Variation of the genetic expression pattern after exposure to estradiol-17beta and 4-nonylphenol in male zebrafish (Danio rerio).

There is much concern about the increasing presence in the environment of synthetic chemicals that are able to disrupt the endocrine system. Among these compounds, 4-nonylphenol (4-NP) is one of the most studied xenoestrogens, due to its widespread accumulation in water sediment and consequent presence in fatty acid of aquatic organisms. Here, we have used a zebrafish microarray representing 16,399 genes to study the effects of 4-NP and estradiol-17beta (E2) in adult male zebrafish in order to elucidate the mechanism of action of 4-NP compared with that of E2. The microarray results showed that both 4-NP and E2 induced a strong expression of vitellogenin (VTG), the sex related precursor of the yolk proteins in oviparous vertebrates. Both treatments induced elevated protein turnover upregulating genes involved in proteolysis and those that are constituents of the ribosome. Many genes regulated by 4-NP and E2 are involved in energy metabolism, oxidative stress defense mechanisms, xenobiotic metabolism, and lipid metabolism. A different pattern of expression in the two treatments was found for genes involved in oxidative stress, since E2 seems to induce the mechanism of detoxification, while 4-NP seems to inhibit this protective mechanism of the cell. Overall, these findings demonstrate that the microarray approach can contribute significantly to the understanding of expression patterns induced by E2 and 4-NP in male zebrafish. The results also demonstrate that 4-NP is able to act through an alternative pattern to that of estradiol-17beta, modulating the expression of the same genes in a different manner.

Partial cloning of CB1 cDNA and CB1 mRNA changes in stress responses in the Solea solea.

Endogenous cannabinoids, through the CB1 receptor, are involved in the control of several functions including stress responses. The aim of this study was to investigate the presence of cannabinoid receptor CB1 in the sole ovary by partial cloning of brain CB1 cDNA; in a stress paradigm of disturbance by handling, which consisted in catching, netting and hand-sorting, changes of CB1 mRNA were related with those of proopiomelanocortin (POMC) mRNA; the trend and timing of stress responses and adaptation were monitored by measuring plasma cortisol levels. We characterized two forms of CB1-like receptor, termed CB1A and CB1B. The two sole CB1 (both 799bp) share 76% identity in their cDNAs, and the deduced amino acid sequences are 80% identical. The handling stress induced a sustained increase in plasma cortisol levels 1h after the handling began and decreased to low levels 12h after initiation of handling, showing the same trend of ovarian POMC mRNA expression. In addition, while CB1A mRNA did not show any significant changes during handling stress, significantly lower levels of CB1B mRNA were found in stressed fish 1h after the beginning of handling, with CB1 expression increased 24h after stress induction, both in the ovary and brain. It can be concluded that endocannabinoid system is involved in the modulation of adaptive responses to environmental conditions.

Assessment of water pollution in the Tronto River (Italy) by applying useful biomarkers in the fish model Carassius auratus.

The Tronto River (southern Marche region of central Italy) is located in an area with neighboring industrial activities and is contaminated with domestic and industrial wastewater. Water quality data analyses revealed the presence of a mixture of low levels of heavy metals and organic compounds. The effects of long-term exposure to Tronto River water on juvenile Carassius auratus were evaluated with an integrated approach using xenoestrogens biomarkers, such as vitellogenin (VTG) and ER beta-1 mRNA expression, and stress parameters (i.e., cortisol and glucose in the blood and glycogen in the liver). Treatment with Tronto River water did not induce VTG synthesis in fish and did not affect ER beta-1 mRNA expression. Moreover, cortisol titers found in the plasma of fish exposed to Tronto River water were lower than those found in the control group. Regarding energy parameters, treatment with Tronto River water induced an increase in plasma glucose and a depletion of liver glycogen reserves.The effects of Tronto River water were studied in parallel with those of 4-NP and CdCl(2). The 4-NP at the dose of 22 microg/L induced the synthesis of peripheral vitellogenin and increase of ER beta-1 titers; on the contrary, CdCl2 exposure at the concentration of 22 microg/L did not induce significant changes on plasma VTG and/or hepatic ER beta-1 levels. In addition, no significant changes in plasma cortisol levels in fish exposed to 4-NP or CdCl(2) were found. Fish exposed to CdCl(2) displayed liver glycogen depletion, but no significant increase in plasma glucose was observed. On the contrary, a 30-day exposure to 4-NP induced only a slight decrease of glycogen reserves without any changes in plasma glucose levels. In conclusion, our study demonstrated that long-term exposure of juvenile goldfish to the water of the Tronto River significantly affects both stress and energy parameters. There is evidence that pollutants, present in Tronto River water, were not able to induce xenoestrogenic effects but caused a functional impairment of the hypothalamum-pituitary-interrenal axis.

Cloning of sole proopiomelanocortin (POMC) cDNA and the effects of stocking density on POMC mRNA and growth rate in sole, Solea solea.

Proopiomelanocortin (POMC) is an important gene implicated in different functions, such as the stress response of the hypothalamus-pituitary-adrenal axis. The aim of the present study was to determine whether farming conditions, such as stocking density, can be considered a powerful stressor influencing in turn the growth rate in juvenile fish. Thus, POMC cDNA expression was investigated during adaptation to farming conditions in sole (Solea solea), as a model for studying the effects of rearing densities on stress response; different stocking densities (50, 100, and 250 animals/m(2)) were applied and, after 7 and 21 days, the fishes were examined for body weight and plasma cortisol levels as indicators of stress. In addition, proopiomelanocortin was cloned and sequenced from the brain of sole, allowing semi-quantitative RT-PCR to be performed to evaluate POMC mRNA expression in brain tissue. There was a significant increase in cortisol levels in fish reared at high stocking densities of 250/m(2) compared to fish reared at control densities of 100 and 50/m(2), in both experimental times, i.e., 7 and 21 days. The high stocking densities were also found to decrease the specific growth rate of fish. Moreover, it was demonstrated that the highest stocking density induced a significant decrease in sole POMC mRNA expression. It is concluded that POMC and cortisol are both involved in the stress response due to high rearing densities, during which cortisol may serve as a negative regulator of POMC.

Role of gonadotropin-releasing hormone (GnRH) in the regulation of gonadal differentiation in the gilthead seabream (Sparus aurata).

It has been proposed that gonadotropin-releasing hormone (GnRH) plays an autocrine/paracrine regulatory role in mammalian and fish ovaries. The marine teleost gilthead seabream is an interesting model since, during the life span of the fish, gonadal tissues develop first as testes, which then regress allowing the development of ovarian follicles. Recent studies carried out in ovaries of the gilthead seabream have demonstrated that various GnRH transcripts as well as GnRH splicing variants are expressed. The mRNA level of several GnRH forms in the female and male areas of the switching gonad, and their possible role in this process, were further investigated. The results here reported show that sGnRH, cGnRH-II, and sbGnRH transcripts are locally expressed during gilthead seabream gonadal differentiation; the expression of the three GnRH forms was found to differ among the morphologically defined areas of the switching gonad, as demonstrated by applying reverse transcription-polymerase chain reaction (RT-PCR), together with in situ hybridization, and semiquantitative PCR analyses. Moreover, the hypothesis that GnRH forms may regulate testicular regression via an apoptotic mechanism was investigated by analyzing the different areas of switching gonads for caspase-3 activity as a measure of apoptosis. Our results showed a marked increase of caspase-3 activity in the area corresponding to the regressing testes in which a significant decrease of testosterone production was also found. The present findings demonstrate that the changes in the endogenous GnRH transcripts could be related with the gonadal differentiation in gilthead seabream, and that exogenous GnRH plays a role by stimulating apoptosis in the degenerating testis.

Changes of gonadal CB1 cannabinoid receptor mRNA in the gilthead seabream, Sparus aurata, during sex reversal.

Two cannabinoid receptor-like genes (CB1-like), named CB1A and CB1B, have been isolated in teleost fish, specifically in the puffer fish, Fugu rubripes. However, information on the physiological roles, such as the control of reproduction and development in fish is still scarce. Therefore, the aim of the present study was to investigate the presence of CB1-like mRNA in the gonads of a marine teleost species, the gilthead seabream, Sparus aurata, a hermaphrodite species in which the gonadal tissues first develop as testes, and then as functional ovary. We isolated an 890 bp fragment (GenBank accession number ); that corresponded to the open reading frame of the teleost CB1 receptor gene, encoding for the central portion of the protein, which was aligned with the other bony fish sequence. Using "in situ" hybridization, CB1-like mRNA was localized in both mature and sex-reversing gonads, and relative changes in CB1-like expression levels were detected through semi-quantitative RT-PCR. In the mature testis and in the testicular part of the sex-reversing gonad, CB1 expression levels were found to be much higher compared to the ovarian portion. This suggests that the CB1 signaling is likely involved in the process of testicular regression of the S. aurata, but its actual role has yet to be determined.

Proopiomelanocortin gene expression and beta-endorphin localization in the pituitary, testis, and epididymis of stallion.

Proopiomelanocortin (POMC) is a precursor protein that contains the sequences of several bioactive peptides including adrenocorticotropin (ACTH), beta-endorphin (beta-EP), and melanocyte-stimulating-hormone (MSH). POMC is synthesized in the pituitary gland, brain, and many peripheral tissues. Immunoreactive POMC-derived peptides as well as POMC-like mRNA have been evidenced in several nonpituitary tissues, thus suggesting that POMC is actively synthesized by these tissues. The present study was aimed at evaluating if also in the case of stallion POMC-derived peptide, beta-EP, is produced locally in the testis, thus playing effects in a paracrine/autocrine fashion. To investigate this hypothesis the POMC gene expression was analyzed using 3' RACE-PCR and Northern Blot approaches in the testis and epididimys of stallion; moreover, immunocytochemical localization for beta-EP was also performed through confocal laser microscopy. The immunofluorescence results showed a positive beta-EP reaction not only in cellular nest of pituitary but also in the testis and genital tract of stallion, which function could be related with sperm mobility. Such role seem not to be no dependent on the peptide synthesized locally, because the molecular biology approach demonstrated the presence of POMC transcript in the pituitary only. In fact the Northern Blot analysis showed the presence of a single POMC transcript in the pituitary while no signal was detected in the testis and epididimys. The same results were obtained by applied 3' RACE-PCR analysis. In conclusion, opioid-derived peptide beta-EP is present in the genital tract of stallion, but is not locally produced as in other mammalian, and nonmammalian models; its possible biological function at testicular level could be linked to a long-loop feed-back mechanisms.

Modulation of vitellogenin synthesis through estrogen receptor beta-1 in goldfish (Carassius auratus) juveniles exposed to 17-beta estradiol and nonylphenol.

Many synthetic chemicals, termed xenoestrogens, have been shown to interact as agonists with the estrogen receptor (ER) to elicit biological responses similar to those of natural hormones. To date, the regulation of vitellogenesis in oviparous vertebrates has been widely used for evaluation of estrogenic effects. Therefore, Carassius auratus juveniles were chosen as a fish model for studying the effects of estradiol-17beta and different concentrations (10(-6) and 10(-7) M) of 4-nonylphenol (4-NP) on the expression of liver ERbeta-1 subtype; plasma vitellogenin and sex steroids (androgens and estradiol-17beta) were also evaluated together with the bioaccumulation process, through mass-spectrometry. C. auratus is a species widespread in the aquatic environment and, on the toxicological point of view, can be considered a good "sentinel" species. Juveniles of goldfish were maintained in tanks with only tap water or water with different concentrations (10(-6) and 10(-7) M) of 4-nonylphenol (4-NP), or 10(-7) M of estradiol-17beta. After 3 weeks of treatment, animals were anesthetized within 5 min after capture, and blood was immediately collected into heparinized syringes by cardiac puncture and stored at -70 degrees C; the gonads were fixed, then frozen and stored at -70 degrees C; the whole fish, liver, and muscle tissues were harvested and immediately stored at -70 degrees C for molecular biology experiments and bioaccumulation measurements. The estrogenic effects of 4-NP were evidenced by the presence of plasma vitellogenin in juveniles exposed both to estradiol-17beta and the two doses of 4-NP; moreover, exposure to 4-NP also increased aromatization of androgens, as suggested by decreasing androgens and increasing estradiol-17beta plasma levels. The changes of these parameters were in agreement with the increasing transcriptional rate of ERbeta-1 mRNA in the liver, demonstrating that both estradiol-17beta and 4-NP modulate the vitellogenin rate through interaction with the ERbeta-1 subtype. The present study also suggests that 4-NP at the concentration of 10(-6) M bioaccumulates in the liver.

Plasma sex steroid and thyroid hormones profile in male water frogs of the Rana esculenta complex from agricultural and pristine areas.

Some chemical compounds used in intensive agriculture have been found to induce estrogenic effects; therefore a histological analysis of the testes and an evaluation of plasma levels of sex steroid, thyroid hormones, and vitellogenin were carried out in adult male water frogs of two coexisting taxa (Rana lessonae and the hemiclonal hybrid Rana esculenta) sampled in agricultural and pristine areas. Differences in seasonal profiles of hormones were found in water frogs living in the agricultural area where the presence of endocrine disrupting compounds was suspected on the basis of a previous study. In R. esculenta, sampled in the pristine area, high androgen levels were found in May; the opposite trend was found for R. esculenta sampled in agricultural areas in which the highest androgen levels were found in September, significantly lower compared with those found in R. esculenta sampled in the pristine area. Low androgen levels were also recorded in R. lessonae males sampled both in pristine and agricultural areas, while the highest levels were found in September. Regarding the trend of estradiol-17beta, an increase of this hormone was found in July both in esculenta and lessonae sampled in the agricultural area, and in the same month an estradiol-17beta peak, even though lower, was also found both in esculenta and lessonae males captured in the pristine area; detectable vitellogenin was found neither in males captured in the agricultural area, nor in those sampled in the pristine one. Moreover, while no significant changes of thyroid hormones were found either in the esculenta or lessonae males sampled in the pristine area, increased T3 and T4 titers were found in July in both esculenta and lessonae captured in the agricultural area. Morphological differences of the testes in males of parental species captured in the agricultural area were also observed. These findings indicate alterations in endocrine and reproductive function in frogs in the agricultural area, that could suggest the presence of endocrine disrupting compounds.

Expression of proopiomelanocortin and its cleavage enzyme genes in Rana esculenta and Xenopus laevis gonads.

Proopiomelanocortin (POMC) is the precursor protein of different hormones and neuropeptides, and the POMC-derived peptides are produced through proteolytic cleavage. Prohormone convertase PC1 and PC2 are enzymes responsible for the cleavage of the POMC prohormone. The coexpression of POMC, PC1, and PC2 genes was previously described in the brain and the pituitary gland of Rana esculenta and Xenopus laevis, but no data are available for the gonad. The present work demonstrates a gonadal POMC convertase gene expression in Rana esculenta and Xenopus laevis.

Apoptosis in sea bream Sparus aurata eggs.

In the present study, the sea-bream Sparus aurata, a pelagic egg spawner, was used as experimental model, in order to establish the occurrence of apoptosis in vertebrates with external reproduction. The same female ovulates floating and nonfloating eggs, but only the former, after fertilization, proceed to embryo development. The eggs were divided into floating and nonfloating and both were analyzed for the presence of several apoptosis markers. The results here reported provide evidence that the nonfloating cells present severe shrinkage and highly express both FAS receptor and FAS ligand on their surface. Furthermore, DNA fragmentation and mitochondria swelling were found, suggesting that the nonfloating eggs were cells programmed to die.

Hormonal mechanisms regulating hepatic vitellogenin synthesis in the gilthead sea bream, Sparus aurata.

Experiments were carried out to study in vitro the effects of 17beta-estradiol (E(2)), homologous pituitary homogenate (HPH), and recombinant red sea bream growth hormone (sbGH) on vitellogenin (VTG) secretion from cultured sea bream liver fragments. Basal secretion of VTG was found to be significantly higher in the prespawning period, compared with sea bream liver in the spawning and postspawning periods. Similarly, the sea bream liver obtained during the prespawning period responded more significantly to treatments with E(2), HPH, or sbGH compared with sea bream liver during spawning. In the postspawning period, treatments with E(2), HPH, or sbGH were without significant effect on VTG secretion level in sea bream liver. The level of E(2) receptors was also analyzed by Western blot analysis. The result demonstrates a significantly higher level of E(2) receptors in the sea bream liver at the prespawning stage compared with those at the spawning and postspawning stages. The findings support the hypothesis that homologous upregulation of estrogen receptors plays an important role in the estrogen-sensitive control of VTG synthesis in the sea bream liver.

Environmental estrogens and reproductive biology in amphibians.

In this paper, the effects of an estrogenic compound, 4-nonyl-phenol (NP), on the amphibians Rana esculenta and Triturus carnifex are described together with those on sexual differentiation in Xenopus laevis. NP increased plasma vitellogenin in male frogs and newts in a dose-related manner; moreover, inhibitory effects on gonadotropin and prolactin (PRL) secretion by pituitary were found together with an elevation of plasma androgens. NP treatment also caused a remarkable increase in number of prolactin-immunolabeled cells, suggesting that xenoestrogen might induce, at least in the newt pituitary, a PRL accumulation possibly due to a reduction of the hormone release. In addition, both NP and bisphenol A caused feminization by increasing the percentage of female phenotypes in X. laevis, and the in vivo effects were more pronounced than those of estradiol-17beta.

Evaluation of ovarian POMC mRNA through quantitative RT-PCR analysis in Rana esculenta.

The evaluation of changes in the expression of specific genes requires accurate measurement of the corresponding mRNA concentration, especially when the gene is expressed at a very low level. We previously showed that the proopiomelanocortin (POMC) gene is expressed in the ovary of the frog Rana esculenta, and, to evaluate its mRNA content in frog ovary, we have now developed a sensitive quantitative RT-PCR method. This study provides evidence for the validation of this method and for the effects of captivity and hypophysectomy on POMC gene expression in the ovary of this anuran. Our data indicate that ovarian POMC gene is involved in short-term captivity stress response and seems not influenced by pituitary. These results are discussed taking into account the knowledge of the role played by opioids in stress response; moreover, a local control of POMC gene expression is also suggested.

Involvement of tyrosine kinase and cAMP in growth hormone-induced vitellogenin synthesis in the anuran, Rana esculenta.

In the frog Rana esculenta, a multihormonal control of vitellogenin synthesis was previously demonstrated. Now in this study, the identity of intracellular second messengers that mediate the GH effects on hepatic VTG synthesis are described. The results clearly indicate that the effect of GH on frog hepatocytes, in vitro, works through a local production of IGF I; in fact, IGF I affects VTG synthesis and its action occurs faster with respect to that of GH. The effect of IGF I was abolished by the anti-estrogen tamoxifen, indicating the involvement of estrogen receptor in VTG induction by IGF I. Furthermore, in vitro treatment of frog hepatocytes with GH resulted in an increase of cAMP with maximum levels after 20 min of treatment. Besides the increase of cAMP, GH induced the appearance of a new phosphotyrosine protein at 20 min, suggesting the occurrence of tyrosine kinase activation. Addition of adenylate cyclase or protein tyrosine kinase inhibitors completely abolished the induction of VTG synthesis, indicating the involvement of cAMP and of a phosphotyrosine protein in VTG synthesis stimulated by both GH and IGF I.

Differential splicing of three gonadotropin-releasing hormone transcripts in the ovary of seabream (Sparus aurata).

Previous studies demonstrated the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the ovary of seabream, Sparus aurata, providing evidence for the role of GnRH as a paracrine/autocrine regulator of ovarian function in this species. In the present study, the expression of three forms of GnRH (salmon, chicken-II, and seabream) genes in this marine teleost species was demonstrated for the first time. Moreover, there is evidence for differential splicing and intronic expression of cGnRH-II and sbGnRH. Treatment of seabream follicle-enclosed oocytes with salmon GnRH stimulated reinitiation of oocyte meiosis, whereas chicken GnRH-II treatment was without effect. Novel information was also provided about organization of cGnRH-II and seabream GnRH transcripts, confirming that GnRH gene organization is maintained through evolution, despite changes in the size and sequence of exons and introns.

Newt prolactin and its involvement in reproduction.

The amino acid sequence of newt (Cynops pyrrhogaster) prolactin deduced from the nucleotide sequence of its cDNA showed a relatively high homology with sequences of chicken and sea turtle prolactins as well as with those of anuran prolactins. Cynops prolactin receptor transcripts were detected in various tissues and organs, suggesting that prolactin plays multiple roles in urodeles. Urodele prolactin was purified from the pituitaries of C. pyrrhogaster. Antiserum against this prolactin was used for radioimmunoassay of plasma prolactin and immunoneutralization experiments. Endogenous prolactin was shown to induce migration to water, courtship behavior, and cessation of spermatocytogenesis in the Cynops newt. The hormone was found to be involved in the development of cloacal glands such as the lateral and abdominal glands, growth of the tail and Mauthner neurons, secretion of oviducal jelly, and enhanced synthesis of a female attracting pheromone (sodefrin), and responsiveness of the olfactory epithelium to sodefrin. In most of these cases, prolactin was found to act synergistically or antagonistically with sex steroids. We also discovered that hypersecretion of prolactin in the newts subjected to cold temperature was induced by hypothalamic stimulation rather than release from hypothalamic inhibition.

Acetyl salmon endorphin-like and interrenal stress response in male gilthead sea bream, Sparus aurata.

The present study investigates the role of melanotrope proopiomelanocortin-derived peptide in the interrenal stress response to different stressors in male gilthead sea bream, Sparus aurata. Plasma cortisol and acetyl salmon endorphin (acetyl s-EP), as well as pituitary acetyl s-EP contents, were measured during two stress paradigms: (a) long-term (1-month) confinement and crowding, and (b) short-term (60-min) confinement, crowding, and manipulation. In addition, naltrexone, a highly specific opioid receptor antagonist, was employed in some experimental groups to evaluate the adaptability of the opioid response to interrenal stress. In the long-term (1-month) confinement and crowding, higher plasma cortisol levels and acetyl s-EP concentrations than in the control group were found. However, although plasma cortisol levels significantly increased in both types of stress paradigm, a significant rise in plasma acetyl s-EP was observed only in the case of confinement plus crowding. These data seem to suggest a direct correlation of acetyl s-EP plasma levels exclusively in cases of specific stress, and support previous observations about the different nature of the pituitary-interrenal stress response in salmonids and in mammals. The results obtained in the short-term (60-min) experiments demonstrate the double activation of both the opioid and corticotrope systems when manipulation plus crowding was applied.

Gilthead seabream (Sparus aurata) vitellogenin: purification, partial characterization, and validation of an enzyme-linked immunosorbent assay (ELISA).

VTG was purified from seabream Sparus aurata plasma by ion exchange chromatography on a DEAE-Sepharose column. The vitellogenin was characterized and its properties were determined. The molecular mass of the native form, obtained by Sephadex G-200 column, was around 450 kDa, whereas an apparent molecular mass of 180 kDa was detected by electrophoresis under denaturing and reducing conditions, suggesting a dimeric form for the native protein. The presence of carbohydrates was determined using concanavalin A, while the presence of phosphate groups was detected by Stains-all, a cationic stain. These data together with the sex specificity, the estrogen inducibility, and the cross-reactivity of the abVTG against the major yolk proteins identifies this protein as vitellogenin. The validated ELISA was used for a rapid and reliable measurement of plasma VTG changes related with those of estradiol-17beta in female broodstock.