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 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.

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

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
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.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

scholarly journals 127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS

2004 ◽  
Vol 16 (2) ◽  
pp. 186
Author(s):  
J.O. Gjørret ◽  
P. Maddox-Hyttel
Keyword(s):  
Production Systems ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Embryo Cell ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Tunel Reaction

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to<0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P<0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general. Table 1

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

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.

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.

scholarly journals Loss of RBBP4 results in defective inner cell mass, severe apoptosis, hyperacetylated histones and preimplantation lethality in mice†

2020 ◽  
Vol 103 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Xiaosu Miao ◽  
Tieqi Sun ◽  
Holly Barletta ◽  
Jesse Mager ◽  
Wei Cui
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lineage Specification ◽  
Mouse Embryo Development ◽  
Evolutionarily Conserved ◽  
Early Mouse

Abstract Retinoblastoma-binding protein 4 (RBBP4) (also known as chromatin-remodeling factor RBAP48) is an evolutionarily conserved protein that has been involved in various biological processes. Although a variety of functions have been attributed to RBBP4 in vitro, mammalian RBBP4 has not been studied in vivo. Here we report that RBBP4 is essential during early mouse embryo development. Although Rbbp4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts cannot hatch from the zona or can hatch but then arrest without further development. We find that while there is no change in proliferation or levels of reactive oxygen species, both apoptosis and histone acetylation are significantly increased in mutant blastocysts. Analysis of lineage specification reveals that while the trophoblast is properly specified, both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification. In summary, these findings demonstrate the essential role of RBBP4 during early mammalian embryogenesis.

scholarly journals Mifepristone Directly Disrupts Mouse Embryonic Development in Terms of Cellular Proliferation and Maturation In Vitro

Toxics ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 294
Author(s):  
Yu-Ting Su ◽  
Jia-Shing Chen ◽  
Yi-Ru Tsai ◽  
Kuo-Chung Lan ◽  
Cheng-Chun Wu ◽  
...  
Keyword(s):  
Potential Risk ◽  
Cellular Proliferation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Direct Effects ◽  
Developmental Problems ◽  
Post Implantation

Mifepristone (RU-486), a synthetic steroid with potent antiprogestogen and anti-glucocorticoid properties, has been widely used in clinical practice. Its effect on the endometrium, ovary, and fallopian tube has been well reported in many human and animal studies. However, its direct impact on post-implantation embryos remains underexplored. Additionally, some women choose to keep their pregnancy after mifepristone treatment fails. Thus, the potential risk remains controversial. Hence, this study investigated the direct effects of mifepristone on the development of mice blastocysts in vitro in terms of implantation and post-implantation. We detected the level of progesterone (P4) associated with ovulation in vivo. The presence of progesterone receptors (PRs) in blastocysts and post-implantation embryos was also evaluated. Cultured embryos were treated directly with mifepristone. We further examined embryonic implantation and post-implantation of blastocysts in vitro to evaluate the direct effects of mifepristone on embryos by the assessment of embryonic outgrowth and differential cell staining. In the oviduct lumen, the P4 level dramatically increased at 48 h and slightly decreased at 72 and 96 h following ovulation. PR was expressed in blastocysts not only in the preimplantation stage but also in the early post-implantation period. In the evaluation of developmental stages, mifepristone significantly reduced the successful ratio of developing into the late egg cylinder and the early somite stage. In addition, it further decreased the cell number of the embryos’ inner cell mass and trophectoderm. We herein provide evidence that mifepristone affects blastocyst viability directly and inhibits post-implantation embryo development in vitro. Furthermore, our data reveal a potential risk of fetus fatality and developmental problems when pregnancies are continued after mifepristone treatment fails.

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