scholarly journals Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos

Reproduction ◽  
1995 ◽  
Vol 104 (2) ◽  
pp. 251-258 ◽  
Author(s):  
T. Tao ◽  
B. Reichelt ◽  
H. Niemann
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblastic Cells

186 LASER CAPTURE MICRODISSECTION FOR GENE EXPRESSION ANALYSIS OF INNER CELL MASS AND TROPHOBLAST FROM BOVINE BLASTOCYSTS

2011 ◽  
Vol 23 (1) ◽  
pp. 194
Author(s):  
M. Filliers ◽  
W. de Spiegelaere ◽  
L. J. Peelman ◽  
K. Goossens ◽  
C. Burvenich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Serial Sections ◽  
Trophoblastic Cells ◽  
Keratin 18 ◽  
Laser Capture

Isolation of pure inner cell mass (ICM) and trophoblast samples from a single blastocyst is necessary to obtain accurate information on the transcriptome of these cells. Microsurgical techniques have been described to separate the ICM and trophoblast, but unfortunately, contamination of the ICM cell population with trophoblastic cells is inevitable with these methods. Alternatively, immunosurgery has been described as a valuable technique to obtain a pure ICM sample, although this technique seems to alter the normal gene expression pattern. Laser capture microdissection (LCM) provides the possibility of isolating small tissue fractions from heterogeneous tissue sections, without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. In this study, a protocol is described for the application of LCM to isolate homogeneous ICM and trophoblast samples from single bovine blastocysts for downstream gene expression analysis. The absence of contaminating trophoblastic fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered a trophoblast-specific marker in bovine blastocysts. Expanded blastocysts were produced by routine in vitro methods described by (Vandaele et al. 2010 Reproduction 139, 505–511) and fixed in a modified methacarn solution for 24 h. After fixation, the blastocysts were embedded in RNase-free soluble agarose 2%, processed in an STP 420D Tissue Processor, embedded in paraffin, cut in serial sections, and adhered to glass slides, followed by deparaffinization in xylene and staining of the sections with 0.1% cresyl violet in a 85% ethanol solution. Laser capture microdissection was performed as described previously by (De Spiegelaere et al. 2008 Anal. Biochem. 382, 72–74). The ICM was isolated by placing the same cap over 3 to 4 serial sections of one blastocyst. Subsequently, the same procedure was performed with a second cap to isolate the trophoblast. Total RNA was isolated from the LCM-derived ICM and trophoblast on the caps and converted into cDNA. Gene-specific primers for KRT18 (5′-GCAGACCGCTGAGATAGGA-3′ and 5′-GCATATCGGGCCTCCACTT-3′) and for 18S rRNA, a commonly used reference gene (5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′), were used and PCR was carried out. Expression of the control gene 18S rRNA was readily detectable in all cell samples. Keratin 18 was detectable in LCM-derived trophoblast, but was absent in the LCM-derived ICM cells, indicative of the successful isolation of ICM cells without contaminating trophoblastic cells. This study demonstrates a novel approach for the application of LCM on small tissue samples that are difficult to handle and which can be used for molecular analysis of specific cell lineages within embryos of different species. Supported by the Fund for Scientific Research–Flanders, Belgium, aspirant 1.1.477.07N00.

97 Loss of aggregation capacity of bovine invitro-produced embryos and blastocyst-derived trophoblasts from Day 6 of development

2020 ◽  
Vol 32 (2) ◽  
pp. 174
Author(s):  
V. Alberio ◽  
M. Yauri Felipe ◽  
D. Salamone
Keyword(s):  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Culture Conditions ◽  
Sodium Pyruvate ◽  
Trophoblastic Cells ◽  
Stereoscopic Observation ◽  
Fetal Bovine ◽  
Humidified Air ◽  
Oviductal Fluid

Embryo aggregation consists of placing more than one zona-free (ZF) embryo in contact during development to obtain a unique structure. It has been reported in different species that aggregated cloned embryos show certain benefits compared with nonaggregated embryos. One way to obtain these benefits in IVF embryos would be to generate a transient chimera by the introduction of trophoblastic cells. Bovine trophoblastic cells can be obtained by embryo bisection of blastocysts, cutting asymmetrically to use trophoblasts (Tr) for aggregation and leaving aside the portion that contains the inner cell mass (ICM). Taking all this into account, the objectives of this work are to study the aggregation of Tr at different days of development and to determine the appropriate time of aggregation. To this aim, cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10µgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. We performed IVF with 16×106 spermatozoa per mL for 5h. Afterwards, presumptive zygotes were cultured in synthetic oviductal fluid (SOF) for 7 days in a humidified atmosphere at 38.5°C, 5% O2, 5% CO2, and 90% N2. In Experiment 1, embryo bisection of Day 7 blastocysts was performed manually under stereoscopic observation with a microblade to obtain Tr. These were aggregated, with the bisected part containing the ICM (n=22) or with ZF embryos of Days 4 (n=23), 5(n=25), or 6 (n=22) and blastocysts (n=25), and placed in microwells in a 100-μL SOF drop covered by mineral oil (Gambini et al. 2012 Biol. Reprod. 87, 15; https://doi.org/10.1095/biolreprod.112.098855). In Experiment 2, ZF synchronous whole embryos were aggregated in microwells at different developmental days: Day 3 (n=18), 4 (n=18), 5 (n=47), 6 (n=48), and 7 (n=45). In both experiments, aggregation was assessed at Day 8. In Experiment 1, no aggregation was observed between the Tr and the embryos or the bisected ICM. Experiment 2 showed embryo aggregation on Days 3 (55%), 4 (27%), and 5 (61%), whereas on Days 6 and 7 no aggregation was observed. According to these results, we can conclude that, in our culture conditions, Tr obtained by blastocyst bisection have no capacity for aggregation. Day 6 and 7 whole ZF embryos also do not aggregate. As a general conclusion, there is a period from Days 0-5 of the invitro development of bovine embryos in which aggregation is possible. Aggregation of blastocyst-derived Tr to cloned or high-value IVF embryos, aiming for quality improvement, is not an effective strategy.

scholarly journals A preliminary note upon the question of the nutrition of the early embryo, with special reference to the guinea-pig and man

1905 ◽  
Vol 76 (508) ◽  
pp. 164-165 ◽  
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Early Embryo ◽  
Uterine Horn ◽  
Dense Layer ◽  
Preliminary Note ◽  
Trophoblastic Cells ◽  
Decidual Cells ◽  
Uterine Secretion

During the progress of a research into the earliest implantation of the embryo of the guinea-pig, I have been particularly struck with the way in which the nutrition of the embryo is anticipated and provided for during the time it remains free in the uterine horn. The so-called yolk-granules of the ovum are obviously insufficient to provide for the growth of the embryo to the stage prior to differentiation of the inner cell-mass, to which it attains during the five or six days which elapse before it comes into contact with the maternal tissues. It is clear that it must derive nourishment from the medium in which it lies―the product of the secretion of the uterine or other glands, which, during the period of pro-œstrum, exhibit such marked activity. I suggest that this secretion, which consists of mucus and probably albumin, is assimilated by the embryo after having undergone a process of digestion, the result of a secretory activity on the part of the outermost cells of the embryo―the cells of the Trophoblast. This suggestion I base on my observations in the guinea-pig, where I am able to demonstrate a breaking-down of maternal cells before the Trophoblastic cells are in actual contact; likewise in human placentation where a more or less dense layer of fibrin and broken-down leucocytes and decidual cells, the result of Trophoblastic activity, affords a barrier interposed between the invading Trophoblastic cells and the Decidua. This layer I purpose naming the “Protective Layer.” Looked at from a comparative point of view, there is in all probability a close analogy between the uterine secretion of mammals, and the secretion of the oviducts of the lower vertebrata. In the case of birds the analogy is very striking, on account of the direct and important share in the nutrition of the embryo afforded by this secretion, commonly known as the white of the egg. In the case of the frog the ovum receives in its passage down the oviduct, corresponding to the uterine horn of the guinea-pig, a coating of mucus and probably albumin, comparable to the uterine secretion referred to above; when it reaches the water and becomes fertilised, this swells up by absorption, forming a gelatinous covering. The embryo for nutriment depends upon the yolk contained in the ovum before fertilisation, upon the covering of mucus and probably albumin, and lastly upon the water in which it lies. In certain mammals, as, for example, the rabbit and the mole, a distinct gelatinous envelope is described as surrounding the embryo before implantation occurs; this envelope is, I suggest, possibly due to some digestive action of the cells of the Trophoblast upon the adjacent medium, producing a form of coagulation.

Preferential paternal X inactivation in extraembryonic tissues of early mouse embryos

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 127-135
Author(s):  
Mary I. Harper ◽  
Mandy Fosten ◽  
Marilyn Monk
Keyword(s):  
X Chromosome ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Phosphoglycerate Kinase ◽  
X Inactivation ◽  
Preferential Expression ◽  
Trophoblastic Cells ◽  
Extraembryonic Membranes ◽  
Early Mouse

The preferential expression of the maternal X chromosome seen in certain extraembryonic membranes of the mouse was studied by investigating the tissues from which these membranes are derived during early development. The electrophoretic variant of the X-coded enzyme PGK-1 (phosphoglycerate kinase) was used to distinguish the expression of the maternal from the paternal X chromosome in heterozygous females. Both the extraembryonic ectoderm and primary endoderm of 6½-day female egg cylinders gave almost exclusive expression of the maternal form of the enzyme whereas the epiblast gave near equal expression of the two parental alleles. No paternal PGK-1 band could be detected in samples of pooled 3½-day blastocysts, but after 3 or 4 days of culture in vitro a faint paternal band was seen in the resultant outgrowths. The activity of the maternal band in these latter samples had increased greatly from that of the blastocysts, consistent with preferential expression of the maternal Pgk-1 allele in the trophoblastic cells of the outgrowths, while both alleles are expressed in inner-cell-mass cells. The results strongly support the idea that non-random X-chromosome expression is due to preferential paternal X inactivation in trophectoderm (from which extraembryonic ectoderm is derived) and in primary endoderm, and not to cell selection.

scholarly journals Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

Reproduction ◽  
2007 ◽  
Vol 134 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Gnanaratnam Giritharan ◽  
Said Talbi ◽  
Annemarie Donjacour ◽  
Francesca Di Sebastiano ◽  
Anthony T Dobson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Preimplantation Embryos ◽  
Global Pattern ◽  
Vitro Fertilization ◽  
Trophoblastic Cells

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 μm2, 113 μm2, and 86 μm2 respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

scholarly journals Selenium Attenuates HPV-18 Associated Apoptosis in Embryo-Derived Trophoblastic Cells but Not Inner Cell Mass In Vitro

2015 ◽  
Vol 2015 ◽  
pp. 1-7
Author(s):  
Jennifer A. Tolen ◽  
Penelope Duerksen-Hughes ◽  
Kathleen Lau ◽  
Philip J. Chan
Keyword(s):  
Cell Viability ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Human Papillomaviruses ◽  
Embryonic Cells ◽  
Trophoblastic Cells ◽  
Group 2 ◽  
Apoptosis Process ◽  
Hpv 18

Objectives. Human papillomaviruses (HPV) are associated with cell cycle arrest. This study focused on antioxidant selenomethionine (SeMet) inhibition of HPV-mediated necrosis. The objectives were to determine HPV-18 effects on embryonic cells and to evaluate SeMet in blocking HPV-18 effects.Methods. Fertilized mouse embryos were cultured for 5 days to implanted trophoblasts and exposed to either control medium (group 1), HPV-18 (group 2), combined HPV-18 and 0.5 µM SeMet (group 3), or combined HPV-18 and 5.0 µM SeMet (group 4). After 48 hrs, trophoblast integrity and, apoptosis/necrosis were assessed using morphometric and dual-stain fluorescence assays, respectively.Results. HPV-18 exposed trophoblasts nuclei (253.8 ± 28.5 sq·µ) were 29% smaller than controls (355.6 ± 35.9 sq·µ). Supplementation with 0.5 and 5.0 µM SeMet prevented nuclear shrinkage after HPV-18 exposure. HPV-18 infected trophoblasts remained larger with SeMet supplementation. HPV-18 decreased cell viability by 44% but SeMet supplementation sustained cell viability. Apoptosis was lower when SeMet was present. HPV-18 decreased inner cell mass (ICM) viability by over 60%.Conclusions. HPV-18 decreased nuclear size and trophoblast viability but these effects were attenuated by the antioxidant SeMet. SeMet blocked HPV-18 associated apoptosis process in trophoblasts but not ICM cells suggesting involvement of different oxidative stress pathways.

scholarly journals The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice

2001 ◽  
Vol 155 (4) ◽  
pp. 571-580 ◽  
Author(s):  
Keisuke Tateishi ◽  
Masao Omata ◽  
Keiji Tanaka ◽  
Tomoki Chiba
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Aberrant Expression ◽  
Cycle Progression ◽  
Trophoblastic Cells ◽  
Cycle Arrest ◽  
Essential Components ◽  
F Box Protein

NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)–like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3−/− mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57Kip2. These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. β-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3−/− cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

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.

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