RESUMEN
In vitro embryo production (IVP) in cattle is crucial for advancing genetic enhancement and preserving valuable genetic lineages, enabling precise genetic modifications and gene studies through modern techniques. Successful genetic manipulation in cattle embryos requires efficient delivery of exogenous DNA/RNA molecules. This research investigates the efficacy of a single embryo culture system for developing genetically modified zona-free (ZF) embryos and examines the use of liposome-based SAMTOR target siRNA transfer in these individually cultured ZF embryos. The findings indicated that the individual culture system resulted in increased cleavage rates, and blastocyst rates were minimally impacted. The new culture system effectively achieved SAMTOR silencing, with 8-16 cell embryos exhibiting reduction in transcript levels compared to control. Measurement of total protein content in the spent culture media was performed to validate the single-culture approach for further analytical applications. Total protein content analysis demonstrated the system's suitability for comprehensive evaluation of the embryo-media interaction, enhancing the scope for in-depth genetic research and applications. This research sheds light into an innovative method to improve genetic editing techniques in reproduction research.
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Técnicas de Cultivo de Embriones , Técnicas de Transferencia de Gen , Liposomas , Animales , Bovinos/embriología , Técnicas de Transferencia de Gen/veterinaria , Técnicas de Cultivo de Embriones/veterinaria , ARN Interferente Pequeño , Embrión de Mamíferos , Blastocisto , Femenino , Animales Modificados Genéticamente , Desarrollo Embrionario , Fertilización In Vitro/veterinariaRESUMEN
STUDY QUESTION: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? SUMMARY ANSWER: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. WHAT IS KNOWN ALREADY: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. STUDY DESIGN, SIZE, DURATION: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. MAIN RESULTS AND THE ROLE OF CHANCE: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). LIMITATIONS, REASONS FOR CAUTION: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.
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Blastocisto , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Humanos , Femenino , Estudios Retrospectivos , Desarrollo Embrionario/fisiología , Embarazo , Adulto , Citoplasma/metabolismo , Aneuploidia , Vitrificación , Transferencia de Embrión/métodos , Fertilización In Vitro , Implantación del Embrión/fisiologíaRESUMEN
Context In vitro embryo production in pigs is an important tool for advancing biomedical research. Intracytoplasmic sperm injection (ICSI) circumvents the polyspermy problems associated with conventional IVF in porcine. However, the suboptimal efficiency for ICSI in pigs requires new strategies to increase blastocyst formation rates. Aim To investigate novel methods for assisted activation using the zinc chelator 1,10-phenanthroline (PHEN), and to improve embryo developmental competence and quality of ICSI porcine blastocyst. Methods ICSI embryos were treated with PHEN after or before sperm injection, recording pronuclear formation, blastocyst rate and the expression of SMARCA4, OCT4, SOX2 and CDX2. Key results Neither electrical nor PHEN significantly improves pronuclear formation rates before or after ICSI. Following in vitro culture to the blastocyst stage, no significant differences were observed in developmental rates among the groups. Moreover, the use of PHEN did not alter the total cell number or the expression of OCT4, SOX2 and CDX2 in pig ICSI blastocysts. Conclusions Assisted oocyte activation with PHEN does not affect the preimplantation development of ICSI-derived pig embryos. Implications These results hold significance in refining and advancing the application of assisted oocyte activation techniques. They offer insights into addressing fertility issues and propelling advancements in human and animal reproductive medicine.
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Quelantes , Desarrollo Embrionario , Oocitos , Fenantrolinas , Inyecciones de Esperma Intracitoplasmáticas , Animales , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Inyecciones de Esperma Intracitoplasmáticas/métodos , Porcinos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Fenantrolinas/farmacología , Femenino , Quelantes/farmacología , Desarrollo Embrionario/efectos de los fármacos , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Zinc/farmacología , Técnicas de Cultivo de Embriones/veterinaria , MasculinoRESUMEN
Cloning by somatic cell nuclear transfer (SCNT) remained challenging for Rhesus monkeys, mostly due to its low efficiency and neonatal death. Genome-scale analyses revealed that monkey SCNT embryos displayed widespread DNA methylation and transcriptional alterations, thus including loss of genomic imprinting that correlated with placental dysfunction. The transfer of inner cell masses (ICM) from cloned blastocysts into ICM-depleted fertilized embryos rescued placental insufficiency and gave rise to a cloned Rhesus monkey that reached adulthood without noticeable abnormalities.
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Clonación de Organismos , Metilación de ADN , Macaca mulatta , Técnicas de Transferencia Nuclear , Animales , Técnicas de Transferencia Nuclear/veterinaria , Macaca mulatta/genética , Femenino , Embarazo , Impresión Genómica , Blastocisto/citología , Blastocisto/metabolismo , GenomaRESUMEN
This study aimed to evaluate the effect of chemical gasification and HEPES as alternative systems to pH control during in vitro maturation on bovine oocytes competence. Groups of 20 bovine cumulus oocytes complexes (COCs) were randomly distributed and cultured for 24 h in one of the following experimental groups: (i) chemical reaction (ChRG) system: CO2 generated from sodium bicarbonate and citric acid reaction (ii) culture media TCM-HEPES (HEPES-G); and (iii) control group (CNTG) in conventional incubator. After in vitro maturation (IVM), the COCs were in vitro fertilized (IVF), and in vitro cultivated (IVC) in a conventional incubator. We evaluated oocyte nuclear maturation, cleavage and blastocyst rates, in addition to the relative mRNA expression of BAX, BMP-15, AREG and EREG genes in oocytes and cumulus cells. The proportion of oocytes in metaphase II was higher in CNTG and ChRG (77.57% and 77.06%) than in the HEPES-G (65.32%; p = .0408 and .0492, respectively). The blastocyst production was similar between CNTG and ChRG (26.20% and 28.47%; p = .4232) and lower (p = .001) in the HEPES-G (18.71%). The relative mRNA expression of BAX gene in cumulus cells was significantly higher (p = .0190) in the HEPES-G compared to the CNTG. Additionally, the relative mRNA expression of BMP-15 gene was lower (p = .03) in oocytes from HEPES-G compared to the CNTG. In conclusion, inadequate atmosphere control has a detrimental effect on oocyte maturation. Yet, the use of chemical gasification can be an efficient alternative to bovine COCs cultivation.
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Fertilización In Vitro , Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Animales , Bovinos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/efectos de los fármacos , Fertilización In Vitro/veterinaria , Femenino , Medios de Cultivo , Blastocisto/efectos de los fármacos , Células del Cúmulo/efectos de los fármacos , Dióxido de Carbono/farmacología , Bicarbonato de Sodio/farmacología , Ácido Cítrico/farmacología , Técnicas de Cultivo de Embriones/veterinariaRESUMEN
PURPOSE: Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos. METHODS: Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls. RESULTS: Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos. CONCLUSION: Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.
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Blastocisto , Elementos de Nucleótido Esparcido Largo , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Blastocisto/metabolismo , Femenino , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Mutagénesis Insercional/genética , Aneuploidia , Genoma Humano/genética , Fertilización In Vitro , Masculino , Variación Genética/genética , Ratones , Mapeo Cromosómico/métodosRESUMEN
PURPOSE: To assess the impact of maternal age on the association between maternal basal FSH and aneuploidy. METHODS: A retrospective study including data from 1749 blastocysts diagnosed as euploid or aneuploid by PGT-A (preimplantation genetic testing for aneuploidy). Aneuploidy incidence was compared between embryos from mothers with high vs. low basal FSH levels (above and below the group median, respectively) in total, pre-AMA (advanced maternal age; < 35 years, 198 embryos) and AMA (≥ 35 years, 1551 embryos) patient groups, separately. To control for the interference of potentially confounding variables, the association between aneuploidy and high basal FSH levels was assessed by multivariate logistic analysis in overall, pre-AMA and AMA patient groups. RESULTS: Overall, aneuploidy rate was 9% higher (p = 0.02) in embryos from patients with high basal FSH (63.7%) compared to those with low basal FSH (58.4%). In the pre-AMA subgroup, aneuploidy incidence was 35% higher (p = 0.04) in embryos from patients with high basal FSH (53.5%) compared to those with low basal FSH (39.4%). Differently, aneuploidy occurrence did not vary between embryos from AMA patients with low (61.0%) and high (64.8%) basal FSH (p = 0.12). The multivariate analysis revealed that, in pre-AMA embryos, the association between aneuploidy occurrence and high basal FSH is independent of potential confounding variables (p = 0.04). CONCLUSION: Maternal basal FSH values are associated with embryo aneuploidy in pre-AMA but not in AMA patients. The present findings suggest that basal FSH is a useful parameter to assess aneuploidy risk in pre-AMA patients and reinforce the hypothesis that excessive FSH signalling can predispose to oocyte meiotic errors.
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Aneuploidia , Hormona Folículo Estimulante , Edad Materna , Humanos , Femenino , Adulto , Hormona Folículo Estimulante/sangre , Embarazo , Diagnóstico Preimplantación , Estudios Retrospectivos , Incidencia , Blastocisto/metabolismo , Fertilización In Vitro , Transferencia de Embrión , Pruebas Genéticas , Índice de EmbarazoRESUMEN
The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimination of identification redundancies among search tools (at three levels, based on identifiers, peptides, and cross-database mapping), 1977 proteins were reliably identified in the samples of equine embryos. Proteomic analysis unveiled robust metabolic activity in the 8-day equine embryo, highlighted by an abundance of proteins engaged in key metabolic pathways like the TCA cycle, ATP biosynthesis, and glycolysis. The prevalence of chaperones among highly abundant proteins suggests that regulation of protein folding, and degradation is a key process during embryo development. These findings pave the way for developing new strategies to improve equine embryo media and optimize in vitro fertilization techniques.
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Blastocisto , Proteoma , Animales , Caballos/embriología , Femenino , Blastocisto/metabolismo , Desarrollo Embrionario , Estudios Prospectivos , Proteómica , Fertilización In Vitro/veterinariaRESUMEN
Integrating in vitro embryo production with embryonic microsurgery facilitates the generation of monozygotic twins. However, despite their potential benefits, these methods have not been widely adopted in commercial settings because of their substantial costs. Hence, there is a need to streamline the bisection procedure while ensuring efficient production of viable demi-embryos. In this study, we investigated the impact of different orientations of microsurgical incisions in relation to inner cell mass on embryonic development, morphology, viability, and expression of cell fate protein markers using a simplified microsurgery approach. Ovaries were transported from the slaughterhouse to the laboratory and aspirated to obtain oocytes that were selected and subjected to in vitro embryo production. The selected expanded blastocysts (n = 204) underwent microsurgery. The blastocysts were immobilized to facilitate incision using an adapted microblade, yielding demi-embryos (vertical incision) and viable embryonic fragments (transverse incision). The structures were then re-cultured for 12 h. Viability was assessed by measuring the re-expansion rate after re-culture, followed by immunofluorescence analysis of proteins (CDX2 and NANOG) and apoptosis analysis using terminal deoxynucleotyl transferase dUTP nick end-labeling (TUNEL). Microsurgically derived embryos exhibited remarkable plasticity, as evidenced by a slight reduction (P < 0.05) in the re-expansion rate (transverse 64.2 % and vertical 57.2 %) compared to that of the control group (blastocysts without microsurgery) (86.7 %). They also demonstrated the ability of morphological reconstitution after culturing. Despite the anticipated decrease (P < 0.05) in the total number of cells and embryo volume, microsurgery did not result in a significant increase (P > 0.05) in the number of apoptotic cells. Furthermore, microsurgery led to higher (P < 0.05) expression of markers associated with pluripotency, indicating its efficiency in preserving regenerative capacity. Moreover, microsurgery, whether followed by immunosurgery or not, made the isolation of embryonic cells easier. In conclusion, both transverse and vertical microsurgery incisions enabled the production of identical demi-embryos and served as tools for isolating embryonic cells without compromising the resumption of development and the apoptotic index.
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Técnicas de Cultivo de Embriones , Microcirugia , Animales , Microcirugia/métodos , Microcirugia/veterinaria , Técnicas de Cultivo de Embriones/veterinaria , Fertilización In Vitro/veterinaria , Desarrollo Embrionario , Femenino , Embrión de Mamíferos/fisiología , Blastocisto/fisiología , Bovinos/embriologíaRESUMEN
The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.
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Blastocisto , Blastómeros , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Animales , Bovinos/embriología , Femenino , Técnicas de Cultivo de Embriones/veterinaria , Blastómeros/citología , Fertilización In Vitro/veterinaria , MicroARNs/genética , MicroARNs/metabolismo , Regulación del Desarrollo de la Expresión Génica , EmbarazoRESUMEN
The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF.
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Blastocisto , Técnicas de Cocultivo , Técnicas de Cultivo de Embriones , Células Epiteliales , Fertilización In Vitro , Animales , Técnicas de Cocultivo/veterinaria , Porcinos/embriología , Femenino , Técnicas de Cultivo de Embriones/veterinaria , Fertilización In Vitro/veterinaria , Células Epiteliales/citología , Células Epiteliales/fisiología , Blastocisto/fisiología , Blastocisto/citología , Desarrollo Embrionario/fisiología , Trompas Uterinas/citología , Oviductos/citología , Embrión de Mamíferos/fisiologíaRESUMEN
BACKGROUND: Genetically modified pigs are considered ideal models for studying human diseases and potential sources for xenotransplantation research. However, the somatic cell nuclear transfer (SCNT) technique utilized to generate these cloned pig models has low efficiency, and fetal development is limited due to placental abnormalities. RESULTS: In this study, we unprecedentedly established putative porcine trophoblast stem cells (TSCs) using SCNT and in vitro-fertilized (IVF) blastocysts through the activation of Wing-less/Integrated (Wnt) and epidermal growth factor (EGF) pathways, inhibition of transforming growth factor-ß (TGFß) and Rho-associated protein kinase (ROCK) pathways, and supplementation with ascorbic acid. We also compared the transcripts of putative TSCs originating from SCNT and IVF embryos and their differentiated lineages. A total of 19 porcine TSCs exhibiting typical characteristics were established from SCNT and IVF blastocysts (TSCsNT and TSCsIVF). Compared with the TSCsIVF, TSCsNT showed distinct expression patterns suggesting unique TSCsNT characteristics, including decreased mRNA expression of genes related to apposition, steroid hormone biosynthesis, angiopoiesis, and RNA stability. CONCLUSION: This study provides valuable information and a powerful model for studying the abnormal development and dysfunction of trophoblasts and placentas in cloned pigs.
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Blastocisto , Técnicas de Transferencia Nuclear , Trofoblastos , Animales , Trofoblastos/metabolismo , Porcinos , Diferenciación Celular , Femenino , Células Madre , Fertilización In Vitro/métodosRESUMEN
Adverse pregnancy outcomes have been associated with the presence of glyphosate (G) in umbilical cord, serum, and urine samples from pregnant women. Our aim was to study the effect of G on blastocyst implantation using an in vitro mouse model, and the migration and acquisition of endothelial phenotype of the human trophoblastic HTR8/SVneo (H8) cells. In mouse blastocysts, no differences in attachment time and implantation outgrowth area were observed after G exposure. H8 cell migration was stimulated by 0.625 µM G without cytotoxicity. After 6 h, the mRNA expression of vascular endothelial growth factor (VEGF) and C-C motif chemokine ligand 2 (CCL2) was upregulated in H8 cells exposed to 1.25 µM G when compared vehicle-treated cells (p ≤ 0.05). No differences were observed in interleukin 11, VEGF receptor 1, and coagulation factor II thrombin receptor in H8 cells exposed to different concentrations of G for 6 h compared to the vehicle. Interestingly, exposure to G did not alter angiogenesis as measured by a tube formation assay. Taken all together, these results suggest that G exposure may contribute as a risk factor during pregnancy, due to its ability to alter trophoblast migration and gene expression.
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Blastocisto , Movimiento Celular , Implantación del Embrión , Glicina , Glifosato , Trofoblastos , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Movimiento Celular/efectos de los fármacos , Humanos , Animales , Femenino , Ratones , Glicina/análogos & derivados , Glicina/toxicidad , Glicina/farmacología , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Implantación del Embrión/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Línea Celular , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Embarazo , Herbicidas/toxicidad , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , AngiogénesisRESUMEN
Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts.
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Blastocisto , Roturas del ADN de Doble Cadena , Factores de Crecimiento de Fibroblastos , Animales , Femenino , Bovinos , Blastocisto/efectos de los fármacos , Blastocisto/fisiología , Embarazo , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Desarrollo Embrionario/efectos de los fármacos , Técnicas de Cultivo de Embriones/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacosRESUMEN
OBJECTIVE: Cryopreservation has some adverse effects on embryos including cell metabolism reduction, mitochondria and plasma membrane damage, excess production of 'Reactive Oxygen Species' and damage to DNA. In the present study. In this study we assessed the effect of coenzyme Q10 as an exogenous antioxidant on mouse embryos following cryopreservation. METHODS: We collected mice embryos at the morula stage from uterine horns on the third day of gestation. The morulae were divided into 9 groups (1 control, 2 vehicles and 6 experimental), then vitrified. The culture and/or vitrification media of the experimental groups were supplemented by 10 or 30 µM of CoQ10. After one week, the embryos were warmed and then cultured. After 48 hours of embryo culture, the blastocyst rate, total cell number, viability; and after 72 hours of embryo culture, we assessed the hatching rate. RESULTS: Blastocyst rate and hatching rate were significantly reduced in the groups containing 30 µM CoQ10 supplemented culture media compared to other groups (p<0.05). The hatching rate in the groups containing 10 µM CoQ10 supplemented in both culture and vitrification media was significantly higher than in the other groups (p<0.05). In groups containing 10 µM CoQ10 supplemented culture media, the viability was higher than that in the other groups (p<0.05). CONCLUSIONS: It seems that CoQ10 in a dose-dependent manner is able to improve hatching rate and viability following cryopreservation through its antioxidant and anti-apoptotic properties, and through the production of ATP.
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Criopreservación , Ubiquinona , Animales , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Ratones , Femenino , Técnicas de Cultivo de Embriones , Desarrollo Embrionario/efectos de los fármacos , Blastocisto/efectos de los fármacos , Vitrificación/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Antioxidantes/farmacología , EmbarazoRESUMEN
Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 µM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 µM, with no embryo formation occurring at 24 µM. Further analysis showed that embryos treated with 12 µM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells.
Asunto(s)
Apoptosis , Autofagia , Blastocisto , Desarrollo Embrionario , Hidroxicloroquina , Animales , Bovinos , Hidroxicloroquina/toxicidad , Desarrollo Embrionario/efectos de los fármacos , Autofagia/efectos de los fármacos , Apoptosis/efectos de los fármacos , Blastocisto/efectos de los fármacos , Femenino , Antimaláricos/toxicidad , Fertilización In Vitro , Técnicas de Cultivo de EmbrionesRESUMEN
Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.
Asunto(s)
Blastocisto , Proteómica , Zona Pelúcida , Animales , Blastocisto/metabolismo , Zona Pelúcida/metabolismo , Gatos , Proteómica/métodos , Técnicas de Cultivo de Embriones , Secretoma/metabolismo , Femenino , Fertilización In Vitro , Proteoma/metabolismo , Desarrollo Embrionario , Espectrometría de Masas en Tándem , Cromatografía LiquidaRESUMEN
Mammalian oocytes undergo maturation and fertilization in the low-oxygen (O2) environment of the oviduct. To evaluate the effect of O2 tension during in vitro maturation and fertilization on embryo yield, quality, cryotolerance, and gene expression, we matured and fertilized bovine cumulus-oocyte complexes under low (5%) or high (20%) O2 tension. Presumptive zygotes from both groups were cultured at 5% O2 for 8 days. Blastocysts were vitrified, and then warmed, and cultured for further 24 h to assess their cryotolerance. Our findings indicate that low O2 during maturation and fertilization enhances embryo development and cell count in both fresh and vitrified/warmed blastocysts. In this study, the interaction of O2 tension and status (fresh or vitrified/warmed) affected the transcript abundance of SOD2, AQP3, and BAX in blastocysts. These results highlight the role of low O2 tension during bovine maturation and fertilization and provide support to using 5% O2 throughout all stages of bovine in vitro embryo production.
Asunto(s)
Fertilización In Vitro , Vitrificación , Bovinos , Animales , Fertilización In Vitro/veterinaria , Fertilización In Vitro/métodos , Oocitos , Blastocisto , Oxígeno/farmacología , Criopreservación/veterinaria , Criopreservación/métodos , MamíferosRESUMEN
The in vivo fertilization process occurs in the presence of follicular fluid (FF). The aim of this study was to evaluate the effect of in vitro fertilization medium supplementation with 5% or 10% bovine follicular fluid (BFF) on the production of in vitro bovine embryos. FF was collected from ovarian follicles with a diameter of 8-10 mm, and cumulus-oocyte complexes (COCs) were co-incubated with sperm for 24 h in the commercial medium BotuFIV® (BotuPharma©), being distributed among the experimental groups: oocytes fertilized in a control medium; oocytes fertilized in a medium supplemented with 5% BFF; and oocytes fertilized in a medium supplemented with 10% BFF. After fertilization, the zygotes were cultured in vitro for 8 days. Embryo development was assessed through cleavage rates (day 2) and blastocyst formation rates (day 8). The relative expression of the genes OCT4, IFNT2, BAX, HSP70 and SOD2 was measured using the real-time polymerase chain reaction method. There was no difference (p > .05) among the different experimental groups in terms of cleavage rates and blastocyst formation rates. Regarding the gene expression results, only the blastocysts from oocytes fertilized with 10% BFF showed significantly lower expression of IFNT2 (p = .003) and SOD2 (p = .01) genes compared to blastocysts from oocytes fertilized in control medium alone, while there was no difference between blastocyst from oocytes fertilized in control medium and the ones from oocytes fertilized with 5% BFF. In addition to this, the blastocysts from oocytes fertilized with 5% BFF showed significantly reduced levels of expression of the heat shock protein HSP70 (p < .001) and the pro-apoptotic protein BAX (p = .015) compared to blastocysts from oocytes fertilized with control medium. This may indicate that lower supplementation of BFF to the IVF medium creates a more suitable environment for fertilization and is less stressful for the zygote.