the spermatogonia and spermatozoa densities). Our results clearly showed that nonylphenol and letrozole co-exposure could induce profound effects on fish, and highlighted the importance of adopting multiple toxicological endpoints when evaluating the combined effects of endocrine disrupting compounds.

Vitellogenin gene characterization and expression of Asian paddle crabs (Charybdis japonica) following endocrine disrupting chemicals

Vitellogenin (VTG), the yolk-precursor lipoprotein, has been widely recognized as a biomarker for the detection of estrogenic activity in water-borne chemical pollutants. The Asian paddle crab, Charybdis japonica, is a potential bio-indicator for monitoring marine environments. The aim of this study was to identify the possibility of using C. japonica VTG as biomarkers of stress caused by endocrine disrupting chemicals (EDCs). We characterized a partial sequence of the VTG cDNA in the C. japonica crab and evaluated the crab’s mRNA expression profiles following exposure to different concentrations of bisphenol A (BPA) and 4-nonylphenol (NP) for 24 or 96 h. The sequence homology of C. japonica VTG is over 93% in nucleotide and over 98% in amino acid with the corresponding gene of other crabs. Phylogenetic analysis revealed that the C. japonica VTG is an ortholog of other species of lobster and shrimp. Tissue distribution analysis of the C. japonica VTG mRNA revealed that the expression of VTG mRNA was highest in the ovary of females and hepatopancreas. The expression of the C. japonica VTG gene in various BPA or NP concentrations during shorter and longer times was assessed. The expression of VTG transcripts was significantly increased in the C. japonica crab exposed to BPA and NP at different concentrations for 24 h. The mRNA expression of the VTG gene was significantly induced in concentration- and time-dependent manners after BPA or NP exposures for 96 h. These results indicate that crab C. japonica VTG could be used as a potential biomarker of EDCs in marine environment monitoring.

The shrimp heat shock cognate 70 functions as a negative regulator in vitellogenin gene expression.

Within the 2.6-kb 5' flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2), several clusters of putative heat shock factor (HSF) response elements were identified. Deletion of these response elements has caused significant increases in MeVg2 promoter activity, suggesting that the HSF and Hsc70 complex may regulate vitellogenin gene expression in a negative manner. To confirm the role of Hsc70 in the regulation of vitellogenin gene expression, the ovary cDNA for Hsc70 was cloned and characterized. The Hsc70 transcript level was high in the ovary and hepatopancreas of females at the early vitellogenic stage but dropped during ovarian maturation. In addition, Western blot analysis revealed the presence of Hsc70 in the nuclear but not in the cytoplasmic fraction during the early stage of ovary maturation. Electrophoretic mobility shift assay (EMSA) results showed that ovary nuclear extract contained a factor that binds to the HSF response element. Since the addition of ATP caused a decrease in the binding of Hsc70, Hsc70 may form a repressor complex with HSF to inhibit MeVg2 expression. An in vitro RNA interference technique was used to study the gene function of Hsc70. Hsc70 gene knockdown resulted in an increased MeVg2 mRNA level in the ovary (54%) and hepatopancreas (62%). In summary, this report describes the first study of vitellogenin gene regulation at the transcription level in crustaceans and provides strong evidence that Hsc70 acts as a molecular chaperone to negatively regulate MeVg2 gene expression in shrimp.

20‐hydroxyecdysone mediates non‐canonical regulation of mosquito vitellogenins through alternative splicing

Vitellogenesis is one of the most well-studied physiological processes in mosquitoes. Expression of mosquito vitellogenin genes is classically described as being restricted to female adult reproduction. We report premature vitellogenin transcript expression in three vector mosquitoes: Culex tarsalis, Aedes aegypti and Anopheles gambiae. Vitellogenins expressed during non-reproductive stages are alternatively spliced to retain their first intron and encode premature termination codons. We show that intron retention results in transcript degradation by translation-dependent nonsense-mediated mRNA decay. This is probably an example of regulated unproductive splicing and translation (RUST), a mechanism known to regulate gene expression in numerous organisms but which has never been described in mosquitoes. We demonstrate that the hormone 20-hydroxyecdysone (20E) is responsible for regulating post-transcriptional splicing of vitellogenin. After exposure of previtellogenic fat bodies to 20E, vitellogenin expression switches from a non-productive intron-retaining transcript to a spliced protein-coding transcript. This effect is independent of factors classically known to influence transcription, such as juvenile hormone-mediated competence and amino acid signalling through the target of rapamycin pathway. Non-canonical regulation of vitellogenesis through RUST is a novel role for the multifunctional hormone 20E, and may have important implications for general patterns of gene regulation in mosquitoes.

Evidence for the presence of sex steroid hormones in Zhikong scallop, Chlamys farreri

To obtain evidence of the presence of sex steroid hormones in mollusks, hormone variation in the gonads of the Zhikong scallop Chlamys farreri was analyzed using UPLC-MS/MS. These were found, as expected, with concentrations of estrone (E1), 17beta-estradiol (E2), and testosterone (T) in the testes ranging from not detected (ND) to 0.07±0.10, ND to 3.10±2.00, and ND to 2.67±1.55ng/g wet weight, respectively. In the ovaries, these hormones ranged from ND to 2.45±1.22, ND to 27.90±4.23, and ND to 2.38±1.56ng/g ww, respectively. The levels of T in males and E2 in females followed a trend similar to the gonadal-somatic index over the course of the reproductive period. In addition, the gene expression of vitellogenin and calmodulin-2 showed similar patterns to T and E2, while the estrogen receptors and calmodulin-1 did not. These results indicate that sex steroids are present in the scallop and that they may regulate endocrine functions during the reproductive process.

Temporal Dynamics of Oocyte Growth and Vitellogenin Gene Expression in Zebrafish (Danio Rerio)

Little is known about how hepatic vitellogenin gene (vtg) expression relates to oogenesis in fish, especially among fractional spawners. The objective of this study was to relate hepatic vtg 1A/B expression to stage-specific oocyte development in zebrafish (Danio rerio), an asynchronous spawning fish. Liver samples were collected at seven time points postspawning (1-32 days) and fish were preserved for subsequent histological analyses. Relative vtg 1A/B expression among liver samples was quantified by reverse transcription-quantitative PCR and oogenesis was evaluated following standard hematoxylin and eosin staining of serial ovarian sections. Histological analyses indicate that a subset of previtellogenic oocytes (stages 1-2) transitioned into postvitellogenic oocytes (stages 3-4) within 4 days (96 h) postspawning. By 8 days postspawning (192 h), the majority of the ovary was occupied by mature (stage 4) oocytes, a trend that continued through 32 days postspawning. Hepatic vtg 1A/B gene expression was upregulated 3.89-fold 1-h postspawning relative to the average gene expression across all time points, but was not correlated to stage-specific oogenesis. Follicular atresia among fish sampled 32 days postspawning highlights the importance of regular spawning in zebrafish and suggests that the event of spawning itself may be integral to the regulation of oocyte development.