179 RELATIONSHIP BETWEEN GENE EXPRESSION IN INDIVIDUAL BLASTOMERES OF 2-CELL STAGE BOVINE EMBRYOS AND THE NORMALITY OF FIRST CLEAVAGE

2016 ◽  
Vol 28 (2) ◽  
pp. 220
Author(s):  
S. Matoba ◽  
M. Kaneda ◽  
T. Somfai ◽  
T. Nagai ◽  
M. Geshi
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Calf Serum ◽  
Transcript Abundance ◽  
Pcr Primers ◽  
Developmental Competence ◽  
Cleavage Pattern ◽  
Bovine Embryos ◽  
Culture Dish ◽  
Cell Stage

Previously early first and second cleavages after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high development competence (Sugimura et al. 2012 PLOS One 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern and the transcript abundance in each blastomere in 2-cell stage bovine embryos. IVF-derived bovine embryos were cultured individually in microwells culture dish in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. First cleavage and cleavage patterns were categorised as being either normal (the first cleavage within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF at the first cleavage). Next, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting. Individual blastomeres (n = 71, 10 replicates) were analysed for gene expression by quantitative RT-PCR. Primers were designed for 19 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation and fetal growth (NANOG, OCT4, PLAC8, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, G6PDH, Glut8, MNSOD-3end, SOX2, DYNLL1, IGF1R, IGF2, IGF2R, IGFBP2, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in both of individual blastomeres of cleaved embryos was examined in embryos that cleaved early with a normal cleavage pattern and in those that showed abnormal cleavage pattern. Values were normalised to the average values of the reference genes and means were compared by the student t-test. Transcript abundance of OCT4, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, MNSOD-3end, IGF2R, and IGFBP2 was significantly higher in blastomeres associated with all categorised abnormal blastomeres compared towith an early normal cleavage (P < 0.05). Furthermore, the expression of PLAC8, IGF1R, and PMSB1 in embryos having 2 uneven blastomeres, Glut8 and SOX2 in 2 uneven blastomeres with fragment/protrusion was higher than that in normal cleavage (P < 0.05). However, the level of G6PDH was lower in embryos having 2 uneven blastomeres than that in those showing normal cleavage (P < 0.05). Our results reveal blastomere gene expression in bovine embryos at the first cleavage may correlated with oocyte developmental competence. This study was supported by JSPS KAKENHI (26450388).

138 THE RELATIONSHIP BETWEEN THE NORMALITY OF FIRST CLEAVAGE, THE GENE EXPRESSION IN BLASTOMERES, AND THE ABILITY TO DEVELOP TO THE BLASTOCYST STAGE IN IVF-DERIVED BOVINE 2-CELL STAGE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 177
Author(s):  
S. Matoba ◽  
M. Kaneda ◽  
T. Somfai ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Target Genes ◽  
Calf Serum ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Embryonic Cleavage ◽  
The Relationship

Early first and second cleaved embryos after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLOS ONE 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern, the transcript abundance, and their development to the blastocyst stage in each blastomere in 2-cell stage bovine embryos. The IVF-derived bovine embryos were cultured individually in well-of-the-well culture dishes in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL−1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. The first embryonic cleavage was categorized as being either normal (occurring within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF). Then, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting (n = 85; 4 replicates). In each embryo, one blastomere was subjected to quantitative RT-PCR to analyse the expression of developmentally important genes. The remaining blastomere was subsequently cultured in an individually identifiable manner to verify their ability to develop to the blastocyst stage. Primers were designed for 12 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation, and fetal growth (OCT4, ATP1A1, CCNB1, CDH1, COX1, CTNNB1, GLUT8, MNSOD-3, SOX2, DYNLL1, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in individual blastomeres was compared between embryos showing normal and abnormal cleavage. Values were normalized to the average values of the reference genes and all the means were compared by the Student t-test. Blastomeres resulted from normal cleavage developed to the blastocyst stage on Day 7 to 8 (Day 0 = IVF) at significantly higher rates than those resulted from abnormal cleavage (65.7% v. 37.5%, respectively, P < 0.05). Transcript abundance of OCT4 was significantly higher in blastomeres associated with all abnormal cleavage than in those associated with normal cleavage (P < 0.05). The expression of CCNB1, COX1, ATP1A1, GLUT8, and PMSB1 in blastomeres associated with normal cleavage and blastocyst development was higher than that in those of abnormal cleavage (P < 0.05). However, the level of OCT4, CCNB1, COX1, ATP1A1, and PMSB1 was lower in blastomeres associated with normal cleavage but failure of blastocyst development than those in blastomeres showing abnormal cleavage (P < 0.05). Our results reveal that significantly higher expression of CCNB1, COX1, ATP1A1, and PMSB1 in blastomeres at the 2-cell stage in bovine embryos with superior developmental competence compared with those showing abnormal cleavage and low competence. Research was supported by JSPS KAKENHI (26450388).

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

285 RELATIONSHIP BETWEEN FOLLICLE FLUID STEROID CONCENTRATIONS, GRANULOSA CELL GENE EXPRESSION, AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 299
Author(s):  
S. Matoba ◽  
S. Mamo ◽  
E. Gallagher ◽  
A. G. Fahey ◽  
T. Fair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Target Genes ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Competence ◽  
Cell Gene Expression ◽  
The Relationship

The ability to culture oocytes and embryos in an individually identifiable manner facilitates the study of the relationship between follicle param- eters and oocyte development, in order to identify markers of competent oocytes. The aim of this study was to examine the predictive value of intrafollicular steroid concentrations and granulosa cell transcript abundance on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles (n = 214, 11 replicates, 49 animals) were dissected from the ovaries of slaughtered animals. Following measure- ment of diameter, follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba and Lonergan 2009 Reprod. Fertil. Dev. 21, 160). Cleavage and blastocyst rates were assessed on Days 2 and 9, respectively. Follicular fluid was recovered and stored at -80°C until analysis for concentrations of the steroids estradiol, progesterone, and testosterone by RIA. Granulosa cells were collected from each follicle for analysis of gene expression by quantitative RT-PCR. Primers were designed for 7 target genes (AMH, CYP19A, ESR1, ESR2, FSHR, HSD3B1 and LHCGR) and 2 reference genes (PPIA and H2AZ). Transcript abundance of target genes in granulosa cells associated with embryos that cleaved and developed to the blastocyst stage (competent) and those that cleaved but failed to develop (incompetent) was examined. Mean steroid concentrations were compared by ANOVA and Spearman correlations, and logistical regression were used to test the relationship between follicle size and steroid con- centration and the ability of steroid concentration to predict developmental competence. Gene expression data were analyzed using the delta-delta CT (cycle threshold) method. Values were normalized to the average values of the reference genes and means were compared by the Student’s t-test In total, 79.1% of oocytes cleaved after IVF and 28.3% developed to the blastocyst stage. The mean (±SEM) follicular concentrations of testosterone (62.8 ± 4.8 ng mL-1), progesterone (616.8 ± 31.9 ng mL-1), or estradiol (14.4 ± 2.4 ng mL-1 were not different (P ≥ 0.05) between competent and incompetent oocytes. Follicular diameter was negatively correlated with testosterone, progesterone, testosterone:estradiol, and pro- gesterone:estradiol (P ≤ 0.01) and positively correlated with estradiol (P ≤ 0.01). Logistical regression analysis showed that steroid concentrations or the ratio of steroids were not satisfactory predictors of oocyte competence. Transcript abundance of AMH, ESR1, ESR2, FSHR, and HSD3B1 was significantly higher (P ≤ 0.05) in granulosa cells associated with competent compared with incompetent oocytes. In conclusion, follicular steroid concentrations were not associated with oocyte development. In contrast, granulosa cell gene expression may be a useful predictor of oocyte competence. Supported by Science Foundation Ireland (07/SRC/B1156).

56 Nobiletin affects gene expression profiles of the ERK1/2 pathway in bovine embryos produced invitro

2021 ◽  
Vol 33 (2) ◽  
pp. 135
Author(s):  
Y. N. Cajas ◽  
K. E. Cañón-Beltrán ◽  
C. L. V. Leal ◽  
A. Gutierrez-Adán ◽  
E. González ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Cell Cycle ◽  
Embryo Development ◽  
Expression Profiles ◽  
Calf Serum ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
And Oxidative Stress

During embryo development the embryonic genome activation (EGA) is one of the most important events and in bovine embryos it occurs at the 8- to 16-cell stage. Invitro embryo production increases the levels of reactive oxygen species (ROS), which leads to the low quality of the produced blastocysts, possibly by affecting EGA. Nobiletin is an antioxidant that affects cell cycle regulation (Huang et al. 2016 Evid. Based. Complement. Alternat. Med. 2016, 2918796, https://doi.org/10.1155/2016/2918796). Therefore, we aimed to evaluate the effect of nobiletin supplementation, in two key periods of early embryo development, on blastocyst yield and expression of selected genes of the ERK1/2 pathway and oxidative stress on produced embryos. Invitro zygotes were cultured in synthetic oviductal fluid (SOF) with 5% fetal calf serum (control, C); C with 5 or 10µM nobiletin (MedChemExpress) (N5, N10); or C with 0.03% dimethyl sulfoxide (CDMSO; vehicle for nobiletin dilution) during the minor (21–54h post-insemination (hpi): 2- to 8-cell; MNEGA; 12 replicates) or major (54–96 hpi: 8- to 16-cell; MJEGA; 10 replicates) phase of EGA. The speed of development was considered and embryos that reached ≥8 cells at 54 hpi from MNEGA phase and ≥16 cells at 96 hpi from MJEGA phase, were selected and further cultured in control medium until Day 7. Embryos at ≥8 cell (MNEGA), ≥16 cell (MJEGA) stage, and Day 7 blastocysts from both periods were snap-frozen in liquid N2 for gene expression analysis (3 pools of 10 embryos/treatment). The expression of genes related to ERK1/2 pathway (H3–3B, H3–3A, NFE2L2) and oxidative stress (GPX1) were measured by quantitative PCR; H2AFZ and ACTB were used as housekeeping genes. Statistical analysis was assessed by one-way ANOVA. At 54 hpi, irrespective of nobiletin supplementation, no differences were found in the proportion of embryos that reached the 8-cell stage between groups in both phases (≈60%). At 96 hpi, nobiletin during MJEGA showed a higher proportion of embryos reaching the 16-cell stage than control groups (≈70% vs. ≈60%, respectively; P&lt;0.001). Blastocyst yield for MNEGA and MJEGA was higher (P&lt;0.001) for N5 (40.0±0.8% and 46.7±0.8%) and N10 (41.0±0.9% and 54.5±1.1%) compared with C (32.0±0.6% and 38.4±1.1%) and CDMSO (31.2±0.4% and 35.8±1.0%) groups, while N10 was higher (P&lt;0.05) compared to N5 group in MJEGA. The expression of H3–3B and H3–3A were higher (P&lt;0.05) in 8-cell embryos from N5 and N10 groups during MNEGA; while in 16-cell embryos, H3–3B and NFE2L2 were higher (P&lt;0.05) only in the N10 group compared with both controls during MJEGA. GPX1 was upregulated in nobiletin-supplemented groups from both phases (8- and 16-cell embryos and blastocysts) compared with controls (P&lt;0.05). In conclusion, nobiletin supplementation during minor or major EGA has a positive effect in pre-implantation embryo development and modifies the transcription of cell cycle and oxidative stress genes in early embryos. These benefits can be attributed to its bioactivity and indicate that it might be a tool to overcome EGA and ROS disorders in bovine invitro-produced embryos.This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; SENESCYT-Ecuador; FAPESP-Brazil 2017/20339-3, CNPq-Brazil 304276/2018-9.

96 EFFICIENT PRODUCTION OF MONOZYGOTIC TWIN BOVINE EMBRYOS USING BLASTOMERE SEPARATION TECHNIQUE WITH COMMERCIAL WELL OF THE WELL CULTURE DISH

2016 ◽  
Vol 28 (2) ◽  
pp. 178
Author(s):  
Y. Hashiyada ◽  
Y. Aikawa ◽  
S. Sugimura ◽  
H. Matsuda ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Statistical Significance ◽  
Cell Mass ◽  
Calf Serum ◽  
Monozygotic Twin ◽  
Efficient Production ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage

Monozygotic twin embryos can be produced using the technique of blastomere separation and well of the well (WOW) dish having handmade micro-wells by the needle depression (Tagawa et al. 2008). We have recently reported that developed commercial WOW dish enhances embryo competence in individual culture (Sugimura et al. 2010). The present study was conducted to evaluate the availability of commercial WOW dish for production of monozygotic twin embryos in bovine. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and IVC. For each culture, TCM-199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10 mg mL–1 BSA, and CR1aa containing 5% CS were used. To evaluate the adaptability of dishes on culture of isolated blastomeres from different cell stage, 2- (n = 63), 4- (n = 94), 8- (n = 137), and 10- to 14- (n = 116) cell stages were obtained on 24–27 h, 30–36 h, 48–54 h, and 48–54 h from the beginning of fertilization, respectively. The zona pellucida was removed by exposure of 0.25% pronase, followed by gentle pipetting by inspiration and expiration in the IVC medium. Then, two halves separated from the original number of blastomeres were randomly allocated to the conical micro-wells of commercial dish (Dai Nippon Printing, Tokyo, Japan) or created micro-wells by pressing the bottom of the dish with an eyeleteer (control). The approximate diameter and depth of each 25 wells in a commercial dish was 287 and 168 μm, and each 20 wells in the control were 800 and 600 μm. The blastomeres were cultured in wells covered with a droplet of 2.5 μL well–1 IVC medium until Day 8 (IVF = Day 0). Expanded blastocysts (n = 28) derived from tetra-blastomeres of 8-cell stage were stained to determine the number of the inner cell mass (ICM) and trophectoderm (TE) in each group. Statistical significance of the blastocyst formation rates and the number of cells were analysed by the chi-square test and the Student’s t-test, respectively. In the 2-cell stage, blastocyst formation rate in commercial dish tended to be higher than that in the control (60.0% v. 46.1%). The rate of monozygotic blastocyst pairs in commercial dish was higher compared with the control (48.0% v. 26.3%, P < 0.05). In the 4-cell stage, rates of blastocyst formation (50.0% v. 33.0%, P < 0.05) and the pairs (39.5% v. 12.5%, P < 0.01) in the commercial dish, both were higher compared with the control. In the 8-cell stage, there were no differences between two groups in rates of blastocyst formation (53.1% v. 59.0%) and the pairs (41.8% v. 48.7%), similarly in the 10- to 14-cell stage (47.9% v. 56.8% and 36.2% v. 40.9%, respectively). The ICM, TE, and total cell numbers were not different between the commercial and the control dish (28.0 ± 3.2 v. 26.0 ± 3.8, 64.6 ± 4.3 v. 76.0 ± 7.9, and 92.6 ± 6.2 v. 102.0 ± 11.0, respectively). These results indicate that separated blastomeres could be developed to blastocysts efficiently and stably regardless of embryo cell stage with a commercial WOW culture dish.

98 SUCCESSFUL PREGNANCIES FOLLOWING TRANSFER OF VITRIFIED PORCINE EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 157 ◽  
Author(s):  
K. Hiruma ◽  
H. Ueda ◽  
H. Saito ◽  
C. Tanaka ◽  
N. Maeda ◽  
...  
Keyword(s):  
Calf Serum ◽  
Developmental Competence ◽  
Penicillin G ◽  
Cell Stage ◽  
Epidermal Growth ◽  
Potassium Penicillin ◽  
The One ◽  
Parthenogenetic Embryos

To date only in vivo-produced embryos have successfully produced live piglets after cryopreservation. In this study, we aimed to produce piglets from vitrified embryos derived from in vitro matured (IVM) oocytes. Cumulus-oocyte complexes collected from ovaries obtained at a local slaughterhouse were matured for 44 to 45 h in NCSU23 MEDIUM supplemented with 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 �g/mL potassium penicillin G, 50 �g/mL streptomycin sulfate, and 10 IU/mL eCG/ hCG. These IVM oocytes were either activated for parthenogenesis or in vitro-fertilized (IVF). For IVF, oocytes were incubated with 5 � 106/mL of cryopreserved epididymal sperm in PGM-tac medium (Yoshioka et al. 2003 Biol. Reprod. 69, 2092-2099) for 20 h. Embryos were treated for removal of cytoplasmic lipid droplets (delipation; Nagashima et al. 1995 Nature 374, 416) at the 4- to 8-cell stages, around 50 to 54 h after activation or insemination. After culture in NCSU23 for 15 h, they were vitrified by the minimum volume cooling (MVC) method. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO), and 20% (v/v) calf serum for 4 min, followed by exposure to vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% calf serum. Embryos were then loaded onto a Cryotop (Kitazato Supply Co., Tokyo, Japan) and immediately plunged into liquid nitrogen. Vitrified embryos were examined for viability in vitro and in vivo after warming. Their in vitro developmental competence was compared to that of corresponding control (nonvitrified) embryos. Vitrified 4- to 8-cell stage embryos, both parthenogenetic and IVF, showed developmental competence into blastocysts comparable to that of control embryos (parthenogenetic: 46.8%, 36/77 vs. 51.7%, 31/60; IVF: 40.0%, 30/75 vs. 44.3%, 35/79). Of four surrogate gilts that received a total of 251 vitrified parthenogenetic embryos, three became pregnant and had 20 fetuses (8.0%, 22 to 23 days old). Three surrogates gilts that received 267 vitrified IVF embryos all became pregnant. Of those, the one that received 47 embryos was confirmed to have eight fetuses (17.0%, 22 days old) by autopsy. The other two were examined by ultrasonography at 56 and 95 days of gestation and found to be pregnant. These results suggest that porcine embryos derived from IVM oocytes have a potential to develop into live offspring after delipation and MVC vitrification. This study was supported by PROBRAIN.

72 HISTONE METHYLATION AND GENE EXPRESSION PATTERNS IN PRE-IMPLANTATION EMBRYOS DERIVED FROM INTRA- AND INTER-SPECIES NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 144
Author(s):  
W. Shi ◽  
F. Yang ◽  
E. Wolf ◽  
V. Zakharchenko
Keyword(s):  
Gene Expression ◽  
Histone Methylation ◽  
Methylation Level ◽  
Dynamic Change ◽  
Expression Patterns ◽  
Mouse Embryos ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Rabbit Embryos

The differential epigenetic changes in embryos from different species provide a model to study how the nucleus from one species interacts with cytoplasm from another species. In this study we examined histone methylation at lysine 9 of histone 3 (K9H3) and lysine 20 of histone H4 (K20H4) and the expression levels of three early development-related genes (Oct-4, Hsp 70.1 and Hprt) in individual intra- and inter-species cloned and control embryos at the 1-, 2-, 4- and 8-cell stages. Mouse fetal fibroblast (MFF) nuclei were transferred into mouse, bovine, or rabbit oocytes. As control, we used in vivo derived (mouse and rabbit) or in vitro-produced (bovine) embryos. Histone methylation was detected by anti-MeK9H3 and anti-MeK20H4 antibodies. Gene expression analysis was performed using a quantitative RT-PCR technique (Daniels et al. 2000 Biol. Reprod. 63, 1034-1040). Data were analyzed by Student's t-test. No embryos from inter-species cloning (MFF-bovine and MFF-rabbit) survived beyond the 8-12 cell stage. MFF-mouse and MFF-bovine embryos exhibited demethylation of K9H3 and K20H4 at the 2-cell stage and the methylation level was increased at the 4-cell stage, but no demethylation was observed at the 2-cell stage of MFF-rabbit embryos and the methylation level in these embryos was significantly higher than that of in vivo rabbit embryos. The level of Oct-4 mRNA was low at the 1- and 2-cell stages of in vivo mouse embryos and increased at the 8-cell stage. No significant increase in Oct-4 transcript was detected at the 8-cell stage of inter-species cloned embryos. The expression of Hsp 70.1 in in vivo mouse embryos was increased at the 2-cell stage and decreased to a level similar to that in the zygote at the 8-cell stage. In cloned embryos, Hsp 70.1 transcripts were also increased at the 2-cell stage, but there was no significant decrease of Hsp70.1 mRNA abundance at the 8-cell stage of inter-species embryos as compared to the corresponding 2-cell stage. For MFF-mouse embryos, Hsp 70.1 expression was increased at the 2-cell stage, but at the 8-cell stage the transcript level was at the level similar to that in inter-species clones. Hprt expression was increased at the 8-cell stage of in vivo mouse embryos. The dynamic change of Hprt transcript in MFF-mouse embryos was not significantly different from that of in vivo mouse embryos, but no significant change of Hprt expression occurred in the development of MFF-bovine and MFF-rabbit embryos. Differential epigenetic characteristics of mouse somatic nucleus after transfer into oocytes from different species suggest the existence of incompatibilities of nuclear-cytoplasm interaction between distantly related species. This abnormal interaction at the time of genome activation may affect normal development. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

251 COMPARATIVE ANALYSIS OF GENE EXPRESSION PATTERNS IN PORCINE PRE-IMPLANTATION EMBRYOS DERIVED FROM DIFFERENT ORIGINS

2006 ◽  
Vol 18 (2) ◽  
pp. 233
Author(s):  
J.-G. Kim ◽  
H.-F. Jin ◽  
B.-K. Mohana ◽  
H.-J. Song ◽  
Y.-J. Jeong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Expression Patterns ◽  
Rna Isolation ◽  
Developmental Competence ◽  
Histone H2a ◽  
Cell Stage ◽  
Real Time Quantitative Pcr ◽  
Morula Stage ◽  
Different Origins

The amount of information gathered on the kinetics and quantitative profile of gene expression in nuclear transferred (NT) pre-implantation embryos is still scarce and limited to a handful of genes in pig. In the present study, we compared the relative abundance (RA) of six development-related genes of pre-implantation embryos from different origins. Cumulus-oocyte complexes (COCs) were matured, fertilized and cultured by the method of Abeydeera et al. (2000 Theriogenology 54, 787-797). Parthenogenetic (PA) and NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Sets of 10 embryos each at 4-cell, 8-16-cell, morula, and Day 7 blastocyst stages were used to extract total RNA for analyzing the expression pattern of Bax (pro-apoptotic), Bcl-xl (anti-apoptotic), Oct-4 (pluripotent transcription), Stat3 (cytoplasmic transcription), IFN-tau (implantation) and VEGF (vasculogenesis) genes (three replicates) with real-time quantitative PCR (LightCycler�; Roche Diagnostics, Mannheim, Germany) following RNA isolation (with Dynabeads� mRNA Direct" kit; Dynal, Oslo, Norway) and cDNA amplification (Oligo (dT)12-18 primer and Superscript" III cDNA synthesis kit; Invitrogen, Carlsbad, CA, USA). The expression of each gene was normalized to Histone H2A expression. Statistically significant (P < 0.05) differences in gene expression were analyzed by ANOVA. Oct-4, Stat3, Bax, and Bcl-xl were expressed at the 4-cell stage. However, IFN-tau and VEGF were expressed at the morula stage. The expression patterns of all genes throughout the embryonic development in all types of embryos were similar. However, the relative abundance (RA) of Bax in the 8-16 cell stage of NT and PA was significantly (P < 0.05) increased as compared to that in IVP counterparts. The RA of Oct-4 and Bcl-xl did not differ throughout all stages in NT and PA embryos. There were no significant differences in all types of embryos with respect to the RA of Oct-4 and Bcl-xl with exception of the blastocysts in NT (significantly decreased, P < 0.01). The RA of Stat3 was significantly (P < 0.05) increased in the 8-16 cell stage (NT and PA) and in blastocysts (NT). Similarly, the RA of IFN-tau was significantly (P < 0.05) increased in NT blastocysts. The RA of VEGF was not significantly different in all stages and types of embryos except NT blastocysts (decreased expression, P < 0.01). These results suggest that expression patterns of Bax, Bcl-xl, Oct-4, Stat3, IFN-tau, and VEGF can be used as markers to assess the developmental competence of porcine nuclear transfer embryos. This work was supported by Grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

69 GLOBAL H3k9ac AND H3k27me3 EXPRESSION IN BLASTOMERES FROM 8- TO 16-CELL STAGE BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 148
Author(s):  
C. S. Oliveira ◽  
N. Z. Saraiva ◽  
L. Z. Oliveira ◽  
R. V. Serapião ◽  
M. R. de Lima ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Histone Modifications ◽  
Pearson Correlation ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Group A ◽  
Genome Activation

Embryonic genome activation is a crucial step in early embryo development, and is accompanied by a dramatic change in the epigenetic profile of blastomeres. Histone modifications related to euchromatin and heterochromatin can be important parameters to infer developmental competence, as they are affected by manipulation and environmental stress conditions. The aim of this study was to characterise permissive (H3k9ac) and repressive (H3k27me3) histone modifications during the embryonic genome activation cell cycle in bovine embryos, regarding correlation between those marks and variance among blastomeres. For that, bovine embryos were produced by IVF and cultured in SOF medium supplemented with 5 mg mL–1 of BSA and 2.5% FCS in 5% O2 in an air atmosphere for 5 days (70 h after IVF). The 8 to 16 cell embryos were fixed in 4% paraformaldehyde and submitted to H3k9ac and H3k27me3 immunofluorescence assay (mouse anti-H3K9ac monoclonal antibody, 1 : 200; Sigma; rabbit anti-H3k27me3 monoclonal antibody, 1 : 200; Upstate, Charlottesville, VA, USA). Nuclei were counterstained with Hoechst 33342. Images of each embryo were captured (AxioCam, Carl Zeiss, São Paulo, Brazil) and measured for nuclear fluorescence intensity in each blastomere using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA). Mean levels were compared using the Mann-Whitney test and variances were compared using F-test (SAS 9.1, SAS Institute Inc., Cary, NC, USA; P = 0.05). We evaluated 2 replicates and 12 embryos during the transition from the 8 to 16 cell stages, totaling 169 blastomeres. Global H3k27me3 levels varied accordingly to H3k9ac levels, as indicated by a high Pearson correlation coefficient (r = 0.913). Levels of each blastomere were normalized to the lowest level obtained within each embryo. Some embryos displayed a high variation between blastomeres, and, for further analysis, we divided the embryos into groups: group A for embryos that presented similar H3k9ac levels between blastomeres (8 embryos, 66%), and group B for embryos that exhibited higher heterogeneity between blastomeres (at least 2 blastomeres presenting a 2-fold increase compared to the lowest blastomere; 4 embryos, 33%). Mean H3k9ac and H3k27me3 normalized levels were lower for group A [H3k9ac: 1.35 ± 0.29 (A), 1.94 ± 1.02* (B); H3k27me3: 1.33 ± 0.24 (A), 1.99 ± 0.77 (B)], and group A displayed lower variance values (H3k9ac: 0.07 (A), 1.05* (B); H3k27me3: 0.06 (A), 0.60 (B)]. Within each embryo, blastomeres were sorted in ascending order for H3k9ac level (1 to 16), and compared between groups A and B. We detected that mean levels differed (P < 0.05) between groups from blastomere 9 to 16 for H3k9ac and 10 to 16 for H3k27me3. Therefore, in 8- to 16-cell stage embryos, the H3k27me3 repressive mark is highly correlated with the H3k9ac permissive mark. Also, our results describe the presence of 2 distinguishable populations of bovine embryos at this stage, considering their epigenetic status. One population presented similar levels of repressive and permissive marks among blastomeres, whereas the second one displayed a remarkable variation among their blastomeres. This observation should be further studied, as it might reflect distinct cleavage pattern embryos and blastomere competence. The authors acknowledge FAPESP, FAPERJ and CNPq for financial support.

242 PROMISING TWO-STEP EVALUATION SYSTEM FOR SELECTING HIGH DEVELOPMENTAL COMPETENCE IN VITRO FERTILIZED EMBRYOS IN CATTLE USING WELL-OF-THE-WELL DISH

2015 ◽  
Vol 27 (1) ◽  
pp. 210
Author(s):  
M. Taniai ◽  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Evaluation System ◽  
Evaluation Method ◽  
Calf Serum ◽  
Time Lapse ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test

Bovine IVF embryos are evaluated morphologically using light microscopy just before transfer. However, this evaluation method is subjective, and an objective method with more certainty is needed. Sugimura et al. (PLoS ONE 2012 7, e36627) reported a promising system for selecting healthy IVF bovine embryo by using time-lapse cinematography and 5 prognostic factors. This study was to investigate the efficacy of a 2-step evaluation system of IVF embryos using microscopy for selecting high developmental competence IVF embryos. Cumulus-oocyte complexes (COC) were collected by ovarian follicular aspiration (2 to 5 mm diameter) obtained from a local abattoir. The COC (n = 488) were matured in TCM-199 medium supplemented with 5% calf serum (CS) and 0.02 IU mL–1 of FSH at 38.5°C for 20 h in an atmosphere of 5% CO2 (20 COC 100 µL–1 droplets). After 10 h of gametes co-culture (5.0 × 106 sperm cells mL–1), the presumptive zygotes were cultured in 125 µL of CR1 aa medium supplemented with 5% CS in well of-the-well culture dishes (AS ONE, Japan; 25 zygotes well–1) at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days. Two-step evaluations of embryos were done at 27 and 55 h post-IVF (hpi). In the first step of evaluation, cleavage patterns at 27 hpi were categorized as mono-cell, 2-cell with even blastomeres and without fragments (normal cleavage), 2-cell with uneven blastomeres, and ≥3 blastomeres. During the second step of evaluation, embryos were classified by their number of blastomeres (2 to 5 cells, 6 to 8 cells, and >8 cells) and the absence or presence of multiple fragments (<20 or >20%) at 55 hpi. The data were analysed by chi-square test. The blastocyst rate (BL%) of embryos cleaved before 27 hpi (56.6%, n = 106) was higher (P < 0.01) than those of embryos cleaved after 27 hpi (37.0%, n = 235). A greater percentage (P < 0.05) of 2-cell embryos with normal cleavage (68.0%, n = 50) developed to blastocysts than from with =3 blastomeres at 27 hpi (40.6%, n = 32). Superior BL% (P < 0.01) was obtained from embryos categorized as 6- to 8-cell stage (58.6%, n = 140) and >8 cell stage (70.6%, n = 25) compared with those embryos at the 2- to 5-cell stage at 55 hpi (26.1%, n = 176). Embryos with no fragments (58.0%, n = 467) had higher BL% (P < 0.01) compared with those with <20% fragments (30.7%, n = 127) and having with >20% fragments (17.5%, n = 25) at 55 hpi. The highest of BL% was observed in embryos showing a normal cleavage to 2-cells with at 27 hpi and having >6 cells with no fragments at 55 hpi (95.2%, n = 21, P < 0.01). These results demonstrate that the 2-step evaluation system at 27 and 55 hpi using microscopy is an effective method for selecting IVF embryos with high developmental competence.

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