RESUMEN
The anti-Müllerian hormone (Amh) is a protein belonging to the TGF-ß superfamily, the function of which has been considered important for male sex differentiation in vertebrates. The Japanese flounder (Paralichthys olivaceus) is a teleost fish that has an XX/XY sex determination system and temperature-dependent sex determination. In this species, amh expression is up-regulated in genetic males and in temperature-induced masculinization during the sex differentiation period. However, to the best of our knowledge, no reports on the Amh receptor (Amhr2) in flounder have been published, and the details of Amh signaling remain unclear. In this study, we produced amhr2-deficient mutants using the CRISPR/Cas9 system and analyzed the gonadal phenotypes and sex-related genes. The results revealed that the gonads of genetically male amhr2 mutants featured typical ovaries, and the sex differentiation-related genes showed a female expression pattern. Thus, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. Moreover, the treatment of genetically male amhr2 mutants with an aromatase inhibitor fadrozole, which inhibits estrogen synthesis, resulted in testicular formation. These results strongly suggest that Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in the flounder.
Asunto(s)
Lenguado , Hormonas Peptídicas , Animales , Masculino , Femenino , Hormona Antimülleriana/genética , Hormona Antimülleriana/metabolismo , Diferenciación Sexual/genética , Lenguado/metabolismo , Gónadas/metabolismo , Hormonas Peptídicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estrógenos/metabolismoRESUMEN
Using a recombinant chimeric single-chain follicle stimulating hormone (FSH), we established a radioimmunoassay (RIA) for red seabream (Pagrus major) FSH (pmFSH) which became a powerful tool for studying reproductive physiology. We studied the profiles in plasma and pituitary concentrations of FSH and luteinizing hormone (LH) during sexual maturation. A pre-established RIA for red seabream LH was used for the LH measurements. The regulation of FSH and LH secretion from the pituitary was investigated using a gonadotropin-releasing hormone analog (GnRHa) in vivo and in vitro. Marked differences in plasma and pituitary FSH levels were observed between males and females; pituitary FSH content in males was much higher than that in females during all seasons, and plasma FSH levels in males were high during the spawning season, whereas those in females were unchanged. In contrast, plasma and pituitary levels of LH were elevated before and during the spawning season in males and females. Injecting or implanting (cholesterol pellet) a GnRHa into adult and juvenile red seabream resulted in significant increases in plasma LH concentrations; however, no significant change was observed in plasma FSH. Moreover, GnRHa stimulated only LH secretion in an in vitro experiment using dispersed pituitary cells. The discrete FSH and LH secretion profiles revealed suggest differential roles for the two gonadotropins during red seabream gametogenesis. In addition, the marked difference in pituitary FSH levels in males and females suggests the relative significance of FSH in male reproduction.
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Hormona Folículo Estimulante/análisis , Hormona Liberadora de Gonadotropina/fisiología , Gonadotropinas/análisis , Gonadotropinas/metabolismo , Dorada/metabolismo , Animales , Femenino , Hormona Folículo Estimulante/sangre , Gametogénesis/fisiología , Hormona Luteinizante/análisis , Hormona Luteinizante/sangre , Masculino , Hipófisis/metabolismo , Radioinmunoensayo/métodos , Dorada/fisiología , Estaciones del Año , Maduración Sexual/fisiologíaRESUMEN
Radiotherapy-related medical accidents are frequently caused by planning problems, excessive irradiation during radiotherapy, or patient movement. This is partly because the local exposure dose cannot be directly monitored during radiotherapy. This article discusses the development of our recent real-time radiation exposure dosimetry system that uses a synthetic ruby for radiation therapy. Background noise was observed before the measurement of the short-term characteristic features. Regarding the relationship between the number of photons and dose rate, using 100 monitor units (MU)/min as the measurement value, the counts decreased by approximately 10% at 600 MU/min. A clear correlation was observed between the MU value and the number of photons (R2 = 0.9987). The coefficient of variation (%CV) was less than ± 1.0% under all the irradiation conditions. Slight differences were observed between the ion chamber and the synthetic ruby dosimeters in the measurement of the percentage depth dose. However, this difference was almost matched by correcting for the Cherenkov light. Although some problems were observed with the synthetic ruby dosimeter system, our results indicate that the developed dosimeter can be used to measure the irradiation dose of patients in real time, with no significant impact on the data, as any effect would be masked by the larger effect of the ruby; however, the impact requires a detailed assessment in the future.
Asunto(s)
Exposición a la Radiación , Planificación de la Radioterapia Asistida por Computador , Humanos , Dosificación Radioterapéutica , Planificación de la Radioterapia Asistida por Computador/métodos , Radiometría/métodos , Fotones/uso terapéutico , RadioterapiaRESUMEN
The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C or 27°C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Lenguado/crecimiento & desarrollo , Células Germinativas/crecimiento & desarrollo , Calor , Meiosis , Caracteres Sexuales , Diferenciación Sexual , Animales , Clonación Molecular , Sistema Enzimático del Citocromo P-450/clasificación , Sistema Enzimático del Citocromo P-450/genética , Femenino , Lenguado/genética , Lenguado/metabolismo , Células Germinativas/efectos de los fármacos , Células Germinativas/enzimología , Hidrocortisona/farmacología , Masculino , Filogenia , ARN Mensajero/biosíntesis , Ácido Retinoico 4-Hidroxilasa , Complejo Sinaptonémico/enzimologíaRESUMEN
Japanese flounder (Paralichthys olivaceus) is an important marine fish species of both fisheries and aquaculture in Northeast Asia. The commercial interest for all-female progenies due to several sex-related traits has prompted basic research on the mechanisms of sex determination in this species. By conducting a linkage analysis of the sex-determining locus, we initially identified 12 microsatellite markers linked to sex in 11 scaffolds, whose localization was restricted to a specific region of linkage group 9. Sequence analysis of this region identified 181 genes based on the UniProt database annotations. Among them, the amh gene was considered a potential candidate for sex determination because this gene is known to have taken over the role of sex determination in many teleosts. An in-depth sequence analysis of both the coding and non-coding regions of amh in XX and XY individuals detected nine SNPs linked with maleness. However, because these substitutions were synonymous, the upstream and downstream regions of amh were also investigated and a male-specific variant with deletions in the promoter region was detected. This truncated Y-specific amh variant was named amhy, and the amh shared by both sexes was named amhx. The association analysis using both females and males of the genotypic sex inferred by the presence/absence of amhy found complete association with phenotypic sex and genotype. Gene expression analysis in larvae derived from a single-pair progeny by quantitative real-time PCR detected amhy transcripts in the larval trunks between 20 and 100 days after hatching only in XY larvae. Localization of amhy by in situ hybridization was detected in presumptive Sertoli cells of XY gonads. Expression of amhx was almost undetectable in both XX and XY genotypes. Loss of Amh function by CRISPR-Cas9 induced male-to-female sex reversal, indicating that this gene was necessary for the masculinization of XY individuals. In conclusion, the complete linkage of amhy with males, its early expression in XY gonads before testicular differentiation, and the induction of sex reversal by loss-of-function mutation support the view that amhy is the sex-determining gene in this species.
RESUMEN
In poikilothermic vertebrates, sex determination is sometimes influenced by environmental factors such as temperature. However, little is known about the molecular mechanisms underlying environmental sex determination. The medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex-reversed into phenotypic males by high water temperature (HT; 32-34 degrees C) treatment during the sex differentiation period. Here we report that cortisol caused female-to-male sex reversal and that metyrapone (an inhibitor of cortisol synthesis) inhibited HT-induced masculinization of XX medaka. HT treatment caused elevation of whole-body levels of cortisol, while metyrapone suppressed the elevation by HT treatment during sexual differentiation. Moreover, cortisol and 33 degrees C treatments inhibited female-type proliferation of germ cells as well as expression of follicle-stimulating hormone receptor (fshr) mRNA in XX medaka during sexual differentiation. These results strongly suggest that HT induces masculinization of XX medaka by elevation of cortisol level, which, in turn, causes suppression of germ cell proliferation and of fshr mRNA expression.
Asunto(s)
Trastornos del Desarrollo Sexual , Calor , Hidrocortisona/metabolismo , Oryzias/fisiología , Sexo , Animales , Antimetabolitos/farmacología , Femenino , Hidrocortisona/fisiología , Masculino , Metirapona/farmacología , Oryzias/genética , Procesos de Determinación del Sexo , Diferenciación Sexual/efectos de los fármacos , Diferenciación Sexual/genética , Diferenciación Sexual/fisiología , Regulación hacia Arriba/fisiología , Cromosoma X/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
OBJECTIVES: Alternative normalization methods were proposed to solve the biased information of SPM in the study of neurodegenerative disease. The objective of this study was to determine the most suitable count normalization method for SPM analysis of a neurodegenerative disease based on the results of different count normalization methods applied on a prepared digital phantom similar to one obtained using fluorodeoxyglucose-positron emission tomography (FDG-PET) data of a brain with a known neurodegenerative condition. METHODS: Digital brain phantoms, mimicking mild and intermediate neurodegenerative disease conditions, were prepared from the FDG-PET data of 11 healthy subjects. SPM analysis was performed on these simulations using different count normalization methods. RESULTS: In the slight-decrease phantom simulation, the Yakushev method correctly visualized wider areas of slightly decreased metabolism with the smallest artifacts of increased metabolism. Other count normalization methods were unable to identify this slightly decreases and produced more artifacts. The intermediate-decreased areas were well visualized by all methods. The areas surrounding the grey matter with the slight decreases were not visualized with the GM and VOI count normalization methods but with the Andersson. The Yakushev method well visualized these areas. Artifacts were present in all methods. When the number of reference area extraction was increased, the Andersson method better-captured the areas with decreased metabolism and reduced the artifacts of increased metabolism. In the Yakushev method, increasing the threshold for the reference area extraction reduced such artifacts. CONCLUSION: The Yakushev method is the most suitable count normalization method for the SPM analysis of neurodegenerative disease.
RESUMEN
In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Atún/fisiología , Animales , Acuicultura/métodos , Femenino , Regulación de la Expresión Génica , Larva , Masculino , Mutagénesis Sitio-Dirigida , Platino (Metal) , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Natación/fisiología , Atún/genéticaRESUMEN
The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES), a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR). Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC) upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP) also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.
Asunto(s)
Oocitos/citología , Oocitos/efectos de los fármacos , Ovulación/efectos de los fármacos , Animales , Dietilestilbestrol/farmacología , Femenino , Hidroxiprogesteronas/farmacología , Oogénesis/efectos de los fármacos , Pez CebraRESUMEN
In vertebrates, sex is normally determined by genotype. However, in poikilothermal vertebrates, including reptiles, amphibians, and fishes, sex determination is greatly influenced by environmental factors, such as temperature. Little is known about the molecular mechanisms underlying environmental sex determination in these species. The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. However, XX flounder can be induced to develop into predominantly either phenotypic females or males, by rearing at 18 or 27 C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that an aromatase inhibitor, an antiestrogen, and 27 C treatments cause masculinization of XX flounder, as well as suppression of mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, estrogen administration completely inhibits masculinization by these treatments, suggesting suppression of cyp19a1 mRNA expression, and the resultant estrogen biosynthesis may trigger masculinization of the XX flounder induced by high water temperature. Here, we demonstrated that cortisol causes female-to-male sex reversal by directly suppressing cyp19a1 mRNA expression via interference with cAMP-mediated activation and that metyrapone (an inhibitor of cortisol synthesis) inhibits 27 C-induced masculinization of XX flounder. Moreover, cortisol concentrations in 27 C-reared juveniles were significantly higher than in 18 C-reared fishes during sexual differentiation. These results strongly suggest that masculinization by high water temperature is ascribable to elevation of cortisol concentration during gonadal sex differentiation in the flounder.
Asunto(s)
Lenguado/fisiología , Hidrocortisona/farmacología , Procesos de Determinación del Sexo , Temperatura , Animales , Aromatasa/genética , Aromatasa/metabolismo , Células Cultivadas , Trastornos del Desarrollo Sexual , Relación Dosis-Respuesta a Droga , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Técnicas de Cultivo de Órganos , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/fisiología , Diferenciación Sexual/fisiología , Razón de MasculinidadRESUMEN
Japanese flounder (Paralichthys olivaceus), a teleost fish that has XX (female)/XY (male) sex determination system, exhibits temperature-dependent sex determination. We have previously shown that high water temperature or an aromatase inhibitor treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that high water temperature treatment suppresses specifically mRNA expression of the forkhead transcription factor gene foxl2, and follicle-stimulating hormone receptor (fshr) in gonads during early sex differentiation. Moreover, transient transfection assay shows that the flounder Foxl2 and cAMP analog can activate the cyp19a1 gene transcription in vitro. These results strongly suggest that FSH signaling and Foxl2 are involved in the transcriptional regulation of cyp19a1 gene during gonadal sex differentiation in Japanese flounder with temperature-dependent sex determination.