Vtg Levels Research Articles

Psoralen and Isopsoralen, Two Estrogen-Like Natural Products from Psoraleae Fructus, Induced Cholestasis via Activation of ERK1/2.

With the increasing use of oral contraceptives and estrogen replacement therapy, the incidence of estrogen-induced cholestasis (EC) has tended to rise. Psoralen (P) and isopsoralen (IP) are the major bioactive components in Psoraleae Fructus, and their estrogen-like activities have already been recognized. Recent studies have also reported that ERK1/2 plays a critical role in EC in mice. This study aimed to investigate whether P and IP induce EC and reveal specific mechanisms. It was found that P and IP increased the expression of esr1, cyp19a1b and the levels of E2 and VTG at 80 μM in zebrafish larvae. Exemestane (Exe), an aromatase antagonist, blocked estrogen-like activities of P and IP. At the same time, P and IP induced cholestatic hepatotoxicity in zebrafish larvae with increasing liver fluorescence areas and bile flow inhibition rates. Further mechanistic analysis revealed that P and IP significantly decreased the expression of bile acids (BAs) synthesis genes cyp7a1 and cyp8b1, BAs transport genes abcb11b and slc10a1, and BAs receptor genes nr1h4 and nr0b2a. In addition, P and IP caused EC by increasing the level of phosphorylation of ERK1/2. The ERK1/2 antagonists GDC0994 and Exe both showed significant rescue effects in terms of cholestatic liver injury. In conclusion, we comprehensively studied the specific mechanisms of P- and IP-induced EC and speculated that ERK1/2 may represent an important therapeutic target for EC induced by phytoestrogens.

Gonad pathology, sex hormone modulation and vitellogenin expression in Chrysichthys nigrodigitatus from Lagos and Epe lagoons within the southern-lagoon system, Nigeria.

Introduction: Estrogenic chemicals in aquatic environments impact fish reproductive health, with vitellogenin protein levels serving as a crucial biomarker for xenoestrogen exposure. Limited knowledge exists on estrogenic effects in tropical environments, prompting an investigation into the influence of environmental estrogens on Chrysichthys nigrodigitatus in Lagos and Epe lagoons. Methods: A total of 195 fish samples underwent analysis for vitellogenin protein, sex hormones (testosterone and 17 β-estradiol), and gonad pathology in effluent-receiving areas of the specified lagoons. Results: Gonadal alterations were observed in male and female fish, including empty seminiferous tubules and distorted ovaries. Intersex occurred in 3.81% of Lagos and 3.33% of Epe. Testosterone levels were generally higher in females and males from both lagoons, while E2 levels were higher in females from both lagoons, with Lagos showing higher levels than Epe. Vtg levels were higher in males than females in Lagos samples but showed no significant difference in Epe samples. Discussion: Contaminant analysis revealed similar trends in metals (Hg, As, Cr) and phthalates (DEHP, DBP, DEP) in both sexes in the Epe population. Multivariate depictions from the PCA showed sex-specific patterns of metal uptake (Cd) in male fishes at the Lagos Lagoon. The positive association between higher pH loadings and metal and DBP levels in sediment at the Lagos lagoon suggests the influence of higher alkalinity in lower bioavailability of contaminants. Conclusion: Endocrine disrupting effects were observed in male and female Chrysichthys nigrodigitatus in Lagos and Epe lagoons populations, with notable differences in hormone and contaminant concentrations between the two lagoon systems. Identification of specific contaminants and their spatial and temporal trends can inform targeted management and remediation efforts to protect and restore these valuable aquatic ecosystems.

The Role of Cholesterol during the Ovarian Maturation and Lipid Metabolism of Female Chinese Mitten Crab (Eriocheir sinensis)

In previous study, we found that the cholesterol requirement of Eriocheir sinensis was 0.27%, to further investigate the effects of cholesterol on health status, ovarian maturation, and lipid metabolism of female Eriocheir sinensis broodstock. Two diets containing 0% and 0.25% (actually 0.05% and 0.27%) cholesterol were fed to the female crabs (average weight: 49.21 ± 0.11 g) for 4 months and sampled once a month. The results showed that the body weight (BW), survival rate (SR), meat yield (MY), condition factor (CF), hepatosomatic index (HSI), and gonadosomatic index (GSI) of Eriocheir sinensis were significantly affected by treatment time and compared with the cholesterol deficient group, supplementing cholesterol significantly increased BW, HSI, and GSI (P<0.05). In addition, long-term lack of cholesterol will lead to a significant decrease in the activity of ACP, AKP, and SOD and a significant increase in the content of MDA. The histological results showed that cholesterol significantly increased the volume of oocytes (P<0.05). Further studies found that 0.27% cholesterol significantly increased the transcription levels of vtg and vgr in hepatopancreas and ovaries, which may be the main reason for the increase of oocyte size (P<0.05). When fed with 0.27% cholesterol diet, the contents of nutrients in hepatopancreas and ovaries increased significantly, especially lipids and cholesterol (P<0.05). Through the analysis of mRNA expression level of genes related to lipid metabolism, it was found that cholesterol enhanced the transcription level of genes related to lipid synthesis and transport in hepatopancreas, thereby promoting the accumulation of lipid in the organism. Furthermore, compared with control group, the levels of juvenile hormone (JH), 17β-estradiol (E2), methyl farnesoate (MF), and ecdysone in the organism were significantly increased after feeding a diet with 0.27% cholesterol (P<0.05). In summary, supplementing an appropriate amount of cholesterol in the diet can improve the growth performance of Eriocheir sinensis broodstock, enhance the body’s antioxidant and immune system, and promote the accumulation of nutrients in the ovaries, thereby promoting ovarian maturation.

Sequence identification and expression characterization of leptin in the spotted scat, Scatophagus argus.

Scatophagus argus is an important marine culture fish in South and South-East Asia, including Southeast coastal areas of China. Artificial propagation technology for S. argus is not optimum; thus further studies on its reproduction biology are required. Although previous studies have shown that leptin (Lep) can regulate fish reproduction, the role of lep genes in S. argus is unknown. Herein, in silico analysis showed that S. argus has two lep genes (lepa and lepb). Protein 3D-structure prediction showed that Lepa has four α-helices (similar to mammals), while Lepb only has three. Tissue distribution analysis showed that lepa is highly expressed in the liver, whereas lepb was not detected in any tissue. Notably, lepr was expressed in all tissues. Lepa mRNA expression levels in the liver and serum Lep, estradiol (E2) and vitellogenin (Vtg) levels of female fish were significantly higher in ovaries at stage IV than in ovaries at stage II. Serum E2 levels were significantly positively correlated with Vtg levels in female fish at different development stages, while serum E2 was not correlated with Lep levels. Consistently, in vitro incubation of the liver with E2 significantly up-regulated vtga, while it did not affect lepa expression. Recombinant Lep (10nM) significantly up-regulated chicken gonadotropin-releasing hormone (cGnRH/GnRH-II) in the hypothalamus and GnRH receptor (GnRHR) and luteinizing hormone beta (Lhb) in the pituitary. These results suggest that lepa regulates female reproduction in S. argus.

Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate.

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study. Exposures targeting 0.1, 0.6, 3.2, 20, and 100 µg/L in parental (P) and first filial (F1) generations lasted for 180 days post fertilization (dpf) and the second filial generation (F2) through 16 dpf. Survival decreased significantly in P and F2 generation exposures, but not in F1, at the highest PFOS treatment (100 µg/L nominal, 94-205 µg/L, measured). Significant adverse effects on body weight and length were infrequent, of low magnitude, and occurred predominantly at the highest exposure treatment. Finally, PFOS had no significant effects on P or F1 egg production and survival or whole-body VTG levels in P or F1 male fish. Overall, the predominance and magnitude of adverse PFOS effects at <1 µg/L reported in prior research were largely nonrepeatable in the present study. In contrast, the present study indicated a threshold for ecologically relevant adverse effects in zebrafish at 117 µg/L (SE 8 µg/L, n = 10) for survival and 47 µg/L (SE 11 µg/L, n = 19) for all statistically significant negative effects observed. Environ Toxicol Chem 2024;43:115-131. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Activation or inhibition of NF-κB from the juvenile stage results in skewed sex ratios in common carp (Cyprinus carpio L.)

Due to sexual dimorphism in growth traits, all-female populations and populations with a high proportion of females hold significant economic value in common carp (Cyprinus carpio L.). However, the underlying mechanisms governing gonadal differentiation remain inadequately understood. NF-κB, as a crucial regulator of anti-apoptotic processes, plays a significant role in mammalian gonad formation and zebrafish oocyte maintenance. To investigate the potential roles of the NF-κB pathway in the gonad differentiation of common carp, juveniles with undifferentiated gonads were treated with an activator (DOC) or inhibitor (NAI) of NF-κB. Our findings revealed that juveniles exposed to DOC exhibited a significantly increased female-to-male ratio (from 0.97:1 to 4.30:1) and substantial increases in weight and body length. Conversely, inhibiting NF-κB with NAI resulted in a decreased female-to-male ratio (from 1.06:1 to 0.33:1). Furthermore, NAI also induced the occurrence of ovotestis. Additional in vivo and ex vivo experiments demonstrated that DOC activation of the NF-κB pathways led to inhibition of apoptosis, significant increases in serum 17β-estradiol (E2) levels and the E2/T ratio, and upregulation of the female-biased genes foxl2 and cyp19a1a. Simultaneously, it significantly suppressed serum 11-ketotestosterone (11-KT) levels and the expression of male-biased genes amh, dmrt1, and gsdf and induced VTG levels in the hepatopancreas of male carp. However, NAI exhibited contrasting effects. Both DOC and NAI disrupted the balance of the hypothalamus-pituitary-gonad (HPG) reproductive axis at multiple potential loci, including changes in the expression levels of steroid hormone synthesis-related genes in gonads. Transcriptomic analysis revealed comprehensive molecular changes in undifferentiated gonads during DOC-induced feminization and NAI-induced masculinization. The transcriptome results were consistent with the observed alterations in hormone content and the expression of genes involved in sex hormone synthesis, inflammation, and apoptosis following exposure to DOC or NAI. These findings suggest the involvement of NF-κB in the sex differentiation of common carp by regulating apoptosis and disrupting the HPG reproductive axis. Our study also explores a DOC-induced method of producing high ratios of females and discovers the underlying mechanism of NF-κB's contribution to sex differentiation in common carp.

Integrated transcriptomic and metabolomic analyses identify key factors in the vitellogenesis of juvenile Sichuan bream (Sinibrama taeniatus)

Vitellogenesis is the most important stage of ovarian maturation in fish, involving the synthesis and transport of essential yolk substances and their complex mechanisms and coordination process. The liver is the main tissue involved in the vitellogenesis of oviparous animals, but studies of vitellogenesis in fish rarely include the liver and ovary as a whole. The aim of this study was to explore the molecular mechanism and associated regulatory factors behind vitellogenesis in Sichuan bream (Sinibrama taeniatus). The different stages of oogenesis were first identified by successive histological observations. Then, ovary and liver tissues that developed to 115 days (stage II, previtellogenesis stage), 165 days (stage III, vitellogenesis stage) and 185 days (stage IV, late-vitellogenesis stage) were collected for transcriptomic and metabolomic analyses, and serum testosterone (T), 17β-estradiol (E2), vitellogenin (Vtg), triiodothyronine (T3), and thyroxine (T4) levels were measured at the corresponding stages. We found that energy redistribution during vitellogenesis is mainly regulated through glycolysis, fatty acid biosynthesis and the citrate cycle pathway. In the liver, energy metabolism was promoted by activating glucolipid metabolic pathways to provide sufficient ATP, but at the same time, the ovary tends to retain nutrients rather than decompose them to produce energy. In addition, we have identified several key factors involved in the metabolism of neutral lipids, polar lipids, amino acids and vitamins, which are involved in the assembly and transport of important yolk nutrients. The initiation of vitellogenesis was found to be associated with a surge in serum E2 levels, but the sustained increase in Vtg levels in the late stage may be due more to upregulation of the estrogen receptor. These results provide valuable information about the regulation of ovarian development in cultured fish.

Dietary arachidonic acid supplementation promotes the growth, steroidogenesis and ovarian development in mud crab Scylla paramamosain

A 22-week feeding experiment was executed to assess the impact of dietary ARA supplementation on the growth, tissue proximate composition, hemolymph biochemical, tissue fatty acids profile, ARA metabolite and sex steroid hormone, and the mRNA expression of ovarian development related gene of female mud crab in the pre-devolopmental stage. Crabs were fed with six diets containing different ARA levels (0.04%, 0.25%, 0.46%, 0.89%, 1.25%, and 1.57%, dry matter). The results showed that the increasing dietary ARA levels had significant effects on survival, percent weight gain (PWG), specific growth rate (SGR), and feed efficiency (FE), which showed an increasing trend initially and then showed a decreasing trend. According to the two-slope broken-line model and quadratic curve model for the relationship between PWG and dietary ARA levels, optimal dietary ARA levels were estimated to be 0.46% and 0.70%. The lipid contents in muscle and hepatopancreas decreased significantly with the increasing dietary ARA levels. Similarly, total cholesterol (T-CHO) and triglyceride (TG) concentrations were significantly decreased with the increasing dietary ARA levels. In the hepatopancreas and muscle, ARA contents significantly increased with the increasing dietary ARA levels. In hemolymph and hepatopancreas, progesterone (Prog), pregnenolone (Preg), estradiol (E2) and vitellogen (Vtg) concentrations presented earlier increase and later decrease trend. In the hepatopancreas, prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE) concentrations showed an upward trend. In the muscle, PGE2 concentration presented earlier increasing and later decreasing trend. In the ovary, the increasing dietary ARA levels produced similar effects on the expressions of genes, including steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (3βhsd), vitellogenin (vtg) and vitellogenin receptor (vtgr). Thus, moderate dietary ARA levels promoted growth, improved ARA deposition and metabolism, increased sex steroid hormone and Vtg levels, and enhanced ovarian development of female mud crab in the pre-developmental stage.

A case study: temporal trends of environmental stressors and reproductive health of smallmouth bass (Micropterus dolomieu) from a site in the Potomac River Watershed, Maryland, USA

Decades of poor reproductive success and young-of-the-year survival, combined with adult mortality events, have led to a decline in the smallmouth bass (SMB; Micropterus dolomieu) population in sections of the Potomac River. Previous studies have identified numerous biologic and environmental stressors associated with negative effects on SMB health. To better understand the impact of these stressors, this study was conducted at the confluence of Antietam Creek and the Potomac River from 2013 to 2019 to identify temporal changes associated with SMB reproductive health. Surface water samples were collected and analyzed for over 300 organic contaminants, including pesticides, phytoestrogens, pharmaceuticals, hormones and total estrogenicity (E2Eq). Adult SMB were collected and sampled for multiple endpoints, including gene transcripts associated with reproduction (molecular), histopathology (cellular), and organosomatic indices (tissue). In males, biomarkers of estrogenic endocrine disruption, including testicular oocytes (TO) and plasma vitellogenin (Vtg) were assessed. Numerous agriculture-related contaminants or land use patterns were associated with gene transcript abundance in both male and female SMB. Positive associations between pesticides in the immediate catchment with TO severity and E2Eq with plasma Vtg in males were identified. In males, the prevalence of TO and detectable levels of plasma Vtg, liver vitellogenin transcripts (vtg) and testis vtg were high throughout the study. Peaks of complex mixtures of numerous contaminants occurred during the spring/early summer when spawning and early development occurs and to a lesser extent in fall/winter during recrudescence. Management practices to reduce exposure during these critical and sensitive periods may enhance reproductive health of these economically important sportfishes.

Persistent impairment of gonadal development in rare minnow (Gobiocypris rarus) after chronic exposure to chlorinated polyfluorinated ether sulfonate

The delayed and persistent adverse effects caused by developmental exposure to per- and poly-fluorinated substances are of significant concern. Juvenile rare minnows (Gobiocypris rarus), were exposed to chlorinated polyfluoroalkyl ether sulfonate (Cl-PFESA) at measured medium concentrations of 86.5 μg/L, 162 μg/L and 329 μg/L, for 4 weeks followed by 12 weeks of depuration. After 4 weeks of exposure, the body weight and length of the juvenile fish were increased compared to controls. Gene expression of gnrh3, lhβ, and cyp19a was decreased, and ar and erα were upregulated. Transcriptomic analysis revealed enrichment of multiple pathways related to gonadal development. After 12 weeks of depuration, the gonadosomatic indices were decreased in female fish in a concentration-dependent manner, with a significant decrease to 59% of control in 329 μg/L group. Histological analysis found increasing numbers of degenerating oocytes and perinucleolar oocytes, and decreasing numbers of mature vitellogenic oocytes in female fish treated by Cl-PFESA. Enlarged interstitial space of the testis was observed in the exposed male fish. Gene expression levels of gnrh3, lhβ, ar, erα, and vtg were upregulated in the adult fish. Chronic developmental exposure to Cl-PFESA caused persistent effects on gonadal development of fish, highlighting the necessity of a comprehensive ecological risk assessment.

Transgenerational effects of co-exposure to cadmium and carbofuran on zebrafish based on biochemical and transcriptomic analyses

The combined toxicity of heavy metals and pesticides to aquatic organisms is still largely unexplored. In this study, we investigated the combined impacts of cadmium (Cd) and carbofuran (CAR) on female zebrafish (F0 generation) and their following F1 generation. Results showed that mixtures of Cd and CAR induced acute synergistic effects on both zebrafish adults of the F0 generation and embryos of the F1 generation. Combined exposure to Cd and CAR could obviously alter the hepatic VTG level of females, and the individual exposures increased the relative mRNA levels of vtg1 and vtg2. Through maternal transmission, co-exposure of Cd and CAR caused toxicity to 4-day-old larvae of the F1 generation, evidenced by the significant changes in T4 and VTG levels, CYP450 activity, and the relative transcriptional levels of genes related to the hormone, oxidative stress, and apoptosis. These effects were also reflected by the global gene expression pattern to 7-day-old larvae of F1 generation using the transcriptomic analysis, and they could also affect energy metabolism. Our results provided a more comprehensive insight into the transgenerational toxic impacts of heavy metal and pesticide mixtures. These findings highlighted that it was highly necessary to consider transgenerational exposures in the ecological risk assessment of chemical mixtures.

Vaccination of Gilthead Seabream After Continuous Xenoestrogen Oral Exposure Enhances the Gut Endobolome and Immune Status via GPER1.

In fish culture settings, the exogenous input of steroids is a matter of concern. Recently, we unveiled that in the gilthead seabream (Sparus aurata), the G protein-coupled estrogen receptor agonist G-1 (G1) and the endocrine disruptor 17α-ethinylestradiol (EE2) are potent modulators in polyreactive antibody production. However, the integral role of the microbiota upon immunity and antibody processing in response to the effect of EE2 remains largely unexplored. Here, juvenile seabreams continuously exposed for 84 days to oral G1 or EE2 mixed in the fish food were intraperitoneally (i.p.) immune primed on day 42 with the model antigen keyhole limpet hemocyanin (KLH). A critical panel of systemic and mucosal immune markers, serum VTG, and humoral, enzymatic, and bacteriolytic activities were recorded and correlated with gut bacterial metagenomic analysis 1 day post-priming (dpp). Besides, at 15 dpp, animals received a boost to investigate the possible generation of specific anti-KLH antibodies at the systemic and mucosal interphases by the end of the trial. On day 43, EE2 but not G1 induced a significant shift in the serum VTG level of naive fish. Simultaneously, significant changes in some immune enzymatic activities in the serum and gut mucus of the EE2-treated group were recorded. In comparison, the vaccine priming immunization resulted in an attenuated profile of most enzymatic activities in the same group. The gut genes qPCR analysis exhibited a related pattern, only emphasized by a significant shift in the EE2 group’s il1b expression. The gut bacterial microbiome status underwent 16S rRNA dynamic changes in alpha diversity indices, only with the exposure to oral G1, supporting functional alterations on cellular processes, signaling, and lipid metabolism in the microbiota. By the same token, the immunization elevated the relative abundance of Fusobacteria only in the control group, while this phylum was depleted in both the treated groups. Remarkably, the immunization also promoted changes in the bacterial class Betaproteobacteria and the estrogen-associated genus Novosphingobium. Furthermore, systemic and mucosal KLH-specific immunoglobulin (Ig)M and IgT levels in the fully vaccinated fish showed only slight changes 84 days post-estrogenic oral administration. In summary, our results highlight the intrinsic relationship among estrogens, their associated receptors, and immunization in the ubiquitous fish immune regulation and the subtle but significant crosstalk with the gut endobolome.

The effects of PBB153 on the levels of E2, T and Vtg in zebrafish

Polybrominated biphenyls (PBBs) are a class of industrial chemicals with the characteristics of persistent organic pollutant, which can cause pathological effects on the endocrine system such as the thyroid and adrenal glands. At the present study, the experiment was designed to obtain the effects of PBB153 on the levels of E2, T and Vtg in zebrafish. The zebrafish were exposed to increasing concentrations of PBB153 solution (0, 0.1 mg/L, 0.2 mg/L, 0.5 mg/L) for 21 days. The results showed that PBB153 had a certain inhibitory effect on the secretion of E2, T and Vtg in zebrafish. However, there are relatively few studies on the harm of PBBs and others environmental chemical pollutants to the endocrine system of aquatic organisms, and further research is needed.

Vitellogenesis in the Patagonian toothfish (Dissostichus eleginoides) conditioned to a recirculating aquaculture system

The Patagonian toothfish (Dissostichus eleginoides) is a new promising fish species for diversifying the aquaculture industry in Chile because of its high economic value and high international demand. However, when attempting to start aquaculture of a new species, one of the major challenges is successfully achieving conditions to reproduce them. This is particularly difficult when the information on the biology and physiology of the reproduction process of the species in question is scarce, as is the case with D. eleginoides. Additionally, female reproductive dysfunction is more prevalent under culture conditions and it is very important to have tools to evaluate the progress of oocyte maturation. Therefore, evaluation of the vitellogenesis process in addition to measuring gonadosomatic index (GSI) and oocyte diameter is an important parameter for allowing the monitoring of females from a broodstock that will spawn in the reproductive season.This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) specific for the Patagonian toothfish (D. eleginoides) vitellogenine (Vtg) and quantify the plasma level in the fishes, maintained in a recirculation aquaculture system (RAS), throughout their reproductive cycle.A polyclonal antibody was prepared using the isolated major egg protein as antigen. This antibody was specific to the major plasma phosphoprotein identified as Vtg and was used to develop and standardize an indirect ELISA assay. The assay standard curve was linear from 0.1 to 1 µg/ml purified egg yolk protein and the average r2 was 0.995. We corroborated our ELISA assay by demonstrating a strong correlation between high levels of plasma Vtg obtained by the assay and the intensity of the corresponding bands in both SDS-PAGE coomassie stained gels and Western Blot.During the two reproductive seasons analyzed, the highest Vtg plasma level was obtained in the majority of the females in the last three months before spawning (December-January). This differs from the wild population in which the spawning occurs during the austral winter (June-September). Therefore, the RAS condition established to maintain in captivity the D. eleginoides allows females to develop mature oocytes normally, as was evidenced by picks of Vtg plasma levels.

Sex steroids and steroidogenesis-related genes in the sea cucumber, Holothuria scabra and their potential role in gonad maturation

The sea cucumber Holothuria scabra is an economically valuable marine species which is distributed throughout the Asia–Pacific region. With the natural population declining due to over fishing, aquaculture of this species is deemed necessary. Hence, it is essential to understand the mechanisms regulating the reproduction in order to increase their populations. Sex steroids, including estrogens, androgens and progestogens, play an important role in reproduction in most vertebrates and several invertebrates. It has been proposed that sea cucumbers have the same sex steroids as vertebrates but the steroidogenic pathway in the sea cucumbers is still unclear. In this study, we demonstrated by using liquid chromatography-tandem mass spectrometry (LC–MS/MS) that sex steroids (estradiol, progesterone, and testosterone) were present in H. scabra neural and gonadal tissues. In silico searches of available sea cucumber transcriptome data identified 26 steroidogenesis-related genes. Comparative analysis of encoded proteins for the steroidogenic acute regulatory protein (HscStAR), CYP P450 10, 17 and 3A (HscCYP10, HscCYP17, HscCYP3A) and hydroxysteroid dehydrogenases (Hsc3β-HSD, Hsc17β-HSD) with other species was performed to confirm their evolutionary conservation. Gene expression analyses revealed widespread tissue expression. Real-time PCR analysis revealed that HscStAR, HscCYP10, Hsc3β-HSD, and Hsc17β-HSD gene expressions were similar to those in ovaries and testes, which increased during the gonad maturation. HscCYP17 mRNA was increased during ovarian development and its expression declined at late stages in females but continued high level in males. The expression of the HscCYP3A was high at the early stages of ovarian development, but not at other later stages in ovaries, however it remained low in testes. Moreover, a role for steroids in reproduction was confirmed following the effect of sex steroids on vitellogenin (Vtg) expression in ovary explant culture, showing upregulation of Vtg level. Collectively, this study has confirmed the existence of steroids in an echinoderm, as well as characterizing key genes associated with the steroidogenic pathway. We propose that sex steroids might also be associated with the reproduction of H. scabra, and the identification of biosynthetic genes enables future functional studies to be performed.

Combined endocrine disruptive toxicity of malathion and cypermethrin to gene transcription and hormones of the HPG axis of male zebrafish (Danio rerio)

Cypermethrin (CYP) and malathion (MAT) have been widely used and are frequently detected in surface waters. The purpose of the present study was to investigate the endocrine disrupting toxicity of CYP, MAT, and CYP + MAT to 5-month-old male zebrafish (Danio rerio). After exposure, the hepatosomatic index (HSI) and gonadosomatic index (GSI) did not change significantly. Following exposure to the combination of 0.1 μg/L CYP +25 μg/L MAT, the E2 and VTG levels of male zebrafish were significantly increased compared to those after individual pesticide treatments. The molecular level of the hypothalamic-pituitary-gonadal (HPG) axis in zebrafish was studied; it was found that the expression of the estrogen-related genes, especially the vtg1 gene, was significantly altered in 0.1 μg/L CYP + 25 μg/L MAT. Overall, our observation indicated that CYP or MAT could disturb the hormonal balance, and their combination of 0.1 μg/L CYP +25 μg/L MAT could significantly enhance the estrogenic effect.

17\u03b1-ethynylestradiol prevents the natural male-to-female sex change in gilthead seabream (Sparus aurata L.)

Exposure to 17α-ethynylestradiol (EE2, 5 μg/g food) impairs some reproductive events in the protandrous gilthead seabream and a short recovery period does not allow full recovery. In this study, spermiating seabream males in the second reproductive cycle (RC) were fed a diet containing 5 or 2.5 μg EE2/g food for 28 days and then a commercial diet without EE2 for the remaining RC. Individuals were sampled at the end of the EE2 treatment and then at the end of the RC and at the beginning of the third RC, 146 and 333 days after the cessation of treatment, respectively. Increased hepatic transcript levels of the gene coding for vitellogenin (vtg) and plasma levels of Vtg indicated both concentrations of EE2 caused endocrine disruption. Modifications in the histological organization of the testis, germ cell proliferation, plasma levels of the sex steroids and pituitary expression levels of the genes coding for the gonadotropin β-subunits, fshβ and lhβ were detected. The plasma levels of Vtg and most of the reproductive parameters were restored 146 days after treatments. However, although 50% of the control fish underwent sex reversal as expected at the third RC, male-to female sex change was prevented by both EE2 concentrations.

Fluoride impairs ovary development by affecting oogenesis and inducing oxidative stress and apoptosis in female zebrafish (Danio rerio)

Previous studies have shown that waterborne fluoride exposure has adverse effects on the reproductive system of zebrafish. However, the underlying toxic mechanisms were still not clear. In the present study, female zebrafish were exposed to different concentrations of 0.787 (Control), 18.599, 36.832 mg/L of fluoride for 30 d and 60 d, and the effects of different doses of fluoride on ovary development, reproductive hormones, oogenesis, ROS content, antioxidant levels, and the expression of apoptosis-related genes and proteins in the ovaries of female zebrafish were analyzed. The results showed that ovarian weight and GSI were significantly decreased, FSH, LH and VTG levels were significantly reduced, the transcriptional profiles of oogenesis-related genes (tgfβ1, bmp15, gdf9, mprα, mprβ, ptg2β) were remarkably altered, ROS levels was notably increased, the SOD, CAT, GPx activities and GSH content as well as their mRNA expressions were significantly decreased, MDA content was remarkably increased, the expressions of apoptosis-related genes and proteins (caspase3, caspase8, caspase9, Fas-L, Cytochrome C, Bax and Bcl-2) were significantly changed, the ratio of Bax/Bcl-2 protein levels were notably increased. Taken together, this study demonstrated that fluoride exposure significantly affected ovarian development, decreased the reproductive hormones, affected oogenesis, induced oxidative stress, caused apoptosis through both extrinsic and intrinsic pathways in ovary of zebrafish. Indicating that oogenesis, oxidative stress, and apoptosis were responsible for the impairment of ovarian development.

No evidence that vitellogenin protein expression is induced in marine mussels after exposure to an estrogenic chemical

A wide variety of endocrine disrupting chemicals reach the marine environment and can cause harmful effects in different marine organisms. Vitellogenin (Vtg), the egg-yolk precursor, is a commonly used endocrine disruption biomarker in fish and more recently in marine invertebrates under the assumption of high expected similarities in the endocrine system of vertebrates and invertebrates. However, this assumption has been recently questioned. The results from previous studies focused on bivalve molluscs showed that Vtg induction could be misleading because of the use of either non-robust or indirect techniques to measure Vtg. In this study, mussels (Mytilus galloprovincialis) were exposed to either 10 or 100 ng/L of the synthetic hormone 17α-ethinylestradiol (EE2) at different exposure times (4 and 24 days) and under different feeding regimes (representing different energy balances), and Vtg levels in both male and female mussel gonads were quantified by label free shotgun LC-MS/MS proteomic analysis. Vtg protein was not detected in male gonads. In female gonads, Vtg levels were not significantly affected by EE2 at any exposure time or EE2 concentration tested, whereas a significant correlation was found between the degree of maturation of the gonad and Vtg levels in females. Results obtained in the present study critically question the use of Vtg as a biomarker of endocrine disruption in marine mussels, and show that the degree of maturation of the gonad can be an important confounding factor in the attempts to evaluate estrogenic effects through Vtg measurement in mussel gonads.

Development and validation of a competitive hybrid ELISA for Seriola lalandi Vitellogenin

The main objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for Seriola lalandi vitellogenin (Vtg). Nucleotide sequences corresponding to VtgAa, VtgAb and VtgC were obtained from S. lalandi liver transcriptomic data. Two peptides (12 amino acids each) were synthesized according to the predicted conserved regions of VtgAa and VtgAb, and used to generate two polyclonal antibodies in rabbits: anti-S. lalandi VtgA and anti-S. lalandi VtgB. Plasma from male S. lalandi treated with 17β-estradiol (E2) was fractionated to produce the Vtg standard and analysed through SDS-PAGE and Western blot. LC-MS/MS analysis showed that the fraction corresponding to the highest peak consisted mainly of VtgAa and VtgAb. Two homologous antibodies (anti-S. lalandi VtgA and anti-S. lalandi VtgB) and one heterologous antibody (anti-Mugil cephalus Vtg) were tested to develop the S. lalandi ELISA. While the antibodies against the Vtg peptides were not effectively recognizing the Vtg standard, parallelism was confirmed between the M. cephalus Vtg standard and the S. lalandi Vtg standard. The ELISA developed is highly specific (no cross-reaction with other proteins present in plasma when samples were diluted over 1:20), precise (the inter-assay coefficient of variation at 50% binding was 13%) and sensitive (19 ng/ml). The assay was validated by quantifying the plasma Vtg levels in females at the start of the reproductive season and in E2-treated males.