Quantitation of the spatial distribution of ‘prespore vacuoles’ in pseudoplasmodia of Dictyostelium discoideum

Development ◽  
1976 ◽  
Vol 35 (3) ◽  
pp. 499-505
Author(s):  
Paul A. Farnsworth ◽  
William F. Loomis
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Axial Distribution ◽  
Total Cell Number ◽  
Total Complement ◽  
Number Of Cells

The axial distribution of an organelle, the prespore vacuole (PV), previously reported absent from the prestalk region, was determined in pseudoplasmodia of varying sizes, under differing conditions of photostimulation of migration. The distribution of these organelles, determined quantitatively by electron microscopy ofsections from known axial locations, was found to have a spatial pattern which varied with pseudoplasmodial size. The total complement of these organelles appeared constant for any size of pseudoplasmodium under similar conditions of illumination. Increased illumination decreased the total number of the organelles. The spatial distribution of PV varies with total cell number, and the size of the region with no PV bears no relationship to the proportion of the cell mass which would form stalk cells. Similarly, the number of cells containing PV bears no fixed relationship to the number of cells which will form spores. On these grounds, the reported role of PV, that of directing or reflecting spore differentiation, appears unlikely.

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.

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.

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.

60 THE EFFECT OF L-ASCORBIC ACID DURING CULTURE, CRYOPRESERVATION, OR BOTH ON PORCINE EMBRYOS PRODUCED IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 177 ◽  
Author(s):  
M. Castillo-Martín ◽  
M. Yeste ◽  
R. Morató ◽  
T. Mogas ◽  
S. Bonet
Keyword(s):  
Ascorbic Acid ◽  
Embryo Quality ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Fertilisation ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Apoptosis Index ◽  
Number Of Cells

The benefits of adding l-ascorbic acid during the cryopreservation procedure have been reported before in mouse and bovine. In this study, the effects of l-ascorbic acid (AC) supplementation during culture, cryopreservation, or both procedures on the developmental ability and embryo quality of in vitro produced porcine blastocysts were examined. Embryo quality criteria consisted of total cell number, percentage of apoptosis, and cryotolerance. After in vitro fertilisation, presumptive zygotes were randomly assigned to 2 culture treatments in which the culture medium NCSU23 was supplemented with 100 µM AC (n = 1162) or nonsupplemented (n = 1163) for a 144-h period. On Day 6, blastocyst formation was assessed by stereomicroscopy, and a representative fraction of Grade I- and II-blastocysts of each culture treatment was evaluated using 4′,6-diamidino-2-phenylindole-TUNEL co-staining and considered as fresh-control. The remaining fraction of Grade I- and II-blastocysts was vitrified/warmed following the Cryotop® method. To determine the effect of AC supplementation during cryopreservation procedures, each culture treatment was divided into 2 groups: (1) embryos exposed to 100 µM AC, and (2) nonexposed embryos (vitrified-control). Survival was determined according to reexpansion rates after 24 h of recovery in NCSU23 medium. After 24 h, reexpanded blastocysts were co-stained using the 4′,6-diamidino-2-phenylindole-TUNEL technique, and total number of cells and apoptosis indexes were determined. Experiment was replicated 9 times for each group. Data were analyzed by t-test for independent variables and a 2-way ANOVA. Results are expressed as means ± SE, and the significant level was set at 5% (Table 1). After culture, supplementing NCSU23 medium with AC showed no significant differences in blastocyst formation (fresh-control 11.6 ± 7.8 v. AC 11.6 ± 7.7), in number of cells (fresh-control 36.7 ± 15.8 v. AC 36.1 ± 15.9), or in apoptosis index (fresh-control 2.9 ± 5.7 v. AC 3.5 ± 4.7). On the other hand, only when both culture and vitrified media were supplemented with AC was there a significant increase of blastocyst survival. In contrast, no significant differences in embryo survival were observed when only 1 of these 2 media (culture or vitrification) was supplemented. Supplementing culture media or cryopreservation solutions with AC did not affect the total cell number or apoptosis index in vitrified blastocysts. In conclusion, the addition of 100 µM l-ascorbic acid to the culture and cryopreservation solutions improves the cryotolerance of in vitro-produced porcine blastocysts. Table 1.Survival of blastocysts (24 h), total cell number, and percentage of apoptosis after vitrification/warming

76 Extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced invitro

2020 ◽  
Vol 32 (2) ◽  
pp. 164
Author(s):  
C. Leal ◽  
K. Cañon-Beltrán ◽  
Y. Cajas ◽  
P. Gallego ◽  
P. Beltrán-Breña ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Lipid Content ◽  
Luteal Phase ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Transmission Electron

Extracellular vesicles (EVs) are released by cells and transport cargo that affect functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during invitro culture (Hamdi et al. 2017 Reprod. Fertil. Dev. 30, 935-945) which may be due to their content of EV (Lopera-Vásquez et al. 2017 Reproduction 153, 461-470). Thus, the aim of this study was to evaluate the effect of EVs from OF and UF on a sequential invitro culture system on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF) supplemented with 3mgmL−1 bovine serum albumin (BSA; n=1228) or 5% EV-depleted fetal calf serum (dFCS, n=1261) in the presence (BSAEV, n=1265 and dFCSEV, n=1253) or absence of 3×105 EVmL−1 from OF (Day 1 to Day 4) and UF (Day 4 to Day 9), mimicking invivo conditions. The EVs pooled from 5 oviducts (early luteal phase) and 5 uterine horns (middle luteal phase) from slaughtered heifers were isolated by a Size Exclusion Chromatography kit (Hansa BioMed). The EV size and concentration were assessed by the nanotracking analysis system and morphology by transmission electron microscopy. Embryo development was recorded on Days 7/9. Day 7/8 blastocysts were assessed for quality by staining with (a) Hoechst 33342 (10 µgmL−1, 30min) for total cell number, (b) Bodipy 493/503 (20 µgmL−1, 1h) for lipid content (lipid droplet area in µm2), and (c) for survival rate after vitrification/warming. Data were analysed by one-way ANOVA and Tukey test. The EV concentration was 2.97 and 7.98×1010 particlesmL−1, and mode size 137.2 and 151.2nm for OF and UF, respectively. Transmission electron microscopy confirmed EV presence and size, showing typical cup-shaped morphology. Blastocyst yield was lower (P&lt;0.05) on Day 7 in the BSA groups (BSA: 15.7±1.9 and BSAEV: 15.2.4%) compared with serum groups (dFCS: 28.1±2.6 and dFCSEV: 30.1±2.9%) irrespective of EV supplementation; however, these differences were compensated at Days 8 and 9 (range: 30.0±3.2-40.8±3.9%). The EVs increased (P&lt;0.05) blastocyst total cell number in dFCSEV (152.6±2.9) and BSAEV (140.5±1.5) compared with dFCS (117.9±2.0) and BSA groups (122.4±1.1). However, lipid content was decreased (P&lt;0.05) in the presence of EVs only in dFCSEV (0.231±0.05µm2) compared with BSA (0.393±0.03µm2) and BSAEV (0.379±0.03µm2) groups. The dFCS did not differ from any group (0.371±0.05µm2; P&gt;0.05). Blastocyst survival after vitrification/warming was high in all groups up to 72h (range: 80.0±3.8-100%; P&gt;0.05). In conclusion, mimicking physiological conditions using EV from OF and UF during invitro culture does not affect development but improves embryo quality by increasing blastocyst total cell numbers and decreasing lipid contents. These results provide evidence of the association of the reproductive tract environment and developing embryo, confirming embryo-maternal communication. Funding was provided by MINECO-Spain AGL2015-70140-R; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brazil 2017/20339-3.

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.

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.

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