RESUMEN
A series of polyethylenimine (PEI) and γ-polyglutamic acid (PGA) nanocomposites (PPGA) was prepared and evaluated in terms of their cell viability and transfection efficiency in vitro and in vivo. On complexion with pDNA, the positively charged PPGA/DNA nanocomposites resulted in a higher level of in vitro reporter gene transfection (2.7-7.9-fold) as compared to native PEI, and selected commercial reagents and >95% cell viability in HEK293, HeLa and HepG2 cell lines. Further, PPGA-5 nanocomposite (the best working system in terms of transfection efficiency among the series) was found to efficiently transfect primary mouse keratinocytes up to 22% above the control level. PPGA-5, when tested for in vivo cytotoxicity in Drosophila, did not induce any stress in the exposed larvae in comparison with control. In vivo gene expression using PPGA-5 showed the highest transfection efficiency in spleen of mouse closely followed by heart tissues after intravenous injection through tail vein. Besides, these nanocomposites also delivered siRNA efficiently into mammalian cells, resulting in ⼠80% suppression of EGFP expression. These results together demonstrated the potential of the projected nanocomposites for in vivo gene delivery.
Asunto(s)
Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Nanocompuestos , Ácido Poliglutámico/química , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , ADN/administración & dosificación , ADN/genética , Drosophila/efectos de los fármacos , Portadores de Fármacos/toxicidad , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos , Polietileneimina/química , Polietileneimina/toxicidad , Ácido Poliglutámico/toxicidad , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Electricidad Estática , TransfecciónRESUMEN
Industrialization and indiscriminate use of agrochemicals have increased the human health risk. Recent epidemiological studies raised a concern for male reproduction given their observations of reduced sperm counts and altered semen quality. Interestingly, environmental factors that include various metals, pesticides and their metabolites have been causally linked to such adversities by their presence in the semen at levels that correlate to infertility. The epidemiological observations were further supported by studies in animal models involving various chemicals. Therefore, in this review, we focused on male reproductive toxicity and the adverse effects of different environmental chemicals on male reproduction. However, it is beyond the scope of this review to provide a detailed appraisal of all of the environmental chemicals that have been associated with reproductive toxicity in animals. Here, we provided the evidence for reproductive adversities of some commonly encountered chemicals (pesticides/metals) in the environment. In view of the recent thrust for an alternate to animal models in research, we subsequently discussed the contributions of Drosophila melanogaster as an alternate animal model for quick screening of toxicants for their reproductive toxicity potential. Finally, we emphasized the genetic and molecular tools offered by Drosophila for understanding the mechanisms underlying the male reproductive toxicity.
Asunto(s)
Drosophila melanogaster , Contaminantes Ambientales/toxicidad , Modelos Animales , Reproducción/efectos de los fármacos , Animales , Drosophila melanogaster/genética , Contaminantes Ambientales/análisis , Perfilación de la Expresión Génica , Humanos , Infertilidad Masculina/inducido químicamente , Masculino , Metales Pesados/toxicidad , Pruebas de Mutagenicidad , Plaguicidas/toxicidad , Reproducción/genética , Semen/químicaRESUMEN
Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.
Asunto(s)
Sistemas de Liberación de Medicamentos , Terapia Genética/métodos , Nanocompuestos/química , Polietileneimina/química , Polisacáridos Bacterianos/química , Animales , Técnicas de Cultivo de Célula , ADN/análisis , ADN/metabolismo , Evaluación Preclínica de Medicamentos , Excipientes/química , Femenino , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Nanocompuestos/análisis , Plásmidos , Polietileneimina/metabolismo , Polisacáridos Bacterianos/metabolismo , ARN Interferente Pequeño/genética , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Drosophila seminal fluid protein (SFP) sex peptide (SP) elicits numerous post-mating responses, including increased egg laying and decreased sexual receptivity, in the mated female. Unlike other SFPs, which are detectable in mated females for only a few hours post mating, SP is maintained-and its effects are sustained-for several days. The persistence of SP in the mated female's reproductive tract is thought to be a consequence of its binding to, and gradual release from, sperm in storage, which maintains SP's ability to act within the female reproductive tract. Recent studies have shown that several other SFPs, acting in a network, are needed for SP's localization to sperm and are necessary for the efficient release of sperm from storage. This result suggested an additional new role for SP modulating the release of sperm from storage. We tested for this possibility by examining sperm storage parameters in mated females that did not receive SP. We found that while sperm accumulation into storage was unaffected, sperm depletion from storage sites was significantly decreased (or impaired) in the absence of SP. Mates of males expressing a modified SP that is unable to be released from sperm showed a similar phenotype, indicating that release of sperm-bound SP is a necessary component of normal sperm depletion. Additionally, SP null males were more successful in a sperm competitive environment when they were first to mate, which is likely a consequence of higher retention of their sperm due to defective sperm release. Our findings illustrate a direct role for SP in the release of sperm from storage.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Péptidos/fisiología , Semen/fisiología , Espermatozoides/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Masculino , Mutación , Oviposición , Péptidos/genética , ReproducciónRESUMEN
Rapid industrialization has increased the burden of chemicals in the environment. These chemicals may be harmful to development and reproduction of any organism. We therefore analyzed the adverse effects of leachates from a tannery solid waste on development and reproduction using Drosophila. We show a significant delay in mean emergence of flies observed at the higher concentrations of the leachates, indicating their effect on the organism's development. Significant leachate-induced effect on reproduction of the organism was also observed. Sub-organismal analyses revealed Hsp70 expression and tissue damage in a sex-specific manner. Refractoriness of Hsp70 expression in accessory glands of male flies and ovaries of females was concurrent with tissue damage. Genes encoding certain seminal proteins (Acp70A and Acp36DE) from accessory glands were significantly down-regulated at higher concentrations of the leachates. The study suggests that (i) sub-organismal adverse responses are reflected at organismal level, (ii) tannery waste leachates cause adverse effects on the expression of genes encoding seminal proteins that facilitate normal reproduction and (iii) Hsp70 may be used as a marker of cellular damage for reproductive organs.
Asunto(s)
Drosophila melanogaster/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Residuos Industriales/análisis , Curtiembre , Contaminantes Químicos del Agua/toxicidad , Animales , Biomarcadores/metabolismo , Daño del ADN/efectos de los fármacos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Monitoreo del Ambiente , Femenino , Expresión Génica/efectos de los fármacos , Genes de Insecto/efectos de los fármacos , Sedimentos Geológicos/química , Proteínas HSP70 de Choque Térmico/genética , Péptidos y Proteínas de Señalización Intercelular , Masculino , Metales Pesados/toxicidad , Ovario/citología , Ovario/efectos de los fármacos , Ovario/metabolismo , Péptidos/genética , Péptidos/metabolismo , Reproducción/efectos de los fármacos , Contaminantes del Suelo/toxicidad , Testículo/citología , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
Male accessory gland secretory protein polymorphism was analysed in natural populations of Drosophila nasuta nasuta and D. sulfurigaster neonasuta for the first time, using SDS-PAGE to score polymorphism of these proteins in 2788 individuals of D. n. nasuta and 2232 individuals of D. s. neonasuta from 12 different populations from southern India. A total of 25 and 18 variant protein phenotypes were identified in D. n. nasuta and D. s. neonasuta, respectively. Protein fractions of group III were more polymorphic than those from groups I and II. The results show that accessory gland secretory proteins show high levels of polymorphism, irrespective of species or habitat. Moreover, we have used the variation in the accessory gland proteins to assess the extent of divergence between the species and to infer their population structure. The study suggests that though both D. n. nasuta and D. s. neonasuta belong to the same subgroup, they differ in population structure, as far as accessory gland protein polymorphism is concerned.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Animales , Drosophila/clasificación , Drosophila/fisiología , Proteínas de Drosophila/metabolismo , Genética de Población , Genitales Masculinos/metabolismo , India , Masculino , Fenotipo , Polimorfismo Genético , Especificidad de la EspecieRESUMEN
Successful reproduction requires contributions from both the male and the female. In Drosophila, contributions from the male include accessory gland proteins (Acps) that are components of the seminal fluid. Upon their transfer to the female, Acps affect the female's physiology and behavior. Although primary sequences of Acp genes exhibit variation among species and genera, the conservation of protein biochemical classes in the seminal fluid suggests a conservation of functions. Bioinformatics coupled with molecular and genetic tools available for Drosophila melanogaster has expanded the functional analysis of Acps in recent years to the genomic/proteomic scale. Molecular interplay between Acps and the female enhances her egg production, reduces her receptivity to remating, alters her immune response and feeding behavior, facilitates storage and utilization of sperm in the female and affects her longevity. Here, we provide an overview of the D. melanogaster Acps and integrate the results from several studies that bring the current number of known D. melanogaster Acps to 112. We then discuss several examples of how the female's physiological processes and behaviors are mediated by interactions between Acps and the female. Understanding how Acps elicit particular female responses will provide insights into reproductive biology and chemical communication, tools for analyzing models of sexual cooperation and/or sexual conflict, and information potentially useful for strategies for managing insect pests.
RESUMEN
Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Animales , Mapeo Cromosómico , Drosophila melanogaster/genética , Genoma de los Insectos , Masculino , Datos de Secuencia Molecular , Familia de Multigenes/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Cromosoma X/genéticaRESUMEN
Male accessory gland proteins (Acps) in Drosophila are components of the seminal fluid and are transferred to females during copulation. In mated females, Acps enhance egg production, augment sperm storage, induce refractory mating behaviors, and affect the female's longevity. To address the functions of eight previously uncharacterized Acps and further analyze five others, we determined the tissues to which they target after transfer to females. Each Acp has multiple targets and is unique in its pattern of localization. Within the reproductive tract, Acps target to the uterus, oviduct, sperm storage organs, ovary and oocytes. Some Acps also leave the reproductive tract, to enter the hemolymph. Some Acps are detected on the surface of eggs laid by mated females but were not detectable within those eggs. Our results can help to identify the likely functions of these Acps as well as to create models for the mechanism of action of Acps.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Animales , Copulación/fisiología , Femenino , Masculino , Reproducción/fisiologíaRESUMEN
Male accessory gland in Drosophila is a secretory tissue of the reproductive system. The proteins synthesized in the accessory gland are tissue specific, stage specific-seen only during the adult stage and sex specific in the sense of male limited expression. These secretions that form a component of the seminal fluid are transferred to the female at the time of copulation and play an important role in reproduction. In conjunction with sperm, these secretory proteins assure reproductive success by reducing the female's receptivity to mating and escalating the rate of egg laying. Some of these proteins are antibacterial in nature with a likely function of protecting the female's genital tract against microbial infection during/after mating. Most of the genes involved in the synthesis of accessory gland proteins are autosomal but a few are still X-linked. Their male specific expression is achieved at the time of sex determination. The level of expression of these genes is dose dependent and they follow Mendelian pattern of segregation. Further, majority of these proteins are rapidly evolving with high rates of non-synonymous substitutions. In this review, by considering the work carried out in different fields, we have tried to generate a comprehensive picture about the male accessory gland and the role of its proteins in the reproduction of Drosophila.
Asunto(s)
Genitales Masculinos/metabolismo , Proteínas/metabolismo , Animales , Drosophila , Regulación de la Expresión Génica , Masculino , Proteínas/genéticaRESUMEN
The quantity of male accessory gland secretory proteins in relation to the number of cells in the gland, size of the gland and the duration of copulation has been studied in seven members of the nasuta subgroup of Drosophila. The study revealed that the difference in the quantity of secretions is independent of the number of secretory cells in the gland. However, a positive correlation exists between the quantity of secretions and size of the gland; while there is no correlation between the copulation duration and the quantity of secretions. Further, there is an increase in the values of all the parameters studied, with increasing distance of the species from the ancestor.