RESUMEN
The oestrogen receptor-related receptors (ERRs) are orphan nuclear receptors that were originally identified on the basis of their close homology to the oestrogen receptors. The three mammalian ERR genes participate in the regulation of vital physiological processes including reproduction, development and metabolic homeostasis. Although unique ERRs have been found in insects, data on the function and regulation of these receptors remain sparse. In the present study, a 2095-bp full-length cDNA encoding an ERR, termed AiERR, was isolated from males of the moth Agrotis ipsilon and deposited in the GenBank database under the accession number KT944662. The predicted AiERR protein shared an overall identity of 47-82% with other known insect and mammalian ERR homologues. AiERR exhibited a broad tissue expression pattern with the detection of one transcript of approximately 2 kb in the primary olfactory centres, the antennal lobes (AL). In adult males, the amount of AiERR mRNA in the AL increased concomitantly with age and responses to the female-emitted sex pheromone. Moreover, AiERR knockdown induced an inhibition in the sex pheromone-orientated flight of male. Using A. ipsilon as a model, our study demonstrates that the insect ERR is critical for the performance of male sexual behaviour, probably by acting on central pheromone processing.
Asunto(s)
Proteínas de Insectos/metabolismo , Mariposas Nocturnas/metabolismo , Receptores de Estrógenos/metabolismo , Atractivos Sexuales/fisiología , Conducta Sexual Animal/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Nervioso Central/metabolismo , Técnicas de Silenciamiento del Gen , Masculino , Datos de Secuencia MolecularRESUMEN
Uridine diphosphate UDP-glycosyltransferases (UGTs) are detoxification enzymes widely distributed within living organisms. They are involved in the biotransformation of various lipophilic endogenous compounds and xenobiotics, including odorants. Several UGTs have been reported in the olfactory organs of mammals and involved in olfactory processing and detoxification within the olfactory mucosa but, in insects, this enzyme family is still poorly studied. Despite recent transcriptomic analyses, the diversity of antennal UGTs in insects has not been investigated. To date, only three UGT cDNAs have been shown to be expressed in insect olfactory organs. In the present study, we report the identification of eleven putative UGTs expressed in the antennae of the model pest insect Spodoptera littoralis. Phylogenetic analysis revealed that these UGTs belong to five different families, highlighting their structural diversity. In addition, two genes, UGT40R3 and UGT46A6, were either specifically expressed or overexpressed in the antennae, suggesting specific roles in this sensory organ. Exposure of male moths to the sex pheromone and to a plant odorant differentially downregulated the transcription levels of these two genes, revealing for the first time the regulation of insect UGTs by odorant exposure. Moreover, the specific antennal gene UGT46A6 was upregulated by insecticide topical application on antennae, suggesting its role in the protection of the olfactory organ towards xenobiotics. This work highlights the structural and functional diversity of UGTs within this highly specialized tissue.
Asunto(s)
Antenas de Artrópodos/enzimología , Glicosiltransferasas/genética , Spodoptera/enzimología , Spodoptera/genética , Uridina Difosfato/genética , Xenobióticos/metabolismo , Secuencia de Aminoácidos , Animales , Etiquetas de Secuencia Expresada/química , Femenino , Regulación de la Expresión Génica , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Cinética , Masculino , Datos de Secuencia Molecular , Odorantes , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Spodoptera/metabolismo , Uridina Difosfato/química , Uridina Difosfato/metabolismoRESUMEN
Cytochrome P450 enzymes (P450s) are involved in many physiological functions in insects, such as the metabolism of signal molecules, adaptation to host plants and insecticide resistance. Several P450s have been reported in the olfactory organs of insects, the antennae, and have been proposed to play a role in odorant processing and/or xenobiotic metabolism. Despite recent transcriptomic analyses in several species, the diversity of antennal P450s in insects has not yet been investigated. Here, we report the identification of 37 putative P450s expressed in the antennae of the pest moth Spodoptera littoralis, as well as the characterization of a redox partner, cytochrome P450 reductase (CPR). Phylogenetic analysis revealed that S. littoralis P450s belong to four clades defined by their conservation with vertebrate P450s and their cellular localization. Interestingly, the CYP3 and CYP4 clans, which have been described to be mainly involved in the metabolism of plant compounds and xenobiotics, were largely predominant. More surprisingly, two P450s related to ecdysteroid metabolism were also identified. Expression patterns in adult and larval tissues were studied. Eight P450s appeared to be specific to the chemosensory organs, ie the antennae and proboscis, suggesting a specific role in odorant and tastant processing. Moreover, exposure of males to a plant odorant down-regulated the transcript level of CPR, revealing for the first time the regulation of this gene by odorants within insect antennae. This work suggests that the antennae of insects are a key site for P450-mediated metabolism of a large range of exogenous and endogenous molecules.
Asunto(s)
Antenas de Artrópodos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Insectos/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Spodoptera/enzimología , Animales , Secuencia de Bases , Femenino , Larva/metabolismo , Masculino , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , OlfatoRESUMEN
In the male moth Agrotis ipsilon behavioural response and antennal lobe (AL) neuron sensitivity to the female-produced sex pheromone increase with age and juvenile hormone (JH) level. We recently showed that the neuromodulator, octopamine (OA), interacts with JH in this age-dependent olfactory plasticity. To further elucidate its role, we cloned a full cDNA encoding a protein that presents biochemical features essential to OA/tyramine receptor (AipsOAR/TAR) function. The AipsOAR/TAR transcript was detected predominantly in the antennae, the brain and, more specifically, in ALs where its expression level varied concomitantly with age. This expression plasticity indicates that AipsOAR/TAR might be involved in central processing of the pheromone signal during maturation of sexual behaviour in A. ipsilon.
Asunto(s)
Envejecimiento/genética , Regulación del Desarrollo de la Expresión Génica , Mariposas Nocturnas/genética , Receptores de Amina Biogénica/genética , Maduración Sexual/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Perfilación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Filogenia , Receptores de Amina Biogénica/química , Receptores de Amina Biogénica/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
The molecular characterization of post-receptor actors involved in insect olfactory transduction has yet to be understood. We have investigated the presence of a Transient Receptor Potential (TRP) channel in the peripheral olfactory system of the moth Spodoptera littoralis. A cDNA encoding a Lepidopteran TRP channel (TRPgamma) was identified by analysis of a male-antennal EST database and subsequently cloned by RACE PCR. In adult males, the TRPgamma transcript was detected in antennae, at the base of olfactory sensilla. Moreover, TRPgamma was observed in antennae in both pupal and adult stages. This work is the first step in understanding the involvement of TRPgamma in signalling pathways involved in the development and function of the insect olfactory system.
Asunto(s)
Estructuras Animales/metabolismo , Vías Olfatorias/metabolismo , Transducción de Señal , Spodoptera/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
In the aim of the characterization of the molecular actors of insect olfactory transduction, we have cloned the full cDNA encoding a Spodoptera littoralis diacylglycerol kinase (DGK) named SlDGK. In male adults, SlDGK transcript was detected predominantly in the brain and in the olfactory sensilla trichodea located on the antennae. SlDGK expression was first detected at day 3 of the pupal stage, then reached a maximum at the end of this stage and was maintained at this level during the adult period. These data provide the first molecular characterization of a DGK potentially involved in the regulation of signalling pathways responsible for the establishment and/or the functioning of the olfactory system in Lepidoptera.
Asunto(s)
Diacilglicerol Quinasa/genética , Perfilación de la Expresión Génica , Vías Olfatorias/enzimología , Spodoptera/enzimología , Spodoptera/genética , Secuencia de Aminoácidos , Estructuras Animales/enzimología , Estructuras Animales/ultraestructura , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/aislamiento & purificación , Diacilglicerol Quinasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Spodoptera/ultraestructuraRESUMEN
To dissect the steroid hormone signaling pathway involved in insect cell morphological differentiation, we extended the application of the double-stranded RNA-mediated interference (dsRNAi) method to the epidermal IAL-PID2 cell line from Plodia interpunctella Lepidoptera. We first demonstrated that dsRNA was capable of efficiently blocking the steroid hormone 20-hydroxyecdysone (20E) inducibility of proteins that belong to the nuclear receptor superfamily, including the ecdysone receptor (EcR), its partner Ultraspiracle (USP), the insect homolog of the vertebrate retinoid X receptor and the HR3 transcription factor. We then showed that inhibiting the 20E induction of EcR, USP or HR3 proteins prevented the increased synthesis of beta tubulin and consequently the morphological transformation of cells. Thanks to this functional approach, we have shown, for the first time, the participation of EcR, USP and HR3 in a 20E signaling pathway that directs morphological differentiation in insect cells by regulating beta tubulin expression.
Asunto(s)
Diferenciación Celular , Ecdisterona/metabolismo , Insectos/citología , Transducción de Señal , Animales , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Ecdisterona/farmacología , Células Epidérmicas , Epidermis/efectos de los fármacos , Proteínas de Insectos/metabolismo , Insectos/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
Rapid degradation of odours after interaction with olfactory receptors is a critical step of the signal reception process. However, the implied mechanisms are still largely unknown in vertebrates as well as in insects. Involvement of odourant-degrading enzymes in odourant degradation within the antennae has been shown in some insect species and, in particular, esterases could play a key role in degradation of sex pheromones from Lepidoptera. Using a PCR-based strategy, we isolated cDNAs encoding two new esterases from two moths which used acetates as pheromone compounds: the Egyptian armyworm Spodoptera littoralis and the Mediterranean corn borer Sesamia nonagrioides. In antennae, both transcripts were clearly restricted to olfactory sensilla, suggesting their involvement in the degradation of odourant acetate components.
Asunto(s)
Esterasas/genética , Esterasas/metabolismo , Mariposas Nocturnas/enzimología , Mariposas Nocturnas/genética , Odorantes , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Regulación Enzimológica de la Expresión Génica , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Masculino , Datos de Secuencia Molecular , Filogenia , Órganos de los Sentidos/metabolismo , Órganos de los Sentidos/ultraestructuraRESUMEN
Signal termination is a crucial step in the dynamic of the olfactory process. It involves different classes of odorant-degrading enzymes. Whereas aldehyde oxidase enzymatic activities have been demonstrated in insect antennae by previous biochemical studies, the corresponding enzymes have never been characterized at the molecular level. In the cabbage armyworm Mamestra brassicae, we isolated for the first time an aldehyde oxidase partial cDNA specifically expressed in chemosensory organs, with the strongest expression in antennae of both sexes. In these organs, expression was restricted to the olfactory sensilla. Our results suggest that the corresponding enzyme could degrade aldehyde odorant compounds, such as pheromones or plant's volatiles.
Asunto(s)
Aldehído Oxidasa/química , Aldehído Oxidasa/metabolismo , Células Quimiorreceptoras/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Mariposas Nocturnas/enzimología , Olfato/fisiología , Aldehído Oxidasa/análisis , Aldehído Oxidasa/genética , Aldehídos/metabolismo , Secuencia de Aminoácidos , Animales , Hombres , Datos de Secuencia Molecular , Especificidad de Órganos , Homología de Secuencia de Aminoácido , Distribución Tisular , MujeresRESUMEN
Using the IAL-PID2 cell line established from pupally committed imaginal wing discs of Plodia interpunctella, we have investigated the dynamics of cellular and molecular events involved in the G2/M arrest. We have first cloned a cDNA sequence named PIUSP-2 that likely encodes a homologue of the Ultraspiracle-2 isoform of Manduca sexta. When the IAL-PID2 cells were exposed to a 8 h 20E treatment applied at different times of the cell cycle, an optimal period of sensitivity of cells to 20E, in inducing G2 arrest, was determined at the S/G2 transition. Using cDNA probes specifically designed from Plodia B cyclin (PcycB), ecdysone receptor B1-isoform (PIEcR-B1) and HR3 transcription factor (PHR3), we provide evidence that the 20E-induced G2 arrest was correlated to a high induction of PHR3, PIEcR-B1, PIUSP-2 mRNAs at the S/G2 transition and a decrease in PcycB mRNA level at the end of G2 phase.
Asunto(s)
Fase G2/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Mariposas Nocturnas/genética , ARN Mensajero/metabolismo , Alas de Animales/metabolismo , Secuencia de Aminoácidos , Animales , Anisomicina/farmacología , Secuencia de Bases , Northern Blotting , Línea Celular , Ciclina B/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila , Ecdisterona/farmacología , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Mariposas Nocturnas/metabolismo , Técnica del ADN Polimorfo Amplificado Aleatorio , Receptores de Esteroides/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We have investigated the molecular and cellular mechanisms involved in the control of insect cell cycle by 20-hydroxyecdysone (20E) using the IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. We first defined conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, in order to synchronize the IAL-PID2 cells in their division cycle. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at 1 mm for 36 h spaced 16 h apart. Under these conditions, flow cytometry analysis demonstrated that 20E at 10(-6) m induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. Using cDNA probes specifically designed from E75 and HR3 nuclear receptors of Plodia interpunctella, we showed that PiE75 and PHR3 were highly induced by 20E through S and G2 phases with maximal enhancement just before the G2/M arrest of cells. These findings suggest that PiE75 and PHR3 could be involved in a 20E-induced genetic cascade leading to G2/M arrest.
Asunto(s)
Mariposas Diurnas/fisiología , Ciclo Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Ecdisterona/farmacología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/genética , Receptores de Esteroides/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Ciclo Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/aislamiento & purificación , Citometría de Flujo , Hidroxiurea/farmacología , Proteínas de Insectos/aislamiento & purificación , Datos de Secuencia Molecular , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
A set of DNA primers was designed within the DNA-binding domain of the Manduca hormone receptor 3 (MHR3) cDNA. These primers were used in RT-PCR to isolate a 204 bp cDNA fragment from IAL-PID2 cells exposed to 10(-6) M 20-hydroxyecdysone (20E) for 12 h. The amino acid sequence deduced from the cDNA fragment presented 100% identity with the zinc finger domain of Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Choristoneura hormone receptor 3 (CHR3). This cDNA fragment was used as a probe on total RNA from IAL-PID2 cells exposed to 20E and hybridized to mRNA, the size of which was close to 4.5 kb and named Plodia hormone receptor 3 (PHR3). Kinetics of induction of PHR3 mRNA were similar to that of HR3 genes but varied according to the position of cells in their cell cycle. The non-steroidal ecdysone agonist, RH-5992 induced the expression of PHR3 at lower concentrations than 20E. From sequence similarity, mRNA size, 20E and RH-5992 inducibilities, we conclude that PHR3 transcript could encode a Plodia hormone receptor 3 involved in the genetic signalling cascade of 20E. Thanks to its periodic expression, this putative orphan nuclear receptor could serve as a suitable cellular marker for studying changes of epidermal cell sensitivity to 20E during the cell cycle.
Asunto(s)
Ecdisterona/farmacología , Expresión Génica , Receptores de Esteroides/genética , Secuencia de Aminoácidos , Animales , Anisomicina/farmacología , Secuencia de Bases , Ciclo Celular , Línea Celular , Clonación Molecular , ADN Complementario , Hidrazinas/farmacología , Hormonas Juveniles/farmacología , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Mariposas Nocturnas/metabolismo , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Three sisters with male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency are described. On the basis of a 46 XY karyotype and female phenotype all subjects were thought to have the testicular feminization syndrome. At puberty the two older patients developed signs of virilization and gynaecomastia. In these patients the plasma androstenedione level was 4-5 times higher than normal, whilst the plasma testosterone level was low compared to the normal range and, under basal conditions, their plasma androstenedione to testosterone ratio was 20-25 times higher than normal. Interestingly, in the third, prepubertal case, the basal androstenedione to testosterone ratio was normal but became six times higher than normal after hCG stimulation. These data support the diagnosis of male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency and underline the diagnostic value of the hCG stimulation test prepubertally.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/deficiencia , Trastornos del Desarrollo Sexual/genética , Ginecomastia/enzimología , Adolescente , Niño , Trastornos del Desarrollo Sexual/complicaciones , Trastornos del Desarrollo Sexual/enzimología , Hormonas Esteroides Gonadales/sangre , Ginecomastia/complicaciones , Humanos , MasculinoRESUMEN
Urinary testosterone and 3 alpha-androstanediol (3 alpha diol G) glucuronides together with plasma testosterone, 5 alpha-dihydrotestosterone (DHT), and delta 4-androstenedione (delta 4) were measured in 43 normal young men (18-36 yr old), 23 elderly men without clinically evident prostatic pathology (54-89 yr old), 68 elderly men with benign prostatic hyperplasia (BPH group; 54-91 yr old), and 26 elderly men with well differentiated cancer of the prostate (K group; 63-97 yr old). Plasma testosterone decreased slightly with age in all 3 elderly groups (from 591 to 438, 479, and 444 ng/100 ml, respectively). Plasma DHT, on the contrary, was significantly (P less than 0.01) higher in the BPH group than in the other three groups (68 vs. 30, 37, and 32 ng/100 ml, respectively). Plasma delta 4 was significantly lower (P less than 0.01) in the elderly K group than in all other groups (59 vs. 109, 83, and 78 ng/100 ml, respectively). Urinary testosterone glucuronide decreased with age in all 3 elderly groups (from 109 to 55, 38, and 44 micrograms/24 h, respectively) as a result of decreased androgen production rates with age. All 3 elderly groups also had decreased urinary 3 alpha diol G, from 194 to 123, 55, and 118 micrograms/24 h, respectively. The group of elderly patients with BPH had the lowest mean urinary 3 alpha diol G excretion together with the highest mean plasma DHT. This low urinary 3 alpha diol G excretion, which reflects a decrease in both androgen production and DHT metabolism, suggests a decrease in 3 alpha-hydroxysteroid dehydrogenase activity, which, in turn, could explain the increased DHT availability and tissue retention in most target organs. Moreover, the extent of these modifications in androgen metabolism specific to the BPH condition raises the question of an overall alteration of androgen metabolism in patients with BPH which could be the cause of the disease.