RESUMEN
Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120-kDa band released following gel-into-gel SDS-PAGE of both glycoproteins share the same N-terminal amino acid sequence, which itself is similar to the N-termini of Xenopus liver-synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120-kDa band is part of both gps 200 and 270/260. A 117-kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC-MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120-kDa band and is responsible for the formation of the 200-270-kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti-vitellogenin antibodies label only the surface of the dimple.
Asunto(s)
Anuros/metabolismo , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Interacciones Espermatozoide-Óvulo/fisiología , Secuencia de Aminoácidos , Animales , Anuros/fisiología , Western Blotting , Cromatografía Liquida , Dimerización , Electroforesis en Gel de Poliacrilamida , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Microscopía Electrónica , Datos de Secuencia Molecular , Oocitos/ultraestructura , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Fenoles/farmacología , Pez Cebra/genética , Animales , Disruptores Endocrinos/farmacología , Perfilación de la Expresión Génica , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Contaminantes Químicos del Agua/farmacologíaRESUMEN
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.
Asunto(s)
ADN Complementario/genética , Peces Planos/genética , Receptor Cannabinoide CB1/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Regulación de la Expresión Génica , Hidrocortisona/sangre , Datos de Secuencia Molecular , Ovario/metabolismo , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB1/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Biomarcadores/análisis , Monitoreo del Ambiente , Carpa Dorada/metabolismo , Ríos/química , Contaminación Química del Agua/análisis , Análisis de Varianza , Animales , Glucemia/análisis , Cloruro de Cadmio/metabolismo , Ensayo de Inmunoadsorción Enzimática , Receptor beta de Estrógeno/sangre , Expresión Génica , Glucógeno/metabolismo , Hidrocortisona/sangre , Italia , Hígado/metabolismo , Fenoles/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiempo , Vitelogeninas/sangre , Xenobióticos/análisisRESUMEN
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.
Asunto(s)
Aglomeración/fisiopatología , Peces Planos/crecimiento & desarrollo , Peces Planos/genética , Proopiomelanocortina/genética , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Peso Corporal/fisiología , Clonación Molecular , ADN Complementario/aislamiento & purificación , Peces Planos/sangre , Regulación de la Expresión Génica , Hidrocortisona/sangre , Datos de Secuencia Molecular , Densidad de Población , Proopiomelanocortina/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Organismos Hermafroditas , Receptor Cannabinoide CB1/genética , Dorada/genética , Procesos de Determinación del Sexo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Gónadas/fisiología , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor Cannabinoide CB1/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Dorada/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Genes de Cambio , Hormona Liberadora de Gonadotropina/fisiología , Gónadas/crecimiento & desarrollo , Dorada/crecimiento & desarrollo , Diferenciación Sexual/genética , Empalme Alternativo , Animales , Femenino , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/farmacología , Gónadas/efectos de los fármacos , Gónadas/metabolismo , Masculino , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Dorada/anatomía & histología , Dorada/genética , Testosterona/biosíntesis , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Epidídimo/metabolismo , Caballos/metabolismo , Hipófisis/metabolismo , Proopiomelanocortina/biosíntesis , Proopiomelanocortina/genética , Testículo/metabolismo , betaendorfina/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Humanos , Masculino , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Agroquímicos/envenenamiento , Hormonas Esteroides Gonadales/sangre , Rana esculenta/sangre , Testículo/efectos de los fármacos , Hormonas Tiroideas/sangre , Contaminantes Químicos del Agua/envenenamiento , Agricultura , Andrógenos/sangre , Animales , Estradiol/sangre , Histocitoquímica , Italia , Masculino , Tiroxina/sangre , Triyodotironina/sangre , Vitelogeninas/sangreRESUMEN
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.
Asunto(s)
Estradiol/farmacología , Receptor beta de Estrógeno/metabolismo , Fenoles/farmacología , Vitelogeninas/biosíntesis , Andrógenos/sangre , Animales , Relación Dosis-Respuesta a Droga , Estradiol/sangre , Estradiol/farmacocinética , Receptor beta de Estrógeno/genética , Cromatografía de Gases y Espectrometría de Masas , Carpa Dorada , Hígado/efectos de los fármacos , Hígado/metabolismo , Fenoles/sangre , Fenoles/farmacocinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución Tisular , Vitelogeninas/sangreRESUMEN
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.
Asunto(s)
Ovario/metabolismo , Proopiomelanocortina/biosíntesis , Proproteína Convertasa 1/genética , Proproteína Convertasa 2/genética , Rana esculenta/metabolismo , Testículo/metabolismo , Xenopus laevis/metabolismo , Animales , Femenino , Gónadas/metabolismo , Masculino , Proopiomelanocortina/genética , Proproteína Convertasa 1/biosíntesis , Proproteína Convertasa 2/biosíntesis , Rana esculenta/genética , Xenopus laevis/genéticaRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Oocitos/fisiología , Folículo Ovárico/metabolismo , Óvulo/fisiología , Dorada/fisiología , Animales , Fragmentación del ADN , Proteína Ligando Fas , Femenino , Glicoproteínas de Membrana/metabolismo , Mitocondrias/patología , Folículo Ovárico/citología , Óvulo/ultraestructura , Proteínas/metabolismo , Dorada/anatomía & histología , Receptor fas/metabolismoRESUMEN
The possible effect of proopiomelanocortin-derived peptide, beta-endorphin on frog gonadotrope cells was investigated. Binding and internalization of beta-endorphin to pituitary pars distalis cultured cells were visualized by immunofluorescence and analyzed by means of confocal laser scanning microscopy. Using biotinylated endorphin, the time-course of beta-binding showed that this opioid was internalized through receptor-mediated endocytosis, the mechanism in which actin and clathrin were involved; then, the lysosomal degradation program occurred at later stages. The beta-endorphin binding was well antagonized by Naloxone, the opiate receptor antagonist, and up-regulated since more rapid response was obtained in the previously primed cells. The double immunostaining reaction for beta-endorphin and LH beta-subunit revealed that half the beta-endorphin labeled cell population was positively immunostained for LH beta-subunit, and beta-endorphin was able to induce an increasing trend of LH secretion in cultured pars distalis cells. Therefore, it seems that beta-endorphin acts directly on pituitary pars distalis and influences gonadotropin secretion through the interaction with its own receptor.
Asunto(s)
Hormona Luteinizante/metabolismo , Hipófisis/metabolismo , Rana esculenta/metabolismo , betaendorfina/metabolismo , Actinas/metabolismo , Análisis de Varianza , Animales , Sitios de Unión , Clatrina/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Masculino , Hipófisis/citología , Factores de TiempoRESUMEN
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.
Asunto(s)
Hormonas/fisiología , Hígado/metabolismo , Vitelogeninas/biosíntesis , Animales , Western Blotting , Línea Celular , Medios de Cultivo Condicionados/química , Estradiol/farmacología , Femenino , Hormona del Crecimiento/farmacología , Hormonas/farmacología , Humanos , Técnicas In Vitro , Hígado/efectos de los fármacos , Hipófisis/química , Hormonas Hipofisarias/farmacología , Receptores de Estradiol/análisis , Receptores de Estradiol/metabolismo , Proteínas Recombinantes/farmacología , Dorada , Estaciones del Año , Extractos de Tejidos/farmacologíaRESUMEN
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.
Asunto(s)
Anfibios/fisiología , Exposición a Riesgos Ambientales , Estrógenos no Esteroides/farmacología , Fenoles/farmacología , Animales , Aromatasa/metabolismo , Femenino , Hormonas Esteroides Gonadales/sangre , Gonadotropinas/metabolismo , Hipotálamo/enzimología , Masculino , Prolactina/metabolismo , Rana esculenta/fisiología , Reproducción , Diferenciación Sexual/efectos de los fármacos , Triturus/fisiología , Vitelogeninas/sangre , Xenopus laevis/fisiologíaRESUMEN
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.
Asunto(s)
Ovario/metabolismo , Proopiomelanocortina/genética , ARN Mensajero/genética , Transcripción Genética , Animales , Secuencia de Bases , Femenino , Hormona Folículo Estimulante/farmacología , Técnicas In Vitro , Hormona Luteinizante/farmacología , Datos de Secuencia Molecular , Hipófisis , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Rana catesbeiana , Rana esculenta , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Extractos de Tejidos/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
In pelagic egg spawners, the production of large numbers of sinking eggs, unable to develop into embryos, represents one of the major limiting factors in controlled reproduction. The aim of this study is to elucidate the molecular differences between floating and nonfloating eggs at cytoplasmic and nuclear level. Comparison of analyses between floating and nonfloating sea bream Sparus aurata eggs evidenced differences in vitelline envelope protein components, such differences being probably related with the hydration process but not with fertilization as supported by the assessment of DNA that doubled after in vitro insemination. These data clearly indicated that the absence of embryo development in nonfloating eggs is not due to lack of fertilization. The cytoplasmic composition was also different, the number of protein components being higher in floating eggs, and these extra components may generate the appropriate osmotic pressure at the base of the hydration process. Some lysosomal enzymes, such as cathepsin D and L both involved in yolk proteolysis, in virgin nonfloating eggs were significantly higher with respect to floating ones; the levels of these two enzymes significantly increased in the latter after fertilization. On the contrary, in nonfloating eggs cathepsin L significantly decreased after fertilization. These changes may be related with a series of metabolic processes vital for the production of viable offspring. The capacity of egg transcription and the protein synthesis in these two types of eggs, indicated by the RNA/DNA and RNA/protein ratios, evidenced that the status of cell transcription rate and protein synthesis capacity is significantly higher in floating eggs. This, in turn, suggested that the lack of embryo development may be due to low levels of proteins involved in cell cycle regulation.
Asunto(s)
Proteínas del Huevo/análisis , Óvulo/química , Óvulo/fisiología , Dorada/fisiología , Animales , Western Blotting , ADN/análisis , Electroforesis en Gel de Poliacrilamida , Femenino , Fertilización , ARN/análisisRESUMEN
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.
Asunto(s)
AMP Cíclico/metabolismo , Hormona del Crecimiento/farmacología , Proteínas Tirosina Quinasas/metabolismo , Rana esculenta/metabolismo , Vitelogeninas/biosíntesis , AnimalesRESUMEN
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.
Asunto(s)
Hormona Liberadora de Gonadotropina/genética , Ovario/fisiología , Perciformes/fisiología , Empalme del ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Femenino , Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/farmacología , Meiosis , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Especificidad de la Especie , Transcripción GenéticaRESUMEN
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.