RESUMEN
Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra-embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4-treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4-treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.
Asunto(s)
Apoptosis/efectos de los fármacos , Blastocisto/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Desarrollo Embrionario/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Blastocisto/citología , Técnicas de Cultivo de Embriones , HumanosRESUMEN
STUDY QUESTION: What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? SUMMARY ANSWER: Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. WHAT IS KNOWN ALREADY: Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in vitro implantation model. STUDY DESIGN, SIZE, DURATION: Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Six days post-fertilization (6dpf) human embryos were co-cultured with Ishikawa cells for 12, 24 (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 145 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expressions were validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This in vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. STUDY FUNDING AND COMPETING INTEREST(S): A.A. was supported by a grant from the 'Instituut voor Innovatie door Wetenschap en Technologie' (IWT, 121716, Flanders, Belgium). This work was supported by the 'Wetenschappelijk Fonds Willy Gepts' (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.
Asunto(s)
Blastocisto/metabolismo , Moléculas de Adhesión Celular/genética , Técnicas de Cocultivo/métodos , Técnicas de Cultivo de Embriones/métodos , Implantación del Embrión/genética , Proteínas de la Matriz Extracelular/genética , Blastocisto/citología , Blastocisto/fisiología , Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Embrión de Mamíferos , Desarrollo Embrionario/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , HumanosRESUMEN
STUDY QUESTION: Is the spindle assembly checkpoint (SAC) active during human preimplantation development? SUMMARY ANSWER: Mitotic spindle disruption during mitosis activates the SAC from at least Day 3 of human preimplantation development, but this does not lead to apoptosis until Day 5. WHAT IS KNOWN ALREADY: Human preimplantation embryos frequently acquire chromosomal abnormalities, but the mechanisms behind this are poorly understood. It has been speculated that a dysfunctional SAC could be responsible. Although research has shown that the SAC components are present during early human development, functional studies are lacking. STUDY DESIGN, SIZE, DURATION: In vitro study using human preimplantation embryos in a university research laboratory. We studied a total of 38 Day-3, 38 Day-4, 29 Day-5 and 21 Day-6 human preimplantation embryos, donated for research, during 16 h of incubation. PARTICIPANT/MATERIALS, SETTING, METHODS: We cultured human preimplantation embryos overnight in a time-lapse imaging system, in control or in a nocodazole-containing medium that prevents the formation of a proper mitotic spindle. The embryos were subsequently fixed and analysed by immunocytochemistry for tubulin or mitotic and apoptotic markers, or by FISH. MAIN RESULTS AND THE ROLE OF CHANCE: All embryos showed an increase in M-phase cells from 4.1-8.8% to 21.4-53.5% when exposed to nocodazole (P < 0.05; two-way ANOVA for all groups except Day-4 embryos, P = 0.128) suggesting SAC functionality. Apoptosis, which was rarely detected between Day 3 and Day 6 in good-quality control embryos, increased from Day 5 onwards in nocodazole-treated embryos and became statistically different from Day 6 (P < 0.01; two-way ANOVA). The FISH data suggest that in compacted Day-4 embryos, approximately one in six cells started a polyploid new cell cycle rather than to go in apoptosis after the failure to maintain the SAC-mediated M-phase arrest. These results suggest that during early embryo development, blastomeres with unresolved chromosome misalignments during M-phase can escape SAC-mediated apoptosis, continue cell division which can then result in aneuploid daughter cells. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study used nocodazole to inhibit microtubule polymerization, a drug that is regularly used to induce metaphase arrest and SAC activation. Results should be extrapolated to naturally occurring chromosome misalignments with care. WIDER IMPLICATIONS OF THE FINDINGS: Our results provide functional data that can help explain the high aneuploidy rates seen in human cleavage-stage embryos and suggest that this is due to their unusual cell cycle control. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen) and the Methusalem grant to Karen Sermon of the Research Council of the Vrije Universiteit Brussel. The authors declare no competing financial interests.
Asunto(s)
Aberraciones Cromosómicas/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Nocodazol/farmacología , Huso Acromático/efectos de los fármacos , Moduladores de Tubulina/farmacología , Apoptosis/efectos de los fármacos , Blastocisto , Técnicas de Cultivo de Embriones , Embrión de Mamíferos , Desarrollo Embrionario/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Embarazo , Huso Acromático/genética , Huso Acromático/patología , Imagen de Lapso de TiempoRESUMEN
During human preimplantation development the totipotent zygote divides and undergoes a number of changes that lead to the first lineage differentiation in the blastocyst displaying trophectoderm (TE) and inner cell mass (ICM) on Day 5. The TE is a differentiated epithelium needed for implantation and the ICM forms the embryo proper and serves as a source for pluripotent embryonic stem cells (ESCs). The blastocyst implants around Day 7. The second lineage differentiation occurs in the ICM after implantation resulting in specification of primitive endoderm and epiblast. Knowledge on human preimplantation development is limited due to ethical and legal restrictions on embryo research and scarcity of materials. Studies in the human are mainly descriptive and lack functional evidence. Most information on embryo development is obtained from animal models and ESC cultures and should be extrapolated with caution. This paper reviews totipotency and the molecular determinants and pathways involved in lineage segregation in the human embryo, as well as the role of embryonic genome activation, cell cycle features and epigenetic modifications.
Asunto(s)
Blastocisto/citología , Linaje de la Célula/fisiología , Desarrollo Embrionario/fisiología , Células Madre Embrionarias/citología , Células Madre Totipotentes/citología , Epigénesis Genética , Femenino , Humanos , EmbarazoRESUMEN
The authors report a case of severe ST elevation after defibrillation which persisted for 6 days, suggesting acute myocardial infarction. The possible mechanisms of ST elevation after cardioversion and the unusually long duration of the ECG changes in this case are reviewed. The protective role of verapamil against myocardial damage is discussed.