RESUMO
Polarization of the mammalian embryo at the right developmental time is critical for its development to term and would be valuable in assessing the potential of human embryos. However, tracking polarization requires invasive fluorescence staining, impermissible in the in vitro fertilization clinic. Here, we report the use of artificial intelligence to detect polarization from unstained time-lapse movies of mouse embryos. We assembled a dataset of bright-field movie frames from 8-cell-stage embryos, side-by-side with corresponding images of fluorescent markers of cell polarization. We then used an ensemble learning model to detect whether any bright-field frame showed an embryo before or after onset of polarization. Our resulting model has an accuracy of 85% for detecting polarization, significantly outperforming human volunteers trained on the same data (61% accuracy). We discovered that our self-learning model focuses upon the angle between cells as one known cue for compaction, which precedes polarization, but it outperforms the use of this cue alone. By compressing three-dimensional time-lapsed image data into two-dimensions, we are able to reduce data to an easily manageable size for deep learning processing. In conclusion, we describe a method for detecting a key developmental feature of embryo development that avoids clinically impermissible fluorescence staining.
Assuntos
Polaridade Celular , Aprendizado Profundo , Embrião de Mamíferos/citologia , Animais , Corantes/química , Embrião de Mamíferos/química , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Fertilização in vitro , Humanos , Camundongos , Coloração e RotulagemRESUMO
We have developed a protocol to quantify the position of a cell in a branched structure based on two-dimensional microscopy images of tissue sections. Biological branched structures include organs such as the lungs, kidneys, and pancreas. In these organs, cell fate has been correlated with position, based on a qualitative estimate. However, a quantitative means of evaluating the cell position has been lacking. With this protocol, the correlation between cell fate and cell position was measured in mouse embryonic pancreas. For complete details on the use and execution of this protocol, please refer to Nyeng et al. (2019).
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Microscopia/métodos , Animais , Embrião de Mamíferos/química , Embrião de Mamíferos/citologia , Feminino , Rim/citologia , Pulmão/citologia , Masculino , Camundongos , Pâncreas/citologiaRESUMO
Somatic cell nuclear transfer (SCNT) is a method with unique ability to reprogram the epigenome of a fully differentiated cell. However, its efficiency remains extremely low. In this work, we assessed and combined two simple strategies to improve the SCNT efficiency in the bovine. These are the use of less-differentiated donor cells to facilitate nuclear reprogramming and the embryo aggregation (EA) strategy that is thought to compensate for aberrant epigenome reprogramming. We carefully assessed the optimal time of EA by using in vitro-fertilized (IVF) embryos and evaluated whether the use of adipose-derived mesenchymal stem cells (ASCs) as donor for SCNT together with EA improves the blastocyst rates and quality. Based on our results, we determined that the EA improves the preimplantation embryo development per well of IVF and SCNT embryos. We also demonstrated that day 0 (D0) is the optimal aggregation time that leads to a single blastocyst with uniform distribution of the original blastomeres. This was confirmed in bovine IVF embryos and then, the optimal condition was translated to SCNT embryos. Notably, the relative expression of the trophectoderm (TE) marker KRT18 was significantly different between aggregated and nonaggregated ASC-derived embryos. In the bovine, no effect of the donor cell is observed on the developmental rate, or the embryo quality. Therefore, no synergistic effect of the use of both strategies is observed. Our results suggest that EA at D0 is a simple and accessible strategy that improves the blastocyst rate per well in bovine SCNT and IVF embryos and influence the expression of a TE-related marker. The aggregation of two ASC-derived embryos seems to positively affect the embryo quality, which may improve the postimplantation development.
Assuntos
Blastocisto/citologia , Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Células-Tronco Mesenquimais/citologia , Animais , Bovinos , Embrião de Mamíferos/química , Feminino , Fertilização in vitro , GravidezRESUMO
During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.
Assuntos
Ciclina B1/genética , Embrião de Mamíferos/química , Oócitos/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-mos/genética , Imagem Individual de Molécula/métodos , Animais , Citoplasma/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , Camundongos , Oócitos/química , Polirribossomos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA não Traduzido/genéticaRESUMO
The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.
Assuntos
Blastocisto/metabolismo , Bovinos/fisiologia , Embrião de Mamíferos/química , Prenhez/metabolismo , Animais , Biomarcadores , Meios de Cultura/análise , Feminino , GravidezRESUMO
Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, invitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n=80) and vitrified (n=80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell-sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.
Assuntos
Blastocisto/metabolismo , Bovinos/embriologia , Criopreservação/veterinária , Expressão Gênica , Animais , Transferência Embrionária/veterinária , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência de RNA , Transdução de Sinais/genéticaRESUMO
The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.
Assuntos
Embrião de Mamíferos/química , Proteoma/análise , Carneiro Doméstico/embriologia , Animais , Feminino , Inseminação Artificial/veterinária , Masculino , MicroRNAs/genética , Proteoma/genética , Carneiro Doméstico/genética , Carneiro Doméstico/metabolismoRESUMO
BACKGROUND: The GABA-A receptor signaling pathway regulates proliferation, differentiation, apoptosis, and responses to overt DNA damage during embryonic development. METHODS: To analyze the gene expression after intracytoplasmic sperm injection (ICSI) and in in vivo mouse embryos, the global pattern of gene expression dataset, GSE23009, was obtained from the Gene Expression Omnibus database. Genes with differential expression were identified using the R software package, and RT-qPCR was performed to confirm the microarray results. RESULTS: Mouse blastocysts derived from ICSI fertilization had decreased expression of GABA-A receptor signaling pathway genes. However, the mechanisms underlying these changes were not elucidated. The gene expression of the GABA-A pathway was not significantly different between blastocysts obtained from IVF and in vivo fertilization. However, microinjection after IVF significantly reduced the expression of the GABA-A pathway gene to levels similar to those in the ICSI group. CONCLUSION: Based on our results, decreased gene expression is a result of the microinjection manipulation performed during ICSI.
Assuntos
Desenvolvimento Embrionário/genética , Microinjeções/efeitos adversos , Receptores de GABA-A , Transdução de Sinais/genética , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Animais , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Receptores de GABA-A/análise , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Transcriptoma/genéticaRESUMO
Oocyte proteins play an important role in oocyte maturation, fertilization and embryonic development. However, the protein composition of mouse germinal vesicle (GV) oocytes is still unclear. Using one-dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) and Reverse-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS), we constructed a protein profile of mouse GV oocytes. First, our proteomics profile identified 1,405 different proteins from 11,000 mouse GV oocytes lacking zona pellucida. Second, with detailed bioinformatics analysis, a group of proteins that play an essential role in oocyte maturation was screened. In addition, the expression and localization of suppressor of G2 allele of skp1(SUGT1, also called SGT1), heterogeneous nuclear ribonucleoprotein K (Hnrpk), Seruin, Cullin1(Clu1) and nuclear distribution protein C (Nudc) in mouse ovaries and early embryos were also captured and investigated in this study. Moreover, the protein profile was submitted to the Proteomics Identifications Database (PRIDE) and is available via ProteomeXchange with the identifier PXD014314. Our research provides valuable resources for the study of oocyte proteins and oocyte maturation and helps to clarify the mechanisms of oocyte maturation.
Assuntos
Oócitos/química , Ovário/química , Proteoma/análise , Animais , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Feminino , Camundongos Endogâmicos ICR , Oócitos/metabolismo , Ovário/metabolismo , Proteoma/genéticaRESUMO
DNA methylation in mammals is essential for numerous biological functions, such as ensuring chromosomal stability, genomic imprinting, and X-chromosome inactivation through transcriptional regulation. Gene knockout of DNA methyltransferases and demethylation enzymes has made significant contributions to analyzing the functions of DNA methylation in development. By applying epigenome editing, it is now possible to manipulate DNA methylation in specific genomic regions and to understand the functions of these modifications. In this review, we first describe recent DNA methylation editing technology. We then focused on changes in DNA methylation status during mammalian gametogenesis and preimplantation development, and have discussed the implications of applying this technology to early embryos.
Assuntos
Metilação de DNA , Embrião de Mamíferos/química , Edição de Genes/métodos , Animais , Blastocisto/química , DNA (Citosina-5-)-Metiltransferases/genética , Técnicas de Inativação de Genes , Impressão Genômica , HumanosRESUMO
Parthenogenesis, a unique form of reproduction, is normally inhibited in mammals and a human embryo with parthenogenetic origin is not considered capable of producing offspring. The aim of this report is to analyze a parthenogenetic oocyte retrieved from a patient so as to have a better understanding on parthenogenesis and causes of infertility. A 38-year-old woman presented at our center with a history of primary infertility for 10 years and underwent an IVF-ICSI cycle. Three MII oocytes retrieved and one of which presented with 1 pronucleus before conducting ICSI and developed into an embryo 30 h post-retrieval. Blastomere biopsy, genome amplification, copy number variation (CNV) analysis and MultiSNPs analysis was performed on the embryo. The results showed that only one blastomere contains DNA and CNV analysis indicated a genotype of 48, XX, +17, +17 and the genetic contribution of biopsied embryo was of exclusively maternal origin. Such analysis might be beneficial for patients with a history of oocyte spontaneous activation in diagnosing case-specific aberrations and providing individualized therapeutic strategies such as preimplantation genetic diagnosis to choose a genetic normal embryo to transplant.
Assuntos
Blastômeros/patologia , Oócitos/fisiologia , Partenogênese/genética , Diagnóstico Pré-Implantação , Adulto , Biópsia , Blastômeros/química , Blastômeros/metabolismo , Variações do Número de Cópias de DNA , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fertilização in vitro , Testes Genéticos , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , Infertilidade Feminina/terapia , Injeções de Esperma Intracitoplásmicas , Falha de TratamentoRESUMO
In mammals, chromatin organization undergoes drastic reorganization during oocyte development. However, the dynamics of three-dimensional chromatin structure in this process is poorly characterized. Using low-input Hi-C (genome-wide chromatin conformation capture), we found that a unique chromatin organization gradually appears during mouse oocyte growth. Oocytes at late stages show self-interacting, cohesin-independent compartmental domains marked by H3K27me3, therefore termed Polycomb-associating domains (PADs). PADs and inter-PAD (iPAD) regions form compartment-like structures with strong inter-domain interactions among nearby PADs. PADs disassemble upon meiotic resumption from diplotene arrest but briefly reappear on the maternal genome after fertilization. Upon maternal depletion of Eed, PADs are largely intact in oocytes, but their reestablishment after fertilization is compromised. By contrast, depletion of Polycomb repressive complex 1 (PRC1) proteins attenuates PADs in oocytes, which is associated with substantial gene de-repression in PADs. These data reveal a critical role of Polycomb in regulating chromatin architecture during mammalian oocyte growth and early development.
Assuntos
Cromatina/química , Oócitos/crescimento & desenvolvimento , Oogênese/genética , Proteínas do Grupo Polycomb/fisiologia , Animais , Blastocisto/química , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Embrião de Mamíferos/química , Inativação Gênica , Código das Histonas , Camundongos , Oócitos/química , Transcrição Gênica , CoesinasRESUMO
Developing effective gene therapies for disorders of the central nervous system (CNS) is extremely challenging due to the lack of safe and efficient gene delivery methods to neurons and glial cells, hampering the study of CNS physiology and the identification of novel therapeutic targets. Current gene transfer methodologies for neuronal cultures rely on synthetic nanoparticles or viral transduction. These approaches present low gene transfer efficiency, are highly toxic, and may induce adverse immune responses. Electroporation has been implemented as an alternative approach; however, this method is restricted for the most part to cells in suspension, and electrical overstimulation of the neuronal membrane may have detrimental consequences. To overcome these barriers, here we describe the implementation of nanochannel-based electroporation for gene delivery into primary neural cultures safely and efficiently. We outline the preparation of viable primary neuronal cultures from the hippocampus of E18.5 mouse embryos and describe the optimal parameter for transfection using a nanochannel-based electroporation platform.
Assuntos
Eletroporação/métodos , Neurônios/citologia , Cultura Primária de Células/métodos , Animais , Células Cultivadas , Embrião de Mamíferos/química , Embrião de Mamíferos/citologia , Técnicas de Transferência de Genes , Camundongos , Nanotecnologia , Neurônios/químicaRESUMO
In vitro produced embryos are still sensitive to the freezing process which can be explained, in part, by the high-lipid accumulation that characterizes these embryos. Therefore, we aimed to evaluate the effect of delipidating agents, L-carnitine and the trans-10 cis-12 conjugated linoleic acid (CLA) isomer, on blastocyst development, lipid content, gene expression and cryotolerance when added to embryo culture media. Embryos were cultured in four different media: T1: control (n = 616), synthetic oviduct fluid (SOF) media with 5% foetal bovine serum (FBS); T2: L-carnitine (n = 648), SOF medium with 5% FBS and 0.6 mg/ml of L-carnitine; T3: CLA (n = 627), SOF medium with 5% FBS and 100 µM trans-10 cis-12 CLA; and T4: L-carnitine + CLA: (n = 597), SOF medium with 5% FBS plus 0.6 mg/ml L-carnitine and 100 µM trans-10 cis-12 CLA. Supplementation of culture medium with either or both delipidating agents reduced (p < .05) blastocyst rate on D7 (T1 = 49 ± 3.5; T2 = 39 ± 3.0; T3 = 42 ± 3.9 and T4 = 39 ± 3.9), but did not affected gene expression (p > .05). Although embryos cultured in the presence of L-carnitine contained fewer (p < .05) lipid droplets than the control embryos, they showed a lower re-expansion rate 24 hr post-thaw than those (p < .05). In conclusion, although L-carnitine reduced the amount of lipids in cultured embryos, the use of L-carnitine and CLA during in vitro culture was not able to improve the embryo production and the response to cryopreservation.
Assuntos
Carnitina/farmacologia , Bovinos/embriologia , Técnicas de Cultura Embrionária/veterinária , Ácidos Linoleicos Conjugados/farmacologia , Animais , Blastocisto/fisiologia , Criopreservação/métodos , Criopreservação/veterinária , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Lipídeos/análiseRESUMO
Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB-) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB- oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB- embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB- embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB- oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.
Assuntos
Blastocisto/química , Núcleo Celular/química , Embrião de Mamíferos/química , Oócitos/química , Oxazinas/química , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Núcleo Celular/ultraestrutura , Tamanho Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Feminino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Reprodutibilidade dos Testes , Coloração e Rotulagem/métodos , SuínosRESUMO
The effect of all-trans retinoic acid (RA) on embryogenesis is tissue specific and highly concentration dependent. Using a liquid chromatography/mass spectrometry-based method to quantify trace amounts of RA in embryonic tissue requires expensive specialist facilities. Here, we describe the use of a RA response element (RARE)-lacZ reporter cell-based method, which is simple and cost effective, to measure RA levels in small pieces of tissue from the embryo. We further apply this method to quantitatively assay activities of RA-synthesizing and RA-catabolizing enzymes, the key regulators of RA bioavailability in tissues and developing organs of the embryo.
Assuntos
Embrião de Mamíferos/química , Genes Reporter , Tretinoína/análise , Aldeído Desidrogenase/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Família 26 do Citocromo P450/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Tretinoína/farmacologiaRESUMO
RNA in situ hybridization is a practical technique that allows investigators to observe temporal and spatial gene expression at the RNA level in the context of whole embryos or tissues. One powerful application of in situ hybridization is to observe the consequences of genetic, toxicologic, or environmental perturbations on gene expression or morphogenesis during development. Herein, I will review the procedure to perform nonradioactive, in situ hybridization on whole-mount mouse or chick embryos.
Assuntos
Embrião de Mamíferos/química , Embrião não Mamífero/química , Perfilação da Expressão Gênica/métodos , Animais , Embrião de Galinha , Hibridização In Situ , Camundongos , Coloração e RotulagemRESUMO
As oocytes and embryos of pigs have greater lipid content in the cytoplasm than those of other species, supplementation of the medium for in vitro maturation (IVM) of oocytes with omega-3 polyunsaturated fatty acids (PUFA) may help to improve embryo development. This study was conducted to evaluate effects of the inclusion of the docosaexaenoic (DHA) and of the eicosapentaenoic acids (EPA) in the IVM medium on the development of pig oocytes and on the lipid content of oocytes and embryos. In all experiments, control media consisted of porcine follicular fluid and oocytes were activated through parthenogenesis. In Experiment 1, there were four treatments for each PUFA: one control; and three treatments including EPA or DHA in the IVM medium at 12.5 µM, 25.0 µM and 50.0 µM). In Experiment 2, inclusion of 50 µM DHA was compared against the control. Cleavage rates in the IVM medium including 12.5 µM EPA and blastocyst development rates in media at any EPA concentration were less than for the control in Experiment 1 (P < 0.05). Compared to the control, inclusion of 50 µM DHA in the IVM medium was related to greater cleavage rates and greater number of embryo cells, in Experiment 1, and lesser lipid content in oocytes after 22 and 44 h and in embryos after 7 days, in Experiment 2 (both P < 0.05). Addition of DHA in the IVM medium may benefit the development of pig oocytes, but EPA appears to be cytotoxic.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Embrião de Mamíferos/química , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Suínos/embriologia , Animais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Relação Dose-Resposta a Droga , Ácido Eicosapentaenoico/administração & dosagem , Feminino , Metabolismo dos Lipídeos , Partenogênese , Suínos/fisiologiaRESUMO
The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .
Assuntos
Embrião de Mamíferos/metabolismo , Estrogênios/metabolismo , Manutenção da Gravidez/fisiologia , Prenhez , Reconhecimento Psicológico/fisiologia , Suínos , Animais , Animais Geneticamente Modificados , Aromatase/genética , Aromatase/metabolismo , Células Cultivadas , Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Embrião de Mamíferos/química , Desenvolvimento Embrionário/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Fertilização/fisiologia , Troca Materno-Fetal/efeitos dos fármacos , Troca Materno-Fetal/fisiologia , Técnicas de Transferência Nuclear , Gravidez , Manutenção da Gravidez/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Suínos/embriologia , Suínos/genética , Suínos/metabolismoRESUMO
Increased use of pyrethroids and the exposure to pyrethroids for pregnant women and children have raised the concerns over the potential effect of pyrethroids on developmental cardiotoxicity and other abnormalities. The purpose of this study was to investigate whether long term perinatal deltamethrin exposure altered embryonic cardiac electrophysiology in mice. Pregnant mice were administered with 0 or 3 mg/kg of deltamethrin by gavage daily from gestational day (gd) 10.5 to gd 17. 5. Whole cell patch-clamp technique was used in electrophysiological study, and real time RT-PCR was applied to analyze the molecular changes for the electrophysiological properties. Deltamethrin exposure resulted in increased mortality of pregnant mice and decreased viability of embryos. Moreover, deltamethrin slowed the maximum depolarization velocity (Vmax), prolonged the action potential duration (APD) and depolarized the maximum diastolic potential (MDP) of embryonic cardiomyocytes. Additionally, perinatal deltamethrin exposure decreased the mRNA expression of Na+ channel regulatory subunit Navß1, inward rectifier K+ channel subunit Kir2.1, and delayed rectifier K+ channel subunit MERG while the L-type Ca2+ channel subunit, Cav1.2 expression was increased. On the contrary, deltamethrin administration did not significantly alter the regulation of ß-adrenergic or muscarinic receptor on embryonic cardiomyocytes. In conclusion, deltamethrin exposure at perinatal stage significantly alters mRNA expression of embryonic cardiac ion channels and therefore influences embryonic cardiac electrophysiological properties. This highlights the need to understand the persistent effects of pyrethroid exposure on cardiac function during embryonic development due to potential for cardiac arrhythmogenicity.