scholarly journals Apoptosis in rabbit embryos produced by fertilization or nuclear transfer with fibroblasts and cumulus cells

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 359-366 ◽  
Author(s):  
Shu-Zhen Liu ◽  
Li-Juan Yao ◽  
Man-Xi Jiang ◽  
Zi-Li Lei ◽  
Li-Sheng Zhang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Rate ◽  
Cumulus Cells ◽  
Cell Number ◽  
Apoptotic Index ◽  
Total Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Total Cell Number

In this study, we investigated the development, the cell number of the blastocyst, and apoptosis in rabbit nuclear transfer (NT) embryos derived from adult fibroblasts and cumulus cells as compared with embryos derived from in vivo fertilization and in vitro culture. The developmental rate and the total cell number of the blastocyst were significantly lower in NT embryos than in fertilized embryos (FEs). The type of donor cells did not affect the embryonic developmental rate and the total cell number of blastocysts in NT groups. The present study investigated the onset and the frequency of apoptosis in NT embryos and FEs by using a terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The earliest positive TUNEL signals were detected at the eight-cell stage in NT embryos and at the morula stage in FEs. The apoptotic index of the total blastocysts, the inner cell mass and the trophoderm was greatly higher in the NT embryos than in FEs. Moreover, the apoptotic index of the blastocyst from fibroblasts was significantly higher than that of the blastocyst from cumulus cells.

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.

Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (4) ◽  
pp. 309-320 ◽  
Author(s):  
Rabindranath de la Fuente ◽  
W. Allan King
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Similar Proportion ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

scholarly journals Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

2012 ◽  
Vol 81 (3) ◽  
pp. 229-234 ◽  
Author(s):  
Martina Lojkic ◽  
Iva Getz ◽  
Marko Samardžija ◽  
Mario Matkovic ◽  
Goran Bacic ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 129
Author(s):  
G.-S. Im ◽  
J.-S. Seo ◽  
I.-S. Hwang ◽  
S.-W. Kim ◽  
H.-S. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Electric Pulse ◽  
Chemical Activation ◽  
Cell Mass ◽  
Cell Number ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 �s. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 �g/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5�C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P < 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P < 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P < 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P < 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.

Chromosome analysis of single-pronuclear haploid parthenogenetic blastocysts and their inner cell mass derivatives

Development ◽  
1986 ◽  
Vol 98 (1) ◽  
pp. 167-174
Author(s):  
M. T. Schnebelen ◽  
M. H. Kaufman
Keyword(s):  
Cellular Proliferation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Chromosome Analysis ◽  
Cell Number ◽  
Total Cell ◽  
Success Rates ◽  
Total Cell Number ◽  
Mitotic Cells

Single-pronuclear haploid parthenogenetically activated mouse embryos were transferred to the oviducts of suitable recipients. One group of embryos was isolated at the morula stage and subsequently allowed to develop to the expanded blastocyst stage in vitro. Intact embryos were either analysed by the air-drying technique at that stage to determine their total cell number and ploidy, or treated by immunosurgery to isolate their inner cell mass. These were either analysed to establish their total cell number and ploidy, or retained in culture for an additional 24 h or 72 h. The inner cell mass derivatives were then analysed to establish the total cell number and ploidy. A second group of recipients was ovariectomized on the 4th day of pseudopregnancy, treated with Depo-Provera and blastocysts recovered 5 or 6 days later. The ‘delayed’ blastocysts recovered were treated by immunosurgery, and the inner cell masses isolated and either analysed at this time or transferred to culture for 72 h, 96 h or 144h. As in the previous groups, the inner cell mass derivatives were analysed to establish the total cell population present and their ploidy. The analysis of this material was found to be technically particularly difficult, though in general the non-‘delayed’ embryos and their inner cell mass derivatives yielded higher success rates than the ‘delayed’ inner cell mass derivatives. The ‘delayed’ inner cell masses initially contained on average about twice the number of cells compared to the number present in those isolated from the non-‘delayed’ expanded blastocysts. Cellular proliferation occurred in all the groups retained in culture, though only a small proportion of the cells analysed gave ‘scorable’ mitotic cells in which the ploidy could be unequivocally determined. In general, in both the non-‘delayed’ and ‘delayed’ groups, the proportion of diploid mitotic cells observed increased with their duration in culture, though this effect was clearly more marked in the ‘delayed’ series. The present study indicated that the chance of obtaining haploid mouse cell lines in the future might be increased by using inner cell masses derived from non-‘delayed’ rather than ‘delayed’ blastocysts despite their initial reduced cell number at the time of explantation into tissue culture.

70 Energy metabolites induce differential DNA methylation levels and transcription in trophectoderm and inner cell mass

2021 ◽  
Vol 33 (2) ◽  
pp. 142
Author(s):  
J. Ispada ◽  
C. B. de Lima ◽  
E. C. dos Santos ◽  
A. M. da Fonseca Junior ◽  
J. V. Alcantara da Silva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Significant Difference

DNA methylation/demethylation is one of several epigenetic mechanisms by which metabolism regulates gene expression. More specifically, α-ketoglutarate (αKG) and succinate (Suc) are tricarboxylic acid cycle metabolites that may decrease and increase, respectively, the activity of DNA demethylases. Because pre-implantation embryos undergo reprogramming in both DNA methylation and metabolic pathways, it is possible that metabolic changes influence this epigenetic mark. To test that hypothesis, bovine embryos were invitro produced by using standard protocols and, 8h after fertilization, zygotes were transferred to synthetic oviductal fluid (SOF)-based culture medium (control, CO) or culture medium containing 4mM dimethyl-αKG, or 4mM dimethyl-Suc, where they remained until Day 4. Embryos were collected at Day 4 or remained in culture until Day 7, in control medium. Day 4 embryos were evaluated for DNA methylation levels by immunofluorescence detection of 5-methylcytosine (5mC) and cleavage rate. Day 7 embryos were also assessed for DNA methylation by immunofluorescence of 5mC, total cell number, blastocyst rates, and quantification of ACTB (housekeeping), DNMT1, DNMT3A, and DNMT3B transcript by RT-qPCR in trophectoderm (TE) and inner cell mass (ICM) separated by immunosurgery. The mRNA expression levels of were normalized to internal control ACTB and subsequently calculated using the 2−ΔΔCT method, using the control group for comparisons. All data were submitted to outlier detection using ROUT with Q=1% followed by one-way analysis of variance (ANOVA) and Fisher’s least significant difference (l.s.d.) test in GraphPad Prism. αKG and Suc did not influence cleavage or blastocyst rates, total cell number, or cell allocation. αKG supplementation reduced 5mC fluorescence intensity in embryos assessed at Day 4 (CO: 12.8±0.4 AU; αKG: 9.0±0.2AU; P&lt;0.0001) and Day 7 (CO: 36.5±0.7 AU; αKG: 23.5±0.4 AU; P&lt;0.0001), whereas Suc incubation increased DNA methylation levels in embryos at Day 4 (CO: 12.8±0.4 AU; Suc: 15.7±0.3 AU; P&lt;0.0001) and Day 7 (CO: 36.5±0.7 AU; Suc: 70.5±0.5 AU; P&lt;0.0001). αKG increased expression of DNMT1 (P=0.0438) in the ICM and led to lower levels of DNMT1 (P&lt;0.0001), DNMT3A (P=0.0013), and DNMT3B (P=0.0015) in TE cells. The culture with Suc increased DNMT1 (P=0.0074), DNMT3A (P=0.0186), and DNMT3B (P=0.0286) in ICM. Regarding TE, Suc resulted in lower expression of DNMT1 (P&lt;0.0001), DNMT3A (P=0.0017), and DNMT3B (P=0.0052). In conclusion, both supplementations resulted in global DNA methylation changes without affecting embryo development rates or morphology. These changes were accompanied by alterations in transcript profiles between ICM and TE, with differences among treatments being more pronounced in transcripts from ICM. This is the first report of DNA demethylation–induced changes by analogues of TCA cycle metabolites during early reprogramming of the bovine embryo with prolonged effects in TE and ICM cells. This research was funded by FAPESP: 2017/18384-0; 2018/11668-6.

62 Sequential nutrient restriction and provision during bovine in vitro embryo culture differentially affect blastocyst development and quality with oocytes from varied sources

2019 ◽  
Vol 31 (1) ◽  
pp. 156
Author(s):  
R. Pasquariello ◽  
Y. Yuan ◽  
D. Logsdon ◽  
J. Becker ◽  
L. Yao ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Embryo Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Nutrient Concentrations ◽  
Blastocyst Development ◽  
Total Cell Number

We have demonstrated that bovine blastocyst development was improved after culture in medium with only 6.25% of standard carbohydrate and amino acid concentrations, supplemented with fatty acids. However, these blastocysts had lower cell numbers. We hypothesised that this was due to deficiencies in embryo metabolism at the time of blastocyst formation. Thus, our objectives were to (1) determine whether using a sequential combination of nutrient concentrations could rescue blastocyst cell number; and (2) investigate the efficacy of reduced nutrient medium in 2 sources of oocytes. Oocytes were in vitro matured in identical medium either in our laboratory or during shipment from a commercial supplier. Oocytes in our laboratory were derived from feedlot heifers while purchased oocytes were obtained from culled cows. Zygotes were cultured using sequential medium with fraction V BSA. In step 1/step 2, embryos were cultured using 100% (glucose 0.5 mM/fructose 3.0mM, pyruvate 0.3/0.1mM, lactate 10.0/6.0mM, NEEA 1×/1× MEM, EAA 0.25×/0.5× MEM), 25% or 6.25% of standard nutrient concentrations. On Day 3, embryos were moved to step 2 as follows: 100% to 100%, 25% to 25%, 25% to 100%, 6.25% to 25%, or 6.25% to 100%. Lipid content of single mature oocytes from both sources was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS; Thermo Scientific, Waltham, MA, USA). Data (mean±s.e.m.) were analysed using ANOVA (P&lt;0.05). When oocytes from feedlot heifers were used, blastocyst development and cell number did not differ between treatments. When oocytes from culled cows were used, blastocyst development was improved after embryo culture in 25-25% (45.1±3.3%) and 6.25-25% (46.6±3.2%) compared with 100-100% (34.2±3.2%). However, inner cell mass number of blastocysts cultured in 25-25% (25.6±2.5) and 6.25-25% (26.0±2.6) was reduced compared with 100-100% (41.4±4.5); TE and total cell number did not differ. Embryos cultured in 100-100%, 25-100%, and 6.25-100% were equivalent. Metabolomics revealed that 10 lipid compounds (polyunsaturated fatty acids, glycosyldiacylglycerols, and glycerophospholipids) differed in abundance between the two sources of oocytes. These results show that oocytes from different sources lead to different experimental outcomes, likely due to a combination of age, body condition, diet, and hormone treatment of the female. Oocytes from culled cows result in embryos that develop to blastocysts better in a reduced nutrient environment, although these embryos have fewer inner cell masses, suggesting that quality may be reduced. Embryos from feedlot heifer oocytes are relatively immune to nutrient fluctuations. Different endogenous fatty acid reserves in the oocyte may lead to differing metabolic strategies in the subsequent embryo, altering their response to substrate availability during in vitro culture. These results also demonstrate that reduction of nutrients during culture has no detrimental effect on blastocyst development or total cell number in either oocyte source, but that inner cell mass formation requires increased nutrient provision.

Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos

1990 ◽  
Vol 2 (1) ◽  
pp. 51 ◽  
Author(s):  
GR Somers ◽  
AO Trounson ◽  
LJ Wilton
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Mouse Embryos ◽  
Total Cell Number ◽  
Cell Stage ◽  
Single Blastomere ◽  
Number Of Cells

The allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) was investigated at 6-h intervals from 78 h to 102 h after hCG injection in 3/4 mouse embryos to determine the effect of removal of a single blastomere at the 4-cell stage on early differentiation. The procedures used to produce 3/4 embryos had little effect on embryo development. Embryos that had a single blastomere removed and then re-aggregated (RA embryos) had the same total number of cells as untreated (UT) embryos except at 78 h (P less than 0.05) and 102 h (P less than 0.01) post hCG where there were slightly less cells in RA embryos. Three-quarter embryos always had significantly fewer cells than RA embryos (P less than 0.001), with an average of 74% of the total cell number of RA embryos. As expected, 3/4 embryos always had significantly fewer cells in the ICM and TE compared with RA embryos (P less than 0.001). However, the ICM:TE ratio was also significantly lower in 3/4 embryos compared with RA embryos at 84, 96, and 102 h post hCG, indicating that the allocation of cells to the ICM and TE was disturbed. The ICM:TE ratio of 3/4 embryos could not be manipulated if either an early- or late-dividing blastomere was selectively biopsied at the 4-cell stage; this suggests that the known preferential contribution of an early-dividing blastomere to the ICM is not cell autonomous.

Effects of changing culture medium on preimplantation embryo development in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Haixia Wang ◽  
Wenbin Cao ◽  
Huizhong Hu ◽  
Chenglong Zhou ◽  
Ziyi Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Apoptotic Index ◽  
Total Cell ◽  
Toxic Substances ◽  
Total Cell Number ◽  
Preimplantation Embryo Development

Summary Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

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