Vitellogenin Research Articles

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.

Impact of Mycotoxin Contaminations on Aquatic Organisms: Toxic Effect of Aflatoxin B1 and Fumonisin B1 Mixture.

(1) Background: Multiple contaminations of several mycotoxins have been detected in human and veterinary food and feed worldwide. To date, a number of studies on the combined effects of mycotoxins have been conducted on cell and animal models, but very limited studies have been done on aquatic organisms. (2) The purpose of the present study was to evaluate the combined toxic effects of Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1) on zebrafish (Danio rerio) embryos. (3) Results: Our results showed that the combination of AFB1 and FB1 at nontoxic concentrations exerted a negative effect on the lethal endpoints analyzed, such as survival, hatching, and heart rate. In addition, the mixture of mycotoxins caused an increase in the levels of enzymes and proteins involved in the antioxidant process, such as superoxide dismutase (SOD) and catalase (CAT), both in terms of protein levels and gene expression, as well as an increase in the levels of the detoxification enzymes glutathione s-transferases (GST) and cytochromes P450 (CYP450). Furthermore, we showed that the mycotoxin mixture induced an increase in pro-apoptotic proteins such as bax and caspase 3, and at the same time reduced the gene expression of the anti-apoptotic bcl-2 protein. Finally, a significant decrease in thyroid function was observed in terms of triiodothyronine (T3), thyroxine (T4), and vitellogenin (VTG) levels. (4) Conclusion: We can say that the mixture of mycotoxins carries a greater risk factor than individual presences. There is a greater need for effective detoxification methods to control and reduce the toxicity of multiple mycotoxins and reduce the toxicity of multiple mycotoxins in feed and throughout the food chain.

Effect of Swimming on the Induction of Vitellogenin in Conger Eel (Conger myriaster)

Vitellogenin (VTG) plays a very important role in the development of oocytes. This study aims to explore the effect of swimming on the VTG synthesis of conger eel (Conger myriaster). Circulating water flow and hormone injection regimen were two factors set in the following trials: A1 (no injection, no water flow), A2 (no injection, water flow), B1 (injection, no water flow), and B2 (injection, water flow). The flow velocity of the flowing water was 0.4 m/s (12 h, 60 days). We examined VTG, estrogen receptor (Er) gene expression, and VTG content in the liver and serum on the 30th and 60th days. VTG gene expression in A1 and A2 gradually decreased and was not significantly different between the two groups. The expression of Er gene in A1 was significantly higher than that in A2. The expression of VTG gene in group B1 was significantly higher than that in group B2, whereas the expression level of Er gene in group B1 was slightly higher than that in group B1. The VTG content in the liver in group B1 reached 1,396.93 μg/L, which was significantly higher than that in group B2 (1,302.06 μg/L). Results showed that the flowing water factor can inhibit the expression of VTG and Er genes in the liver and reduce the synthesis of VTG in the liver. Stimulation of flowing water can inhibit the yolk accumulation during the ovarian development of conger eel.

Integrated estrogenic effects and semi-volatile organic pollutants profile in secondary and tertiary wastewater treatment effluents in North China

Endocrine-disrupting effects on aquatic organisms caused by wastewater discharging have raised extensive concerns. However, the efficiency of various wastewater treatment processes to remove estrogenic activity in effluents and the association with organic micropollutants was not well known. We evaluated the estrogenic activity using a well-characterized in vivo bioassay featuring the Chinese rare minnows (Gobiocypris rarus) and analyzed 886 semi-volatile organic compounds (SVOCs) in effluents from four secondary wastewater treatment plants (SWTP A-D) and a tertiary wastewater treatment plant (TTP E) that utilized various common treatment processes in northern China. The final effluents from SWTPs and TTP E all exhibited estrogenic effects, increasing male fish plasma vitellogenin (VTG) contents and estradiol/testosterone (E2/T) ratios. Key regulating genes in the male fish liver including vtg1, vtg3, era, erβ, and cyp19a were upregulated. TTP E demonstrated high performance in reducing estrogenic activity in the effluents, with a reduction of 64% in integrative biomarkers of estrogenic response (IBR). UV disinfection at SWTPs removed IBR by 14%− 33%, while ozone disinfection at TTP E did not reduce IBR. Several SVOCs including alkanes, chlorobenzenes, and phthalates, detected at ng/L to µg/L level, significantly correlated with effluent estrogenic activity. Our findings suggest the necessity and the potential means to improve the efficiency of current wastewater treatment approaches to achieve better protection for aquatic organisms against the joint effects of mixtures of various categories of micropollutants in effluents.

Characterization of vitellogenin concentration in male fathead minnow mucus compared to plasma, and liver mRNA.

The objective of this study was to characterize vitellogenin (VTG) protein in male fathead minnow (Pimephales promelas) mucus compared with more conventional measures in plasma and mRNA isolated from liver. To assess the intensity and duration of changes in mucus VTG concentrations, male fathead minnows were exposed to 17α-ethinylestradiol (EE2) for 7 days with a subsequent depuration period of 14 days. The experiment was conducted in a flow-through system to maintain a consistent concentration of EE2 at a nominal EC50 concentration of 2.5 ng/L and high concentration of 10 ng/L as a positive control. Mucus, plasma and liver were sampled at regular intervals throughout the study. Relative abundance of vtg mRNA increased after 2 days of exposure and returned to control levels after 4 days of depuration. VTG protein concentration displayed similar induction kinetics in both mucus and plasma, however, it was found to be significantly increased after 2 days of exposure using the mucus-based assays and 7 days with the plasma-based assay. Significantly elevated levels of VTG were detected by both assays throughout the 14-day depuration period. The elimination of the laborious plasma collection step in the mucus-based workflow allowed sampling of smaller organisms where blood volume is limiting. It also resulted in significant gains in workflow efficiency, decreasing sampling time without loss of performance.

Effects of dietary protein level on the growth, reproductive performance, and larval quality of female yellow catfish (Pelteobagrus fulvidraco) broodstock

This study was conducted to evaluate the effects of dietary protein levels on the growth, spawning, and larval quality of female yellow catfish (Pelteobagrus fulvidraco) broodstock through a six-week feeding trial. Five isoenergetic diets were formulated, and the crude protein levels were 34.4 (DP34), 38.8 (DP39), 44.2 (DP44), 49.3 (DP49), and 52.7% (DP53). The results showed that the growth and reproductive performance, including the specific growth rate (SGR), feed efficiency (FE), gonadosomatic index (GSI), and hatching rate of yellow catfish broodstock in the DP44, DP49, and DP53 groups were significantly higher than those of other groups (P < 0.05). The optimal dietary protein levels for yellow catfish broodstock were 45.8% and 44.1% based on SGR and GSI. The lowest condition factor (CF), GSI, and smallest egg size were found in the DP34 group, while the total egg production was the highest in the DP53 group. Vitellogenin (VTG) contents in eggs and plasma were enhanced significantly with the increasing dietary protein levels from 34% to 49%, but decreased precipitously in the DP53 group (P < 0.05). The optical densities of androgen receptor (AR) and estrogen receptor (ER) in the DP34 group were significantly lower than those in other groups. The mRNA expression levels of CYP19a, AR, and ERβ were significantly increased in the DP49 group (P < 0.05). For larval quality, the deformity rate decreased significantly with the increasing dietary protein levels (P < 0.05). The larval survival rate at 9 DPH (days post-hatching) as a function of dietary protein level was analyzed by second-order polynomial regression. The results showed that a level of 47.7% was optimal for improving larval quality. Thus, a suitable range of dietary protein of yellow catfish broodstock for growth and reproduction is 44.1–47.7%.

Sex-specific responses of the reproductive system of zebrafish (Danio rerio) to electrolysis

Adult zebrafish (Danio rerio) were electrolyzed at different current densities to explore the effects of electrolysis on their reproductive system, especially on embryo production, and to uncover the molecular mechanism of changes in sex hormone and vitellogenin (VTG) levels. The results showed that embryo reproduction of zebrafish was reduced at a current density of 0.64 A/m2 after 28 days of exposure. In addition, the 17β-estradiol concentration significantly decreased and the testosterone concentration increased in female zebrafish above 0.53 A/m2. However, opposite trends were observed in male zebrafish. The VTG concentration was reduced considerably in the livers of female zebrafish in the 0.64 A/m2 electrolysis group (p < 0.05). In addition, the mRNA expression of hormone-regulating genes was significantly altered in female and male zebrafish when the current density was greater than 0.53 A/m2, and their change trends were sex-dependent. The genes expression levels of vtg1 and esr1 were downregulated in female zebrafish. However, the gene expression of esr1 and cyp19a was upregulated in male zebrafish. These changes were related to disruption in the hormone balance and VTG levels of adult zebrafish. Thus, electrolysis could cause masculinization of female zebrafish and feminization of male zebrafish. Nonetheless, there were few influences on the hormone levels and reproduction rate of adult zebrafish at the threshold of 0.26 A/m2. Thus, the current density of electrolysis needs to be controlled within a specific range to reduce its harmful effects on the reproductive system of aquatic animals.

Vitellogenin Induction in Mucus from Brook Trout (Salvelinus fontinalis)

Induction of vitellogenin (VTG) is widely used as a biomarker of exposure of male or immature fish to chemicals that are agonists of the estrogen receptor (i.e., xenoestrogens). Analysis of VTG in samples of epidermal mucosa collected from fish is a non-invasive method for evaluating whether wild fish are exposed to xenoestrogens. In this study, the mean levels of VTG in the mucus of immature brook trout (Salvelinus fontinalis) collected from the Credit River in Ontario, Canada downstream of aging residential septic systems and in an agricultural watershed were 0.67ng per mg protein, which was significantly elevated relative to the mean VTG levels of 0.22ng per mg protein in the mucus of immature brook trout collected from a less impacted site. To validate the mucus assay, immature brook trout were exposed in the laboratory to 17α-ethinylestradiol (EE2) at nominal concentrations of 10, 50 and 100ng/L and VTG levels in mucus from these fish showed a concentration-dependent increase relative to fish from the control treatment. This study illustrates the utility of this non-lethal method for assessing whether wild fish have been exposed in situ to xenoestrogens. Exposures to xenoestrogens from non-point sources may be impacting brook trout populations in urban watersheds in southern Ontario.

Sea Anemones Responding to Sex Hormones, Oxybenzone, and Benzyl Butyl Phthalate: Transcriptional Profiling and in Silico Modelling Provide Clues to Decipher Endocrine Disruption in Cnidarians.

Endocrine disruption is suspected in cnidarians, but questions remain how occurs. Steroid sex hormones are detected in corals and sea anemones even though these animals do not have estrogen receptors and their repertoire of steroidogenic enzymes appears to be incomplete. Pathways associated with sex hormone biosynthesis and sterol signaling are an understudied area in cnidarian biology. The objective of this study was to identify a suite of genes that can be linked to exposure of endocrine disruptors. Exaiptasia diaphana were exposed to nominal 20ppb concentrations of estradiol (E2), testosterone (T), cholesterol, oxybenzone (BP-3), or benzyl butyl phthalate (BBP) for 4 h. Eleven genes of interest (GOIs) were chosen from a previously generated EST library. The GOIs are 17β-hydroxysteroid dehydrogenases type 14 (17β HSD14) and type 12 (17β HSD12), Niemann-Pick C type 2 (NPC2), Equistatin (EI), Complement component C3 (C3), Cathepsin L (CTSL), Patched domain-containing protein 3 (PTCH3), Smoothened (SMO), Desert Hedgehog (DHH), Zinc finger protein GLI2 (GLI2), and Vitellogenin (VTG). These GOIs were selected because of functional associations with steroid hormone biosynthesis; cholesterol binding/transport; immunity; phagocytosis; or Hedgehog signaling. Quantitative Real-Time PCR quantified expression of GOIs. In silico modelling utilized protein structures from Protein Data Bank as well as creating protein structures with SWISS-MODEL. Results show transcription of steroidogenic enzymes, and cholesterol binding/transport proteins have similar transcription profiles for E2, T, and cholesterol treatments, but different profiles when BP-3 or BBP is present. C3 expression can differentiate between exposures to BP-3 versus BBP as well as exposure to cholesterol versus sex hormones. In silico modelling revealed all ligands (E2, T, cholesterol, BBP, and BP-3) have favorable binding affinities with 17β HSD14, 17β HSD12, NPC2, SMO, and PTCH proteins. VTG expression was down-regulated in the sterol treatments but up-regulated in BP-3 and BBP treatments. In summary, these eleven GOIs collectively generate unique transcriptional profiles capable of discriminating between the five chemical exposures used in this investigation. This suite of GOIs are candidate biomarkers for detecting transcriptional changes in steroidogenesis, gametogenesis, sterol transport, and Hedgehog signaling. Detection of disruptions in these pathways offers new insight into endocrine disruption in cnidarians.

Effect of the concentration of estradiol and vitellogenin in the blood serum of meat and egg breed chickens on egg productivity

The aim of the research is to determine the effect of changes in the concentration of estradiol-17β on the concentration of vitellogenin at the stage of egg shell formation and preparation of the body of meat and egg breed chickens (for example, the Pushkin breed) for ovulation.Materials and methods. The material for the study was the Pushkin breed chickens, in the amount of 28 heads. at the age of 48 weeks. Chickens were kept in individual cages. Light mode - 14C:10T. Drinking - nipple, temperature in the house and feeding - according to the standards adopted in the bioresource collection of RRIFAGB "Genetic collection of rare and endangered chicken breeds." Within 10 days prior to blood sampling, an ultrasound scan of the ovary and oviduct was performed from the studied group of chickens, which made it possible to determine the stage of egg formation. Blood from the studied chickens was taken in the morning on an empty stomach for the simultaneous determination of the concentration of estradiol and vitellogenin at the stage of egg shell formation. The concentration of estradiol and vitellogenin in the blood plasma of chickens was determined by ELISA. To clarify the effect of the concentration of estradiol (E2) on vitellogenin (VTG), the studied flock was divided into 2 groups according to the concentration of vitellogenin: 1st gr. – &gt; Mav. (&gt; 20 μg / ml) and 2nd gr. – &lt; Mav. (&lt;20 μg / ml). During the study period, eggs of chickens were evaluated according to the following morphometric parameters: the weight of the egg, yolk, protein and shell, elastic deformation, shape index, density of protein fractions, thickness of the shell and subshell, marbling. The number of eggs laid was determined in 10-day trial period.Results. It has been established that the duration of the ovulatory cycle in Pushkin breed chickens is 27-29 hours. As a result of such a long ovulatory cycle in the following days, laying of eggs occurs later with a progressive shift, as a result of which the laying cycle on average for the studied group of chickens is 3 days. The E2 level in the 1st group was 258,48 ± 30,60 pg/ml (p &lt; 0,05), which significantly differed from the 2nd group – 181,45 ± 13,81 pg/ml. A positive correlation was found between VTG and E2, r = 0,5 (p &lt; 0,001).Conclusion. The VTG level significantly increases in Pushkin chickens at the age of 48 weeks with an increase in the concentration of E2 in the blood. A positive correlation was noted between these features. According to the results of our research, the high content of estradiol and vitellogenin in the chickens blood serum did not have a significant effect on egg productivity.

Sex-specific effects of bisphenol S with tissue-specific responsiveness in adult zebrafish: The antiandrogenic and antiestrogenic effects

This study investigates the adverse effects and the associated underlying mechanism of bisphenol S (BPS) exposure on reproductive endocrine activity in adult zebrafish. Fish were exposed for 21 days to different BPS concentrations (0, 8, 40, and 200 µg/mL) determined via the lowest observed adverse effect level (LOAEL, i.e., < EC15 = 250 µg/mL) for zebrafish embryos. Exposure to 200 µg/mL BPS in female zebrafish in the absence of vitellogenic oocytes or the presence of degenerated oocytes in the ovary significantly decreased the biosynthesis of hepatic vitellogenin (VTG) mRNA, while hepatic VTG mRNA in male fish abundance was significantly elevated (P < 0.05). The levels of gonadal steroids were significantly increased in female zebrafish, while in male zebrafish, the levels of endogenous androgens were reduced (P < 0.05). However, the activities of 17β-estradiol and aromatase in male zebrafish were significantly elevated in all BPS exposure groups in male zebrafish (P < 0.05). Interestingly, thyroid hormone levels and residual whole-body BPS levels increased in female and male zebrafish with increasing exposure concentrations. A novel finding is that the response to BPS depends on zebrafish sex and tissue-specific responsiveness to the accumulation of BPS, suggesting that BPS may cause long-term environmental problems in adult zebrafish through tissue-specific suppression and hormonal imbalance.

Seasonal Changes in Plasma Hormones, Sex-Related Genes Transcription in Brain, Liver and Ovary during Gonadal Development in Female Rainbow Trout (Oncorhynchus mykiss)

The purpose of this study was to investigate the periodic seasonal changes in endocrine activity and gonadal development of female rainbow trout (Oncorhynchus mykiss) in a high-altitude cold-water environment. The fish were sampled monthly from January to November and the levels of plasma hormones (estradiol (E2), cortisol and thyroid hormones (THS)) and vitellogenin (VTG) were measured by ELISA. Moreover, the transcriptions of sex-related genes in the ovary, brain, and liver were detected by qRT-PCR. The results showed a seasonal fluctuation of plasma hormones and VTG together with the development of the ovary, which reached a peak from August to October. Similarly, the transcription of hypothalamic gonadotropin-releasing hormone-2 (cgnrh-2), hypothalamic gonadotropin-releasing hormone receptors (gnrhr) and follicle-stimulating hormone (fsh) in the brain varied from January to September, but the highest level was detected in September to November. In addition, the transcription of sex-related genes located in the ovary and liver increased significantly during August to October, accompanied by a continuous increase in the gonadosomatic index (GSI) and a decrease in the hepatosomatic index (HSI). Therefore, plasma hormones and sex-related genes regulate the development and maturation of O. mykiss oocytes with the change in seasons and peaked in November. The results of this study provide a reference for improving the efficiency of the artificial reproduction of O. mykiss.

Gonadal maturation in Litopenaeus vannamei fed on four different polychaetes

Broodstock culture of Litopenaeus vannamei plays an important role in the shrimp culture industry; thus, a nutritional and bio-safety food for broodstock culture is necessary. Marine polychaetes, which have a good nutritional content, are an essential and widely used live food in the current shrimp industry. In this study, four polychaete species, Marphysa maxidenticulata, Cheilonereis cyclurus, Nereis vexillosa, and Perinereis aibuhitensis, were fed to broodstock of L. vannamei as the only food, and the gonadal maturity parameters of the broodstock were examined. The results showed that broodstock, which were fed with M. maxidenticulata, had the highest gonadal maturation rate (56.67%). The N. vexillosa food group had the highest gonadal index (5.37%). The content of serum progesterone and vitellogenin (VTG) was highest in the broodstock fed N. vexillosa. The expression of VTG mRNA in the gonads was also highest in the broodstock fed N. vexillosa, followed by those fed M. maxidenticulata. Nutritional content analysis showed that lipid content in the four worms ranged from 13% to 22%. The content of polyunsaturated fatty acids was highest in N. vexillosa, particularly C18:2 n-6, C18:3 n-3, C20:4 n-6, and C20:5 n-3. In addition, the 20:5 n-3 content in N. vexillosa was 2.5–8.0 times higher than that in the other polychaetes. These results indicated that polychaetes had different effects on promoting the gonadal maturation of L. vannamei. N. vexillosa and M. maxidenticulata could be potential live food to promote the gonadal maturation of L. vannamei.

Graphene oxide enhanced the endocrine disrupting effects of bisphenol A in adult male zebrafish: Integrated deep learning and metabolomics studies

In our previous studies, it was found that graphene oxide (GO) reduced the endocrine disruption of bisphenol A (BPA) in zebrafish embryo and larvae, but through different mechanisms. In this study, adult male zebrafish were selected to further understand the interactions between GO and BPA considering that adult zebrafish have different uptake pathways and metabolism from embryo and larvae. BPA was predicted to bind with the estrogen receptor α (ERα) with a probability of 98.1% by training a directed-message passing deep neural network model, and was confirmed by molecular docking analysis. The results were in accordance with the significantly increased vitellogenin (VTG) and estradiol (E2) levels, while decreased testosterone (T) and follicle-stimulating hormone (FSH) levels in the adult male zebrafish after 7 d exposure to 500 μg/L BPA. Compared to BPA single exposure group, the presence of GO led to significantly lower T and FSH levels and fewer spermatozoa, indicating that GO enhanced the endocrine disruption effects of BPA in the adult zebrafish. Metabolomics analysis revealed that 5 μg/L BPA could elicit changes in the metabolome, and the responses were correlated with BPA concentrations. Metabolic pathway analysis revealed more disturbance was caused by the mixture of GO and BPA compared to BPA alone, including three additional pathways and stronger perturbations on carbohydrate, lipid, and amino acid metabolism, fortifying that GO exaggerated the toxic effects of BPA. This was opposite to the depression effect observed in zebrafish embryo and larvae, magnifying that the joint effects of exposure to nanomaterials and endocrine disrupting chemicals are relevant to the life stages of organisms.

Insights into the sex-dependent reproductive toxicity of 2-ethylhexyl diphenyl phosphate on zebrafish (Danio rerio)

As a frequently detected organophosphate ester in various environmental media, the toxic effects of 2-ethylhexyl diphenyl phosphate (EHDPHP) on aquatic organisms of different sexes remain unclear. In this study, adult zebrafish were exposed to 2.5, 50, 250 µg/L of EHDPHP for 21 days to investigate its sex-dependent reproductive toxicity and related mechanisms. EHDPHP exposure significantly inhibited the reproduction of zebrafish, evidenced by the reduced spawning of females, depressed growth and development of their offspring. EHDPHP induced greater impacts on the changes of sex hormones and vitellogenin (VTG) in the males than females. For females, the synthesis of testosterone (T) was inhibited because of the down-regulated gnrhr2, gnrhr3, gnrhr4, gnrh3, gnrh2 and er2β in the brain, while 17β-estradiol (E2) increased in 250 µg/L due to up-regulated cyp19a. For males, the promotion of T was directly related to the up-regulation of fshr, 3βhsd, star, cyp11 and cyp17 in the gonad, and eventually led to the increase of E2. The decrease of plasma 11-KT in both sexes could be mainly attributed to the down-regulation of cyp11b and hsd11b. The plasma VTG decreased in females but increased in males, which was in accordance with the down and up regulation of erα and er2β in the females and males, respectively. All these indicated EHDPHP displayed reproductive toxicity on zebrafish in a sex dependent manner. Molecular docking analysis indicated stronger interaction of EHDPHP with the antagonisms of estrogen receptor (ER) and androgen receptor (AR), as well as the agonism of CYP19A1, which further revealed the sex-dependent reproductive toxicity mechanism of EHDPHP. This study highlights the importance of distinguishing males and females in toxicity evaluation of endocrine disruption chemicals.

Characterization and Gene Expression of Vitellogenesis-Related Transcripts in the Hepatopancreas and Ovary of the Red Swamp Crayfish, Procambarus clarkii (Girard, 1852), during Reproductive Cycle

The major component of the animal egg yolk is the lipoglycoprotein vitellin, derived from its precursor vitellogenin (VTG), which is produced species-specifically in decapod crustaceans in the hepatopancreas and/or in the ovary of reproductive females. Previous studies on Procambarus clarkii vitellogenesis report the existence of two single VTGs. Here, from a multiple tissue transcriptome including ovaries and hepatopancreas of P. clarkii, we characterized four different VTG and two VTG-like transcriptomes encoding for the discoidal lipoprotein-high density lipoprotein/β-glucan binding protein (dLp/HDL-BGBP). The relative expression of the various genes was evaluated by quantitative Real-Time PCR in both the ovary and hepatopancreas of females at different reproductive stages (from immature until fully mature oocytes). These studies revealed tissue-specificity and a reproductive stage related expression for the VTGs and a constitutive expression in the hepatopancreas of dLp/HDL-BGBP independent from the reproductive stage. This study may lead to more detailed study of the vitellogenins, their transcription regulation, and to the determination of broader patterns of expression present in the female hepatopancreas and ovary during the vitellogenesis. These findings provide a starting point useful for two different practical aims. The first is related to studies on P. clarkii reproduction, since this species is highly appreciated on the market worldwide. The second is related to the study of new potential interference in P. clarkii reproduction to delay or inhibit the worldwide spread of this aggressively invasive species.

Endocrine disruption in the sub Antarctic fish Patagonotothen tessellata (Perciformes, Notothenidae) from Beagle Channel associated to anthropogenic impact

Situated in the sub-Antarctic region, Beagle Channel represents a unique marine ecosystem due to the connection between the Pacific and the Atlantic Oceans, and its proximity to the Antarctic Peninsula. Ushuaia city, the biggest settlement on the channel, exerts an increasing anthropogenic pressure by discharges of urban and industrial effluents. In the present work, we use Patagonotothen tessellata, one of the most abundant and widespread species in the channel, as a bioindicator species in order to evidence anthropic impact from Ushuaia Bay and surrounding areas. We first analyzed and characterized real time gene expression of androgen receptor, estrogen receptor and different forms of vitellogenin (VTG), under laboratory conditions. This was achieved by induction with estradiol of P. tessellata males. Then, the selected genes were used as biomarkers for an environmental biomonitoring study. Morphometric indices and circulating sex steroids (estradiol and testosterone) were also quantified in male fish collected from different sites. The qPCR analysis showed that vtgAb form is more inducible than vtgAa or vtgC forms after estrogen induction. The field survey revealed the up-regulation of vtgAb and the androgen receptor in fish from sites with higher anthropogenic influence. Sex steroids followed seasonal variations according to their reproductive cycle, with higher levels of estradiol and testosterone in winter and summer seasons. The use of biomarkers such as gene expression of VTG demonstrates that fish from Ushuaia Bay are likely to be exposed to endocrine disrupting compounds. To our knowledge, this research is the first attempt to assess the endocrine disruption associated to anthropic impact in a widespread fish of the Beagle Channel and contributes to a better understanding of the reproductive physiology of sub Antarctic ichthyofauna.

Gene expression analysis on growth, development and toxicity pathways of male Nile tilapia (Oreochromis niloticus), after acute and sub-chronic benzo (α) pyrene exposures

Benzo[α]pyrene (BaP), a lipophilic polycyclic aromatic hydrocarbon (PAH), is a contaminant widely distributed in aquatic systems. Its presence in freshwater organisms is of great concern; particularly in Nile tilapia (Oreochromis niloticus), due to its economic relevance. The aim of this study is to evaluate the effects of acute and sub-chronic BaP exposures on molecular growth/development responses, toxicity to DNA pathways and xenobiotic metabolism. Negative morphometric changes (the growth condition factor, hepatosomatic and gonadosomatic indices), the fluorescent aromatic compounds (FACs) in bile were also studied in order to understand the mechanisms of action of BaP. Genes involved in the growth hormone GH/insulin-like growth factor 1 (IGF-1) were measured, such as IGF1–2 with the growth hormone receptor gene expression GHR1–2, and the endocrine disruption biomarker vitellogenin (VTG). Acute exposure elicited changes in the GH/IGF axis, mainly in the GHR1 and in IGF1 mRNA levels without affecting the GHR2 expression. While sub-chronic exposure had less effect on both GHR and IGF genes. The most notable tissue-specific effects and morphometric endpoints were observed upon sub-chronic exposure, such as changes in key genes involved in detoxification, DNA damage, and altered reproductive morphological endpoints; showing that sub-chronic BaP doses have longer-lasting toxic effects. This study shows that sub-chronic BaP exposure may compromise the health of Nile tilapia and sheds light on the changes of the GH/IGF axis and the biotransformation of the xenobiotics due to the presence of this contaminant.

Long-Term Caffeine Intake Exerts Protective Effects on Intestinal Aging by Regulating Vitellogenesis and Mitochondrial Function in an Aged Caenorhabditis Elegans Model.

Caffeine, a methylxanthine derived from plants, is the most widely consumed ingredient in daily life. Therefore, it is necessary to investigate the effects of caffeine intake on essential biological activities. In this study, we attempted to determine the possible anti-aging effects of long-term caffeine intake in the intestine of an aged Caenorhabditis elegans model. We examined changes in intestinal integrity, production of vitellogenin (VIT), and mitochondrial function after caffeine intake. To evaluate intestinal aging, actin-5 (ACT-5) mislocalization, lumenal expansion, and intestinal colonization were examined after caffeine intake, and the levels of vitellogenesis as well as the mitochondrial activity were measured. We found that the long-term caffeine intake (10 mM) in the L4-stage worms at 25 °C for 3 days suppressed ACT-5 mislocalization. Furthermore, the level of autophagy, which is normally increased in aging animals, was significantly reduced in these animals, and their mitochondrial functions improved after caffeine intake. In addition, the caffeine-ingesting aging animals showed high resistance to oxidative stress and increased the expression of antioxidant proteins. Taken together, these findings reveal that caffeine may be a potential anti-aging agent that can suppress intestinal atrophy during the progression of intestinal aging.

Study of Heavy Metals Pollution and Vitellogenin Levels in Brown Trout (Salmo trutta trutta) Wild Fish Populations

The objectives of this research were, first, to determine the concentrations of certain heavy metals in the edible tissue of wild brown trout (Salmo trutta trutta) from two different rivers located in the Abruzzi region (Italy), and then, to investigate the levels of variation in vitellogenin (VTG) associated with the presence of metalloestrogens. VTG is an effective indicator for endocrine disturbance, and an increase in the vitellogenin levels in male fish is widely employed as a biomarker of estrogenic contamination in the aquatic environment. The muscles of the trout were analyzed for As, Cd, Co, Cr, Ni, Pb, Al, and Zn using an inductively coupled plasma-mass spectrometer (ICP-MS), and Hg was measured using a direct mercury analyzer (DMA-80). The calculated values of the condition factor confirmed a healthy status for this species, indicating that the aquatic habit in both rivers is suitable for brown trout life. No significant difference in the concentrations of each metal were reported between the trout from the two rivers, and no significant difference for VTG levels were found between male and female fish. It is interesting to note the high concentrations of Al/Zn, while the Pb, Cd, and Hg concentrations in all of the samples were lower than those established by the European Commission.