RESUMO
Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.
Assuntos
Reabsorção Óssea/prevenção & controle , Melatonina/uso terapêutico , Voo Espacial , Animais , Densidade Óssea/efeitos dos fármacos , Calcitonina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Carpa Dourada , Imuno-Histoquímica , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Ausência de Peso/efeitos adversosRESUMO
BACKGROUND: Type and volume of infant feeding determines infant growth, hematological parameters, and serum lipids. METHODS: Study subjects consisted of 103 infants who were born vaginally at term, with birthweight >2200 g. Milk feeding amount, type, and anthropometry were measured at 1 and 6 months. Hematological tests and serum lipid profile were assessed at 1 and 6 months. Thirty-four infants were breast-fed and 36 were formula-fed at 6 months. RESULTS: Breast-fed infants demonstrated similar growth patterns compared with partially breast-fed or formula-fed infants, despite the lower milk intake. Infants with higher breast milk intake at 6 months, however, tended to have lower hemoglobin levels. In contrast, higher formula intake at 6 months was related to lower serum total cholesterol. CONCLUSIONS: Japanese breast-fed infants were more likely to be anemic at 6 months, while formula-fed infants were likely to have low serum lipid levels. Iron-fortified infant foods may be useful to prevent anemia in breast-fed infants. Fat quality of infant formulas should be improved to enhance lipid status of formula-fed infants.
Assuntos
Anemia/epidemiologia , Aleitamento Materno , Desenvolvimento Infantil/fisiologia , Ingestão de Energia/fisiologia , Alimentos Infantis , Lipídeos/sangue , Leite , Anemia/sangue , Anemia/prevenção & controle , Animais , Peso Corporal , Feminino , Seguimentos , Humanos , Recém-Nascido , Japão/epidemiologia , Masculino , Prevalência , Estudos RetrospectivosRESUMO
A worldwide decline in the number of wild salmonids calls for strategies to restore endangered populations. Here we show that germ cells can be transplanted between two different salmonid species, with the subsequent production of xenogenic, donor-derived offspring. This pioneering xenotransplantation technology may eventually find applications in facilitating the production of commercially valuable fish, as well as in species conservation.
Assuntos
Células Germinativas/transplante , Oncorhynchus/fisiologia , Reprodução/fisiologia , Animais , Animais Geneticamente Modificados , Conservação dos Recursos Naturais/métodos , Feminino , Células Germinativas/citologia , Masculino , Oncorhynchus/anatomia & histologia , Oncorhynchus/embriologia , Oncorhynchus/genética , Oncorhynchus mykiss/anatomia & histologia , Oncorhynchus mykiss/embriologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/fisiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Transplante HeterólogoRESUMO
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.
Assuntos
Animais Geneticamente Modificados/fisiologia , Ácidos Graxos Dessaturases/biossíntese , Ácidos Graxos Ômega-3/biossíntese , Tecnologia de Alimentos , Regulação Enzimológica da Expressão Gênica/fisiologia , Peixe-Zebra/fisiologia , Actinas/análise , Ração Animal/análise , Animais , Artemia/química , Peso Corporal/fisiologia , Primers do DNA , Dessaturase de Ácido Graxo Delta-5 , Ácidos Docosa-Hexaenoicos/análise , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/análise , Ácido Eicosapentaenoico/biossíntese , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-3/análise , Feminino , Lipídeos/análise , Masculino , Oncorhynchus/genética , RNA Mensageiro/análiseRESUMO
BACKGROUND: Preemptive therapy against cytomegalovirus (CMV) disease has succeeded in reducing the incidence of CMV disease, but the toxicity of ganciclovir remains problematic. METHODS: We prospectively evaluated the efficacy and toxicity of a preemptive protocol with ganciclovir at a reduced initial dose in 40 patients who achieved engraftment after allogeneic hematopoietic stem cell transplantation. RESULTS: Twenty-three (58%) patients had high-risk features, including transplant from an HLA-mismatched or unrelated donor, or associated acute graft-versus-host disease. CMV antigenemia assay was performed weekly, and ganciclovir was started in a risk-adapted manner, in which the initial dose of ganciclovir was fixed at 5 mg/kg/d and then adjusted based on the results of a weekly CMV antigenemia assay. In this protocol, 23 (58%) patients demonstrated positive antigenemia, and 19 (48%) received a preemptive administration of ganciclovir. Only one patient had CMV disease in the gastrointestinal system, which was successfully treated with a regular therapeutic dose of ganciclovir. Consequently, the total dose of ganciclovir was significantly less than that in a previous protocol using the conventional double dose (5 mg/kg twice daily) of ganciclovir (134 mg/kg vs. 190 mg/kg on average, P=0.046). There were no significant toxicities attributed to ganciclovir, except for neutropenia <0.5 x 109/L, which developed in three patients for 3, 4, and 14 days, respectively, with granulocyte colony-stimulating factor support. CONCLUSION: Preemptive therapy with a low initial dose of ganciclovir appeared to be effective even in high-risk patients. Further randomized controlled trial is warranted.
Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/prevenção & controle , Ganciclovir/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Adulto , Idoso , Antígenos Virais/sangue , Infecções por Citomegalovirus/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/prevenção & controle , Estudos Prospectivos , Transplante HomólogoRESUMO
BACKGROUND: Reactivation of chronic hepatitis B virus (HBV) infection is a major complication when HBV carriers receive immunosuppressive therapy. Recipients of allogeneic hematopoietic stem cell transplantation (HSCT) carry the highest risk of fatal HBV disease (up to 12%). METHODS: In an attempt to identify a suitable procedure for the prevention and management of HBV reactivation, the administration of lamivudine over the course was tested in two patients. RESULTS: Generally, the patients transplant courses were successfully managed despite their difficult clinical situations: a high HBV load before transplant in one patient and intense steroid therapy for complicated acute graft-versus-host disease (GVHD) in the other patient. However, one patient showed a reactivation of HBV after discontinuing lamivudine and the other showed persistently high DNA polymerase activity despite prolonged administration of lamivudine. CONCLUSIONS: We concluded that lamivudine could have a place in the management of patients who suffer from chronic HBV infection and who are undergoing allogeneic HSCT. However, the efficacy of lamivudine seemed to be limited compared with other settings, including solid organ transplantation and autologous HSCT.
Assuntos
Antivirais/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Hepatite B Crônica/tratamento farmacológico , Lamivudina/uso terapêutico , Adulto , Medula Óssea/efeitos dos fármacos , DNA Viral/sangue , Feminino , Humanos , Lamivudina/efeitos adversos , Transplante HomólogoRESUMO
We previously reported that characteristic deformities were induced by retinoic acid (RA) treatment of the Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching (dph). To evaluate the toxic potency of nuclear retinoid receptors in induction of deformities by RA, we here investigated the effects of retinoic acid isomers on postembryonic development of this species. Larvae were exposed to either 25 nM of all-trans RA (atRA), 9-cis RA (9cRA) or 13-cis RA (13cRA) at 6-9 dph. All RA isomers induced deformities in the lower jaw, caudal fin and vertebrae. In the lower jaw, growth retardation of the dentary was evident. In the vertebrae, the major abnormalities were hypertrophy of the centrum, central fusion, and an increase in the number of abdominal vertebrae. Caudal fin deformities included deformity of caudal bone complex and absence of the entire caudal fin. The absence of the hypural primordium at 12 dph was the first sign of abnormality in caudal fin development, and resulted in complete blocking of the caudal fin development. Among the RA isomers, atRA induced the most severe deformity in all skeletons examined. Retinoic acid receptor (RAR) expression was activated by atRA and 9cRA, and pitx2 expression was inhibited in the lower jaw by atRA and 9cRA. Vitamin D receptor (VDR) expression was specifically inhibited by atRA treatment, suggesting that RA inhibits the lower jaw growth by suppressing the expression of these genes. These results suggest that RA exerted toxic effects on the skeletal systems, mainly through the RAR pathway.
Assuntos
Linguado/anormalidades , Linguado/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Tretinoína/análogos & derivados , Tretinoína/toxicidade , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo/efeitos dos fármacos , Isomerismo , Japão , Anormalidades Maxilomandibulares/induzido quimicamente , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coluna Vertebral/anormalidades , Coluna Vertebral/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tretinoína/químicaRESUMO
Gene knock-down technology using antisense molecules has many applications for studying gene function, disrupting undesirable genetic traits, as well as providing effective therapy for a number of viral diseases. Encouraged by these applications, we developed a gene knock-down technique to interfere with gene expression using transgenic rainbow trout expressing the green fluorescent protein (GFP) gene as a model. One of the antisense morpholino phosphorodiamidate oligonucleotides (AMOs) used in this study (AtGFP-1) was 25 nucleotides in length and localized against codons 2 to 8 of GFP messenger RNA. Microinjection of AtGFP-1 into the blastodisc of fertilized eggs decreased the level of GFP gene expression in a dose-dependent manner, A comparison of the effects of various doses of AtGFP-1 suggested that 10 ng of AtGFP-1 was the optimal concentration in that it interfered with specific gene expression without being strongly toxic to trout embryos. Conversely, morpholino phosphorodiamidate oligonucleotides with the inverted AtGFP-1 sequence, which cannot bind to the target mRNA, did not inhibit GFP gene expression. AtGFP-1 did not affect the expression of nontargeted genes such as the skeletal muscle actin and foreign lacZ genes. These results also indicate that AtGFP-1 interfered with the expression of only the targeted gene. Western blot and reverse transcriptase polymerase chain reaction analyses revealed that the amount of GFP protein drastically decreased whereas the mRNA level was not affected by AtGFP-1, suggesting that AtGFP-1 blocked specific gene function at the translational level. Further, this gene inhibition persisted until the hatching stage. Another AMO, which was localized against the junction region between the 5? untranslated region and the starting codon of GFP mRNA (AtGFP-2), also caused inhibition effects. Thus AMOs can have potent and specific gene knock-down effects in trout embryos. This technology may be useful for examining the roles of selected genes and disrupting their expression during embryonic development of salmonid fish.
RESUMO
OBJECTIVE: To investigate how the increase in survival rate in extremely low birth weight (a birth weight of 1,000 g or less) infants had affected the incidence of retinopathy of prematurity (ROP) and the frequency of laser treatment. METHODS: We retrospectively reviewed the medical records of 122 surviving premature infants with birthweights less than 1,000 g to determine the severity of ROP observed at 16 neonatal intensive care units in Tokyo between April and October 2002. RESULTS: The survival rate was 85.6%. The mean gestational age was 26.74 weeks and the mean birth weight was 782.25 g. One-hundred-and-five infants (86.1%) developed ROP, fifty (41.0%) received laser treatment, and six (4.9%) had retinal detachment. The median postmenstrual age (gestational age at birth plus chronological age in weeks, PMA) at the onset of ROP was 32.5 weeks, and the first laser treatment was performed at the median PMA of 35.7 weeks. CONCLUSIONS: In these extremely low birth weight infants, there was an increase in the survival rate and in the incidence of severe ROP that progressed to the stage that required treatment.
Assuntos
Recém-Nascido de muito Baixo Peso , Retinopatia da Prematuridade/epidemiologia , Peso ao Nascer , Idade Gestacional , Humanos , Incidência , Recém-Nascido , Unidades de Terapia Intensiva Neonatal/estatística & dados numéricos , Terapia a Laser , Retinopatia da Prematuridade/terapia , Estudos Retrospectivos , Taxa de Sobrevida , Fatores de Tempo , Tóquio/epidemiologiaRESUMO
Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18alpha-glycyrrhetinic acid (alpha-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20beta-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Junções Comunicantes/fisiologia , Oócitos/fisiologia , Oogênese/fisiologia , Osmeriformes/fisiologia , Animais , Carbenoxolona/farmacologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Junções Comunicantes/efeitos dos fármacos , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Hormônios de Invertebrado/farmacologia , Isoquinolinas/farmacologia , Hormônio Luteinizante/farmacologia , Meiose/efeitos dos fármacos , Modelos Biológicos , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimentoRESUMO
Our understanding of the molecular mechanisms of primordial germ cell (PGC) proliferation in fish is rudimentary, but it is thought to be controlled by the surrounding somatic cells. We assumed that growth factors that are specifically involved in PGC proliferation are expressed predominantly in the surrounding genital ridge somatic cells. In order to isolate these growth factors, we compiled a complementary DNA (cDNA) subtractive library using cDNA from the genital ridges of 40-dpf rainbow trout embryos as the tester and cDNA from embryos without genital ridges as the driver. This approach identified a novel cytokine, designated gonadal soma-derived growth factor (GSDF), which is a member of the transforming growth factor (TGF)-beta superfamily. GSDF was expressed in the genital ridge somatic cells surrounding the PGCs during embryogenesis, and in both the granulosa and Sertoli cells at later stages. Inhibition of GSDF translation by antisense oligonucleotides suppressed PGC proliferation. Moreover, isolated testicular cells that were cultured with recombinant GSDF demonstrated dose-dependent proliferation of type-A spermatogonia; this effect was completely blocked by antiserum against GSDF. These results denote that GSDF, a novel member of the TGF-beta superfamily, plays an important role for proliferation of PGC and spermatogonia.
Assuntos
Células Germinativas/metabolismo , Oncorhynchus mykiss/embriologia , Espermatogônias/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Sequência de Bases , DNA Complementar , Células Germinativas/citologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , FilogeniaRESUMO
An increasing number of wild fish species are in danger of extinction, often as a result of human activities. The cryopreservation of gametes and embryos has great potential for maintaining and restoring threatened species. The conservation of both paternal and maternal genetic information is essential. However, although this technique has been successfully applied to the spermatozoa of many fish species, reliable methods are lacking for the long-term preservation of fish eggs and embryos. Here, we describe a protocol for use with rainbow trout (Oncorhynchus mykiss) primordial germ cells (PGCs) and document the restoration of live fish from gametes derived from these cryopreserved progenitors. Genital ridges (GRs), which are embryonic tissues containing PGCs, were successfully cryopreserved in a medium containing 1.8 M ethylene glycol (EG). The thawed PGCs that were transplanted into the peritoneal cavities of allogenic trout hatchlings differentiated into mature spermatozoa and eggs in the recipient gonads. Furthermore, the fertilization of eggs derived from cryopreserved PGCs by cryopreserved spermatozoa resulted in the development of fertile F1 fish. This PGC cryopreservation technique represents a promising tool in efforts to save threatened fish species. Moreover, this approach has significant potential for maintaining domesticated fish strains carrying commercially valuable traits for aquaculture purposes.
Assuntos
Criopreservação , Oncorhynchus mykiss/crescimento & desenvolvimento , Óvulo/fisiologia , Espermatozoides/fisiologia , Animais , Animais Geneticamente Modificados , Sobrevivência Celular , Transplante de Células , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Masculino , Oncorhynchus mykiss/embriologia , Óvulo/citologia , Espermatozoides/citologiaRESUMO
The post-vitellogenic oocytes of teleost fish are generally unresponsive to maturation-inducing hormone (MIH) until a luteinizing hormone (LH) surge stimulates sensitivity via the acquisition of oocyte-maturational competence (OMC). Heterologous gap junctions (GJs) between granulosa cells and the oocyte have been previously implicated in the regulation of oocyte maturation in various vertebrate species. Although heterologous GJ are present in ovarian follicles of ayu (Plecoglossus altivelis), their role in maturation remains unclear. In the present study, we cloned and characterized complementary DNAs for GJ protein connexin (Cx), and examined the expression pattern of Cx messenger RNAs in the ayu ovary. Four Cx cDNAs with predicted molecular masses of 32.1 (Cx32.1), 34.9 (Cx34.9), 44.1 (Cx44.1), and 44.2 (Cx44.2) kDa, respectively, were cloned. Northern blot analysis revealed that the levels of Cx44.1 and Cx44.2 transcripts were similar during the vitellogenic and ovulatory stages. Cx32.1 transcripts were more abundant during the vitellogenic stage, but their levels declined thereafter. Cx34.9 transcript levels increased during the vitellogenic stage and remained high during the acquisition of OMC. In situ hybridization revealed that Cx44.1 and Cx44.2 signals were restricted to the oocyte, whereas the Cx32.1 and Cx34.9 signals were detected in both cellular fractions. Furthermore, a dye-transfer assay revealed the presence of functional GJs between the oocytes and follicle cells. These results suggest that Cx34.9 contributes to the formation of heterologous GJs between oocytes and granulosa cells. Moreover, GJs formed by Cx34.9 may function during the LH-dependent acquisition of OMC and the MIH-dependent resumption of meiosis in ayu.
Assuntos
Conexinas/metabolismo , Oogênese , Osmeriformes/metabolismo , Ovário/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Clonagem Molecular , Conexinas/análise , Conexinas/genética , DNA Complementar/genética , Feminino , Junções Comunicantes/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/química , Ovário/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Estações do Ano , Transcrição GênicaRESUMO
Understanding the mechanisms that regulate germ-cell development is crucial to reproductive medicine and animal production. Animal gametes originally derive from sexually undifferentiated primordial germ cells (PGCs), which develop into mitotic germ cells (oogonia or spermatogonia) before proceeding to meiosis [Wylie, C. (1999) Cell 96, 165-174]. Spermatogonia are thought to include a population of cells with stem cell activity, which proliferate throughout the lifespan of male animals and produce spermatozoa [Zhao, G. Q. & Garbers, D. L. (2002) Dev. Cell 2, 537-547]. However, the functional differences between PGCs and spermatogonial stem cells are poorly understood. Here we show that transplanted adult testicular germ cells can colonize sexually undifferentiated embryonic gonads and resume gametogenesis. Testicular germ cells containing spermatogonial stem cells isolated from adult male rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of newly hatched embryos of both sexes, and the behavior of the donor cells was observed. The testicular germ cells differentiated into spermatozoa in male recipients and fully functional eggs in female recipients. Furthermore, the donor-derived spermatozoa and eggs obtained from the recipient fish were able to produce normal offspring. These findings indicate that fish testicular germ cells, probably spermatogonial stem cells, possess a high level of developmental plasticity and sexual bipotency, even after the animal reaches maturity. Furthermore, our results suggest that spermatogonial stem cells are at least partly functionally similar to PGCs.
Assuntos
Oncorhynchus mykiss/fisiologia , Oogênese , Espermatogônias/transplante , Animais , Diferenciação Celular , Feminino , Masculino , Oncorhynchus mykiss/embriologia , Espermatogênese , Espermatozoides/citologia , Espermatozoides/fisiologia , Transplante de Células-Tronco , Testículo/citologia , Testículo/embriologiaRESUMO
We report an effective gene knockdown technique in rainbow trout embryos using additional RNA components combined with ribozymes (R(z)s). Chimeric R(z)s (tR(z)Cs) containing tRNA(Val), R(z) against GFP, and a constitutive transport element were microinjected into transgenic embryos. tR(z)Cs induced greater gene interference than R(z)s alone. Control tR(z)Cs did not affect unpaired bases of target RNA, and the tR(z)C did not interfere with non-relevant gene expression, suggesting that the tR(z)C-mediated gene-interference effects were sequence-specific. Furthermore, the tR(z)C-containing expression vector specifically suppressed target GFP expression in transgenic trout. tR(z)Cs enhance R(z) cleavage and could therefore be powerful tools for studying unknown gene function in vertebrates.
Assuntos
Animais Geneticamente Modificados/embriologia , Peixes/embriologia , Peixes/genética , Inativação Gênica , Engenharia Genética/métodos , RNA Catalítico/genética , Animais , Animais Geneticamente Modificados/genética , Quimera/embriologia , Quimera/genéticaRESUMO
The n - 3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have important nutritional benefits in humans. Farmed fish could serve as promising sources of EPA/DHA, but they need these fatty acids or their precursors in their diets. Here we transferred masu salmon delta6-desaturase-like gene in zebrafish to increase its ability for synthesizing EPA and DHA. Expression of this gene in transgenic fish elevated their EPA content by 1.4-fold and DHA by 2.1-fold. On the other hand, the alpha-linolenic acid (ALA) content decreased, it being a substrate of delta6-desaturase, while the total lipid remained constant. This achievement demonstrates that fatty acid metabolic pathway in fish can be modified by the transgenic technique, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food.
Assuntos
Animais Geneticamente Modificados , Ácidos Docosa-Hexaenoicos , Ácido Eicosapentaenoico/biossíntese , Ácido Eicosapentaenoico/genética , Ácidos Graxos Dessaturases/genética , Salmão/genética , Animais , Aquicultura , Ácidos Graxos Dessaturases/biossíntese , Feminino , Perfilação da Expressão Gênica , Linoleoil-CoA Desaturase , Masculino , Estado Nutricional , Peixe-Zebra/genéticaRESUMO
Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.
Assuntos
Células Germinativas/metabolismo , Células Germinativas/transplante , Proteínas de Fluorescência Verde/metabolismo , Salmonidae/fisiologia , Regiões 3' não Traduzidas/genética , Animais , Biotecnologia/métodos , Conservação dos Recursos Naturais/métodos , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Perciformes , RNA/biossíntese , RNA/genética , Salmonidae/genética , Salmonidae/metabolismo , Quimeras de Transplante/genética , Quimeras de Transplante/fisiologia , Peixe-ZebraRESUMO
We report on segmental and full paternal isodisomy for chromosome 14 in three previously unreported Japanese patients. Patient 1 was a 5(6/12)-year-old girl, Patient 2 was a male neonate, and Patient 3 was a -year-old girl. Physical examination at birth showed various somatic features characteristic of paternal uniparental disomy for chromosome 14 (upd(14)pat) such as hairy forehead, protruding philtrum, micrognathia, small thorax, and abdominal wall defects in Patients 1-3, and the constellation of somatic features was persistently observed in Patients 1 and 3. Radiological studies at birth delineated unique bell-shaped thorax with coat-hanger appearance of the ribs in Patients 1-3, but the thoracic deformity ameliorated in Patients 1 and 3 by mid childhood. Chromosome analysis showed a 46,XX karyotype in Patients 1 and 3 and was not performed in Patient 2. Microsatellite analysis indicated full paternal isodisomy for chromosome 14 in Patients 1 and 2 and segmental paternal isodisomy for chromosome 14 distal to D14S981 at 14q23.3 in Patient 3. Methylation specific PCR assay for the differentially methylated region (DMR) of GTL2 at 14q32 yielded positive products with methylated allele specific primers and no products with unmethylated allele specific primers in Patients 1-3. Since clinical phenotype was similar between Patient 3 with segmental upd(14)pat and Patients 1 and 2 with full upd(14)pat, the results are keeping with the 14q32 localized imprinted genes as the critical components of the phenotype observed in upd(14)pat and help narrow the search for additional genes to the approximately 40 Mb region distal to D14S981. Furthermore, it is likely that the characteristic thoracic deformity ameliorates with age.
Assuntos
Cromossomos Humanos Par 14/genética , Dissomia Uniparental , Criança , Bandeamento Cromossômico , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/patologia , Metilação de DNA , Evolução Fatal , Feminino , Humanos , Recém-Nascido , Cariotipagem , Masculino , Repetições de Microssatélites , Proteínas/genética , RNA Longo não CodificanteRESUMO
Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.
Assuntos
Peixes/genética , Inativação Gênica , RNA Interferente Pequeno/fisiologia , Animais , Sequência de Bases , Primers do DNA , Peixes/embriologia , Monofenol Mono-Oxigenase/genética , TransgenesRESUMO
Germ cell transplantation has tremendous applications in transgenic animal production, assisted reproductive technology, and germline stem cell research. Here, we report for the first time the production of individuals from intraperitoneally transplanted primordial germ cells (PGCs) in animals. To trace the behavior of exogenous PGCs in recipients, PGCs visualized by a green fluorescent protein gene were used as donors. The PGCs prepared from the genital ridges of hatching embryos were transplanted into recipients at various developmental stages. The PGCs injected into the peritoneal cavities of hatching embryos had the ability to migrate toward, and to colonize, the genital ridges of recipient embryos. Furthermore, donor-derived PGCs proliferated and differentiated into mature eggs and sperm in the allogenic gonads; the resulting gametes produced live fry, showing the donor-derived phenotype, through fertilization. Combined with in vitro culture, genetic modification, and cryopreservation of PGCs, this technique provides new approaches for fish bioengineering.