RESUMEN
A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.
Asunto(s)
Oncorhynchus mykiss , Oogonios , Espermatogonias , Conservación de Tejido/métodos , Animales , Animales Modificados Genéticamente , Frío , Medios de Cultivo , Femenino , Glutamina , Masculino , SueroRESUMEN
Marine fishes are generally unable to produce sufficient quantities of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) for their normal growth and survival, as the key fatty acid-metabolizing enzymes in the EPA and DHA biosynthetic pathway are limited. It is therefore necessary to supplement cultured marine fish species diets with fish oils in order to supply EPA and DHA. Given that freshwater fishes are capable of synthesizing both EPA and DHA, they presumably express all of the enzymes required for this biosynthetic pathway. Thus, we hypothesize that transgenic marine species carrying these fatty acid-metabolizing enzymes could be reared without the dietary supplementation of fish oil. As the first step toward this goal, we used marine fish, nibe croaker to produce a transgenic line carrying the elongase gene isolated from masu salmon. Fatty acid analysis revealed that the liver EPA (20:5n-3) content in the transgenic fish was lower (3.3% vs. 7.7%). However, docosapentaenoic acid (22:5n-3) content in the transgenic fish was 2.28-fold (4.1% vs. 1.8%) higher than in non-transgenic fish. Further, tetracosapentaenoic acid (24:5n-3) was specifically detected in the transgenic fish. We therefore conclude that the development of transgenic fish lines with these fatty acid-metabolizing enzymes could be a powerful tool for manipulating fatty acid metabolic pathways in fish.
Asunto(s)
Acetiltransferasas/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Ácidos Grasos Insaturados/metabolismo , Perciformes/genética , Salmón/metabolismo , Acetiltransferasas/genética , Animales , Animales Modificados Genéticamente , Vías Biosintéticas , Clonación Molecular , Elongasas de Ácidos Grasos , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Hígado/metabolismo , Perciformes/clasificación , Perciformes/metabolismo , Filogenia , Salmón/genéticaRESUMEN
In this study, we characterized the neuropeptide Y (NPY) mRNA in snakeskin gourami (Trichogaster pectoralis) (TpNPY). TpNPY displayed characteristics typical of previously reported NPYs, and it exhibited a high degree of homology with the NPY proteins of other vertebrates. A phylogenetic analysis demonstrated that TpNPY was closely related to the NPYs found in the acanthomorpha and salmoniformes fish species. TpNPY was found to be ubiquitously expressed in all brain regions when assessed by real-time RT-PCR and in situ hybridization. In addition, a graded expression level of TpNPY was observed in peripheral tissues; for example, a moderate level of TpNPY was found in the gills, liver, kidney, stomach, intestine, spleen and gonads, while a low level of TpNPY was found in the muscle. The change in expression of TpNPY with respect to daily feeding habits was investigated in distinct brain regions, including the telencephalon, mesencephalon, metencephalon, and diencephalon. Fluctuations in the expression level of TpNPY were observed for a 24h post-prandial period. Except for the telencephalon, a reduction in TpNPY expression was found after a meal, while a peak level of TpNPY was observed 1h before the scheduled breakfast. Furthermore, there was a positive correlation between TpNPY and TpMC4R in the telencephalon and diencephalon throughout the circadian feeding cycle, which suggests that there is a connection between the function of NPY and the melanocortin system for the regulation of daily feeding. Fish brains were incubated with an MC4R antagonist (i.e., HS024), and the expression of TpNPY and TpMC4R was measured. Interestingly, there was a significant relationship between the expression of TpNPY and TpMC4R under the effects of HS024, which demonstrates that there are interactions between MC4R and NPY, particularly in a hyperphagic state.
Asunto(s)
Ingestión de Alimentos/fisiología , Neuropéptido Y/fisiología , Perciformes/metabolismo , Receptor de Melanocortina Tipo 4/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Ritmo Circadiano , ADN Complementario/química , Datos de Secuencia Molecular , Neuropéptido Y/biosíntesis , Péptidos Cíclicos/farmacología , Filogenia , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Alineación de Secuencia , Telencéfalo/metabolismoRESUMEN
The influence of 17α-methyltestosterone (MT) on growth responses, biological parameters and the expression of genes involved in the GH-IGF pathway of the hypothalamic-pituitary-liver-gonadal axis were investigated in female, male, and sex-reversed Nile tilapia to evaluate the relationship between sex and MT-induced changes in these parameters. Female fish had a lower growth rate than male and sex-reversed fish, and MT increased growth performance and duodenal villi in females. Most but not all biological parameters of sex-reversed fish were similar to those of male fish. Male fish had higher red blood cell counts and hemoglobin levels than female and sex-reversed fish, suggesting that these hematological indices reflect a higher metabolic rate in male fish. Greater blood triglyceride levels indicated the vitellogenin process in female fish. MT increased the alternative complement activity in female fish (P<0.05). Sex and MT had no significant effects on the hypothalamic mRNAs of GHRH and PACAP. Although not statistically significant, females tended to have higher GH mRNA levels than male and sex-reversed fish. Additionally, MT tended to decrease and increase GH mRNA levels in female and male fish, respectively. There were significant differences among sexes in the expression of GHR, and IGF mRNAs at the peripheral level in the liver and gonads. Females had lower hepatic GHRs and higher ovarian GHRs than male and sex-reversed fish. While the mRNA levels of IGF-1 were lower in the ovary, the levels of IGF-2 were higher compared with those in testes. A significant correlation between GHRs and IGFs was demonstrated in the liver and gonad (except for IGF-1). Multiple regression analysis showed a significant relationship between GH mRNA and both GHRs and IGFs in the liver and gonad. MT exerted androgenic and, to some extent, estrogenic effects on several physiological parameters and GH-IGF action.
Asunto(s)
Cíclidos , Trastornos del Desarrollo Sexual , Gónadas/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Hígado/efectos de los fármacos , Metiltestosterona/farmacología , Maduración Sexual/efectos de los fármacos , Animales , Cíclidos/genética , Cíclidos/metabolismo , Cíclidos/fisiología , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/metabolismo , Trastornos del Desarrollo Sexual/fisiopatología , Trastornos del Desarrollo Sexual/veterinaria , Evaluación Preclínica de Medicamentos , Femenino , Identidad de Género , Gónadas/metabolismo , Gónadas/fisiología , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hígado/metabolismo , Hígado/fisiología , Masculino , Conducta Sexual Animal/efectos de los fármacos , Maduración Sexual/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.
Asunto(s)
Baculoviridae , Bombyx/metabolismo , Anguilas/genética , Gonadotropinas , Proteínas Recombinantes , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Bombyx/crecimiento & desarrollo , Células Cultivadas , Clonación Molecular , Evaluación Preclínica de Medicamentos , Femenino , Vectores Genéticos/administración & dosificación , Gonadotropinas/genética , Gonadotropinas/aislamiento & purificación , Gonadotropinas/metabolismo , Gonadotropinas/farmacología , Larva/metabolismo , Masculino , Modelos Biológicos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Oogénesis/efectos de los fármacos , Oogénesis/fisiología , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Espermatogénesis/efectos de los fármacos , Espermatogénesis/fisiología , Transducción Genética/métodosRESUMEN
Cytochrome P450 aromatase (cyp19) is an enzyme that catalyzes the conversion of androgens to estrogens and may play a role in temperature-dependent sex determination (TSD) of reptiles, amphibians, and fishes. In this study, the ovarian P450 aromatase form (cyp19A1) of pejerrey Odontesthes bonariensis, a teleost with marked TSD, was cloned and its expression profile evaluated during gonadal differentiation at feminizing (17 degrees C, 100% females), mixed-sex producing (24 and 25 degrees C, 73.3 and 26.7% females, respectively), and masculinizing (29 degrees C, 0% females) temperatures. The deduced cyp19A1 amino acid sequence shared high identity (>77.8%) with that from other teleosts but had low identity (<61.8%) with brain forms (cyp19A2), including that of pejerrey itself. The tissue distribution analysis of cyp19A1 mRNA in adult fish revealed high expression in the ovary. Semi-quantitative reverse transcription polymerase chain reaction analysis of the bodies of larvae revealed that cyp19A1 expression increased before the appearance of the first histological signs of ovarian differentiation at the feminizing temperature but remained low at the masculinizing temperature. The expression levels at mixed-sex producing temperatures were bimodal rather than intermediate, showing low and high modal values similar to those at the feminizing and masculinizing temperatures, respectively. The population percentages of high and low expression levels at intermediate temperatures were proportional to the percentage of females and males, respectively, and high levels were first observed at about the time of sex differentiation of females. These results suggest that cyp19A1 is involved in the process of ovarian formation and possibly also in the TSD of pejerrey.
Asunto(s)
Aromatasa/metabolismo , Temperatura Corporal/fisiología , Peces/fisiología , Diferenciación Sexual/fisiología , Secuencia de Aminoácidos , Animales , Aromatasa/genética , Secuencia de Bases , Temperatura Corporal/genética , ADN Complementario/genética , Femenino , Perfilación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Ovario/embriología , Ovario/metabolismo , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Diferenciación Sexual/genética , Razón de Masculinidad , Testículo/embriología , Testículo/metabolismoRESUMEN
Farmed fish could substitute for marine capture fish as a source of fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) beneficial for human health; however, they require these compounds in their diets. In the present study on a model fish species, we modified the EPA/DHA biosynthesis pathway by overexpression of masu salmon Delta5-desaturase-like gene in zebrafish to increase its ability to synthesize EPA and DHA. Expression of this gene in transgenic fish fed a commercial diet and Artemia helped to improve their EPA content by 1.21-fold and DHA by 1.24-fold. In similar fish that were fed only Artemia the increments were 1.14-fold for EPA and 1.13-fold for DHA, compared with nontransgenic fish. In contrast, eicosatetraenoic acid content decreased, as it is a substrate of Delta5-desaturase, while the total lipid remained constant. The results demonstrated that masu salmon Delta5-desaturase is functional in zebrafish and can modify its fatty acid metabolic pathway. The technique could be applied to farmed fish to generate a nutritionally richer product for human consumption.
Asunto(s)
Animales Modificados Genéticamente/fisiología , Ácido Graso Desaturasas/biosíntesis , Ácidos Grasos Omega-3/biosíntesis , Tecnología de Alimentos , Regulación Enzimológica de la Expresión Génica/fisiología , Pez Cebra/fisiología , Actinas/análisis , Alimentación Animal/análisis , Animales , Artemia/química , Peso Corporal/fisiología , Cartilla de ADN , delta-5 Desaturasa de Ácido Graso , Ácidos Docosahexaenoicos/análisis , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/análisis , Ácido Eicosapentaenoico/biosíntesis , Ácido Graso Desaturasas/genética , Ácidos Grasos Omega-3/análisis , Femenino , Lípidos/análisis , Masculino , Oncorhynchus/genética , ARN Mensajero/análisisRESUMEN
Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.