From hatching into fetal life in the pig

Background: Potential adverse effects of assisted reproductive technologies may have long term consequences on embryonic and fetal development. However, the complex developmental phases occurring after hatching from the zona pellucida are less studied than those occurring before hatching. The aim of the present review is to introduce the major post-hatching developmental features bringing the embryo form the blastocyst into fetal life in the pig. Review: In the pre-hatching mouse blastocyst, the pluripotency of the inner cell mass (ICM) is sustained through expression of OCT4 and NANOG. In the pre-hatching porcine blastocyst, a different and yet unresolved mechanism is operating as OCT4 is expressed in both the ICM and trophectoderm, and NANOG is not expressed at all. Around the time of hatching, OCT4 becomes confined to the ICM. In parallel, the ICM is divided into a ventral cell layer, destined to form the hypoblast, and a dorsal cell mass, destined to form the epiblast. The hypoblast gradually develops into a complete inner lining along the epiblast and the trophectoderm. Upon hatching (around Day 7-8 of gestation), the trophectoderm covering the developing epiblast (Rauber´s layer) is lost and the embryonic disc is formed by development of a cavity in the epiblast, which subsequently "unfolds" resulting the establishment of the disc. In parallel, the epiblast initiates expression of NANOG in addition to OCT4. The blastocyst enlarges to a sphere of almost 1 cm around Day 10 of gestation. Subsequently, a dramatic elongation of the embryo occurs, and by Day 13 it has formed a thin approximately one meter long filamentous structure. This elongation is paralleled with the initiation of placentation along with which, the embryonic disc undergoes gastrulation. The latter process is preceded by a thickening of the posterior region of the epiblast, putatively developing as a consequence of an absence of inhibitory signals from a condensed portion of the hypoblast underlying the anterior epiblast. The thickened posterior epiblast expresses the primitive streak marker BRACHYURY. Subsequently, the epiblast thickening extends in an anterior direction forming the primitive streak; also expressing BRACHYURY. Gastrulation is hereby initiated, and epiblast cells ingress through the primitive streak to form mesoderm and endoderm; the latter is inserted into the dorsal hypoblast whereas the mesoderm forms a more loosely woven mesenchyme between the epiblast and the endoderm. The anterior mesoderm, ingressing through the anterior end of the primitive streak, referred to as the node, forms the rod-like notochord interposed between the epiblast and the endoderm. During the subsequent neurulation, which is a process overlapping with gastrulation in time, the notochord induces the overlying epiblast to form neural ectoderm, which sequentially develops into the neural plate, neural groove, and neural tube, whereas the lateral epiblast develops into the surface ectoderm. In parallel with the development of the somatic germ layers, ectoderm, mesoderm, and endoderm, the primordial germ cells, the predecessors of the germ line, develop in the posterior epiblast and initiates a migration finally bringing them to the genital ridges of the developing embryo. In parallel, the ectoderm gives rise to the epidermis and neural tissue, the mesoderm develops into the cardiovascular system as well as the urogenital and musculoskeletal systems, whereas the endoderm forms the gastrointestinal system and related organs as the liver and pancreas. Conclusions: Porcine embryonic and fetal development is controlled by molecular mechanisms that to some degree differ from those operating in the mouse. It is of importance to uncover the molecular control of development in ungulates as it has great implications for assisted reproductive technologies as well as for biomedical model research.

Histopatologia da interação de Bacillus thuringiensis e extratos vegetais no intestino médio de Spodoptera frugiperda (Lepidoptera: Noctuidae) / Histopathology of the interaction of Bacillus thuringiensis and plant extracts in the midgut of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Plantas são fontes naturais de substâncias inseticidas, já que podem ser produzidas pelo vegetal em resposta a ataques de insetos podendo representar uma alternativa no manejo de insetos-praga, como Spodoptera frugiperda. Dessa forma, o presente trabalho objetivou a análise histopatológica do intestino médio de lagartas de S. frugiperda, após a ingestão dos extratos obtidos por maceração e infusão de Petiveria alliacea. Zingiber officinale. Cymbopogon citratus. Malva silvestris. Baccharis genistelloides e Ruta graveolens, assim como a associação desses extratos com Bacillus thuringiensis aizawai. As lagartas foram tratadas in vivo com cada extrato e a bactéria, mais a associação desses extratos com B. thuringiensis e após, uma reação de cinética entre 3 e 27 horas, foram fixadas para o preparo de cortes histológicos, os quais foram corados e analisados comparativamente às testemunhas em microscopia óptica. Os resultados mostraram mudanças na histologia do intestino médio das lagartas de S. frugiperda, 3 horas após a aplicação dos tratamentos à base de alliacea. Z. officinale. C. citratus e M. silvestris, enquanto que para B. genistelloides e R. graveolens só foram observados alterações após 6h. Na interação dos extratos com B. thuringiensis observou-se alterações nas microvilosidades, desorganização do intestino médio e a hipertrofia das células epiteliais que projetaram-se para o lúmen. Os resultados desse trabalho mostram que o efeito histopatológico de Z. officinale. M. silvestris. R. graveolens e B. genistelloides, foram mais ativos quando comparados aos extratos de P. alliacea e C. citratus, os quais apresentaram uma interação positiva com B. thuringiensis.

The natural insecticidal substances produced by plants in response to insect attack may represent an alternative for the management of pest insects such as Spodoptera frugiperda. This study was therefore aimed to histopathologically analyze the midgut of larvae of S. frugiperda, after ingestion of extracts obtained by maceration and infusion of Petiveria alliacea. Zingiber officinale. Cymbopogon citratus. Malva silvestris. Baccharis genistelloides and Ruta graveolens, and the association of these extracts with Bacillus thuringiensis aizawai. The larvae were treated in vivo with each extract, B. thuringiensis, and the combination of these extracts with the bacteria, and after a reaction kinetics of between 3 to 27 hours they were fixed for the preparation of histological sections, which were stained and analyzed in comparison to the controls with light microscopy. The results showed changes in the histology of the midgut of the larvae of S. frugiperda, 3 hours after application of treatments based on P. alliacea. Z. officinale. C. citratus and M. silvestris, while for B. genistelloides and R. graveolens changes were observed only after 6h. In the interaction of extracts with B. thuringiensis the analysis revealed changes in microvilli, disorganization of the midgut and hypertrophy of epithelial cells that projected into the lumen. The findings show that the histopathological effects of Z. officinale. M. silvestris. R. graveolens and B. genistelloides were more active when compared to extracts of P. alliacea and C. citratus, which showed a positive interaction with B. thuringiensis.

Análise proteômica do intestino primitivo de embriões bovinos / Proteomic analysis of primitive gut from bovine embryo

O desenvolvimento de biotecnologia de embriões em animais de produção é prejudicado por perdas no primeiro trimestre da gestação, idade em que o intestino primitivo está sendo estabelecido. O estudo das proteínas contidas no intestino primitivo nesta fase inicial da gestação pode aumentar o conhecimento sobre as vias moleculares envolvidas no desenvolvimento embrionário normal e em perdas de embriões, assim como a sua participação na organogênese e diferenciação celular. Intestino primitivo de embriões de bovinos a partir dos 39 SD ± 4 dias de desenvolvimento (variando de 33 a 45 dias) foram coletados em um matadouro local. As amostras foram processadas e agrupadas para análise proteômica shotgun label-free usando MudPIT (Multidimensional Protein Identification Technology). Análise funcional e de via foram feitas usando FatiGO (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) para identificar as ontologias relevantes e vias canônicas ou não-canônicas representada pelas proteínas expressas no Intestino primitivo. Um total de 74 proteínas ou sequências randômicas foram identificadas, correspondentes a 30 proteínas específicas expressas pelo Intestino primitivo bovino. Das 30 proteínas únicas, 21 proteínas foram utilizadas na ontologia e análise de vias. As análises mostraram um enriquecimento de ontologias relacionadas com a ligação (N = 5); atividade catalítica (N = 6); organela intracelular (N = 6). Houve um enriquecimento de ontologias associado às modificações do citoesqueleto; processo de diferenciação celular (N = 3), a migração celular (N = 4) e no metabolismo celular (N = 6). Além disso, a via e a análise de rede mostraram um enriquecimento de vias de comunicação entre células, tais como junções comunicantes e tight e as vias de adesão focal. Além disso, as vias envolvidas no movimento celular (por exemplo, vias de regulação do citoesqueleto de actina e a migração transendotelial de leucócitos) foram extremamente enriquecimento no grupo de proteínas expressas pelo Intestino primitivo bovino. Nossos resultados sugerem que as células do intestino primitivo tem alto perfil migratório e são compostas de células não totalmente diferenciadas, com alto metabolismo celular. A migração e a diferenciação destas células poderiam determinar o destino do embrião em desenvolvimento. Além disso, a compreensão da função e interação de proteínas expressas pelo embrião normal fornecerá informações sobre o impacto das biotecnologias reprodutivas no desenvolvimento do embrião durante a implantação e placentação.

The development of embryo biotechnology in farm animals is hampered by embryo losses in first trimester of gestation, period in which the primitive gut is being established. The study of proteins contained in the primitive gut at this early stage of pregnancy may increase the knowledge about the molecular pathways involved in normal embryonic development and loss of embryos, as well as their involvement in organogenesis and cell differentiation.primitive gut from bovine embryos on day 39 SD± 4 of development (ranging from 33 to 45 days) were collected at a local slaugtherhouse. The samples were processed and pooled for label-free shotgun proteomics analysis using MudPIT (Multidimensional Protein Identification Technology) tandem MSE acquisition. Functional and pathway analysis using FatiGo (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) and Ingenuity Pathway Analysis (www.ingenuity.com) were used to identify relevant ontologies and canonical or noncanonical pathways represented by the expressed proteins in the primitive gut. A total of 74 protein sequences were identified corresponding to 30 unique proteins expressed by the bovine primitive gut. out of 30 unique proteins, 21 proteins were used on the ontology and pathway analysis. The ontology analysis showed an enrichment of ontologies related to binding (N=5); catalytic activity (N=6); intracellular organelle (N=6). There was an enrichment of ontologies associated to cytoskeleton modifications; cell differentiation process (N=3); cellular migration (N=4) and cell metabolism (N=6). Furthermore, the pathway and the network analysis showed an enrichment of cell-to-cell communication pathways such as gap and tight junction, and focal adhesion pathways. In addition, pathways involved in cellular movement (regulation of actin cytoskeleton and leukocyte transendothelial migration) were extremely enrichment in the group of proteins expressed by the bovine primitive gut. Our results suggested that the cells from primitive gut have high migratory profile and are composed of not fully differentiated cells with high cellular metabolism. The proper migration and differentiation of these cells would dictate the fate of the developing embryo. Moreover, understanding the function and interaction of proteins expressed by normal embryo will give clues of the impact of the reproductive biotechnologies in embryo development during the window between implantation and placentation.

Análise proteômica do intestino primitivo de embriões bovinos / Proteomic analysis of primitive gut from bovine embryo

O desenvolvimento de biotecnologia de embriões em animais de produção é prejudicado por perdas no primeiro trimestre da gestação, idade em que o intestino primitivo está sendo estabelecido. O estudo das proteínas contidas no intestino primitivo nesta fase inicial da gestação pode aumentar o conhecimento sobre as vias moleculares envolvidas no desenvolvimento embrionário normal e em perdas de embriões, assim como a sua participação na organogênese e diferenciação celular. Intestino primitivo de embriões de bovinos a partir dos 39 SD ± 4 dias de desenvolvimento (variando de 33 a 45 dias) foram coletados em um matadouro local. As amostras foram processadas e agrupadas para análise proteômica shotgun label-free usando MudPIT (Multidimensional Protein Identification Technology). Análise funcional e de via foram feitas usando FatiGO (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) para identificar as ontologias relevantes e vias canônicas ou não-canônicas representada pelas proteínas expressas no Intestino primitivo. Um total de 74 proteínas ou sequências randômicas foram identificadas, correspondentes a 30 proteínas específicas expressas pelo Intestino primitivo bovino. Das 30 proteínas únicas, 21 proteínas foram utilizadas na ontologia e análise de vias. As análises mostraram um enriquecimento de ontologias relacionadas com a ligação (N = 5); atividade catalítica (N = 6); organela intracelular (N = 6). Houve um enriquecimento de ontologias associado às modificações do citoesqueleto; processo de diferenciação celular (N = 3), a migração celular (N = 4) e no metabolismo celular (N = 6). Além disso, a via e a análise de rede mostraram um enriquecimento de vias de comunicação entre células, tais como junções comunicantes e tight e as vias de adesão focal. Além disso, as vias envolvidas no movimento celular (por exemplo, vias de regulação do citoesqueleto de actina e a migração transendotelial de leucócitos) foram extremamente enriquecimento no grupo de proteínas expressas pelo Intestino primitivo bovino. Nossos resultados sugerem que as células do intestino primitivo tem alto perfil migratório e são compostas de células não totalmente diferenciadas, com alto metabolismo celular. A migração e a diferenciação destas células poderiam determinar o destino do embrião em desenvolvimento. Além disso, a compreensão da função e interação de proteínas expressas pelo embrião normal fornecerá informações sobre o impacto das biotecnologias reprodutivas no desenvolvimento do embrião durante a implantação e placentação. (AU)

The development of embryo biotechnology in farm animals is hampered by embryo losses in first trimester of gestation, period in which the primitive gut is being established. The study of proteins contained in the primitive gut at this early stage of pregnancy may increase the knowledge about the molecular pathways involved in normal embryonic development and loss of embryos, as well as their involvement in organogenesis and cell differentiation.primitive gut from bovine embryos on day 39 SD± 4 of development (ranging from 33 to 45 days) were collected at a local slaugtherhouse. The samples were processed and pooled for label-free shotgun proteomics analysis using MudPIT (Multidimensional Protein Identification Technology) tandem MSE acquisition. Functional and pathway analysis using FatiGo (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) and Ingenuity Pathway Analysis (www.ingenuity.com) were used to identify relevant ontologies and canonical or noncanonical pathways represented by the expressed proteins in the primitive gut. A total of 74 protein sequences were identified corresponding to 30 unique proteins expressed by the bovine primitive gut. out of 30 unique proteins, 21 proteins were used on the ontology and pathway analysis. The ontology analysis showed an enrichment of ontologies related to binding (N=5); catalytic activity (N=6); intracellular organelle (N=6). There was an enrichment of ontologies associated to cytoskeleton modifications; cell differentiation process (N=3); cellular migration (N=4) and cell metabolism (N=6). Furthermore, the pathway and the network analysis showed an enrichment of cell-to-cell communication pathways such as gap and tight junction, and focal adhesion pathways. In addition, pathways involved in cellular movement (regulation of actin cytoskeleton and leukocyte transendothelial migration) were extremely enrichment in the group of proteins expressed by the bovine primitive gut. Our results suggested that the cells from primitive gut have high migratory profile and are composed of not fully differentiated cells with high cellular metabolism. The proper migration and differentiation of these cells would dictate the fate of the developing embryo. Moreover, understanding the function and interaction of proteins expressed by normal embryo will give clues of the impact of the reproductive biotechnologies in embryo development during the window between implantation and placentation. (AU)