Ratio and number of inner cell mass and trophoblast cells of in vitro and in vivo produced porcine embryos

Correlative scanning electron, transmission electron, and light microscopy were utilized to study the morphogenic events occurring during mouse blastocyst outgrowth and earlyegg- cylinder development in vitro. After hatching and attachment of blastocysts on theplastic surface, the blastocoelic cavity collapses as the mural trophoblasts spread and migrate outward. The inner cell mass is covered with a differentiated endoderm on the blastocoelic cavity side and by the polar trophoblasts on the medium sideat this stage. As the endodermcovered inner cell mass proliferates, being physically restricted from further downward expansion by the plastic coverslip and by lack of space in the collapsed blastocoelic cavity, it migrates upward and protrudes into the culture medium in a breakbetween the polar and mural trophoblast cells. Polar trophoblast cellsapposed to the base of the egg cylinder continue to proliferate forming the ectoplacental cone. Thus, the early egg cylinder lacking a trophoblast barrier begins inverting its growth pattern from towards the culture dish surface to a more upright position. Egg-cylinder development in vitro from the inner cell mass and polar trophoblast cells closely paralleled in vivo. The functional nature of variousembryonic cell types observed in these embryos was revealed by scanning electron microscopy. These studies as well as those of Wiley and Pedersen (1977) suggest that blastocysts can serve as a source of in vitro developing early mouse egg cylinders that appear to resemble their in vivo counterparts and can be used in experimental studies of mouse embryogenesis.

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276 EXPRESSION OF CD9 AND ALPHA6-INTEGRIN IN PORCINE BLASTOCYSTS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab276 ◽

2009 ◽

Vol 21 (1) ◽

pp. 235 ◽

Cited By ~ 1

Author(s):

M. D. Goissis ◽

F. R. O. de Barros ◽

M. G. Marques ◽

C. M. Mendes ◽

M. P. Milazzotto ◽

...

Keyword(s):

Inner Cell Mass ◽

Rna Extraction ◽

Cell Mass ◽

Embryonic Stem ◽

Trophoblast Cells ◽

Pcr Products ◽

Nucleotide Database ◽

First Time

Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers is necessary for validation of a pluripotent cell line. Not all markers observed in ESC from other species are characterized in swine embryos. The objective of this study was to characterize CD9 and α6-integrin expression in porcine blastocysts and to derive porcine ESC using Matrigel. In vitro or in vivo porcine blastocysts were submitted to total RNA extraction for RT-PCR, fixation for immunocytochemistry or immunosurgery for culture of inner cell mass. Expression of Oct-4, CD9, and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass (ICM) and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. This study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts. Financial support: Fapesp 05/57314-0.

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The development of trophoblast in vitro from blastocysts containing varying amounts of inner cell mass

Development ◽

10.1242/dev.33.1.177 ◽

1975 ◽

Vol 33 (1) ◽

pp. 177-185

Author(s):

J. D. Ansell ◽

M. H. L. Snow

Keyword(s):

Giant Cell ◽

Cell Transformation ◽

Inner Cell Mass ◽

Cell Mass ◽

Calf Serum ◽

Foetal Calf Serum ◽

Trophoblast Cells ◽

Intact Mouse

When intact mouse blastocysts are cultured in vitro in medium supplemented with foetal calf serum, trophoblast cells proliferate and undergo giant cell transformation such as occurs in vivo. If the amount of inner cell mass in the blastocyst is decreased by culture with [3H]-thymidine then giant cell transformation occurs normally but proliferation is reduced. In the absence of inner cell mass no proliferation occurs, and giant cell transformation is more rapid than in undamaged blastocysts.

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The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽

10.1242/dev.102.4.793 ◽

1988 ◽

Vol 102 (4) ◽

pp. 793-803 ◽

Cited By ~ 3

Author(s):

V.E. Papaioannou ◽

K.M. Ebert

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Staining Technique ◽

Cell Number ◽

Total Cell ◽

Differential Staining ◽

Morphological Development ◽

Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

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The control of trophoblastic growth in the guinea pig

Development ◽

10.1242/dev.60.1.405 ◽

1980 ◽

Vol 60 (1) ◽

pp. 405-418

Author(s):

E. B. Ilgren

Keyword(s):

Guinea Pig ◽

Inner Cell Mass ◽

Cell Mass ◽

The Other ◽

In Vivo Analysis

The growth of mouse trophectoderm depends upon the presence of the inner cell mass. Whether this applies to other species of mammals is not known. To investigate this problem, the guinea pig was selected for two reasons. Firstly, the growth of guinea-pig trophoblast resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm and the other containing ICM tissue as well. Subsequently, the growth of these mural and polar fragments was followed in vitro since numerous technical difficulties make an in vivo analysis of this problem impracticable. In a manner similar to the mouse, the isolated mural trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig polar fragments formed egg-cylinder-like structures. The latter contained regions structurally similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal growth may occur in a manner similar to the mouse and thus be under ICM control.

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CSF-1 and mouse preimplantation development in vitro

Development ◽

10.1242/dev.121.5.1333 ◽

1995 ◽

Vol 121 (5) ◽

pp. 1333-1339 ◽

Cited By ~ 5

Author(s):

P. Bhatnagar ◽

V.E. Papaioannou ◽

J.D. Biggers

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Preimplantation Development ◽

High Rate ◽

Colony Stimulating Factor ◽

Trophoblast Cells ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

The effects of macrophage colony stimulating factor on the development of the zygote to the blastocyst stage of an outbred strain of mouse have been studied in KSOM, an improved medium that supports a high rate of in vitro development. Macrophage colony stimulating factor accelerates the formation of the blastocyst cavity by day 4 (96 hours post-hCG). It also increases overall embryonic cell number through a differential increase in the number of trophoblast cells, with no significant effect on the number of inner cell mass cells. By day 5 of culture (120 hours post-hCG), colony stimulating factor-treated embryos have about 20 more trophoblast cells than control embryos, an increase of about 30 percent of the total number of cells in a control blastocyst. The maximum response of embryos was obtained at a concentration around 540 U ml-1 colony stimulating factor (identical to 918 Stanley units ml-1), and the cytokine can produce the same effects even if it is present in the medium for only part of the culture period. This in vitro stimulation of preimplantation development with macrophage colony stimulating factor is compatible with continued normal fetal development in vivo.

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Investigation of the determinative state of the mouse inner cell mass

Development ◽

10.1242/dev.33.4.979 ◽

1975 ◽

Vol 33 (4) ◽

pp. 979-990

Author(s):

J. Rossant

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

In Utero ◽

Glucose Phosphate Isomerase ◽

Trophoblast Cells ◽

Electrophoretic Variants ◽

Glucose Phosphate

Inner cell masses (ICMs) were dissected from 3½- and 4½-day blastocysts and cultured in contact with 2½-day morulae. Blastocysts and morulae were homozygous for different electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Aggregation of ICMs and morulae was observed, and such aggregates were able to form blastocysts in vitro and morphologically normal foetuses in utero. GPI analysis of these conceptuses revealed that most were chimaeric. However, donor ICM-type isozyme was only detected in the embryonic and extra-embryonic fractions of the chimaeras and never in the trophoblastic fraction. Thus, ICM cells appear unable to form trophoblast derivatives even when exposed to ‘outside’ conditions as experienced by developing trophoblast cells. This is evidence that ICM cells, although not overtly differentiated, are determined by 3½ days.

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Derivation of human embryonic stem cell lines after blastocyst microsurgery

Biochemistry and Cell Biology ◽

10.1139/o09-188 ◽

2010 ◽

Vol 88 (3) ◽

pp. 479-490 ◽

Cited By ~ 12

Author(s):

Guoliang Meng ◽

Shiying Liu ◽

Xiangyun Li ◽

Roman Krawetz ◽

Derrick E. Rancourt

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Lines ◽

Clinical Medicine ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

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Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽

10.1530/rep.1.0966 ◽

2007 ◽

Vol 133 (1) ◽

pp. 231-242 ◽

Cited By ~ 43

Author(s):

Craig Smith ◽

Debbie Berg ◽

Sue Beaumont ◽

Neil T Standley ◽

David N Wells ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Ectopic Expression ◽

Cell Number ◽

Transcript Levels ◽

Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

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Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

Molecular and Cellular Biology ◽

10.1128/mcb.01188-06 ◽

2007 ◽

Vol 27 (8) ◽

pp. 3123-3130 ◽

Cited By ~ 26

Author(s):

Klaus Fortschegger ◽

Bettina Wagner ◽

Regina Voglauer ◽

Hermann Katinger ◽

Maria Sibilia ◽

...

Keyword(s):

Matrix Protein ◽

Replicative Senescence ◽

Inner Cell Mass ◽

Umbilical Vein ◽

Cell Mass ◽

Preimplantation Embryos ◽

Proliferative Potential ◽

Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

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