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

Development ◽  
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.

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

1995 ◽  
Vol 43 (1) ◽  
pp. 304 ◽  
Author(s):  
D. Rath ◽  
H. Niemann ◽  
T. Tao ◽  
M. Boerjan
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells

Correlative scanning electron, transmission electron, and light microscopic studies of the in vitro development of mouse embryos on a plastic substrate at the implantation stage

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 23-39
Author(s):  
Matthew A. Gonda ◽  
Yu-Chih Hsu
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Plastic Substrate ◽  
Trophoblast Cells ◽  
Electron Transmission ◽  
Transmission Electron ◽  
Development In Vitro ◽  
Scanning Electron

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.

Effect of culture conditions on diploid to giant-cell transformation in postimplantation mouse trophoblast

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 217-227
Author(s):  
J. Rossant ◽  
W. Tamura-Lis
Keyword(s):  
Giant Cell ◽  
Cell Transformation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Formation ◽  
Culture Conditions ◽  
Embryonic Cells ◽  
Giant Cell Formation ◽  
Ectoplacental Cone

Diploid extraembryonic ectoderm and ectoplacental cone from the 7·5-day mouse embryo were grown in vitro under a variety of culture conditions in an attempt to discover conditions which maintain trophoblast in a diploid state and prevent giant-cell formation. It was found that maintenance of tissue integrity was not enough to keep the tissues dividing and diploid, but that the presence of inner-cell-mass derivatives did have some effect. This effect was only apparent when trophoblast cells were entirely enclosed by embryonic tissues. Monolayers of embryonic or embryonal carcinoma cells did not prevent giant-cell formation. Diploid extraembryonic ectoderm and ectoplacental cone responded differently: ectoplacental cells eventually formed trophoblast giant cells even when enclosed by embryonic cells whereas extraembryonic ectoderm cells apparently could be maintained in a diploid condition. This and other differences in properties between extraembryonic ectoderm and ectoplacental cone are discussed with reference to a new model for the postimplantation trophoblast lineage in the mouse.

276 EXPRESSION OF CD9 AND ALPHA6-INTEGRIN IN PORCINE BLASTOCYSTS

2009 ◽  
Vol 21 (1) ◽  
pp. 235 ◽  
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.

scholarly journals Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

2005 ◽  
Vol 17 (8) ◽  
pp. 799 ◽  
Author(s):  
Natalie I. Alexopoulos ◽  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Andrew J. French ◽  
Alan O. Trounson
Keyword(s):  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Collagen Gel ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Term Survival

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

Immunosurgical studies on Inner cell mass development in rat and mouse blastocysts before and during implantation in vitro

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 255-269
Author(s):  
Horst Spielmann ◽  
Ursula Jacob-Müller ◽  
Werner Beckord
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Cell Number ◽  
C57bl Mouse ◽  
Before And After ◽  
Rabbit Complement

Eighty per cent of rat blastocysts (Wistar, SW72) cultured for 96 h in NCTC-109 supplemented with fetal calf serum (FCS) hatched from the zona pellucida and developed a trophoblast giant cell layer. Thirty seven per cent of the rat blastocysts developed an inner cell mass (ICM) which, in about 7 %, consisted of two germ layers (ectoderm and endoderm), compared to 84% in NMRI mice. A significantly better ICM development was obtained with cultured rat blastocysts that had hatched in vivo. Similar to the in vivo situation LDH-5 was present in rat blastocysts after implantation in NCTC-109-FCS. Differentiation of C57BL mouse blastocysts in NCTC-109-FCS proceeded as poorly as in the rat. ICM development of rat and mouse blastocysts in NCTC-109-FCS was studied in detail. ICMs of the two species were isolated immunosurgically using complement from different species, e.g. human, rat and rabbit complement, since guinea-pig complement did not lyse trophectoderm cells of rat blastocysts. All immunosurgically isolated rat ICMs degenerated within 48 h, but mouse ICMs isolated with rat or rabbit complement developed significantly better than mouse ICMs isolated with guinea-pig complement. Determinations of theblastocyst total cell number (BTCN) and of the cell number of immunosurgically isolated ICMs were performed in rat and mouse blastocysts to investigate growth kinetics of the ICM before implantation in vitro. In the mouse an exponential increase in both BTCN and cell number of the ICM was observed during the 48 h before implantation in NCTC-109-FCS and also during the 16-24 h before implantation in vivo. In the rat, doubling of the BTCN was found only during the first 24 h in NCTC-109-FCS and there was hardly any increase in the cell number of the ICM during the first 48 h in culture. ICM growth of blastocysts in NCTC-109-FCS is, therefore, stimulated in the mouse before and after implantation and. in the rat it is inhibited already before implantation.

The immediate postimplantation development of tetraploid mouse blastocysts

Development ◽  
1976 ◽  
Vol 35 (1) ◽  
pp. 81-86
Author(s):  
M. H. L. Snow
Keyword(s):  
Normal Development ◽  
Cell Transformation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mass Function ◽  
Decidual Cell ◽  
Minimum Number ◽  
Implantation Period

The development during and immediately after the implantation period of 143 tetraploid blastocysts was studied both in vitro and in vivo 58·7 % in vitro and 38·8 % in vivo werefound to exhibit the changes associated with the early implanting blastocyst, i.e. giant cell transformation of the trophoblast and induction of the decidual cell reaction in the uterus. Of these 38·7 % in vitro and 19·4% in vivo showed evidence of inner cell mass function during this time but only two in each system could be claimed as showing normal development. Examination of the developing blastocyst leads to the conclusion that lack of cell numbers in the inner cell mass is the most likely reason for the poor development of tetraploid embryos and suggests that the minimum number of ICM cells required to fulfil its role in embryogenesis is between four and eight.

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

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
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.

The control of trophoblastic growth in the guinea pig

Development ◽  
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.

CSF-1 and mouse preimplantation development in vitro

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1333-1339 ◽  
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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