61 DIPLOID (BOS TAURUS) AND TETRAPLOID (BOS INDICUS) EMBRYO AGGREGATION TO PRODUCE BOVINE EMBRYONIC CHIMERAS

2012 ◽  
Vol 24 (1) ◽  
pp. 142
Author(s):  
E. Montanari Razza ◽  
R. A. Satrapa ◽  
I. P. Emanuelli ◽  
C. Moraes Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Single Pulse ◽  
Blastocyst Stage ◽  
Random Pattern ◽  
Cell Stage ◽  
Protease Treatment ◽  
Holstein Bulls

The formation of tetraploid embryos (4n) by electrofusion and their subsequent chimerism with a diploid embryo (2n) must result in a chimeric conceptus, whose inner cell mass (ICM) is entirely 2n. Hence, the aggregation of a zebu embryo (4n, thermotolerant) with a taurine embryo (2n, thermosensitive) would result in an exclusively taurine ICM, but the trophectoderm (future extraembryonic components) would be mostly from the zebu embryo, which could interact with the taurine embryo/fetus in a different way (than it occurs in whole Bos taurus conceptus) during pregnancy in a tropical environment. The purpose of this study was to standardize the production of 4n Nelore embryos (Bos indicus) and the production of embryonic chimeras by aggregation of 1/2 B. taurus (2n) with B. indicus (4n) embryos. Oocytes from Nelore cows from the abattoir were matured, fertilized with semen from Nelore and Holstein bulls and cultured in SOF. Two-cell stage Nelore embryos (30 h post-insemination), with a well-defined inter-blastomeric axis, were selected for the electrofusion procedure (ECM 830-BTX, Harvard Apparatus) to produce 4n embryos. For this procedure, some parameters were tested according to the number of pulses (1 or 2), voltage (40, 50, 75, 100, 140 and 500 V) and duration of electroshock (20, 25, 50 and 60 μs). Nelore 4n embryos produced after electrofusion and 2n taurine embryos, both at 8 to 16-cell stages (72 h post-insemination) were subjected to protease treatment to remove the zona pellucida and subsequently treated with the agglutinant agent phytohemagglutinin. Bos indicus (4n) and B. taurus (2n) embryos were added in pairs (4n+2n) into individual wells (well of the well, WOW) for culture until the blastocyst stage to validate the chimeric embryos formation. Among the tested parameters, the best fusion results (92%) and rates of cleavage after fusion (66%) were obtained with a single pulse of 75 V for 60 μs. The production rate of expanded 4n blastocysts was 31.5% using these parameters. After 4 replicates (still in the final stage of standardization), 4 blastocyst chimeras (4n+2n) were obtained from 31 attempts (13%). The production of bovine embryonic chimeras [Bos indicus (4n) + Bos taurus (2n)], with a non-random pattern of distribution of their cell aggregates, will enable the validation of this technique in applied research, by producing exclusively taurine calves, but with placental elements from the B. indicus breed, following transfer of these chimeras into recipient cows. Financial support received from FAPESP, Brazil.

155 CHARACTERIZATION OF LIPID DROPLETS IN BOS INDICUS AND BOS TAURUS EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 226 ◽  
Author(s):  
E. P. López-Damián ◽  
T. Fiordelisio ◽  
M. A. Lammoglia ◽  
M. Alarcón ◽  
M. Asprón ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Embryo Quality ◽  
Developmental Stages ◽  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Nile Red ◽  
Blastocyst Stage ◽  
Transfer Program

Accurate evaluation of bovine embryos for assessing developmental stage and quality is critical to the success of any embryo transfer program. However, this evaluation process has been reported to be highly subjective in Bos indicus (BI) and can vary as much as 23% compared with that of Bos taurus (BT). These differences in assessment may be related to the quantity of lipid droplets (LD) within the embryo, which has been shown to have a negative effect in cryopreserving embryos. The aim of the present study was to characterize the number and size of LD in different developmental stages of fresh embryos from BI and BT and to compare LD across the three different embryo quality grades (1 = excellent or good, 2 = fair, and 3 = poor). Nonsurgical embryo collection was performed 7 days post-insemination in 10 BI and 10 BT females. Forty-eight embryos were evaluated for stage and grade using stereoscopic microscopy, processed for transmission electron microscopy, and stained with Nile red. Digitalized images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA), contour of lipid droplets were designed, and values of perimeter, area, and fluorescence intensity were assessed. Nonparametric statistical analysis (Mann–Whitney) was utilized. There was no difference in LD number for BT or BI for morulae and blastocyst; however, BI morulae presented larger LD compared with blastocyst stage embryos (286 µm2 v. 223 µm2; P < 0.05). Likewise, BI TF cells had more LD compared with inner cell mass (ICM) cells (48 v. 36; P < 0.05). BT TF cells exhibited larger LD compared with ICM cells (149 µm2 v. 128 µm2; P < 0.05), while BI embryos exhibited a larger area of LD in the ICM compared with the TF (591 µm2 v. 472 µm2; P < 0.05). In all embryos, BI contained more lipid droplets than BT (78 v. 49; P < 0.05). Across all quality grades (good, fair, and poor) there was no difference in the number of LD in BT embryos; however, BI grade-3 embryos presented more LD than grade-1 (36 v. 25). BT embryos LD were larger than BI LD (907 µm2 v. 625 µm2; P < 0.05). Fluorescence images showed higher arbitrary units of fluorescence (auf) for LD in BI. Compared with BT embryos (386 auf v. 280 auf; P < 0.05). These results suggest that BI embryos contain more and smaller LD than BT embryos and the LD described for BI embryo quality grade 1 are larger than those of quality grades 2 and 3, and even though the number of LD in morulae and blastocyst stage embryos are not different LD size is reduced as development occurs. Research funding provided by UNAM-DGAPA-PAPIIT IN200810.

scholarly journals Embryo cell allocation patterns are not altered by biopsy but can be linked with further development

Reproduction ◽  
2017 ◽  
Vol 154 (6) ◽  
pp. 807-814
Author(s):  
L P Sepulveda-Rincon ◽  
N Islam ◽  
P Marsters ◽  
B K Campbell ◽  
N Beaujean ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Strains ◽  
Random Pattern ◽  
Cell Stage ◽  
Cell Counts ◽  
Cell Embryo ◽  
Allocation Patterns ◽  
Further Development

It has been suggested that first embryo cleavage can be related with the embryonic–abembryonic axis at blastocyst stage in mice. Thus, cells of the 2-cell embryo might be already biased to form the inner cell mass or trophectoderm. This study was conducted to observe the possible effects of embryo biopsy on cell allocation patterns during embryo preimplantation in two different mouse strains and the effects of these patterns on further development. First, one blastomere of the 2-cell embryo was injected with a lipophilic tracer and cell allocation patterns were observed at blastocyst stage. Blastocysts were classified into orthogonal, deviant or random pattern. For the first experiment, embryos were biopsied at 8-cell stage and total cell counts (TCC) were annotated. Furthermore, non-biopsied blastocysts were transferred into foster mothers. Then, pups and their organs were weighed two weeks after birth. Random pattern was significantly recurrent (≈60%), against orthogonal (<22%) and deviant (<22%) patterns among groups. These patterns were not affected by biopsy procedure. However, TCC on deviant embryos were reduced after biopsy. Moreover, no differences were found between patterns for implantation rates, litter size, live offspring and organ weights (lungs, liver, pancreas and spleen). However, deviant pups presented heavier hearts and orthogonal pups presented lighter kidneys among the group. In conclusion, these results suggest that single blastomere removal does not disturb cell allocation patterns during pre-implantation. Nonetheless, the results suggest that embryos following different cell allocation patterns present different coping mechanisms against in vitro manipulations and further development might be altered.

129 KNOCKDOWN OF WBP1 DECREASES TROPHECTODERM FORMATION IN THE PRE-IMPLANTATION BOVINE EMBRYO

2017 ◽  
Vol 29 (1) ◽  
pp. 173
Author(s):  
M. S. Ortega ◽  
P. J. Hansen
Keyword(s):  
Embryonic Development ◽  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Fold Change ◽  
Bovine Embryo

A single nucleotide polymorphism (SNP) in WBP1 has been previously associated with embryonic development to the blastocyst stage. WBP1 interacts with WW domain containing proteins including YAP1 from the hippo signalling pathway that is involved in trophectoderm (TE) formation. Here we tested whether reduction in mRNA abundance for WBP1 would reduce development to the blastocyst stage and formation of cells in the inner cell mass (ICM) and TE. Knockdown was performed using a GapmeR LNATM antisense oligonucleotide designed to target WBP1. A scrambled version of the same sequence was used as a control. Embryos were produced in vitro from slaughterhouse oocytes and bulls from Bos taurus and Bos indicus breeds. At 20 to 22 h after insemination (hpi), embryos were treated with 5 µM anti-WBP1 GapmeR (KD), 5 µM scrambled GapmeR (SC), or vehicle (CTL). At 72 to 75 hpi (the time of maximal WBP1 expression), groups of 18 to 20 embryos were collected from each treatment to evaluate WBP1 expression. Other cultured embryos (minimum of 50/treatment for each replicate) were cultured until Day 8 after insemination. Cleavage was assessed at Day 3 and blastocyst formation at Day 7 and 8. Embryos were collected at Day 8 to determine ICM and TE cell number by determining nuclear immunoreactive CDX2. All experiments were replicated 5 times. Fold change was calculated relative to the CTL group. Data were analysed by analysis of variance for gene expression and cell number, and through logistic regression for embryonic development. WBP1 expression was reduced (P = 0.04) in KD embryos compared to CTL (least squares means ± SEM: 1 ± 0.19 v. 0.64 ± 0.19 fold change) or SC (1.05 ± 0.19). There was no difference in expression between CTL and SC. Percent of embryos that cleaved was not affected by treatment (P > 0.05); however, percent of inseminated oocytes that became blastocysts tended to be lower in KD compared to CTL and SC at Day 7 (P = 0.09) [10.8 ± 2.8, 20 ± 3.0, and 16.3 ± 3.1% for KD, CTL, and SC, respectively] and 8 after insemination (P = 0.06) [13.7 ± 3.3, 24.2 ± 3.3, and 22.9 ± 3.6%]. Knockdown of WBP1 caused a reduction in number of total (P = 0.0004) and TE (P < 0.0001) cells with no effect on ICM cell number (P = 0.83). Total cell numbers for KD, SC, and CTL were 124.2 ± 6.4, 157.75 ± 7.4, and 124.28 ± 6.4 and numbers of TE cells were 59.7 ± 3.8, 90.0 ± 4.47, and 90.0 ± 4.4. Results show that reduction in mRNA for WBP1 decreases TE formation and tends to reduce competence of embryos to become blastocysts. This study was supported by USDA AFRI 2013–68004–20365.

213 HOXB9 PROTEIN IS PRESENT THROUGHOUT EARLY EMBRYO DEVELOPMENT IN THE BOVINE

2013 ◽  
Vol 25 (1) ◽  
pp. 255
Author(s):  
C. Sauvegarde ◽  
D. Paul ◽  
R. Rezsohazy ◽  
I. Donnay
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Immature Oocyte ◽  
Cell Stage ◽  
Morula Stage ◽  
Oocyte Stage

Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).

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.

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 125-130 ◽  
Author(s):  
Lei Lei ◽  
Na Guan ◽  
Yan-Ning Xu ◽  
Qing-Hua Zhang ◽  
Jing-Ling Shen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Karyotype Analysis ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lower Number ◽  
Mouse Embryos ◽  
Developmental Pattern ◽  
Cell Stage ◽  
Positive Expression

SummaryPolyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 ± 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 ± 7.2; 18.4 ± 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. β-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

scholarly journals G9a regulates temporal preimplantation developmental program and lineage segregation in blastocyst

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jan J Zylicz ◽  
Maud Borensztein ◽  
Frederick CK Wong ◽  
Yun Huang ◽  
Caroline Lee ◽  
...  
Keyword(s):  
Cell Fate ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Mouse Development ◽  
Cell Stage ◽  
Developmental Progression ◽  
Early Mouse ◽  
The Impact

Early mouse development is regulated and accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2). Previously, we provided insights into its role in post-implantation development (Zylicz et al., 2015). Here we explore the impact of depleting the maternally inherited G9a in oocytes on development shortly after fertilisation. We show that G9a accumulates typically at 4 to 8 cell stage to promote timely repression of a subset of 4 cell stage-specific genes. Loss of maternal inheritance of G9a disrupts the gene regulatory network resulting in developmental delay and destabilisation of inner cell mass lineages by the late blastocyst stage. Our results indicate a vital role of this maternally inherited epigenetic regulator in creating conducive conditions for developmental progression and on cell fate choices.

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.

142 DYNAMICS OF Tet FAMILY DURING PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 183 ◽  
Author(s):  
J. Teson ◽  
K. Lee ◽  
L. Spate ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Profile ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Dna Demethylation ◽  
Dimensional Structure ◽  
Cell Stage ◽  
Donor Cells

One of the key regulators of gene expression in mammals is DNA methylation. The Tet family (Tet1–3) is suggested to be involved in regulating the level of methylation by hydroxylating a methyl group from 5-methylcytosine to form 5-hydroxymethylcystosine. This hydroxylation alters the 3-dimensional structure of the DNA and results in altered gene expression. Previous studies conducted in the mouse have shown that Tet1 is important for inner cell mass specification by regulating the apparent level of methylation on a specific promoter region in blastocysts and Tet3 is related to the apparent paternal DNA demethylation after fertilization by hydroxylating the paternal genome. The objective of this study was to investigate the expression profile of the Tet family in porcine oocytes and pre-implantation-stage embryos derived from IVF and somatic cell nuclear transfer (SCNT). The RNA was isolated from donor cells, germinal vesicle (GV), MII and 2-cell and blastocyst stage embryos (20 oocytes or embryos per group). Levels of mRNA for each Tet gene were measured by quantitative real-time RT-PCR. The levels of each mRNA transcript were compared to YWHAG, a housekeeping gene that shows a constant level of expression throughout pre-implantation embryo development and normalized to the GV stage. The analysis was repeated with 3 biological replications and 2 experimental replications. Differences in gene expression were compared by ANOVA and P < 0.05 was considered significant. No difference was found in the levels of the Tet family members between GV and MII stage oocytes. Compared with GV stage oocytes, up-regulation of Tet3 at the 2-cell stage was detected in both IVF and SCNT embryos, 4.7 and 6.2 fold, respectively. A dramatic increase in Tet1 was also observed at the blastocyst stage in IVF and SCNT embryos when compared with the GV stage, 65.7 and 79.7 fold increases, respectively. Interestingly, the level of Tet3 was down-regulated in blastocyst embryos at a 25 or more fold decrease compared with GV. The level of Tet2 remained constant throughout embryo development. Embryos (2-cell and blastocyst) compared from IVF and SCNT showed no difference in Tet expression levels. Donor cells had significantly lower levels of Tet2 and Tet3 when compared with GV. Our results indicate that the Tet family shows a dynamic expression profile during porcine pre-implantation embryo development. High expression of Tet3 in 2-cell stage embryos suggests its importance during the post-activation demethylation process. The increase of Tet1 transcript in blastocysts suggests that Tet1 is involved in regulating the type of methylation at the blastocyst stage. These results are consistent with results from previous mouse studies. There was no misregulated expression of the Tet family in SCNT embryos compared with IVF embryos, thus indicating successful reprogramming of the Tet family after SCNT. Lower levels of Tet2 and Tet3 would indicate that Tet1 is important for maintaining type of methylation in donor cells. This is the first report on the profile of the Tet family during porcine pre-implantation embryo development and further studies are needed to clarify their role during this stage.

Gp330 is specifically expressed in outer cells during epithelial differentiation in the preimplantation mouse embryo

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3289-3299 ◽  
Author(s):  
C. Gueth-Hallonet ◽  
A. Santa-Maria ◽  
P. Verroust ◽  
B. Maro
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Transcriptional Level ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Extensive Cell ◽  
High Level ◽  
Endocytic Activity

During preimplantation development of the mouse embryo, a layer of outer cells differentiates into a perfect epithelium, the trophectoderm. The divergence between the trophectoderm and the inner cell mass takes place from the 8-cell stage to the 64-cell stage and precedes their commitment at the blastocyst stage. In this work, we have investigated the expression of gp330, a 330 × 10(3) M(r) glycoprotein found in clathrin-coated areas of the plasma membrane of some epithelial cells characterized by a high level of endocytic activity. Our results show that gp330 is first synthesized in 16-cell stage embryos and that its appearance is restricted to outer cells until the blastocyst stage. Furthermore, its expression is repressed in inner cells at a post-transcriptional level, probably through the development of extensive cell-cell contacts.

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