Effects of dietary nucleotides on acute stress response and cannabinoid receptor 1 mRNAs in sole, Solea solea.

The objective of the present study was to evaluate the modulation of acute stress response by dietary nucleotides (NT) in sole, Solea solea. A basal diet was supplemented with levels of 0 (normal diet), or 0.4 g NT/kg dry diet for 8 weeks. At the end of feeding trial, fish fed the normal and NT-supplemented diet were subjected to a standardized protocol of disturbance and sampled over a 24h recovery after the stressor exposure. Modulatory effects of NT on acute stress response (cortisol and glucose), proopiomelanocortin (POMC) and cannabinoid receptor 1 splice variants (CB1A and CB1B) mRNA levels were studied. Both plasma cortisol and glucose levels of fish fed NT-supplemented diet were significantly lower than fish fed the control diet at 1 and 4h post-stress time-points. There are no significant effects of dietary NT on POMC and HSP70 mRNA levels. In our study, both CB1A and CB1B trascript levels were induced in fish fed the normal diet at 1 and 4h post-stress intervals. Collectively, the results obtained suggest that dietary NT modulates the CB1-like receptor mRNA expressions leading to attenuation in stressor-induced plasma cortisol level in sole.

Dietary Aloe vera components' effects on cholesterol lowering and estrogenic responses in juvenile goldfish, Carassius auratus.

Aloes are now considered a very interesting source of bioactive compounds among which phytosterols should play a major role. The present study is an attempt to investigate the hypocholesterolemic activity of Aloe vera associated with its impact on the reproductive status of juvenile goldfish. Therefore, the short- and long-term effects of feeding supplementary diet containing aloe components (20 mg aloe/g diet; 2%) on plasma lipids, plasma vitellogenin, and hepatic estrogen receptor α/ß1 mRNA levels in goldfish were examined. Results of GC-MS for phytosterols show high abundance of ß-sitosterol in freeze-dried powder of Aloe vera whole leaves. Moreover, a 2% aloe powder dietary supplement was not found estrogenic in juvenile goldfish after either 7- or 30-day treatment, but was consistent in plasma hypocholesterolemic effects following long-term exposure. The present data further support that plasma cholesterol modulation induced by phytosterols may not be related to estrogen-like activity.

Xenoestrogens elicit a modulation of endocannabinoid system and estrogen receptors in 4NP treated goldfish, Carassius auratus.

Based on pharmacological, behavioral and neuroanatomical studies, the endocannabinoids appear to be pivotal in some important neuroendocrine regulations of both vertebrates and invertebrates. Interestingly, a well developed endocannabinoid system was recently demonstrated by us in different bonyfish brain areas which control reproduction, energy balance and stress. Fish in particular are very sensitive to different types of stressors which can heavily affect their reproductive activity and negatively reverberate on aquaculture. Since recent new data have been reported on endocrine disruptors (EDs) impact on zebrafish receptor CB1 expression, in the present research we have investigated the response of the endocannabinoid system to acute treatment with an environmental stressor such as the xenoestrogen nonylphenol (4NP) in the brain and peripheral tissues of the goldfish Carassius auratus. First of all the estrogenic effects induced by 4NP were demonstrated by a dose-dependent increase of plasma levels and gene expression of the biomarker vitellogenin, then changes in cannabinoid receptors and anandamide degradative enzyme, the fatty acid amide hydrolase (FAAH), were analysed by means of Real Time PCR. As the exposure to EDs may lead to an activation of estrogen receptors and affects the Aromatase (AROB) transcription, changes in mRNA levels for ER subtypes and AROB were also evaluated. Our results confirm in goldfish the effect of 4NP on ERα and ERß1 receptors and point out a different sensitivity of CB1 and CB2 for this compound, suggesting distinct roles of these cannabinoid receptors in some adaptive processes to contrast stress induced by xenoestrogen exposure.

Modulation of cortisol levels, endocannabinoid receptor 1A, proopiomelanocortin and thyroid hormone receptor alpha mRNA expressions by probiotics during sole (Solea solea) larval development.

In the present study, we investigated whether the use of Enterococcus faecium IMC 511 as a probiotic can modulate neuroendocrine system responses during the larval rearing of Solea solea; to this end, the gene expression patterns of proopiomelanocortin (POMC), endocannabinoid receptor 1A (CB1A), and thyroid receptor alpha (TRα) were quantified, and whole-body cortisol levels were measured. Probiotic treatment up-regulated transcription of all selected genes and cortisol concentrations on day 10 post hatch (ph), while on day 30 ph experimental groups showed significantly lower levels of both POMC and CB1A compared to those of the control group. These changes were no longer evident on day 60 ph, when POMC, CB1A, TRα gene expression and cortisol titers were found to be similar in all experimental groups. Our results suggest that metabolic responses to probiotic treatment can be modulated through the activation of genes selected for functional interaction between the hypothalamic-pituitary-thyroid (HPT) axis and the melanocortin and the endocannabinoid systems. Furthermore, the observed (30 ph) down-regulation of both POMC and CB1A gene expression coupled with up-regulation of TRα mRΝΑ levels suggest the activation of a compensatory mechanism that promotes growth and development and perhaps modulates food intake.

Analysis of endocrine disruption in Southern California coastal fish using an aquatic multispecies microarray.

BACKGROUND: Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. OBJECTIVES: We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. METHODS: We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. RESULTS: Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. CONCLUSIONS: The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Multihormonal control of vitellogenesis in lower vertebrates.

The comparative approach on how and when vitellogenesis occurs in the diverse reproductive strategies displayed by aquatic and terrestrial lower vertebrates is presented in this chapter; moreover, attention has been paid to the multihormonal control of hepatic vitellogenin synthesis as it is related to seasonal changes and to vitellogenin use by growing oocytes. The hormonal mechanisms regulating vitellogenin synthesis are also considered, and the effects of environmental estrogens on the feminization process in wildlife and humans have been reported. It is then considered how fundamental nonmammalian models appear to be, for vitellogenesis research, addressed to clarifying the yolkless egg and the evolution of eutherian viviparity.

Are the cannabinoids involved in bony fish reproduction?

Following the discovery of two CB1 genes in the fish Fugu rubripes, investigations on the phylogeny of endocannabinoids have indicated that this system is highly conserved. Our study demonstrated that CB1 receptors are expressed in the CNS and gonads of two teleosts, Carassius auratus and Pelvicachromis pulcher, and they show a high percentage of sequence identity with Fugu rubripes CB(1A) and Danio rerio CB1. By means of immunohistochemistry for CB1, sGnRH, and TH, we found a codistribution of these signaling molecules in the basal telencephalon/preoptic area, which are key centers for gonadotropic regulation. We therefore suggest that endocannabinoids are possibly involved in modulating fish reproduction at both the central and peripheral levels.

Peptide pheromones in newts.

This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.

Temporary impairment of reproduction in freshwater teleost exposed to nonylphenol.

New born guppies, Poecilia reticulata, were exposed to a sublethal concentration of nonylphenol (NP) for 90 days, with the aim of evaluating the influence that this pollutant has on reproductive functions. At the end of treatment, the gender balance was significantly biased towards females (sex ratio = 0.3 males per female). Treated males displayed anomalous transcription of vitellogenin in correlation with a significant reduction of the gonadosomatic index (GSI); however, an increase of vitellogenin gene transcription, correlating directly with an increase in GSI, was observed in NP treated females. The hepatosomatic index (HSI) was significantly increased in exposed males and females. Females exposed to NP showed no alteration of sexual behavior or reproduction. These findings in guppies suggest that NP has estrogenic potency sufficient to disturb reproduction. Effects were transitory in nature because three months in uncontaminated water allowed males to recover normal sexual efficiency.

PCR-ELISA detection of estrogen receptor ß mRNA expression and plasma vitellogenin induction in juvenile sole (Solea solea) exposed to waterborne 4-nonylphenol.

In this study, the effect of 4-nonylphenol (4NP) on reproductive axis of sole (Solea solea) has been investigated by using selected biomarkers of estrogenic effects: vitellogenin (VTG) and estrogen receptor ß (ERß) mRNA. Furthermore, an enzyme linked immunosorbent assay (ELISA) detection system of reverse transcriptase-polymerase chain reaction (RT-PCR) products for the analysis of sole ERß mRNA expression was developed and validated. The proposed method allows large-scale analyses of different mRNAs in fast and not expensive way. Our results have demonstrated that the PCR-ELISA method developed shows high sensitivity, good reproducibility and also the potential for semi-quantitative analysis of hepatic ERß mRNAs. Both plasma VTG level and ERß mRNA expression were increased in tested animals following a short exposure to environmental relevant concentrations (10(-6)M) of 4NP, confirming the functional role of ERß in the regulation of xenoestrogens-induced production of VTG. The methodology provided in the present study together with the preliminary results on the hepatic expression of ERß may be useful in environmental xenoestrogens monitoring studies, using flatfish as "sentinel" species.

Cortisol response to waterborne 4-nonylphenol exposure leads to increased brain POMC and HSP70 mRNA expressions and reduced total antioxidant capacity in juvenile sole (Solea solea).

4-Nonylphenol (4-NP) is a breakdown product of alkylphenolpolyethoxylates and can be found in almost all environmental water matrices. 4-NP can act as environmental stressor on fish, typically causing modulation of hypothalamic-pituitary-interrenal axis (HPI). To examine the effects of the xenoestrogen 4-NP or 17ß-estradiol (E2) on induction of stress response mechanisms by evaluating the levels of proopiomelanocortin (POMC) mRNA, heat shock protein 70 (HSP70) mRNA and plasma cortisol, we exposed juvenile sole (Solea solea), under static condition for 7 day, to either 10(-6) or 10(-8) M 4-NP, or 10(-8) M E2. In addition, plasma cortisol titers were correlated to the total antioxidant capacity (TAC), one of the oxidative stress parameters. 4-NP treatments resulted in high levels of POMC mRNA, HSP70 mRNA and plasma cortisol. On the contrary, E2 basically down-regulated POMC expression. Moreover, elevated cortisol levels in fish exposed to the highest dose of 4-NP were accompanied by low TAC. These results suggest that 4-NP modulates the sole HPI axis inducing a cortisol-mediated stress response. Specifically, we suggest that 4-NP affects brain POMC mRNA levels via non-estrogen receptor (ER)-mediated mechanism further supporting the ability of 4-NP to target multiple receptor systems.

Effect of dietary supplements of mussel and polychaetes on spawning performance of captive sole, Solea solea (Linnaeus, 1758).

Three diets were compared for the feeding of captive common sole broodstock (Solea solea) kept under ambient photoperiod and temperature conditions. A group of 70 adults were caught in the wild and the 38 males and 32 females distributed at random in six tanks. All the fish were acclimated to the same semi-moist diet (M) in the pre-experimental period from December to February. Three dietary treatments were offered in the experimental period from March to May with two replicates (tanks) per treatment. The treatments were M alone, M supplemented with fresh mussels (Mytilus edulis) (M+M), and M supplemented with live polychaetes (Perinereis cultrifera) (M+P). Spawning occurred during April and May when water temperature was 17 and 18 degrees C, respectively, and salinity around 34-35ppt. Average daily dry matter intake expressed as a proportion of body weight was M 0.65+/-0.34%, M+M 0.43+/-0.18%, and M+P 0.56+/-0.27%, and differed significantly (P<0.05) between treatments. The average daily dry matter intake within a tank ranged from 0.31+/-0.04% in February to 0.98+/-0.26% in May (P<0.05), apparently due to changes in the photo-thermal regime. Diet significantly affected the number of days when spawning occurred, the number of days when hatched eggs were produced, and the proportion of fertilized eggs (P<0.05); and affected the number of days on which viable eggs were produced during April (NS). In all cases, the results were lowest for M+M, while those for the other two treatments did not differ significantly. Differences in hatching rate were not significant in April. During May, no spawning occurred in fish given the M+M treatment, and the differences between the other two treatments were not significant. Values for all variates tended to be higher for M+P than M+M in April and lower for M+P and M in May. These results suggest that supplementing the semi-moist diet with mussels depressed feed intake and, consequently, reproductive performance; the semi-moist diet alone and semi-moist diet supplemented with polychaetes allowed satisfactory food intake and reproductive performance in broodstock sole.

Involvement of endocannabinoid CB1 receptor in the modulation of stress responses related to xenoestrogen exposure.

The endocannabinoid system, through the cannabinoid receptor CB1, is involved in the modulation of adaptive responses to environmental conditions. However, little is known about the role of the cannabinergic system, particularly CB1 receptor expression, in relation to the effects induced by xenoestrogens concerning the reproductive axis. Our results demonstrate that only 10(-8) mol/L of 17beta-estradiol was able to induce significantly higher levels of CB1A mRNA, while no effects were found after treatment with 4-nonylphenol (10(-8) or 10(-6) mol/L); moreover, mRNA expression titers of CB1B did not show any significant change. The estrogenic effects of treatments were evidenced by a dose-dependent induction of plasma hepatic vitellogenin titers. It can be concluded that low doses of estrogens, and possibly of xenoestrogens, may increase endocannabinoid signaling pathways.

Influence of dietary feeding of low monomer content grape seed extract on vitellogenin production and cholesterol levels in goldfish, Carassius auratus.

Grape seed extract (GSE) is a source of naturally occurring compounds known as proanthocyanidins and flavan-3-ols, which are recognized to exert a protective effect on human health, so GSE is widely used mainly as a nutritional supplement. However, polyphenols may have, in some cases, estrogenic effects or may interfere with the endocrine system. For that reason, it was considered of interest to investigate the beneficial or detrimental effects induced by low monomer content grape seed extract (LMC-GSE) in a teleost experimental model, the juvenile goldfish (Carassius auratus); therefore, biomarkers of estrogenic exposure together with cholesterol titers were assessed in both plasma and tissue samples taken from fish fed with different doses of LMC-GSE for 4 weeks. Dietary LMC-GSE (71 or 35 mg/g diet) did not affect vitellogenin (VTG) synthesis; on the contrary, VTG production was exclusively induced in fish fed with an estradiol-17beta (E2)-incorporated diet. In addition, it was found that both plasma E2 levels and hepatic total cholesterol were not affected by LMC-GSE dietary regimens.

Bisphenol A acts differently from and independently of thyroid hormone in suppressing thyrotropin release from the bullfrog pituitary.

The objective of this investigation was to ascertain whether bisphenol A (BPA), which has a structural resemblance to thyroid hormone (TH), acts as a TH agonist or antagonist in terms of affecting the release of thyrotropin (TSH). To this end, we exposed adult bullfrog (Rana catesbeiana) pituitary cells to BPA and/or TH in the presence or absence of corticotropin-releasing factor (CRF), which is known to have a potent TSH-releasing activity in amphibians. BPA (10(-9)-10(-4)M) did not affect the basal release of TSH. However, it suppressed CRF-inducible TSH release at 10(-4)M, but not at 10(-5)M. Triiodothyronine (T(3)) at 10(-7)M and l-thyroxine (T(4)) at 10(-6)M also suppressed the CRF-inducible release of TSH. The combination of T(3) (10(-7)M) or T(4) (10(-6)M) with BPA (10(-4)M) had an additive effect in suppressing TSH release. A comparison of the suppressive effects of BPA and T(3) on the release of TSH following the addition of actinomycin D or cycloheximide to the culture medium revealed that both of the latter compounds blocked T(3)-inducible but not BPA-inducible suppression of TSH release. The results indicate that the mechanism of action of BPA is different from that of T(3) in that T(3) action involves RNA and protein synthesis, whereas BPA action does not involve either of these processes. Furthermore, BPA was found to suppress the thyrotropin-releasing hormone-inducible release of both prolactin (PRL) and TSH. Our results suggest that BPA acts not only as a blocker of TSH secretagogues but also as a blocker of a PRL secretagogue at the pituitary level. Estradiol affected neither the release of TSH nor the release of PRL in the presence or absence of their secretagogues, suggesting that the suppression of the release of TSH and PRL caused by BPA may not be derived from its estrogenic activity.

Hormonal regulation of hepatic IGF-I and IGF-II gene expression in the marine teleost Sparus aurata.

The present work aimed to determine whether GnRH potentiates the effect of growth hormone (GH) on insulin-like growth factors (IGF-I and IGF-II) hepatic gene expression in Sparus aurata liver. Since several hepatic genes were shown to underlie direct regulation via the hepatic estrogen receptor, another aim was to extend our understanding of direct estrogen effects on liver IGFs gene expression. Pre-reproductive sea bream females were treated with GH, GnRH, estradiol-17beta, GH plus GnRH, and estradiol-17beta plus GH. After 72 hr, all treatment induced an increase of plasma estradiol well correlated with the increase of plasma vitellogenin (VTG) levels. IGF-I and IGF-II expression in the liver of treated females was determined by semi-quantitative RT-PCR, using beta-actin as internal standard. The results reported here show that GH significantly stimulates hepatic transcription of IGF-I and IGF-II genes. Surprisingly, E2 and GnRH treatments decreased both IGF-I and IGF-II mRNA levels. In fishes treated with GH plus GnRH, the GnRH contrasted the GH effect: the IGF-I mRNA levels were still significantly higher than in controls, while the effect of GH on IGF-II gene expression was totally abolished. At the same time, in the combined treatment with GH plus E2, the E2 counteracted the stimulatory effect of GH on both IGF-I and IGF-II genes expression.

Temperature-dependent prolactin secretion and reproductive biology of the newt Triturus carnifex Laur.

The effects of temperature on pituitary prolactin (PRL) gene expression and peripheral levels were studied in both male and female newts obtained from wild conditions during reproductive (Experiment I) and nonreproductive (Experiment II) periods; moreover, changes in parameters related to reproductive function are also described. Male and female newts were taken from a pond in February (Experiment I, reproductive period) and maintained for 1 month in tanks at 4 and 18 degrees C. In male newts kept at 4 degrees C, increase of PRL mRNA in the pituitary and plasma PRL was found compared with that measured in those kept at 18 degrees C. The increase in PRL secretion was parallel to that of plasma androgens and related secondary sexual characteristics (SSC) in males and of plasma estradiol-17beta and vitellogenin in females. On the contrary, in nonreproductive newts (Experiment II), taken from the field in May, no significant changes in plasma PRL, androgens, and SSC were found in those maintained at low temperature (4 degrees C), whereas low temperature significantly increased PRL mRNA expression in the male pituitary and PRL mRNA plus plasma PRL levels in females. These findings suggest that low temperature regulates PRL secretion in this urodele species, showing a sex- and season-related control mechanism; moreover, low temperature failed to influence the reproductive biology of newts taken from the field in May, after naturally occurring reproduction during winter months.