221 HOXB9 IS DIFFERENTIALLY EXPRESSED IN THE TWO FIRST CELL LINES OF THE MAMMALIAN EMBRYO

2015 ◽  
Vol 27 (1) ◽  
pp. 200
Author(s):  
C. Sauvegarde ◽  
R. Rezsöhazy ◽  
I. Donnay
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Nuclear Staining ◽  
Mammalian Embryo ◽  
Mean Intensity ◽  
Whitney Test ◽  
Mann Whitney Test ◽  
The Mean

Hox proteins are transcription factors known to be essential for embryo patterning. The detection of some Hox transcripts in oocytes and early embryos suggests that they could play a role before gastrulation. We previously demonstrated Hoxb9 expression in oocytes and from the zygote to the blastocyst stage in the mouse and the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436). The protein is present at all stages and in all cells with a strong nuclear staining in both species. The objective of this study was to perform an in-depth study at the blastocyst stage to compare the level of the nuclear protein between the inner cell mass (ICM) and the trophectoderm (TE) from the early to the expanded blastocyst stage. In vitro produced bovine blastocysts were collected at Day 6, Day 7.5, and Day 8 post-insemination. Hoxb9 proteins were detected by whole-mount immunofluorescence. TE nuclei were strongly stained at all stages while from D6 but especially from D7.5, the level of HOXB9 seemed to decrease in ICM nuclei with an increasing heterogeneity of staining between ICM nuclei. A light and apparently stable staining was also observed in the cytoplasm. Confocal images were quantified (Nis-element 3.1, Nikon). For each cell of TE or ICM, the ratio between the mean intensity of the nucleus and the mean intensity of the corresponding total cytoplasm was calculated. Whatever the stages, TE ratios were significantly (Mann–Whitney test; P < 0.0001) higher than ICM ratios, suggesting that HOXB9 is present in higher amounts in TE than in ICM cells. This observation could be correlated with the reduced HOXB9 relative expression observed in blastocysts. Moreover, the proportion of blastocysts showing a reduction of HOXB9 staining in at least one nucleus significantly increased from Day 6 to Day 7.5 blastocysts and Day 8 blastocysts (from 26% to 74% or 85%, chi-squared test; P < 0.001). Mouse zygotes, collected from superovulated mice, were cultured in vitro and embryos were collected 72 h, 80 h, 92 h and 100 h post-hCG injection. A similar nuclear staining was observed in all cells until 80 h post-hCG injection, while heterogeneity of staining appeared in ICM cells 92 h post-hCG, but especially in 100 h post-hCG embryos. The quantitative study was performed only on this latest stage and confirmed the stronger staining in TE than in ICM nuclei (Mann–Whitney test; P < 0.0001) observed in the bovine. At this stage, 82% of blastocysts presented a reduced Hoxb9 staining in some or all ICM nuclei. In conclusion, Hoxb9 protein is detected in all blastocyst nuclei both in the mouse and in the bovine. However, the protein seems globally less abundant in the ICM than in the TE cells. Moreover, the percentage of bovine blastocysts showing a reduction in HOXB9 staining intensity in ICM nuclei increases with blastocyst expansion. These results suggest an involvement of Hoxb9 in cell lineage differentiation in mammals.C. S. holds a FRIA PhD grant from the FRS-FNRS (Belgium). This study is supported by the FRS-FNRS and by an Action de Recherche Concertée.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

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).

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

scholarly journals 45PRODUCTION OF CHIMERIC TRANSCHROMOSOMIC CALVES

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
P. Kasinathan ◽  
M.F. Nichols ◽  
J.E. Griffin ◽  
J.M. Robl
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Peripheral Blood Lymphocytes ◽  
Blastocyst Stage ◽  
Human Artificial Chromosome ◽  
Fish Analysis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Fetal Fibroblasts

Chimeras have been used for investigating fundamental aspects of early embryonic development, and differentiation, and for introducing foreign genes into mammals (Robertson et al., 1986 Nature 323, 445–448; Cibelli et al., 1998 Science 280, 1256–1258). The main objective of this study was to determine if the transfer of blastomeres from in vitro-produced (IVP) embryos into cloned, transchromosomic embryos improved the efficiency of producing transchromosomic calves. Cloned embryos were produced using in vitro-matured bovine oocytes and bovine fetal fibroblasts containing a human artificial chromosome (HAC) (Kuroiwa et al., 2002 Nat Biotechnol 20, 889–894). IVP embryos were produced using standard procedures and blastomeres were harvested at the 8–16 cell stage by removing the zona pellucida with protease. Cloned embryos were randomly divided on Day 4 into two groups. One group received 3–4 IVP blastomeres while a second group served as a control (nonmanipulated cloned embryos). After transferring the blastomeres, the chimeric and cloned embryos were placed in culture (Kasinathan et al., 2001 Biol. Reprod. 64, 1487–1493) and on Day 7 development to the blastocyst stage was evaluated. Grades 1 and 2 embryos were transferred; two each per synchronized recipient. Pregnancy maintenance, calving, and calf survival were evaluated in both groups. Presence of a HAC in live calves was evaluated in both fibroblasts and peripheral blood lymphocytes (PBLs) using FISH analysis. Embryo development to the blastocyst stage, maintenance of pregnancy and number of calves born were analyzed using Chi-square. There were no differences in the rate of blastocyst development at day 7 or establishment of pregnancy at 40d (P&gt;0.05). However, pregnancy rate at 120d, and number of calves that developed to term and were alive at birth (chimera 14/54 and clone 4/90), and at 1 month of age (chimera 13/54 and clone 1/90) were lower (P&lt;0.01) for cloned embryos. The proportion of cells containing an HAC in PBLs, was higher in cloned calves (100%) compared to chimeric calves (26%). The HAC retension rates in PBLs in HAC-positive chimeric and cloned calves were 84% and 95%, respectively. These data indicate that, although the proportion of calves retaining an HAC was lower in chimeras compared to clones, more HAC-positive calves were produced in the chimeric treatment from fewer cloned embryos. We speculate that higher rates of development in the chimeras may be related to the normality of the placenta. Future studies will be required to determine the contribution of the IVP blastomeres to both the inner cell mass and trophectoderm. Therefore, a chimeric approach may be useful for improving the efficiency of producing cloned transchromosomic calves.

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.

114 VITRIFICATION OF BOVINE EMBRYOS IN MEDIUM WITH POLYVINYL ALCOHOL REPLACING BSA

2006 ◽  
Vol 18 (2) ◽  
pp. 165
Author(s):  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Ethylene Glycol ◽  
Polyvinyl Alcohol ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Sodium Hyaluronate ◽  
Blastocyst Stage ◽  
Embryo Survival ◽  
Better Than

Embryos vitrified in medium supplemented with 4.25 μg/mL sodium hyaluronate (SH) and 0.1% polyvinyl alcohol (PVA) survived vitrification better than embryos vitrified in medium supplemented with 0.25% FAF-BSA (Walker and Seidel 2005 Reprod. Fert. Dev. 17, 153). The purpose of the present study was to determine if the small amount of SH was beneficial to in vitro survival and to examine the effects of different concentrations of PVA in vitrification solutions. Day 7 blastocysts (n = 360) were produced in vitro with semen from three bulls, two replicates each. Cryoprotectant solutions were prepared in a 2 × 3 factorial combination with two SH concentrations (0 or 4.25 μg/mL) and three PVA concentrations (0.05, 0.1%, or 0.2%). For vitrification, embryos were placed into chemically defined HEPES-buffered medium (HCDM-2) at room temperature (22–24°C) and then transferred to V1 (5 m ethylene glycol in HCDM-2) for 3 min. Next, embryos were placed in a 6 μL drop of V2 (7 m ethylene glycol, 0.5 m galactose, and 18% w/v Ficoll 70 in HCDM-2) for 45 s. During these 45 s, dilution medium (0.5 m galactose in HCDM-2) was aspirated into 0.25-mL straws, followed by the 6 μL drop of V2 plus embryos and a final short column of dilution medium. When 45 s had elapsed, the heat-sealed end of straw was dipped into liquid nitrogen to cover the embryo, and then the remainder of the straw was immersed slowly. Straws were thawed in air for 10 s and then in 37°C water for 20 s. Next, straws were shaken like a clinical thermometer four times to mix columns, and held in 37°C water for 10 min before embryos were expelled, rinsed and cultured in CDM-2 + 5% FCS. At 48 h, embryo survival (as determined by expansion of blastocysts), embryo quality (1 = excellent, 2 = fair, 3 = poor), inner cell mass (ICM) quality (1 = large and compact, 2 = clearly visible, 3 = not discernable) and blastocyst stage (5 = early, 6 = full, 7 = expanded, 8 = hatching, 9 = hatched) were evaluated and replicate averages were analyzed by ANOVA. Neither bull nor SH concentration nor PVA concentration significantly affected any response (P > 0.10). Averaged over PVA concentrations, vitrification of embryos in 0 μg/mL or 4.25 μg/mL SH resulted in similar survival rates (67% vs. 62%, respectively). When averaged over SH concentrations, 0.2% PVA had a numerically higher survival rate of blastocysts as compared to 0.1% or 0.05% (71% vs. 63% and 60%, respectively). The main effects of 0 μg/mL SH and 0.2% PVA also resulted in numerically higher, but nonsignificant improvements in quality score, ICM score and blastocyst stage as compared to the other doses of SH and PVA. Vitrification of Day 7 in vitro-produced bovine blastocysts in medium containing 0.2% PVA in the absence of SH resulted in a subclass mean of 80% embryo survival. Results of this experiment show no benefit of 4.25 μg/mL SH and that 0.2% PVA may be slightly better than 0.05% or 0.1% in terms of embryo survival. Therefore, our results indicate that 0.2% PVA can be used alone as an effective alternative to animal products in this vitrification procedure for in vitro-derived bovine blastocysts.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

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.

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.

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