scholarly journals Cell surface fluctuations regulate early embryonic lineage sorting

2020 ◽  
Author(s):  
Ayaka Yanagida ◽  
Christopher Revell ◽  
Giuliano G. Stirparo ◽  
Elena Corujo-Simon ◽  
Irene M. Aspalter ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Sorting ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Spatial Segregation ◽  
Lineage Sorting ◽  
Surface Fluctuations ◽  
Key Factor ◽  
Surface Fluctuation ◽  
Spatial Positioning

In development, lineage segregation of multiple lineages must be coordinated in time and space. An important example is the mammalian inner cell mass (ICM), in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the foetus). The physical mechanisms that determine this spatial segregation between EPI and PrE are still poorly understood. Here, we identify an asymmetry in cell-cell affinity, a mechanical property thought to play a significant role in tissue sorting in other systems, between EPI and PrE precursors (pEPI and pPrE). However, a computational model of cell sorting indicated that these differences alone appeared insufficient to explain the spatial segregation. We also observed significantly greater surface fluctuations in pPrE compared to pEPI. Including the enhanced surface fluctuation in pPrE in our simulation led to robust cell sorting. We identify phospho-ERM regulated membrane tension as an important mediator of the increased surface fluctuations in pPrE. Using aggregates of engineered cell lines with different surface fluctuation levels cells with higher surface fluctuations were consistently excluded to the outside of the aggregate. These cells behaved similarly when incorporated in the embryo. Surface fluctuations-driven segregation is reminiscent of activity-induced phase separation, a sorting phenomenon in colloidal physics. Together, our experiments and model identify dynamic cell surface fluctuations, in addition to static mechanical properties, as a key factor for orchestrating the correct spatial positioning of the founder embryonic lineages.

305. OPPOSING ROLES FOR MUCINS 13 AND 15 IN EARLY PREGNANCY

2010 ◽  
Vol 22 (9) ◽  
pp. 105
Author(s):  
C. E. Poon ◽  
M. Day ◽  
C. R. Murphy
Keyword(s):  
Cell Surface ◽  
Early Pregnancy ◽  
Steric Hindrance ◽  
Inner Cell Mass ◽  
Apical Cell ◽  
Cell Mass ◽  
Cell Fractionation ◽  
Apical Surface ◽  
Adhesive Surface ◽  
Important Player

Successful pregnancy is dependent on the cumulation of numerous changes in both the uterine luminal epithelium (ULE) and the invading blastocyst prior to implantation. The apical surface of the ULE constitutes the first point of contact with the blastocyst; thus forming an important player in the implantation event. A constituent of this surface comprises the Mucin family of proteins, which is suggested to play an important role in implantation. Prior studies have found that some Mucins form an anti-adhesive surface on the uterine epithelium to prevent implantation of the blastocyst. The formation of this anti-adhesive surface relates to their large extracellular domains which act to ‘mask’ adhesive receptors present on the ULE from interaction with corresponding ligands present on the blastocyst surface. This process of contact inhibition has been termed ‘steric hindrance’. This study examined the localisation of previously uncharacterised Mucins 13 and 15 in the uterus and blastocyst during early pregnancy to investigate their role in the implantation event. Western blotting, cell fractionation and immunofluorescence techniques were utilised in this study. It was found that Mucin 13 localised to the apical cell surface of the ULE at time of implantation while conversely Mucin 15 was lost from the apical cell surface. In the blastocyst, both proteins were localised to the trophoblast cells and the inner cell mass, with Mucin 15 additionally present in the zona pellucida. Further, the changes in localisation of these proteins in the ULE corresponded to changes in their glycosylation profiles from pre-implantation to time of implantation. These results demonstrate that Mucins 13 and 15 participate in contrasting roles in implantation. Mucin 13 may promote adhesion between the uterus and blastocyst to facilitate implantation while Mucin 15 may prevent adhesion through the mechanism of steric hindrance, given its absence from the apical cell surface at time of implantation.

P–158 Assisted Hatching on D + 3 in order to facilitate trophectoderm biopsy in blastocyst for PGT-A is not advisable in all patients

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Belchin ◽  
Y Cabello ◽  
M Sanche. d. Burgos ◽  
J Guerrero ◽  
M D Riva ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Assisted Hatching ◽  
Culture Dish ◽  
Key Factor ◽  
Registration Number ◽  
Number Of Cycles

Abstract Study question Is it useful or beneficial to perform Assisted Hatching (AH) on D + 3 previously to biopsy for PGT-A on blastocyst stage on D + 5? Summary answer The routine use of AH on D + 3 to facilitate the embryo biopsy on D + 5 could negatively influence the development of the embryos to blastocyst stage. What is known already The blastocyst stage is the optimal stage for performing biopsies for PGT-A, which has been reported as a key factor determining the growing clinical application of this strategy worldwide. For trophectoderm (TE) biopsy, laser-assisted drilling is used to create a zona opening on D + 3 or D + 5 of development. The method of zona opening on D + 3 allows some of the TE cells to herniate during blastocyst formation and expansion, which facilitates the biopsy process. However, this method may result in herniation of inner cell mass cells instead of TE or maybe could affect the development of the embryo to blastocyst stage. Study design, size, duration A total of 100 PGT-A cycles were performed in 2019 and 2020. In 78 of them laser-assisted drilling was used to create a zona opening on D + 5 only in those embryos which arrived to blastocyst stage for TE biopsy (Group No-AH). In 22 cycles the same drilling was achieved on D + 3 in all embryos, independently of their quality (Group AH). The average of embryos per cycle in each group was 5 and 4.3 respectively. Participants/materials, setting, methods A total of 100 PGT-A cycles coming from 65 patients were studied. The average of the age of the patients was 40.83 (SD 3.45) in the group No-AH vs 42.18 (SD 3.42) in the Group AH (p = 0.108), so the age was not a determining factor for the development of the embryos. We analyzed by χ 2 test differences between groups on fertilization rates, number of embryos, development to blastocyst stage, euploidy and pregnancy rates. Main results and the role of chance The fertilization rate was 74.79% (No-AH group) and 68.53% (AH group) with no significative statistical differences (p = 0.12). In the No-AH group, the TE biopsy was performed on D + 5 in 63 cycles (81%). In the AH group, 41% of cycles didn’t reach the blastocyst stage, obtaining statistical differences between groups (p = 0.035). We found also significant differences in the number of cycles with biopsied blastocyst when we had 1 to 6 embryos/cycle on D + 3 between groups (p = 0.002), without obtaining any blastocyst to be diagnosed in 53% of the cycles in AH group vs 27% in No-AH group. When the number of embryos on D + 3 per cycle was > 6, at least 1 embryo reached the blastocyst stage in both groups, although this number was higher in No-AH group. The rate of biopsied blastocysts was significantly higher in the No-AH group compared to the AH group (46.61 vs 34.69) with a p = 0.031. The rate of euploid embryos analyzed was 23.30% in the No-AH group compared to 29.41% in the AH group, although no significant differences were found (p = 0.44) between groups. In the No-AH group, a clinical pregnancy rate of 52.94% was obtained (n = 34) vs 50% in the AH group (n = 4) (p = 0.91). Limitations, reasons for caution We have recently started to perform AH on D + 3, so the number of cases is smaller than No-AH group. We use a time lapse incubator in all cases, so in the No-AH the culture dish is changed, disturbing the stable incubation environment, while in the other group it is not. Wider implications of the findings: The use of AH on D + 3 in order to facilitate the TE biopsy on D + 5 could affect negatively the development of the embryos to blastocyst stage. Its routine use should be avoided based on laboratory workload, mainly if the patient has less than 7 embryos at D + 3. Trial registration number Not applicable

scholarly journals Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

2010 ◽  
Vol 191 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Daniel Mesnard ◽  
Daniel B. Constam
Keyword(s):  
Cell Surface ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Axis Formation ◽  
Fluorescent Biosensor ◽  
Membrane Bound ◽  
Biosensor Cell

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β–related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface–targeted fluorescent biosensor (cell surface–linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

141 A NOVEL METHOD FOR PURIFICATION OF INNER CELL MASS AND TROPHECTODERM CELLS FROM BOVINE BLASTOCYSTS USING MAGNETIC ACTIVATED CELL SORTING

2011 ◽  
Vol 23 (1) ◽  
pp. 174
Author(s):  
M. Ozawa ◽  
P. J. Hansen
Keyword(s):  
Cell Sorting ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Expression Level ◽  
Hoechst 33342 ◽  
Mammalian Embryo ◽  
Positive Fraction ◽  
Relative Expression Level ◽  
Single Blastomeres ◽  
Magnetic Activated Cell Sorting

The first distinct lineage differentiation in the mammalian embryo occurs at the blastocyst stage when blastomeres are segregated into inner cell mass (ICM) or trophectoderm (TE). Obtaining purified TE or ICM can be useful for understanding regulation of early development and differentiation. Although several methods have been reported to separate TE and ICM (e.g. immunosurgery, mechanical dissection using a micromanipulator, or manual selection following trypsinization), limitations exist with these methods. Here, we describe a simple and effective method to sort cells of the blastocyst using magnetic activated cell sorting (MACS) following disaggregation of the blastocyst into single cells using trypsin. Bovine blastocysts were produced in vitro and the zona pellucida removed with a short exposure to acidic Tyrode’s solution. Zona-free blastocysts were incubated with concanavalin A conjugated to fluorescein isothiocyanate (FITC) to label the outer layer of the blastocyst. The blastocysts were then exposed to Hoechst 33342 to label nuclei of all blastomeres. The blastocysts were treated with 0.05% (wt/vol) trypsin, and then disaggregated into single blastomeres by repeating pipetting using a finely drawn, flame-polished mouth micropipette. Single blastomeres were incubated with magnetic microbeads conjugated to anti-FITC and subjected to MACS separation. A fraction of sorted cells was observed under a fluorescence microscope. The remainder were subjected to mRNA extraction, and NANOG (ICM marker) and CDX2 (TE marker) mRNA were quantified by quantitative PCR. After disaggregation of the blastocyst, 2 types of single blastomeres were observed: cells that were positive for both FITC and Hoechst 33342 (TE cells) and cells that were negative for FITC but positive for Hoechst 33342 (ICM cells). Before MACS, about two-thirds of the disaggregated blastomeres labelled with Hoechst 33342 were also labelled with FITC, while one-third were FITC negative. After MACS, the percent of dual-labelled cells in the FITC positive fraction was 91.2%, whereas the incidence of dual-labelled cells in the FITC negative fraction was only 7.8 ± 3.0%. A total of 11.5 μg of RNA per blastocyst was recovered from cells isolated by MACS. This represents 80% of the RNA present in intact blastocysts and suggests a high rate of recovery of blastomeres during the purification process. Furthermore, relative expression level of NANOG was lower in the FITC-positive fraction than in the FITC-negative fraction (0.30 ± 0.05 v. 3.1 ± 0.6, respectively, relative to gene expression level in whole blastocysts). Conversely, the relative expression level of CDX2 was higher in the FITC-positive fraction than in the FITC-negative fraction (3.2 ± 0.09 v. 0.30 ± 0.9, respectively). Results indicate that highly purified TE cells or ICM cells can be collected using MACS. This simple method can be used to study differentiation of the mammalian embryo as well as to prepare embryonic cells of specific lineages for cell therapy. Research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2009-65203-05732 from the USDA NIFA.

Use of F9 teratocarcinoma STEM cells to study cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 602-603
Author(s):  
Marc Lenburg ◽  
Rulang Jiang ◽  
Lengya Cheng ◽  
Laura Grabel
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Embryoid Bodies ◽  
F9 Cells ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions ◽  
F9 Teratocarcinoma

We are interested in defining the cell-cell and cell-matrix interactions that help direct the differentiation of extraembryonic endoderm in the peri-implantation mouse embryo. At the blastocyst stage the mouse embryo consists of an outer layer of trophectoderm surrounding the fluid-filled blastocoel cavity and an eccentrically located inner cell mass. On the free surface of the inner cell mass, facing the blastocoel cavity, a layer of primitive endoderm forms. Primitive endoderm then generates two distinct cell types; parietal endoderm (PE) which migrates along the inner surface of the trophectoderm and secretes large amounts of basement membrane components as well as tissue-type plasminogen activator (tPA), and visceral endoderm (VE), a columnar epithelial layer characterized by tight junctions, microvilli, and the synthesis and secretion of α-fetoprotein. As these events occur after implantation, we have turned to the F9 teratocarcinoma system as an in vitro model for examining the differentiation of these cell types. When F9 cells are treated in monolayer with retinoic acid plus cyclic-AMP, they differentiate into PE. In contrast, when F9 cells are treated in suspension with retinoic acid, they form embryoid bodies (EBs) which consist of an outer layer of VE and an inner core of undifferentiated stem cells. In addition, we have established that when VE containing embryoid bodies are plated on a fibronectin coated substrate, PE migrates onto the matrix and this interaction is inhibited by RGDS as well as antibodies directed against the β1 integrin subunit. This transition is accompanied by a significant increase in the level of tPA in the PE cells. Thus, the outgrowth system provides a spatially appropriate model for studying the differentiation and migration of PE from a VE precursor.

Decreased inner cell mass proportion in blastocysts from diabetic rats

Diabetes ◽  
1990 ◽  
Vol 39 (4) ◽  
pp. 471-476 ◽  
Author(s):  
S. Pampfer ◽  
R. de Hertogh ◽  
I. Vanderheyden ◽  
B. Michiels ◽  
M. Vercheval
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Diabetic Rats ◽  
Mass Proportion
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