Microinjection of Bovine Embryos with a Foreign Gene and Its Detection at the Blastocyst Stage

Metabolic and molecular profiles were reported as different for bovine embryos with distinct kinetics during the first cleavages. In this study, we used this same developmental model (fast vs slow) to determine if the relationship between metabolism and developmental kinetics affects the levels of acetylation or tri-methylation at histone H3 lysine 9 (H3K9ac and H3K9me3, respectively). Fast and slow developing embryos presented different levels of H3K9ac and H3K9me3 from the earliest stages of development (40 and 96 hpi) and up to the blastocyst stage. For H3K9me3, both groups of embryos presented a wave of demethylation and de novo methylation, although it was more pronounced in fast than slow embryos, resulting in blastocysts with higher levels of this mark. The H3K9ac reprogramming profile was distinct between kinetics groups. While slow embryos presented a wave of deacetylation, followed by an increase in this mark at the blastocyst stage, fast embryos reduced this mark throughout all the developmental stages studied. H3K9me3 differences corresponded to writer and eraser transcript levels, while H3K9ac patterns were explained by metabolism-related gene expression. To verify if metabolic differences could alter levels of H3K9ac, embryos were cultured with sodium-iodoacetate (IA) or dichloroacetate (DCA) to disrupt the glycolytic pathway or increase acetyl-CoA production, respectively. IA reduced H3K9ac while DCA increased H3K9ac in blastocysts. Concluding, H3K9me3 and H3K9ac patterns differ between embryos with different kinetics, the second one explained by metabolic pathways involved in acetyl-CoA production. So far, this is the first study demonstrating a relationship between metabolic differences and histone post-translational modifications in bovine embryos.

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

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab136 ◽

2005 ◽

Vol 17 (2) ◽

pp. 219 ◽

Cited By ~ 5

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.

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Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage

Theriogenology ◽

10.1016/0093-691x(94)90682-9 ◽

1994 ◽

Vol 42 (3) ◽

pp. 445-453 ◽

Cited By ~ 47

Author(s):

L. Ferry ◽

P. Mermillod ◽

A. Massip ◽

F. Dessy

Keyword(s):

Conditioned Medium ◽

Blastocyst Stage ◽

Bovine Embryos

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

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab153 ◽

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.

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78 NONINVASIVE CELL LINEAGE TRACING IN BOVINE EMBRYOS FROM 2-CELL STAGE UP TO BLASTOCYST STAGE

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab78 ◽

2015 ◽

Vol 27 (1) ◽

pp. 132

Author(s):

L. P. Sepulveda-Rincon ◽

D. Dube ◽

P. Adenot ◽

L. Laffont ◽

S. Ruffini ◽

...

Keyword(s):

Cell Mass ◽

Cell Lineage ◽

Blastocyst Stage ◽

Lineage Tracing ◽

Specific Cell ◽

Bovine Embryos ◽

Cell Stage ◽

Daughter Cells ◽

Significant Difference ◽

Allocation Patterns

The first lineage specification occurs during pre-implantation mammalian development. At the blastocyst stage, 2 cell lineages can be distinguished: the inner cell mass (ICM) and the trophectoderm (TE). The exact timing when embryo cells are skewed to these lineages is not clearly determined in mammalian species. In murine embryos, it has been suggested that the first cleavage plane might be related to the embryonic-abembryonic (Em-Ab) axis at blastocyst stage. Thus, the daughter cells of the 2-cell embryo might already be predisposed to a specific cell lineage further on development. The objective of the present study was to observe how the first cleavage in bovine embryos may be related to cell lineage allocation at the blastocyst stage, using a noninvasive tracing approach. Bovine oocytes were harvested, in vitro matured, and fertilised. At the 2-cell stage, embryos were injected in one blastomere with the membrane tracer DiI. At the blastocyst stage, embryos (n = 346) were classified as orthogonal when the Em-Ab axis was orthogonally divided by the borderline between labelled and non-labelled cells; as deviant if the borderline was overlapping the Em-Ab axis; and as random when the labelled and non-labelled cells were randomly distributed. Total cell count (TCC) and the ICM/TE ratio was allowed by DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunostaining of the ICM with Sox2 antibody. Analysis of variance was performed by one-way ANOVA employing IBM SPSS v21 (SPSS Inc., Chicago, IL, USA) to determine any difference between the cell lineage allocation patterns, TCC, and the ICM/TE ratio. P-values = 0.05 were considered significant. All values are reported as mean ± standard error of mean. Within 40 repetitions, the blastocyst classification was as follows: orthogonal 14.9% (±2.32, n = 56), deviant 22.2% (±2.58, n = 80), and random 62.9% (±2.64, n = 210). A significant difference was found in the incidence between the random group against the orthogonal and deviant, but not between the latter two. Regarding TCC, a significant difference was observed only between the orthogonal (99.6 ± 11.7 cells, n = 15) and deviant (135 ± 7.3 cells, n = 25) groups, but not with random embryos (116 ± 5.5 cells, n = 42). Finally, no significant difference was found among the groups concerning the ICM/TE ratio (0.43 ± 0.07 for orthogonal, n = 7; 0.54 ± 0.06 for deviant, n = 14; and 0.40 ± 0.03 for random embryos, n = 26). In conclusion, bovine embryos present a marked tendency for a random distribution of the daughter cells derived from the 2-cell blastomeres. However, around 37% of the blastocysts present a patterned cell division, where the daughter cells remain together through pre-implantation development. The effect of these cell lineage allocation patterns on implantation and further embryo development needs to be addressed.The authors acknowledge Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73) and CONACyT Mexico for funding.

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Expression of a Foreign Gene Introduced Into Bovine Embryos at Early Stages of Development

Animal Cell Technology: Basic & Applied Aspects ◽

10.1007/978-94-011-0848-5_38 ◽

1994 ◽

pp. 255-258

Author(s):

T. Muramatsu ◽

A. Nakamura ◽

J. Okumura

Keyword(s):

Foreign Gene ◽

Bovine Embryos ◽

Stages Of Development ◽

Early Stages

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MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

International Journal of Molecular Sciences ◽

10.3390/ijms21238888 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8888

Author(s):

Bárbara Melo-Baez ◽

Yat S. Wong ◽

Constanza J. Aguilera ◽

Joel Cabezas ◽

Ana C. F. Mançanares ◽

...

Keyword(s):

Extracellular Vesicles ◽

Fatty Acid Biosynthesis ◽

Culture Media ◽

Blastocyst Stage ◽

Developmental Competence ◽

Target Cells ◽

Bovine Embryos ◽

Developmental Potential ◽

Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

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In vitro culture and non-invasive metabolic profiling of single bovine embryos

Reproduction Fertility and Development ◽

10.1071/rd17446 ◽

2019 ◽

Vol 31 (2) ◽

pp. 306

Author(s):

Monika Nõmm ◽

Rando Porosk ◽

Pille Pärn ◽

Kalle Kilk ◽

Ursel Soomets ◽

...

Keyword(s):

Molecular Weight ◽

Metabolic Profiling ◽

Culture Medium ◽

Culture Media ◽

Blastocyst Stage ◽

Growth Media ◽

Low Molecular Weight ◽

Bovine Embryos ◽

Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

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190 SUPPRESSION OF A TRANSCRIPTION FACTOR MSX1 GENE IN BOVINE PREIMPLANTATION EMBRYOS AND ITS EFFECT ON mRNA, PROTEIN, AND EXPRESSION OF DOWNSTREAM GENES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab190 ◽

2008 ◽

Vol 20 (1) ◽

pp. 174

Author(s):

D. Tesfaye ◽

A. Regassa ◽

M. Hoelker ◽

F. Rings ◽

C. Phatsara ◽

...

Keyword(s):

Transcription Factor ◽

Cell Growth ◽

Protein Expression ◽

Cdna Array ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Bovine Embryos ◽

Expression Levels ◽

Treatment Groups ◽

Zygote Stage

MSX1 is a transcription factor gene that orchestrates gene expression and regulates cell growth, proliferation, differentiation, cell-to-cell communication, and the apoptotic pathway during pattern formation in vertebrate embryogenesis. However, its role in bovine preimplantation embryo is not known. Here we aim to investigate the effects of suppressing MSX1 transcript on the development of in vitro-produced bovine embryos, study the expression of mRNA and protein products of the gene, and identify downstream genes using microarray analysis. In the first experiment, IVP zygotes were injected with 341 bp-long dsRNA (LdsRNA) (n = 384), 19 bp small interfering RNA (siRNA) (n = 374), and scrambled sequence RNA (scRNA) (n = 388). Uninjected zygotes (n = 313) were used as control. Developmental phenotype data were collected during culture until Day 8. The mRNA and protein expression levels of the different treatment groups were validated at the 8-cell and blastocyst stages using quantitative real-time PCR and immunohistochemistry, respectively. Developmental phenotype and mRNA data were analyzed using ANOVA under statistical package SPSS (SPSS, Inc., Chicago, IL, USA). In the second experiment, custom SMARTpool siRNA (Dharmacon Inc., Chicago, IL, USA) targeting bovine MSX1 (NM_174798) was used for microinjection together with siRNA and uninjected control. Following treatment at zygote stage, 8-cell embryos were used for mRNA isolation and subsequent array hybridization using bovine cDNA array containing 2000 clones. Array data analysis was performed using statistical analysis of microarray (SAM) procedure. While 33% and 29% of the zygotes from the control and scRNA treatment groups, respectively, reached blastocyst stage, only 20% and 19% of the zygotes from the LdsRNA and siRNA treatment groups, respectively, reached the same stage. Injection of LdsRNA and siRNA at the zygote stage reduced the mRNA expression level by 52% and 33% at the 8-cell stage and by 77% and 87%, respectively, at the blastocyst stage as compared to the control. Similarly, cellular protein expression levels in LdsRNA- and siRNA-injected treatment groups were found to be lower than the control groups at each stage. In all cases, injection of scRNA had no effect on mRNA and protein levels. SAM analysis revealed that, of the total 2000 clones, 3.5% and 5.4% were found to be differentially expressed in embryos injected with SMARTpool and siRNA, respectively, compared to the control. Genes involved in various activities including transcription factors (ALF), cell growth (BMP-15), metabolism (RIOK3), and cytokinasis (AURKA) were found to be down-regulated in 8-cell embryos treated with SMARTpool siRNA compared to the controls. On the other hand, genes involved in protein synthesis (RPL23), energy metabolism (COQ1), cell growth (MNS1) and skeletal development (LGALS3) were found to be upregulated in the same samples. In conclusion, suppression of MSX1 at the mRNA and protein level significantly affected the development of bovine embryos, and our study revealed list of downstream genes regulated by the activity of MSX1.

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133 CRISPR/Cas9 gene editing of invivo-fertilized bovine embryos via endoscopic oviductal flushing and electroporation of zygotes

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab133 ◽

2020 ◽

Vol 32 (2) ◽

pp. 193

Author(s):

D. Miskel ◽

L. Beunink ◽

M. Poirier ◽

V. Havlicek ◽

F. Rings ◽

...

Keyword(s):

Bovine Serum ◽

Gene Editing ◽

Statistical Significance ◽

Blastocyst Stage ◽

Culture Conditions ◽

Bovine Embryos ◽

Embryo Survival ◽

Guide Rna ◽

Endoscopic Method ◽

Developmental Rates

In recent years, CRISPR/Cas9 has been used to efficiently edit the genomes of embryos in many animal models. Due to smaller anatomy, lower costs, and multiple ovulations, it is relatively simple to derive large numbers of invivo fertilized zygotes for gene editing experiments in small mammal models. In cattle, however, harvesting invivo fertilized zygotes generally requires a highly invasive surgical procedure. Here, we use the combination of a minimally invasive endoscopic method for harvesting invivo fertilized zygotes by oviductal flushing of superovulated heifers and the subsequent electroporation of zygotes with CRISPR/Cas9 ribonucleoproteins (RNP). After superstimulation of 21 heifers, on average 12 zygotes were flushed per animal with fetal bovine serum, then stored in synthetic oviductal fluid (SOFaa) before electroporation. Targeting exon 1 of the tyrosinase (Tyr) gene, zygotes were electroporated in 1-mm gap cuvettes (Biorad) in groups of ~20 in 20μL of OptiMEM media containing 3μM Cas9 RNP (IDT Cas9 protein pre-incubated with anti-Tyr guide RNA). Electroporation was performed in 3 replicates of 3 electrical potentials, namely 20, 25, and 30V using a Biojet CF 50. The other electroporation parameters were fixed at 5 repetitions of 2-ms square wave pulses at 100-ms intervals. The zygotes were than cultured under standard embryo culture conditions (SOFaa + 0.3% bovine serum albumin, 5% CO2, 5% O2, 39°C, humidified air). Embryo survival, cleavage, and developmental rates to the blastocyst stage were tracked. Statistical significance between groups was determined by pairwise one-way ANOVA using Sidak correction for multiple comparisons. Electroporation of invivo-derived zygotes using 20V yielded significantly higher survival (83.6% vs. 42.8% vs. 20.7% for 20, 25, and 30V, respectively), cleavage (65.6% vs. 37.9% vs. 40.0%), and developmental rates (47.5% vs. 21.4% vs. 16.5%) than 25 or 30V. There was no statistical difference between 25 and 30V. Subsequently, editing rates were determined using the T7 mismatch assay and verified with Sanger sequencing followed by sequence alignment and analysis using Tracking of Indels by Decomposition (TIDE) software (https://tide.nki.nl/). Although there was high variance between electroporation groups, blastocyst editing rates of up to 80.0% were achieved using 30V. To our knowledge, these are the first confirmed gene-edited bovine embryos produced from invivo fertilized zygotes. This method offers the ability to utilise the embryos of high-value cows or cows with known genotypes for genetic engineering experiments. In addition, given that electroporated bovine zygotes can be transferred back to the oviduct endoscopically, our future attempts will focus on genome editing in bovine embryos developed nearly completely within the physiological invivo environment.

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