RESUMO
RNA interference caused by exogenous double-stranded RNA (dsRNA) is used to downregulate crucial genes to control insects. The reproductive success of all oviparous species depends on vitellogenin (Vg) biosynthesis and its accumulation in the developing oocytes. Adult females of Triatoma infestans were independently injected with two Vg dsRNAs (Vg1 dsRNA or Vg2 dsRNA) or nuclease-free water (control) 24 hours before feeding, and a group of adult females not injected was also analyzed (control). Vg1 and Vg2 messenger RNAs silencing was verified by reverse transcription quantitative polymerase chain reaction. The transcript levels of the Vg1 and Vg2 genes were significantly reduced after dsRNA treatment in fat body and ovary of T. infestans in relation to those detected in individuals injected with nuclease-free water and not injected (controls). Moreover, the present study demonstrated that the silencing of the Vg1 or Vg2 genes inhibits oviposition in the Chagas disease vector T. infestans. These findings may have important implications for the development of novel vector control strategies.
Assuntos
Doença de Chagas/transmissão , Inativação Gênica , Insetos Vetores/genética , Oviposição/genética , Triatoma/genética , Vitelogeninas/biossíntese , Vitelogeninas/genética , Animais , Feminino , Expressão Gênica , Genes de Insetos , Controle de Insetos/métodosRESUMO
The importance of juvenile hormone regulating insect oogenesis suggests looking for genes whose expression is regulated by this hormone. SPARC is a calcium-binding glycoprotein that forms part of the extracellular membranes, which in vertebrates participates in bones mineralization or regulating cell proliferation in some cancer types. This large number of functions described for SPARC in different species might be related to the significant differences in its structure observed when comparing different species-groups. Indeed, these structural differences allow characterizing the different clades. In the cockroach Blattella germanica, a SPARC homolog emerged from ovarian transcriptomes that were constructed to find genes responding to juvenile hormone. In insects, SPARC functions have been studied in oogenesis and in embryo development of Drosophila melanogaster. In the present work, using RNAi approaches, novel functions for SPARC in the B. germanica panoistic ovaries are described. We found that depletion of SPARC does not allow to the follicular cells to complete mitosis, resulting in giant follicular cells nuclei and in a great alteration of the ovarian follicle cytoskeleton. The SPARC contribution to B. germanica oogenesis occurs stabilizing the follicular cell program and helping to maintain the nuclear divisions. Moreover, SPARC is necessary to maintain the cytoskeleton of the follicular cells. Any modification of these key processes disables females for oviposition.
Assuntos
Blattellidae/embriologia , Citoesqueleto/metabolismo , Epitélio/fisiologia , Oogênese/fisiologia , Osteonectina/metabolismo , Folículo Ovariano/embriologia , Animais , Proliferação de Células , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Mitose/fisiologia , Oogênese/genética , Osteonectina/genética , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Vitelogeninas/biossínteseRESUMO
BACKGROUND: Protein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control. METHODOLOGY/PRINCIPAL FINDINGS: We conducted an analysis to identify the ortholog of mammalian PTP1B in the Aedes aegypti genome. We identified eight genes coding for classical PTPs. In silico structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production. CONCLUSIONS/SIGNIFICANCE: The data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel targets to block egg formation in mosquitoes.
Assuntos
Aedes/enzimologia , Genoma de Inseto , Oviposição/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Vitelogeninas/genética , Aedes/efeitos dos fármacos , Aedes/genética , Sequência de Aminoácidos , Animais , Corpo Adiposo/efeitos dos fármacos , Corpo Adiposo/enzimologia , Feminino , Regulação da Expressão Gênica , Himecromona/análogos & derivados , Himecromona/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ovário/efeitos dos fármacos , Ovário/enzimologia , Oviposição/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vanadatos/farmacologia , Vitelogeninas/antagonistas & inibidores , Vitelogeninas/biossínteseRESUMO
This study evaluated the condition factor, gonadosomatic, and hepatosomatic indexes, occurrence of plasmatic vitellogenin (Vg), and frequency of spermatogenic maturity stages in male Chilean flounders, Paralichthys adspersus, caught at three different coastal sites off the Bio-Bio region, central Chile, during 1 year. The Vg was detected by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Western blot analyses using an antibody against Chilean flounder Vg. The spermatogenic maturity stages were analyzed by histological gonadic diagnostic. The prevalence of plasmatic Vg induction in male fish differed significantly among sites. The flounders sampled from the Itata area were the most affected. Evaluations of biometric data, plasmatic Vg induction, and spermatogenic maturity stages of the flounder showed the following: (1) lower gonadosomatic index, (2) greater hepatosomatic index, (3) greater prevalence of plasmatic Vg, and (4) delayed development of the gonad. The results suggest that estrogenic endocrine-disruption compounds are introduced into the marine environment, negatively affecting the fish studied. The relevance of this report is discussed in relation to estrogenic compounds introduced by industrial and municipal wastewater effluents in the areas studied.
Assuntos
Disruptores Endócrinos/toxicidade , Linguado , Reprodução/efeitos dos fármacos , Vitelogeninas/biossíntese , Poluentes Químicos da Água/toxicidade , Animais , Western Blotting/métodos , Chile , Eletroforese em Gel de Poliacrilamida/métodos , Disruptores Endócrinos/análise , Monitoramento Ambiental/métodos , Estrogênios , Gônadas/efeitos dos fármacos , Gônadas/crescimento & desenvolvimento , Masculino , Poluentes Químicos da Água/análiseRESUMO
Girardinichthys viviparus is a Mexican endangered endemic fish species living only in Lake Texcoco (LTX), one of two extant type localities for this species. The other type locality is Lake Zumpango (LZ). LTX and LZ are fed by wastewater treated at secondary level that contains several endocrine disrupting chemicals. Our goal was to assess the estrogenic and anti-estrogenic effects elicited in G. viviparus by water from the two type localities and by these same matrices enriched with PCBs in order to understand potential damage due to increased xenobiotic levels. Estrogenic and anti-estrogenic effects were evaluated in vitro by E-screen assay in MCF-7 cells and cytotoxicity by MTT assay. PCBs were quantified in type localities. In vivo vitellogenin (VTG) induction was determined by a hybrid ELISA in adult laboratory-born fish exposed during 1, 2, 4, 8 and 16 days to LTX or LZ water in static exposure systems, and by the same matrices enriched with PCBs. We found PCBs only in LTX, but the water from both type localities elicit estrogenic and anti-estrogenic effects in vitro. Cytotoxicity was not observed in MCF-7 cells exposed to LTX or LZ water. VTG induction was higher with LTX water than with LZ water; also the response of males was greater than in females. In the PCB-enriched matrices, VTG induction in both sexes exposed to LTX water was reduced compared to un-enriched matrices. Thus, the sublethal increases in PCB levels may be hazardous to both sexes since they are linked probably to hepatotoxicity.
Assuntos
Disruptores Endócrinos/toxicidade , Antagonistas de Estrogênios/toxicidade , Estrogênios/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Peixes , Masculino , Bifenilos Policlorados/toxicidade , Vitelogeninas/biossíntese , Eliminação de Resíduos Líquidos , Purificação da ÁguaRESUMO
In the honeybee, Apis mellifera, vitellogenin (Vg) expression has been detected in the ovary of queens, but not in that of workers. In addition, larvae of both sexes produce Vg in significant amounts, which suggest that Vg serves for functions additional to oocyte growth and energy supply to the embryo. In vivo hormone treatment experiments suggest that the decrease of 20-hydroxyecdysone concentration occurring in previtellogenic phases allows Vg production. Southern analysis indicates that the Vg gene is present as a single copy in the honeybee genome.
Assuntos
Abelhas/metabolismo , Ovário/metabolismo , Vitelogeninas/biossíntese , Animais , Northern Blotting , Southern Blotting , Western Blotting , Ecdisterona/farmacologia , Feminino , Dosagem de Genes , Hormônios Juvenis/farmacologia , Masculino , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vitelogeninas/genéticaRESUMO
Several investigations have demonstrated that the increase in chemicals in the environment may have caused effects on aquatic life and wildlife. The impact from oil production activities on the benthic community structure and shrimp (Farfantepenaeus duorarum and Litopenaeus setiferus) biochemical markers were studied in the Gulf of Mexico, which is heavily contaminated by polycyclic aromatic hydrocarbons and heavy metals. Shrimp were collected from a control area and from an area close to oil production platforms during October and November 2002. There was no spatial difference in exposure and response probably because shrimp migrate, as results did not show significant differences in cytochrome P4501A (as measured by ethoxycoumarin O-deethylase (ECOD) metabolism) between the two sites. In October, shrimp ECOD activities were higher and statistically different from those measured in the samples taken in November. As for ECOD activities, with the concentration of vitellogenin as another biomarker, there were no differences between the shrimp collected from the control area and the shrimp collected on the oil production platforms. In this case the concentrations were higher in shrimp collected in November vs. shrimp collected in October. However, there are significant correlations between contaminants and responses (biomarkers), indicating an effect of pollution. One of the most important considerations brought up by this kind of study is that, although the majority of groups studying the effects of endocrine disruption have focused almost exclusively on human health or vertebrate wildlife-related issues, it is necessary to increase research focused on understanding the function of hormones in invertebrate species exposed in the field to different kinds of pollutants.
Assuntos
O-Dealquilase 7-Alcoxicumarina/metabolismo , Penaeidae/metabolismo , Vitelogeninas/biossíntese , Animais , Biomarcadores , Sedimentos Geológicos/análise , Hemolinfa/metabolismo , Hepatopâncreas/enzimologia , Masculino , Microssomos/enzimologia , Estações do Ano , Poluentes Químicos da Água/análiseRESUMO
During the last decade, special attention has been focused on the consequences of exposure to environmental estrogens on reproduction in wild fish species. For this reason, characterization of biomarkers of such exposures could result in useful tools for these studies. The detection of vitellogenin (Vtg), a precursor of yolk proteins, is being intensely studied since its synthesis in the liver is regulated by the estradiol-17beta and is influenced by other estrogenic compounds. The aim of this work was to assess the presence of Vtg in the surface mucus of males of Cichlasoma dimerus (Teleostei, Perciformes), a typical South American freshwater cichlid, after hormonal treatment with estradiol-17beta (intraperitoneal injections of 10 microg E(2)/g fish). Plasma and mucus from females and treated males analyzed by Western blot revealed that different heterologous antisera against Vtg bind to putative protein of 180 & 120 kDa and 120 & 110 kDa, respectively, whereas no reaction was found in samples of untreated males. The same profile was observed in mucus samples using Dot blot, a very easy and direct technique. Using immunocytochemistry techniques, immunoreactive Vtg (ir-Vtg) producing cells in the liver of females and treated males were detected. Testes and liver tissues were also assessed by histological techniques. Marked alterations in both organs were observed, such as lower sperm production, presence of immature germ cells in the lobular lumen of testes, and some morphology changes in the hepatocytes due to the accumulation of Vtg. This is the first report about the effects of an estrogen in the Vtg synthesis and their consequences on liver and gonads of a South American fresh water cichlid. These results also support the possibility of using Vtg from surface mucus as a potential biomarker for estrogenic compounds through a noninvasive, useful and easy assay to monitor the presence of endocrine disrupting chemicals in the environment.
Assuntos
Ciclídeos/fisiologia , Estradiol/efeitos adversos , Vitelogeninas/biossíntese , Animais , Biomarcadores/análise , Exposição Ambiental , Feminino , Gônadas/química , Imuno-Histoquímica , Fígado/química , Masculino , Muco/química , Vitelogeninas/análiseRESUMO
Oocyte secondary growth in S. spiloleura corresponds to the period in which different vesicular structures are formed, including the cortical alveoli and the yolk granules. The oocytes with cortical alveolus formation show vesicular structures with filamentous content in the cortical cytoplasmic region, which are the cortical alveolus precursors. In these oocytes, electron-dense vesicles of heterogenous content are dispersed in the inner cytoplasmic region and their nuclei are irregular, showing many nucleoli of different sizes. The oocytes in vitellogenesis are filled with many vesicles. The cortical alveolus precursors are in the peripheral region, and electron-dense granules are seen near to the nucleus. These fuse and form yolk granules. The oocytes in vitellogenesis show a very irregular nucleus that has nucleoli of different sizes. In the oocytes in final vitellogenesis, the yolk granules are scattered throughout the cytoplasm, displacing the cortical alveoli toward cell periphery. The nucleus is similar to the other stages.
Assuntos
Peixes/fisiologia , Oócitos/crescimento & desenvolvimento , Oogênese/fisiologia , Vitelogeninas/biossíntese , Animais , Brasil , Vesículas Citoplasmáticas/ultraestrutura , Feminino , Microscopia Eletrônica , Oócitos/ultraestruturaRESUMO
The caste-specific regulation of vitellogenin synthesis in the honeybee represents a problem with many yet unresolved details. We carried out experiments to determine when levels of vitellogenin are first detected in hemolymph of female castes of Apis mellifera, and whether juvenile hormone and ecdysteroids modulate this process. Vitellogenin levels were measured in hemolymph using immunological techniques. We show that in both castes the appearance of vitellogenin in the hemolymph occurs during the pupal period, but the timing was different in the queen and worker. Vitellogenin appears in queens during an early phase of cuticle pigmentation approximately 60h before eclosion, while in workers the appearance of vitellogenin is more delayed, initiating in the pharate adult stage, approximately 10h before eclosion. The timing of vitellogenin appearance in both castes coincides with a slight increase in endogenous levels of juvenile hormone that occurs at the end of pupal development. The correlation between these events was corroborated by topical application of juvenile hormone. Exogenous juvenile hormone advanced the timing of vitellogenin appearance in both castes, but caste-specific differences in timing were maintained. Injection of actinomycin D prevented the response to juvenile hormone. In contrast, queen and worker pupae that were treated with ecdysone showed a delay in the appearance of vitellogenin. These data suggest that queens and workers share a common control mechanism for the timing of vitellogenin synthesis, involving an increase in juvenile hormone titers in the presence of low levels of ecdysteroids.
Assuntos
Abelhas/crescimento & desenvolvimento , Ecdisona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hormônios Juvenis/farmacologia , Vitelogeninas/biossíntese , Acetona/farmacologia , Animais , Abelhas/química , Abelhas/fisiologia , Western Blotting/métodos , Western Blotting/veterinária , Dactinomicina/farmacologia , Feminino , Hemolinfa/química , Masculino , Inibidores da Síntese de Proteínas/farmacologia , Pupa/química , Pupa/fisiologia , Fatores de Tempo , Vitelogeninas/análise , Vitelogeninas/genética , Vitelogeninas/fisiologiaRESUMO
The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.
Assuntos
Folículo Ovariano/fisiologia , Rhodnius/crescimento & desenvolvimento , Vitelogeninas/biossíntese , Animais , Autorradiografia , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/fisiologia , Feminino , Folículo Ovariano/citologiaRESUMO
Tropical anoline lizards have been shown to undergo massive reproductive retrogression when held in captivity. The present study focused on the effects generated by captivity-induced stress on the hepatic expression of vitellogenin (Vtg), the precursor of the major egg yolk proteins, in Anolis pulchellus. Several hepatic dysfunctions accompanying the regression of the reproductive organs were detected when mature highly vitellogenic females were kept in captivity for long periods. These included decreased synthesis of Vtg to undetectable levels after 4 days of captivity concomitant with a large reduction in the levels of its cognate mRNA. In addition, a drastic reduction in Vtg plasma levels preceding the conspicuous cessation of follicular growth was observed. Results suggest the activation of a specific mechanism for rapid clearance of vitellogenic and other female-specific proteins from plasma. The effects of captivity, both in the liver and in the reproductive tract, were alleviated or even reversed by treatment with 17 Beta-estradiol. Hepatic protein synthesis increased threefold when animals were maintained under estrogen therapy during the captivity period. Also, the levels of Vtg mRNA and Vtg protein synthesis and plasma levels were similar or even higher to the observed in control vitellogenic females. Animals treated with 17 beta-estradiol after long-term captivity recovered the normal vitellogenic levels after 72 to 96 hr. Therefore, our results im this tropical anole strongly suggest that the stress effects upon reproductive behavior previously reported in anoline lizards results from suppression of the estrogen stimulus for the hepatic vitellogenic response.
Assuntos
Expressão Gênica , Lagartos/metabolismo , Estresse Fisiológico/metabolismo , Vitelogeninas/genética , Animais , Proteínas Sanguíneas/biossíntese , Estradiol/farmacologia , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , RNA Mensageiro/metabolismo , Vitelogeninas/biossíntese , Vitelogeninas/sangueRESUMO
It was shown that Rhodnius prolixus vitellogenin (Vg) is synthesized as precursors of 205 and 190 kDa. Each Vg subunit is antigenically related to a domain in the precursor molecules. Since Vg has been previously detected in R. prolixus male adults, protein synthesis by fat bodies from 5th instar male nymphs was investigated and no Vg synthesis could be detected. Also, a 6.1 Kb RNA is present in female adults but not in 5th instar male nymphs. Therefore, cDNAs from female adult and 5th instar male fat bodies were used for differential screening of a female fat body cDNA library leading to the isolation of several female specific clones. All the clones hybridizing to the female specific 6.1 Kb RNA species were identical. We also describe the construction of new expression vectors, pGex-A and pGex-B, derived from the previously described plasmid pGex-1N. The new vectors, together with pGex-3X, comprise a set of expression plasmids with cloning sites in all three possible reading frames that give a fusion polypeptide with the glutathione S-transferase. This carrier protein can be cleaved by digestion with factor Xa in all three plasmids; one of the Vg cDNA clones was subcloned in pGex-A. Antibodies affinity purified from the fusion protein Vg/glutathione S-transferase recognized both large Vg subunits, suggesting an antigenic relationship between them. Furthermore, the small Vg subunits were not recognized, indicating that they may be localized at the N-terminal region of Vg precursors.
Assuntos
Precursores de Proteínas/genética , Rhodnius/genética , Vitelogeninas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Corpo Adiposo , Feminino , Masculino , Dados de Sequência Molecular , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Rhodnius/embriologia , Caracteres Sexuais , Vitelogeninas/biossínteseRESUMO
Insect vitellogenesis involves coordinated activities of the fat body and oocytes. We have studied these activities at the cellular level in the mosquito. During each vitellogenic cycle, the fat body undergoes three successive stages: 1) proliferation of biosynthetic organelles, 2) vitellogenin synthesis, 3) termination of vitellogenin synthesis and degradation of biosynthetic organelles by lysosomes. Analysis with monoclonal antibodies and radiolabelling demonstrated that the mosquito yolk protein consists of two subunits (200-kDa and 65-kDa). Both subunits are glycosylated, their carbohydrate moieties are composed of high-mannose oligosaccharides. The yolk protein subunits are derived from a single 220 kDa precursor detected by an in vitro translation. Oocytes become competent to internalize proteins as a result of juvenile hormone-mediated biogenesis of endocytotic organelles. The yolk protein is then accumulated by receptor-mediated endocytosis. A pathway of the yold protein and factors determining its routing in the oocyte have been studied.
Assuntos
Culicidae/metabolismo , Vitelogênese , Vitelogeninas/biossíntese , Animais , Vitelogeninas/análiseRESUMO
Se estudia al desarrollo y maduración de los ovocitos de triatominos, describiéndose la anatomía y fisiología del aparato reproductor femenino de estos insectos, y analizándose resultados y principales investigaciones realizadas sobre la caracterización, identificación y control hormonal de la síntese de vitelogeninas y vitelinas