In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽  
1978 ◽  
Vol 45 (1) ◽  
pp. 107-121
Author(s):  
Brigid Hogan ◽  
Rita Tilly
Keyword(s):  
Outer Layer ◽  
Normal Mouse ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Visceral Endoderm ◽  
Floating Structures ◽  
Parietal Endoderm ◽  
Vitro Development

This paper describes the development in culture of inner cell masses isolated immunosurgically from C3H/He mouse blastocysts immediately after collection between 3·5 and 4·0 days p.c. By 24–48 h most of the inner cell masses isolated from half-expanded blastocysts, and about 50% of those from expanded blastocysts, regenerate an outer layer of trophectoderm- like cells and so resemble mini-blastocysts. With further in vitro culture these structures attach to the substratum and give rise to trophoblast-like giant cells, together with clusters of parietal endoderm cells or inner cell masses surrounded by visceral endoderm. Many of the inner cell masses from the remaining expanded blastocysts develop into floating structures with an outer layer of endoderm cells, and by 7 days consist of a large fluid filled cyst surrounding a collapsed vesicle of epithelial cells. Mesodermal cells line the cysts and form numerous blood islands. When mechanically disrupted, and grown as attached sheets of cells, these cystic structures give rise to patches of trophoblast-like giant cells similar to those described in the previous paper. These results suggest that the inner cell mass of normal mouse blastocysts contains cells which are capable of giving rise to trophoblast in culture.

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽  
1978 ◽  
Vol 45 (1) ◽  
pp. 93-105
Author(s):  
Brigid Hogan ◽  
Rita Tilly
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Epithelial Layer ◽  
Visceral Endoderm ◽  
Limited Region ◽  
In Vitro Development ◽  
Vitro Development ◽  
Embryonic Ectoderm

This paper describes the in vitro development of inner cell masses isolated immunosurgically from mouse blastocysts which had been collected on 3·5 days p.c. and then incubated for 24 h. The inner cell masses continue to grow in culture and develop through a series of stages with increasing complexity of internal organization. By day 1 all of the cultured ICMs have an outer layer of endoderm, and by day 3 some of them have two distinct kinds of inside cells; a columnar epithelial layer and a thin hemisphere of elongated cells. Later, mesodermal cells appear to delaminate from a limited region of the columnar layer, close to where it forms a junction with the thinner cells. By day 5, about 25% of the cultured ICMs have a striking resemblance to normal 7·5-day p.c. C3H embryos, with embryonic ectoderm, extra-embryonic ectoderm and chorion, embryonic and extra-embryonic mesoderm, and visceral endoderm. When mechanically disrupted and grown as attached clumps of cells in a tissue dish, these embryo-like structures give rise to trophoblast-like giant cells. These results suggest that the inner cell mass of 4·5-day p.c. blastocysts contains cells which can give rise to trophoblast derivates in culture.

In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 43-55
Author(s):  
J. Rossant ◽  
K. M. Vijh
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Parietal Endoderm ◽  
Trophoblast Giant Cells ◽  
Vitro Development ◽  
Ectoplacental Cone

Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6·5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8·5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5·5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives, It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.

Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo

Development ◽  
1982 ◽  
Vol 68 (1) ◽  
pp. 175-198
Author(s):  
R. L. Gardner
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Early Embryo ◽  
Visceral Endoderm ◽  
Primitive Endoderm ◽  
Developmental Potential ◽  
Extraembryonic Endoderm ◽  
Parietal Endoderm ◽  
Endodermal Cells

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

104 BIRTH OF FIRST BUFFALO (BUBALUS BUBALS) CALF FOLLOWING EMBRYO SPLITTING AND POLYMERASE CHAIN REACTION SEXING AT BLASTOCYST STAGE

2012 ◽  
Vol 24 (1) ◽  
pp. 164 ◽  
Author(s):  
M. Zhang ◽  
H. H. Chen ◽  
J. W. Tang ◽  
X. W. Liang ◽  
M. T. Chen ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Buffalo Calf ◽  
Practical Procedure ◽  
Blastocyst Quality ◽  
Grade 3 ◽  
Vitro Development

Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192–196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245–1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211–1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos. This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4).

Cell interactions and endoderm differentiation in cultured mouse embryos

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 379-394
Author(s):  
Brigid L. M. Hogan ◽  
Rita Tilly
Keyword(s):  
Basement Membrane ◽  
Mouse Embryo ◽  
Cell Lineage ◽  
Giant Cells ◽  
Visceral Endoderm ◽  
Extraembryonic Endoderm ◽  
Type Iv ◽  
The Matrix ◽  
Parietal Endoderm

Morphological and biochemical evidence is presented that the visceral extraembryonic endoderm of the 6·5-day mouse embryo will differentiate into parietal endoderm when cultured in contact with extraembryonic ectoderm undergoing transition into trophoblast giant cells. Egg cylinders from 6·5-day embryos were dissected into embryonic and extraembryonic halves and cultured in suspension in vitro for up to 7 days. After 4 days, the endoderm cells of the extraembryonic fragments morphologically resemble parietal endoderm, are associated with a thick basement membrane and synthesize large amounts of the matrix proteins laminin and Type IV procollagen. A similar transition in phenotype is not seen in the endoderm of embryonic fragments, nor in visceral extraembryonic endoderm cells cultured in isolation. In another series of experiments, complete egg cylinders were dissected free of visceral endoderm ovei lying the extraembryonic ectoderm and then cultured in vitro. The visceral endoderm cells which recolonize the surface of the extraembryonic ectoderm develop a parietal endoderm phenotype and lay down a thick basement membrane. These results suggest that the differentiation of the extraembryonic endoderm of the early mouse embryo into visceral and parietal phenotypes can be influenced by local cell—cell or cell—substrate interactions, and is not determined solely by cell lineage.

136 INVESTIGATION OF LEPTIN ON DEVELOPMENT OF MOUSE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 160
Author(s):  
A. C. Taskin ◽  
A. Kocabay ◽  
M. Yucel
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Development Rate ◽  
Mouse Embryos ◽  
In Vitro Development ◽  
Development Rates ◽  
Significant Difference ◽  
Vitro Development

Leptin is a hormone-like protein of 167 amino acids. As an adipocyte-related hormone it has an important role in weight regulation and physical fitness but also has effects on reproductive and other physiological mechanisms. The aim of the present study was to investigate the effects of different concentrations of leptin added to the culture media, the quality, in vitro development rate, and in vivo rate of mouse embryos. Superovulated CB6F1 (C57BL/6XBalb/c) hybrid female mice (5–6 weeks of age) were killed ~18 to 20 h after hCG administration. Single-cell embryos were flushed from the oviducts of the dead mice with human tubal fluid medium supplemented with HEPES and 3 mg mL–1 of BSA. They were cultured in Quinn's cleavage medium supplemented with 4 mg mL–1 of BSA in 5% CO2, 37°C until reaching 2-cell stage. The 2-cell embryos were randomly divided into 4 groups and cultured in Quinn's blastocyst medium supplemented with 4 mg mL–1 BSA + 0, 10, 50, and 100 ng mL–1 leptin (L0, L10, L50, and L100) in 5% CO2, 37°C until the blastocyst stage. Some of the developing blastocysts were used for differential staining for the inner cell mass and trophectoderm (TE) cells [Mallol et al. 2013 Syst. Biol. Reprod. Med. 59,117–122]. Some of them were transferred into pseudopregnant females (CD1) on the 2.5 to 3.5th days and kept until the 13.5th day of pregnancy for the in vivo development rate. The results were evaluated using one-way ANOVA with Bonferroni post-hoc test in SPSS 22.0. The P-values <0.05 were considered statistically significant. Each experiment was repeated at least 4 times. The blastocyst development rates of L0, L10, L50, and L100 were 92.57% (162/175), 97.16% (205/211), 97.80% (178/182), and 97.85% (182/186), respectively. The in vitro development rates were significantly higher in the L10, L50, and L100 compared with L0 (P < 0.05). The inner cell mass cells of L0, L10, L50, and L100 were 13.17, 14, 16, and 15.43. There was no significant difference between the groups in terms of inner cell mass cells (P > 0.05). The TE cells of L0, L10, L50, and L100 were 47, 56.4, 53.7, and 58.57, respectively. The TE numbers were significantly increased in the presence of L10 and L100 compared with L0 (P < 0.05). The in vivo development rates of L0, L10, L50, and L100 were 13.51% (5/37), 48.72% (19/39), 15.38% (6/39), and 41.03% (16/39), respectively. The in vivo development rates of L10 and L100 were significantly higher than for L0 and L50 (P < 0.05). The resorption rates of L0, L10, L50, and L100 were 10.8% (4/37), 30.8% (12/39), 12.8% (5/39), and 20.5% (8/39), respectively. There was no significant difference between the groups in terms of the resorption rates (P > 0.05). This study found that L10, L50, and L100 were supporting the development of the embryos in the in vitro culture. The L10, L50, and L100 significantly increased the total cell numbers. The L10 and L100 were particularly effective on the number of the TE cells. In conclusion, 10 and 100 ng mL–1 leptin have a positive effect on the in vitro, quality and in vivo development of the mouse embryo. Therefore, leptin seems to play an important role on the embryo development and in vivo development. Research supported by TUBITAK-113O223.

Effects of bromodeoxyuridine, cytosine arabinoside and Colcemid upon in vitro development of mouse blastocysts

Development ◽  
1975 ◽  
Vol 34 (2) ◽  
pp. 467-484
Author(s):  
Michael I. Sherman ◽  
Sui Bi Atienza
Keyword(s):  
Cytosine Arabinoside ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells ◽  
Culture Dish ◽  
In Vitro Development ◽  
Vitro Development

Mouse blastocysts in culture have been treated with increasing concentrations of cytosine arabinoside, bromodeoxyuridine or Colcemid. Concentrations of all three antimetabolites have been found which interfere with neither hatching of the blastocysts from their zona pellucidae nor subsequent attachment of the blastocysts to the culture dish, but which eventually result in death of the inner cell mass (ICM) and its derivatives. The effect upon the ICM is selective at these antimetabolite concentrations since many or, in some cases, all trophoblast cells continue to survive, and by a number of criteria, undergo normal patterns of differentiation and development.

scholarly journals Proliferation Failure and Gamma Radiation Sensitivity of Fen1 Null Mutant Mice at the Blastocyst Stage

2003 ◽  
Vol 23 (15) ◽  
pp. 5346-5353 ◽  
Author(s):  
Elisabeth Larsen ◽  
Christine Gran ◽  
Barbro Elisabet Sæther ◽  
Erling Seeberg ◽  
Arne Klungland
Keyword(s):  
Dna Replication ◽  
Gamma Radiation ◽  
Inner Cell Mass ◽  
Excision Repair ◽  
Cell Mass ◽  
Giant Cells ◽  
Radiation Sensitivity ◽  
Blastocyst Stage

ABSTRACT Flap endonuclease 1 (FEN1) has been shown to remove 5′ overhanging flap intermediates during base excision repair and to process the 5′ ends of Okazaki fragments during lagging-strand DNA replication in vitro. To assess the in vivo role of the mammalian enzyme in repair and replication, we used a gene-targeting approach to generate mice lacking a functional Fen1 gene. Heterozygote animals appear normal, whereas complete depletion of FEN1 causes early embryonic lethality. Fen1−/− blastocysts fail to form inner cell mass during cellular outgrowth, and a complete inactivation of DNA synthesis in giant cells of blastocyst outgrowth was observed. Exposure of Fen1−/− blastocysts to gamma radiation caused extensive apoptosis, implying an essential role for FEN1 in the repair of radiation-induced DNA damage in vivo. Our data thus provide in vivo evidence for an essential function of FEN1 in DNA repair, as well as in DNA replication.

BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies

2007 ◽  
Vol 85 (1) ◽  
pp. 121-132 ◽  
Author(s):  
Brock J Conley ◽  
Sarah Ellis ◽  
Lerna Gulluyan ◽  
Richard Mollard
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Transmission Electron ◽  
Spherical Structures ◽  
Bmp Signalling ◽  
Serum Free Conditions

Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, α-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.

Sign in / Sign up

Export Citation Format

Share Document