Vtg Expression Research Articles

Effects of mifepristone, a model compound with anti-progestogenic activity, on the reproduction of African clawed frog (Xenopus laevis)

This is the first study on how a substance with anti-progestogenic activity affects amphibian reproduction. Mifepristone, a synthetic anti-progestin used in abortion pills, was chosen as model compound. African clawed frog (Xenopus laevis) females were exposed to four mifepristone concentrations (0.7, 9, 120, and 1380ng∙L−1) for 30 days. A control group was also included. Egg-laying during the experiment was significantly less at the highest concentration. At the experiment’s end, mifepristone-exposed and control females were randomly mated with sexually mature males. Breeding rate for females exposed to 1380ng∙L−1 mifepristone was only 50%. Histology revealed no significant changes in gonads, thyroid, or liver. Females exposed to 1380ng∙L−1 mifepristone had lower estradiol levels in plasma, lower mRNA expression of lh and fsh in brain–pituitary complex, and p450scc in ovaries. In liver, mRNA level of npr was significantly increased in females exposed to 120ng∙L−1 mifepristone. mRNA expression of mpr, erβ, dio2, and dio3 were upregulated in animals exposed to 9ng∙L−1 and 120ng∙L−1 mifepristone, whereas vtg expression was significantly downregulated in females exposed to 1380ng∙L−1 mifepristone. All these findings show that exposure to compounds with anti-progestogenic activity affects the hypothalamus–pituitary–gonad axis and decreases reproductive success.

Use of All-Male cyp17a1-Deficient Zebrafish (Danio rerio) for Evaluation of Environmental Estrogens.

Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all-testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1-deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3-day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1-deficient fish. Compared with the control fish, a higher E2-mediated vtg upregulation observed in cyp17a1-deficient zebrafish exposed to 0.1 μg/L E2 is known to be estrogen receptor-dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1-deficient zebrafish was observed when exposed to 200 and 2000 μg/L bisphenol A (BPA) and perfluoro-1-octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1-deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all-male cyp17a1-deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062-1074. © 2024 SETAC.

Pyriproxyfen Contamination in Daphnia magna: Identifying Early Warning Biomarkers.

Pyriproxyfen is an insecticide currently employed in numerous countries for the management of agricultural and indoor pests. Several studies indicate that this insecticide has been detected in multiple rivers, with concentrations reaching as high as 99.59 ng/L in the Júcar River in Spain. Therefore, the determination of some biochemical and genetic effects of this insecticide on aquatic organisms could serve as an early warning mechanism to identify potential disruptions in various biomarkers. Based on this, Daphnia magna organisms were exposed to pyriproxyfen sublethal concentrations for 21 days. Some biochemical parameters, including cholesterol, triglycerides, glucose, lactate, and LDH activity, were determined. Additionally, some genetic biomarkers associated with oxidative stress, heat shock proteins, lipid metabolism, hemoglobin, metallothioneins, and vitellogenin synthesis were evaluated in daphnids exposed to the insecticide for 21 days. LDH activity increased significantly in those daphnids exposed to the highest insecticide concentration (14.02 µg/L), while cholesterol levels decreased significantly. In contrast, glucose, total proteins, and triglycerides remained unaffected in D. magna exposed to pyriproxyfen. On the other hand, exposure to the insecticide led to notable alterations in gene expression among individuals. Specifically, genes associated with lipid metabolism and reproduction exhibited a significant reduction in gene expression. Fabd expression was decreased by approximately 20% in exposed daphnids, while vtg expression was suppressed as much as 80% when compared to control values. Furthermore, it was observed that the hgb1 and hgb2 genes, associated with hemoglobin synthesis, exhibited significant overexpression. Notably, the dysfunction observed in both hemoglobin genes was linked to an increase in pigmentation in Daphnia magna during the course of the experiment. These alterations in gene expression could serve as effective indicators of early contamination even at low pesticide concentrations.

Fermented Aronia melanocarpa pomace improves the nutritive value of eggs, enhances ovarian function, and reshapes microbiota abundance in aged laying hens.

There is a decline in the quality and nutritive value of eggs in aged laying hens. Fruit pomaces with high nutritional and functional values have gained interest in poultry production to improve the performance. The performance, egg nutritive value, lipid metabolism, ovarian health, and cecal microbiota abundance were evaluated in aged laying hens (320 laying hens, 345-day-old) fed on a basal diet (control), and a basal diet inclusion of 0.25%, 0.5%, or 1.0% fermented Aronia melanocarpa pomace (FAMP) for eight weeks. The results show that 0.5% FAMP reduced the saturated fatty acids (such as C16:0) and improved the healthy lipid indices in egg yolks by decreasing the atherogenicity index, thrombogenic index, and hypocholesterolemia/hypercholesterolemia ratio and increasing health promotion index and desirable fatty acids (P < 0.05). Additionally, FAMP supplementation (0.25%-1.0%) increased (P < 0.05) the ovarian follicle-stimulating hormone, luteinizing hormone, and estrogen 2 levels, while 1.0% FAMP upregulated the HSD3B1 expression. The expression of VTG II and ApoVLDL II in the 0.25% and 0.5% FAMP groups, APOB in the 0.5% FAMP group, and ESR2 in the 1% FAMP group were upregulated (P < 0.05) in the liver. The ovarian total antioxidant capacity was increased (P < 0.05) by supplementation with 0.25%-1.0% FAMP. Dietary 0.5% and 1.0% FAMP downregulated (P < 0.05) the Keap1 expression, while 1.0% FAMP upregulated (P < 0.05) the Nrf2 expression in the ovary. Furthermore, 1.0% FAMP increased cecal acetate, butyrate, and valerate concentrations and Firmicutes while decreasing Proteobacteria (P < 0.05). Overall, FAMP improved the nutritive value of eggs in aged laying hens by improving the liver-blood-ovary function and cecal microbial and metabolite composition, which might help to enhance economic benefits.

Increased Endocrine Activity of Xenobiotic Chemicals as Mediated by Metabolic Activation.

The US Environmental Protection Agency (USEPA) is faced with long lists of chemicals that require hazard assessment. The present study is part of a larger effort to develop in vitro assays and quantitative structure-activity relationships applicable to untested chemicals on USEPA inventories through study of estrogen receptor (ER) binding and estrogen-mediated gene expression in fish. The present effort investigates metabolic activation of chemicals resulting in increased estrogenicity. Phenolphthalin (PLIN) was shown not to bind rainbow trout (Oncorhynchus mykiss) ER (rtER) in a competitive binding assay, but vitellogenin (Vtg) expression was induced in trout liver slices exposed to 10-4 and 10-3.7 M PLIN. Phenolphthalein (PLEIN), a metabolite of PLIN, was subsequently determined to be formed when slices were exposed to PLIN. It binds rtER with a relative binding affinity to 17β-estradiol of 0.020%. Slices exposed to PLEIN expressed Vtg messenger RNA (mRNA) at 10-4.3 , 10-4 , and 10-3.7 M, with no detectable PLIN present. Thus, Vtg expression noted in PLIN slice exposures was explained by metabolism to PLEIN in trout liver slices. A second model chemical, 4,4'-methylenedianiline (MDA), was not shown to bind rtER but did induce Vtg mRNA production in tissue slices at 10-4.3 , 10-4 , and 10-3.7 M in amounts nearly equal to reference estradiol induction, thus indicating metabolic activation of MDA. A series of experiments were performed to identify a potential metabolite responsible for the observed increase in activity. Potential metabolites hydroxylamine-MDA, nitroso-MDA, azo-MDA, and azoxy-MDA were not observed. However, acetylated MDA was observed and tested in both ER-binding and tissue slice Vtg induction assays. Environ Toxicol Chem 2023;42:2747-2757. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Transcriptome profiling of banana shrimp (Fenneropenaeus merguiensis) ovaries and testes: Insights into FoxL2.

The banana shrimp is found in the Pacific and Indian Oceans. Female shrimp are preferred for consumption because they are larger than males. Understanding the mechanism of sex differentiation is important for developing techniques to increase the number of female shrimp for economic benefits. This study investigates the reproductive development of F. merguiensis using transcriptome analysis. Sxl2, dsx, AGH, FEM-1, and Nrg-X2 were classified as essential genes for testes development during the juvenile stage. Several genes were required for both juvenile and adult male development. Additionally, the expression of several genes was shown to be required for juvenile and adult ovarian development, including SOP1, SOP2, Ptgr1, EST, Vgr, Vmol1, and TR-beta A. Interestingly, high levels of FoxL2 expression were observed in the testes, in contrast to previous studies in humans and other mammals. The binding of FoxL2 to the Vtg promoter was demonstrated in silico with the highest relative binding score (RS = 0.89) using the JASPAR program. Knock-down of the FoxL2 gene with dsRNA significantly suppressed FoxL2 at 2, 4, and 6 d. As a result, Vtg expression increased when compared with the control at 2, 4, and 6 d, indicating that FoxL2 plays an important role in Vtg expression in the ovary. Our findings highlight the role of FoxL2 in banana shrimp reproduction and provide valuable information on the genes associated with the F. merguiensis reproductive system.

Triazophos exposure on maternal Daphnia magna at environmental-related concentrations revealed toxic effects to its offspring

Due to chemical and photochemical stability, triazophos has been frequently detected in rivers and oceans over the years with extensive use for pest control in agriculture, and it has become a worldwide ecological concern to the aquatic environment. Until now, fewer data are available regarding the potential long-term adverse effects of triazophos on aquatic invertebrates, which plays an essential role in aquatic food webs, as a key group for water ecosystems. In this experiment, the F1- and F2 progenies of Daphnia magna were recovered when daphnias (F0) exposure to triazophos at environmental-related concentrations (0.1 and 1.0 μg/L) for 21 d; and the indexes related to phenotypic traits, reproduction and gene expression were measured in tested animals. The results showed that heart rate and total number of neonates in exposed F0-daphnias were significantly lower than those of control group, and the detoxification genes (HR96 and P-gp) were up-regulated while genes related reproduction (Vtg) and molting (Nvd and Shd) were significantly down-regulated. The heart rate and individual size of F1-daphnias (<24 h) were significantly reduced in the treatment group. After 21-d recovery, the heart rate and expression of HR96, P-gp, Vtg, Nvd and Shd were declined in F1-daphnias. There was no obvious difference of morphological traits and heart rate between treatment and control in F2-daphnias (<24 h). In summary, daphnias (F0) exposure to triazophos with environmental dose could raise toxic effects on its offspring (F1), which is mainly manifested by reduced heart rate, the accumulated number and individual size of offspring and decreased expression of genes related to molting and reproduction.

Effect of hexavalent chromium exposure on the reproductive status and biomarker responses of female Geloina erosa.

Hexavalent chromium (Cr (VI)) is widely distributed in the marine environment of Hainan Province, China and poses a potential threat to its mangrove ecosystems. However, the mechanisms underlying Cr-induced stress and reproductive toxicity in clams remain largely unknown. In this study, the clams, Geloina erosa, were exposed to 4.34, 8.69, 17.38 and 34.76 mg/L Cr (VI) for 24, 48 and 72 h. The gonad-somatic index (GSI) was determined and histological alterations of the ovaries were quantified by light microscopy. The micronucleus test was performed which quantifies the genotoxic presence of small cytoplasmic bodies in eukaryotic cells. Enzymatic assays for catalase (CAT), glutathione reductase (GR), and malondialdehyde (MDA) activities were done. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of glutathione-S-transferase (GST), heat shock protein 70 (HSP70) and vitellogenin (Vtg) in ovaries of G. erosa. The results showed that the micronucleus frequency was significantly increased when clams were exposed to Cr (VI). Cr (VI) exposure induced the accumulation of MDA and affected CAT and GR enzyme activities. The high Cr (VI) concentration of 34.76 mg/L significantly increased the levels of GR activity, GST expression and HSP70 expression and inhibited Vtg expression and CAT activity. MDA content was significantly increased after 72 h at the high Cr (VI) exposure (34.76 mg/L). Therefore, Cr (VI) exposure may be toxic to the development of ovaries of G. erosa.

Estrogenic Activity of Perfluoro Carboxylic and Sulfonic Acids in Rainbow Trout Estrogen Receptor Binding and Liver Slice Vtg mRNA Expression Assays.

Perfluoroalkylated substances (PFAS) such as carboxylic acids, and sulfonic acids were manufactured in high quantities and are ubiquitous environmental contaminants. These chemicals persist in the environment and tend to bioaccumulate. In the current study, the estrogenic potential of a series of perfluoro carboxylic acids and select perfluoro sulfonic acids were assessed in an in vitro rainbow trout estrogen receptor (rtER) binding assay and an ex vivo rtER dependent vitellogenin (Vtg) expression rainbow trout liver slice assay. Perfluoro carboxylic acids with perfluoroalkyl chain lengths of four to six did not significantly bind to the rtER or induce Vtg expression in liver slices. Perfluoro carboxylic acids with chain lengths of seven to ten, and sulfonic acids with seven and eight carbon chains bound to the rtER, but with low relative binding affinities. While affinity for the rtER increased with increasing chain length the highest affinity measured was only 0.0025% relative to the endogenous hormone 17ß-estradiol at 100%. Both the eight-carbon carboxylic acid and eight-carbon sulfonic acid induced Vtg expression in ex vivo liver slices. However, toxicity did not allow expression to achieve maximum efficacy relative to estradiol.

Studying paraben-induced estrogen receptor- and steroid hormone-related endocrine disruption effects via multi-level approaches

Increasing concerns have been raised on the health risks of parabens in the regard of their widespread applications and potential endocrine disrupting activities. In this study, four typical parabens, including methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), and butyl paraben (BuP) were systematically investigated for their estrogen receptor- and steroid hormone-related endocrine disruptions using multi-level approaches. Paraben exposure promoted the proliferation of MCF-7 cells, increased the luciferase activity in MVLN cells, and induced the vitellogenin (vtg) expression in zebrafish larvae, showing the typical estrogenic effects. The in vitro protein assays further revealed that PrP and BuP could bind with two isoforms of estrogen receptors (ERs). The estrogenic activities of parabens were predicted to be positively correlated with their chemical structure complexity by using molecular docking analysis. Furthermore, the synthesis and secretion of estradiol (E2) and testosterone (T) were significantly disturbed in H295R cells and zebrafish larvae, which could be regulated by paraben-induced transcriptional disturbance in both in vitro steroidogenesis and in vivo hypothalamic-pituitary-gonadal (HPG) axis. Parabens could disturb the endocrine system by activating the ERs and disrupting the steroid hormone synthesis and secretion, suggesting their potential deleterious risks to the environment and human health.

Evaluating the alterations of the estrogen-responsive genes in Cyprinodon variegatus larvae for biomonitoring the impacts of estrogenic endocrine disruptors (EEDs)

Currently, endocrine disruptors (EDs) can be found in all the environmental compartments. To understand the effects of estrogenic EDs (EEDs), adults of Cyprinodon variegatus have been classically used as a marine model. However, it is during development that exposure to contaminants may generate permanent consequences. Thus, the aim of this study was to verify the effects produced by acute exposure to 17α-ethinylestradiol (EE2) in C. variegatus larvae. Quantitative PCR (qPCR) results revealed the induction of vtg and zp gene expression on exposure to 1000 ng/L EE2 and the induction of vtgc, zp2, zp3 and cyp19a2, and inhibition of vtgab, wap and cyp1a1 on exposure to 100 ng/L EE2. Lower concentrations inhibited the gene expression of vtgab and wap (50 ng/L), cyp1a1 (25 ng/L) and zp2 (12.5 ng/L). These alterations in gene expression allow us to affirm that larvae of C. variegatus are an efficient and sensitive model for biomonitoring EEDs.

Effect of dietary cholesterol on ovarian development of Chinese mitten crabs (Eriocheir sinensis)

This study was conducted to evaluate the effect of cholesterol (CHO) on ovarian development of Eriocheir sinensis through a feeding trial. Crabs (average weight 43.35 ± 0.05 g) were randomly fed with one of three diets: 0% CHO, 0.4% CHO and 1.6% CHO for 16 weeks. Results indicated that the hepatosomatic index (HSI) and gonad index (GSI) of crabs fed with 0.4% CHO diet were significantly higher than those in the control group. (P &amp;lt; 0.05). Meanwhile, 0.4% CHO can significantly increase the gene expression of vtg and vgr in ovary (P &amp;lt; 0.05), thus promoting the accumulation of Vtg. The protein expression of MEK1/2, ERK, p-ERK1/2, Cyclin B and CDC2 were significantly increased in the 0.4% CHO group, therefore dietary CHO might promote oocyte maturation by activating MAPK signal pathway and cooperating with maturation promoting factor (MPF). Further exploration results showed that 0.4% CHO was able to significantly up-regulate the protein expression of STAR and SR-B1 to promote the transport of CHO to ovary, thereby providing sufficient substrates for estradiol (E2) synthesis. In addition, the results in vivo and in vitro shown that CHO could regulate the activities of enzymes such as CYP11A1 and CYP19A1 through the cAMP-PKA-CREB/SF-1 signal pathway and then affects the level of E2 in the organism. In summary, supplementing the appropriate amount of CHO in the diet can improve the E2 level of the organism, thus increasing the accumulation of nutrients in the ovary, promoting the completion of meiosis, and finally achieving the purpose of promoting ovarian maturation.

Bisphenol A alters sexual dimorphism and gene expression in marine medaka Oryzias melastigma.

Bisphenol A (BPA) is an endocrine disruptor that is present in freshwater and marine environments. However, conclusive evidence for the toxicity of chronic BPA exposure to marine fishes remains lacking. Therefore, we investigated the influence of BPA on male marine medaka (Oryzias melastigma). BPA exposure induced formation of testis-ova at 2610µg/L, and male-type anal fins became more female type in a concentration-dependent manner. Some males with female-type anal fins had normal testes, indicating that anal fin shape is more sensitive to BPA. Gonadal soma-derived factor (gsdf) expression decreased after BPA exposure in the 746 and 2610µg/L exposure groups, although the changes were not statistically significant. Additionally, liver vitellogenin (vtg) expression increased in a dose-dependent manner and was significantly higher in all exposure groups. vtg and gsdf are likely to be useful biomarkers for the impact of estrogenic endocrine disrupters in O. melastigma.

Chronic effects of nano and microplastics on reproduction and development of marine copepod Tigriopus japonicus

This study aimed to examine the impact of chronic (30 days) exposure to polystyrene microplastics (PS-MPs) of different sizes (50 nm and 2 µm) and at different concentrations (0.5 μg/L and 100 mg/L) to marine copepod Tigriopus japonicus. Polystyrene microplastics affected survival rates in size- and concentration-dependent manners. The LC50s values of 50 nm and 2 µm PS-MPs were 0.10 mg/L and 3.92 mg/L, respectively. The developmental time was delayed by 50 nm PS-MPs, and Usp expression was downregulated. Reproduction was negatively affected by 2 µm PS-MPs even at environmentally relevant concentrations; however, the expression of Vtg was not altered. The production rates of reactive oxygen species and nitric oxide also increased after exposure to PS-MPs; but this effect was independent of particle size. The expression levels of Cat and Tnf, genes related to oxidative stress and inflammation, respectively, were upregulated by exposure to PS-MPs, independently of particle size. Meanwhile, the level of oxidative stress in T. japonicus was not significantly affected by PS-MPs at environmentally relevant concentrations. This study suggests that nano-sized PS-MPs are not always more toxic than micro-sized PS-MPs, and that oxidative stress is a key factor in determining the toxic effect on T. japonicus at high concentrations.

Distinct Roles of Estrogen Receptors in the Regulation of Vitellogenin Expression in Orange-Spotted Grouper (Epinephelus coioides).

During their breeding season, estrogen induces vitellogenin (VTG) production in the liver of teleost fish through estrogen receptors (ERs) that support oocyte vitellogenesis. There are at least three ER subtypes in teleost fish, but their roles in mediating E2-induced VTG expression have yet to be ascertained. In this study, we investigated the expression of vtgs and ers in the liver of orange-spotted grouper (Epinephelus coioides). Their expression levels were significantly increased in the breeding season and were upregulated by an estradiol (E2) injection in female fish, except for the expression of erβ1. The upregulation of vtgs, erα and erβ2 by E2 was also observed in primary hepatocytes, but these stimulatory effects could be abolished by ER antagonist ICI182780 treatment. Subsequent studies showed that ERβ antagonist Cyclofenil downregulated the E2-induced expression of vtg, erα, and erβ2, while the ERβ agonist DPN simulated their expression. Knockdown of erβ2 by siRNA further confirmed that ERβ2 mediated the E2-induced expression of vtgs and erα. To reveal the mechanism of ERβ2 in the regulation of erα expression, the erα promoter was cloned, and its activity was examined in cells. E2 treatment simulated the activity of the erα promoter in the presence of ERβ2. Deletions and site-directed mutations showed that the E2 up-regulated transcriptional activity of erα occurs through a classical half-estrogen response element- (ERE) dependent pathway. This study reveals the roles of ER subtypes in VTG expression in orange-spotted grouper and provides a possible explanation for the rapid and efficient VTG production in this species during the breeding season.

Regulation of vtg and VtgR in mud crab Scylla paramamosain by miR-34.

Vitellogenin (Vtg) is the precursor of major yolk protein and plays a crucial role in the maturation of oocytes and the production of eggs in oviparous animals. Vitellogenin receptor (VtgR) mediates the transport of Vtg explicitly to oocytes in the membrane. In a previous study, we found that miR-34 can regulate the expression of some eyestalk genes and affect reproduction in mud crab Scylla paramamosain, one of the most important economic crabs on the coasts of southern China. In this study, firstly, we found that miR-34 can target at 3'-UTR of Vtg and VtgR genes by using bioinformatic tools and predicted miR-34 might depress the expression of Vtg and VtgR. Secondly, the relative luciferase activity of HEK293T cells co-transfected with miRNA mimic and pmir-RB-REPORTTM-Vtg/VtgR-3'UTR was significantly lower than those of cells co-transfected with mimic NC and pmir-RB-REPORTTM-Vtg/VtgR-3'UTR. Finally, in vivo experiments showed that agomiR-34 could repress the expression of Vtg and VtgR genes, while Antigomir-34 could promote the expression of these two genes. These results confirm our hypothesis and previous published results that miR-34 may indirectly regulate ovarian development by binding to the 3'-UTR of Vtg and VtgR genes and inhibiting their expression.

Preself-Feeding Medaka Fry Provides a Suitable Screening System for in Vivo Assessment of Thyroid Hormone-Disrupting Potential.

Endocrine-disrupting chemicals are assessed based on their physiological potential and their potential associated adverse effects. However, suitable end points for detection of chemicals that interfere with the thyroid hormone (TH) system have not been established in nonmammals, with the exception of amphibian metamorphosis. The aims of the current study were to develop an in vivo screening system using preself-feeding medaka fry (Oryzias latipes) for the detection of TH-disrupting chemicals and elucidate the underlying molecular mechanism. 17α-Ethinylestradiol (EE2: <100 ng/L) did not induce mRNA expression of estrogen-responsive genes, vitellogenins (vtgs) mRNA. Meanwhile, coexposure with thyroxin (T4) induced an increase of vtg expression. TH-disrupting chemicals (thiourea (TU), perfluorooctanoic acid (PFOA), and tetrabromobisphenol A (TBBPA)) significantly suppressed EE2 (1,000 ng/L)-induced vtg1 expression, while T4 rescued their expression as well as that of thyroid hormone receptor α (tRα) and estrogen receptors (esrs). These results were supported by in silico analysis of the 5'-transcriptional regulatory region of these genes. Furthermore, the esr1 null mutant revealed that EE2-induced vtg1 expression requires mainly esr2a and esr2b in a TH-dependent manner in preself-feeding fry. Application of preself-feeding medaka fry as a screening system might help decipher the in vivo mechanisms of action of TH-disrupting molecules, while providing an alternative to the traditional animal model.

Gene expression changes in Daphnia magna following waterborne exposure to cyanobacterial strains from the genus Nostoc

Cyanobacteria can produce highly potent cyanotoxins, however, limited information is provided about their toxicity mechanisms in exposed aquatic invertebrates at the molecular level. In the present study, the effects of cyanobacterial strains from the genus Nostoc (Nostoc Z1 and Nostoc 2S3B) in Daphnia magna after waterborne exposure were investigated. Examined endpoints included immobilization (survival) in acute toxicity tests and selected gene expression changes (cyp314, cyp360A8, gst, p-gp, vtg) analyzed by the quantitative real-time polymerase chain reaction (RT-PCR). In addition, enzyme-linked immunosorbent assay (ELISA) was performed to determine whether the observed changes could be due to the presence of microcystins, the most widespread group of cyanotoxins. The results of acute toxicity tests have shown only minor changes in survival rates, which have not exceeded 20% after 48 h of exposure to either strain. On the other hand, significant changes were recorded in molecular responses of Daphnia to tested strains. Treatment with the aquatic strain Nostoc Z1 altered the expression levels of all analyzed genes. Both strains caused a significant p-glycoprotein (p-gp) induction at 75 µg ml−1 which suggests the involvement of p-gp mediated multixenobiotic resistance mechanism (MXR) in facilitating excretion of toxic cyanobacterial compounds in daphnids. Additionally, these strains caused an increase in the expression levels of cyp360A8, indicating that genes related to detoxification processes could be sensitive indicators of cyanobacterial toxicity. Statistically significant induction of cyp314, as well as increases in expression of gst and vtg, were observed only after exposure to Nostoc Z1. This study indicates the potential of certain cyanobacterial metabolites to modify the expression of toxicant responsive genes involved in phase I and phase III of the xenobiotic metabolism, as well as possible interference with growth and reproduction in D. magna. Low microcystin concentrations found in both samples suggest that these cyanotoxins were not responsible for the detected toxic effects.

Comparative Transcriptome Reveals the Potential Modulation Mechanisms of Spfoxl-2 Affecting Ovarian Development of Scylla paramamosain.

Previously, we reported the identification, tissue distribution and confirmed the roles of Spfoxl-2 in regulating vitellogenin (vtg) expression in Scylla paramamosain. Here, we primally analyzed its potential target genes in the ovary with RNAi and RNA-Seq technology. By comparing the transcriptome data of two groups (ovaries that injected with EGFP and Foxl-2 siRNA, respectively), we found 645 DEGs (differentially expressed genes), including several conserved crucial genes involved in ovarian development, such as vtg, vitellogenin receptor (vtgR), adenylate cyclase (AC), cyclinB, and cell division cycle 2 (cdc2). In addition, these DEGs were also enriched in pathways related to ovary development, including relaxin signaling pathway, ovarian steroidogenesis, and progesterone-mediated oocyte maturation. Moreover, several genes were selected for qRT-PCR to validate the accuracy of the bioinformatic result. To the best of our knowledge, the current study was the first report about foxl-2 function through comparative transcriptome analysis in crustacean species, which identified not only relevant genes and pathways involved in ovarian development of S. paramamosain, but also provided new insights into the regulatory mechanisms of foxl-2 at the molecular level in crustacean.

Effects of polyvinyl chloride microplastics on reproduction, oxidative stress and reproduction and detoxification-related genes in Daphnia magna

The drastic increase of microplastics (MPs) in aquatic environment has become a serious threat to marine and freshwater ecosystems. However, little information is available regarding the potential detrimental effects of polyvinyl chloride microplastics (PVC-MPs) on aquatic organisms. This study investigated the changes of reproduction parameters, oxidative stress and the expression of reproduction and detoxification-related genes in Daphnia magna after exposed to 2 ± 1 and 50 ± 10 μm PVC-MPs. The results showed that chronic exposure to 2 ± 1 μm PVC-MPs prolonged days to the first brood, increased total number of broods per female and frequency of molting per adult, decreased offspring number at first brood and total number of offspring per female in D. magna. Moreover, 2 ± 1 μm PVC-MPs also disturbed the activities of SOD and CAT, increased GSH and MDA levels. The expression of Vtg, SOD, CAT, CYP314 and CYP360A8 genes also exhibited different response patterns depending on exposure time. Furthermore, 50 ± 10 μm PVC-MPs decreased offspring at first brood and Vtg mRNA level, increased the transcription levels and activities of SOD and CAT. These results suggest that the presence of PVC-MPs in aquatic environment may cause reproduction toxicity by disrupting the reproduction and detoxification-related genes expression and inducing oxidative stress in D. magna.