scholarly journals Identification and regulation of glycogen synthase kinase-3 during bovine embryo development

Reproduction ◽  
2010 ◽  
Vol 140 (1) ◽  
pp. 83-92 ◽  
Author(s):  
I M Aparicio ◽  
M Garcia-Herreros ◽  
T Fair ◽  
P Lonergan
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Blastocyst Stage ◽  
Serine Phosphorylation ◽  
Glycogen Synthase Kinase ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage

The aim of this study was to examine the presence and regulation of glycogen synthase kinase-3α (GSK3A) and GSK-3β (GSK3B) in bovine embryos and their possible roles in embryo development. Our results show that GSK3A and GSK3B are present in bovine embryos at the two-cell stage to the hatched blastocyst stage. Bovine embryo development was associated with an increase in the phosphorylation of both isoforms, being statistically significant at blastocyst and hatched blastocyst stages, compared with earlier stages. Inhibition of GSK3 with CT99021 (3 μM) resulted in a significant increase in the percentage and quality of blastocysts, while inhibition of GSK3 with lithium chloride (LiCl; 20 mM) significantly reduced at the proportion of eight-cell embryos on day 3 and inhibited blastocyst formation. The use of LY294002 (10 μM), a specific inhibitor of phosphatidylinositol-3 kinase, also produced a significant decrease in embryo development. In addition, treatment with LiCl and LY294002 produced a significant decrease in the serine phosphorylation of both isoforms of GSK3. Finally, CT99021 and LiCl reduced the phosphorylation of β-catenin on Ser45 in two-cell embryos, while LY294002 increased it. Despite the fact that LiCl inhibited GSK3 activity, as demonstrated by β-catenin phosphorylation, its effects on the bovine embryo could be mediated through other signaling pathways leading finally to a decrease in the phosphorylation of GSK3 and a reduction in embryo development. Therefore, in conclusion, GSK3A/B serine phosphorylation was positively correlated with embryo development, indicating the importance of an accurate regulation of GSK3 activity during developmental stages to achieve normal bovine embryo development.

Temporally differential protein expression of glycolytic and glycogenic enzymes during in vitro preimplantation bovine embryo development

2018 ◽  
Vol 30 (9) ◽  
pp. 1245 ◽  
Author(s):  
Manuel García-Herreros ◽  
Constantine A. Simintiras ◽  
Patrick Lonergan
Keyword(s):  
Embryo Development ◽  
Glucose Transporter ◽  
Developmental Stages ◽  
Glycogen Synthase ◽  
Differentially Expressed ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Glucose Transporter 1 ◽  
Glut 1

Proteomic analyses are useful for understanding the metabolic pathways governing embryo development. This study investigated the presence of enzymes involved in glycolysis and glycogenesis in in vitro-produced bovine embryos at five developmental stages leading up to blastocyst formation. The enzymes examined were: (1) glycolytic: hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase mutase 1/2 (PKM-1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (2) glycogenic: glycogen synthase kinase-3 isoforms α/ β (GSK-3α/β). Glucose transporter-1 (GLUT-1) was also analysed. The developmental stages examined were: (1) 2–4-cell, (2) 5–8-cell, (3) 16-cell, (4) morula and (5) expanded blastocyst. The enzymes HK-I, PFK-1, PKM-1/2, GAPDH and GLUT-1 were differentially expressed throughout all stages (P < 0.05). GSK-3α and β were also differentially expressed from the 2–4-cell to the expanded blastocyst stage (P < 0.05) and GLUT-1 was identified throughout. The general trend was that the abundance of PFK1, GAPDH and PKM-1/2 decreased whereas HK-I, phospho-GSK3α (P-GSK3α) and P-GSK3β levels increased as the embryo advanced. In contrast, GLUT-1 expression peaked at the 16-cell stage. These data combined suggest that in vitro bovine embryo metabolism switches from being glycolytic-centric to glycogenic-centric around the 16-cell stage, the developmental window also characterised by embryonic genome activation.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

scholarly journals 153EXPRESSION OF TGF-BETA I AND TYPE I AND TYPE II OF TGF-BETA RECEPTORS IN BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
B.K. Kim ◽  
H.J. Chung ◽  
B.C. Yang ◽  
D.H. Kim ◽  
J.H. Woo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Type I ◽  
Bovine Embryo ◽  
Type Ii ◽  
Bovine Embryos ◽  
Mrna And Protein Expression ◽  
Bovine Oocytes

Although the effects of TGFβ1, as an important factor in the mice embryo development have been reported, little information relevant to this subject is known in the bovine embryo. The objectives of this study were to investigate the presence and expression patterns of TGFβ1 and TGFβ1 receptors, types I and II, in unfertilized oocytes and fertilized bovine embryos in normal and NT embryo development. We postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and show different expression patterns at different stages of bovine embryo development. Immature bovine oocytes were aspirated from follicles of ovaries obtained from a local abattoir and they were cultured for up to 24h and fertilized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the presence of TGFβ1 and type I and type II of TGFβ1 receptors (the essential components of the TGFβ1 signaling pathway) in unfertilized oocytes and preimplantation embryos. Also, mRNA and protein expression patterns of TGFβ1 and their receptors at various stages of embryos were examined. It was found that both receptors, as well as TGFβ1, were present in the unfertilized bovine oocytes, indicating that TGFβ1 is a maternally expressed protein. Although the type I TGFβ1 receptor was present at the morulae and blastocyst stages, the type II TGFβ1 receptor was not present at both stages. It was also confirmed that the expression level of TGFβ1 was high at the 8-cell stage, and mRNA and protein expression patterns of TGFβ1 and their receptors were not coincident. Interestingly, TGFβ1 protein was not detected at blastocyst stage of embryos, whereas the mRNA expression level was high at this stage. The results of this experiment indicate that TGFβ1 protein may be needed by embryos after the blastocyst stage and may be expressed in hatched embryos for implantation. These findings support the hypothesis that there may be an interaction between the TGFβ1 and TGFβ1 receptors in the unfertilized oocytes and preimplantation embryos, and that TGFβ1 signaling may be important for the development of the oocytes and the preimplantation embryos.

The requirement for protein kinase C delta (PRKCD) during preimplantation bovine embryo development

2016 ◽  
Vol 28 (4) ◽  
pp. 482 ◽  
Author(s):  
Qi-En Yang ◽  
Manabu Ozawa ◽  
Kun Zhang ◽  
Sally E. Johnson ◽  
Alan D. Ealy
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Embryonic Development ◽  
Embryo Development ◽  
Early Embryonic Development ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Kinase C

Protein kinase C (PKC) delta (PRKCD) is a member of the novel PKC subfamily that regulates gene expression in bovine trophoblast cells. Additional functions for PRKCD in early embryonic development in cattle have not been fully explored. The objectives of this study were to describe the expression profile of PRKCD mRNA in bovine embryos and to examine its biological roles during bovine embryo development. Both PRKCD mRNA and protein are present throughout early embryo development and increases in mRNA abundance are evident at morula and blastocyst stages. Phosphorylation patterns are consistent with detection of enzymatically active PRKCD in bovine embryos. Exposure to a pharmacological inhibitor (rottlerin) during early embryonic development prevented development beyond the eight- to 16-cell stage. Treatment at or after the 16-cell stage reduced blastocyst development rates, total blastomere numbers and inner cell mass-to-trophoblast cell ratio. Exposure to the inhibitor also decreased basal interferon tau (IFNT) transcript abundance and abolished fibroblast growth factor-2 induction of IFNT expression. Furthermore, trophoblast adhesion and proliferation was compromised in hatched blastocysts. These observations provide novel insights into PRKCD mRNA expression profiles in bovine embryos and provide evidence for PRKCD-dependent regulation of embryonic development, gene expression and post-hatching events.

scholarly journals 6ACETYLATION OF HISTONE H4 LYSINE-5 AND LYSINE-8 DURING DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 125
Author(s):  
W.E. Maalouf ◽  
R. Alberio ◽  
K.H.S. Campbell
Keyword(s):  
Dna Methylation ◽  
Cell Mass ◽  
Staining Intensity ◽  
Blastocyst Stage ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Histone H4 ◽  
Cell Stage ◽  
Intense Staining

The oocyte is remarkable in its ability to remodel the parental genomes following fertilization and to reprogram somatic nuclei as in nuclear transfer. While significant research has been carried out on DNA methylation patterns in the early embryo, increased interest in histone acetylation is more recent. The objective of this study was to characterize the pattern of acetylation of histone H4 lysine-5 (H4L5) and lysine-8 (H4L8) in the early pre-implantation bovine embryo. Bovine embryos were produced as previously described (Fouladi Nashta AA et al., 1998 Biol. Rep. 59, 255–262) and collected at different developmental stages, 1-cell (20h), 2-cell (30h), 4- and 8-cell (Day 2), 16-cell (Day 4), and blastocyst (Days 7–8) with an average of 6 embryos per group in two replicates. Embryos were fixed in 2.5% paraformaldehyde, 15min at room temperature (RT), stained with polyclonal rabbit antibodies against H4L5 (1:800) and H4L8 (1:600) residues (Serotec, UK) at 4°C overnight. A polyclonal swine anti-rabbit (1:200; Dako, Denmark) was used as secondary antibody for 40min at RT. Images were examined using a fluorescent microscope (Leica DMR, Germany). Image analysis and quantification were performed using Simple PCI software (Compix Imaging Systems, USA). Changes in intensities within and between different embryo stages were recorded as a ratio of red stain to blue counterstain. Data were corrected for confounding area and absorbance and analysed using a multivariate linear regression model. The intensity of staining for H4L5 appeared higher in 8-cell embryos than 2- and 4-cell embryos but not to a significant level (P≥0.05); 8-cell embryos also appeared higher in stain intensity than 16-cell but of borderline significance (P=0.073). Staining intensity decreased between the 8-cell and blastocyst stage (P≤0.05). In contrast, the intensity of acetylation staining for H4L8 residue decreased slightly between the 1- and 4-cell stages and then decreased significantly between the 4- and 8-cell stages (P≤0.05), increasing significantly by the 16-cell stage (P≤0.05). A significant decrease in staining intensity was observed at the blastocyst stage (P≤0.05). In blastocyst-stage embryos both lysine-5 and lysine-8 showed a differential staining of inner cell mass (ICM) and trophectoderm (TE) cells. ICM cells showed intense staining and TE cells stained very weakly. The intensity results presented are cumulative of ICM and TE intensities, which explains the overall low levels of acetylation in blastocysts when compared to the earlier stages. Acetylation of H4L5 starts high in 1-cell embryo, as it is necessary for protamine replacement (Adenot et al., 1997 Development 124, 4615–4625), decreases when methylation is high and increases when methylation is low (as in the 8-cell stage which corresponds with zygotic gene activation). Acetylation of H4L8 decreases between the 1-and 8-cell stages; however, its association with changes in DNA methylation has yet to be determined.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

131 GENE SILENCING IN BOVINE ZYGOTES: SIRNA TRANSFECTION v. MICROINJECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 224 ◽  
Author(s):  
C. M. O'Meara ◽  
J. D. Murray ◽  
J. F. Roche ◽  
S. Mamo ◽  
E. Gallagher ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Embryo Development ◽  
Relative Abundance ◽  
Gene Function ◽  
Developmental Stages ◽  
Analysis Data ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
E Cadherin

Ribonucleic acid interference (RNAi) has become an effective tool for studying gene function in a variety of cells. The objective of this study was to compare the efficiency of gene silencing when siRNA were introduced into bovine zygotes by microinjection (as done previously; Tesfaye D et al. 2007 Mol. Reprod. Dev. 74, 978-988) v. a novel method of transfection in terms of gene knockdown and embryo development. For microin-jection, in vitro-produced bovine zygotes (16 h post insemination) were randomly assigned to 1 of 3 groups over 2 experiments. In Experiment 1, E-cadherin siRNA was injected at 100 μM (n = 168) and compared with PBS-injected (n = 180) and noninjected controls (n = 152). In Experiment 2, E-cadherin siRNA was injected at 375 μM (n = 154) and compared with PBS-injected (n = 136) and noninjected controls (n = 151). Embryos were subsequently cultured in vitro until Day 7 (day of IVF = Day 0). For transfection, the zona pellucida was removed from in vitro-produced zygotes. Zona-free zygotes were randomly assigned to 1 of 4 groups (i) GAPDH (n = 67), (ii) scrambled (n = 66), (iii) E-cadherin (n = 69) siRNA treatments at 100 nM or (iv) nontransfected controls (n = 66). Zygotes were incubated in transfection medium with siRNA for 1 h at 39°C, cultured individually in the well-of-the-well system to Day 7. The proportion of zygotes undergoing cleavage and developing to the blastocyst stage was recorded, and Day 7 embryos were frozen individually for mRNA analysis. Data for mRNA expression were fitted to a general linear model, and developmental stages were tested using ANOVA. Microinjection of 100 μM E-cadherin siRNA had no effect on phenotype (P > 0.05). Injection of PBS or 375 μM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5%, 45.5%, and 62.9%, respectively) and blastocyst stage (39.0%, 32.5%, and 45%, respectively) compared with noninjected controls (P < 0.05). The mRNA abundance of the target gene was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 μM and 375 μM compared with control and PBS-injected groups (P < 0.05). Transfection with E-cadherin siRNA decreased development of 8-cell embryos (20.3 v. 53.0%, respectively) and blastocysts (7.2 v. 18.2%, respectively) compared with controls (P < 0.05). The mRNA relative abundance was not different between controls (nontransfected, or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100nM E-cadherin siRNA led to a 70% reduction in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05). Zona removal and transfection resulted in decreased embryo development compared with microinjection (P < 0.05). However, transfection yielded more efficient gene silencing of E-cadherin mRNA with reduced embryo development compared with microinjection. This technique of gene silencing could improve the efficiency of gene function studies in early bovine embryogenesis. Supported by Science Foundation Ireland.

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

120 EMBRYO SURVIVAL FOLLOWING LIPID-BASED TRANSFECTION OF 1-CELL STAGE BOVINE EMBRYOS WITH SMALL INTERFERING RNA (siRNA) FRAGMENTS AND/OR DNA

2006 ◽  
Vol 18 (2) ◽  
pp. 168 ◽  
Author(s):  
M. Bertolini ◽  
L. R. Bertolini ◽  
S. G. Petkov ◽  
K. R. Madden ◽  
J. D. Murray ◽  
...  
Keyword(s):  
Embryo Development ◽  
Small Interfering Rna ◽  
Blastocyst Stage ◽  
Qualitative Assessment ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryo Survival ◽  
Interfering Rna ◽  
Higher Doses

The RNA interference (RNAi) technology is a powerful tool for studies in functional genomics. The aim of this study was to evaluate the effects of a cationic lipid-based small interfering RNA (siRNA) and/or DNA delivery to 1-cell-stage bovine embryos on survival to the blastocyst stage. In vitro-produced (IVP) embryos were generated according to Bertolini et al. 2002 (Theriogenology 58, 973), and cloned embryos were produced by the handmade cloning technique (Vajta et al. 2003 Biol. Reprod. 68, 571) using green fluorescent protein (GFP)-expressing fibroblast cells as nuclear donors. Lipofections were performed on zona-free 1-cell-stage IVP embryos at 24–28 h post-fertilization by exposure to 1% (v/v) Lipofectamine 2000 (Invitrogen Co., CA, USA), 0.002% (w/v) GFP plasmid (pEFGP-N1, Clontech Laboratories, CA, USA) and/or various doses of siRNA GFP-specific siRNA oligonucleotide (Invitrogen) or DNA methyltransferase 1 (Dnmt1)-specific siRNA fragments for 60 min at 39°C, according to 5 treatment groups: (1) zona-intact IVP embryos (controls), (2) zona-free control embryos (controls for embryo development after zona removal), (3) embryos treated with GFP + GFP-siRNA at 0, 50, 100, 200, 400, or 800 nm, (4) embryos treated with Dnmt1-siRNA at 0, 50, 100, 250, or 500 nm, and (5) cloned embryos (positive controls for GFP expression). After treatment, embryos were in vitro-cultured in a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256) for 7 days. Cleavage and developmental rates to at least 8-cell and to blastocyst stages were assessed at 48, 96, and 168 h post-fertilization (hpf), respectively. Data were analyzed by the chi-square test. Cleavage rates in embryos treated with higher doses of siRNA were lower than in all other groups (Table 1). Embryo survival to at least 8-cell stage at 48 h, based on cleavage, was similar among all treatments (data not shown), but survival to blastocyst stage was affected by higher doses of GFP- or Dnmt1-siRNA (Table 1). After a qualitative assessment by fluorescence microscopy at 168 hpf, 40 to 63% of GFP-transfected blastocysts showed various levels of fluorescence, irrespective of the siRNA treatments. Fragments of siRNA are known to be short-lived in cultured cells, although we are still uncertain of their behavior and effects in early bovine embryos. We are currently analyzing the effectiveness of the siRNA transfection in the early IVP and clone embryo. In conclusion, liposome transfection of 1-cell-stage embryos did not affect survival and development to the blastocyst stage. However, survival followed an siRNA dose-response effect, with doses higher than 400 nm appearing to be detrimental to embryo development, with a developmental arrest at or close to the embryonic genome activation period. Table 1. Developmental rate of bovine embryos following lipid-based transfection at the 1-cell-stage

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