scholarly journals Adapting CRISPR/Cas9 Lentivirus Technology for Functional Studies of GATA6 & GATA4 during Bovine Preimplantation Embryogenesis

2021 ◽  
Author(s):  
◽  
Shicheng Ni
Keyword(s):  
Regulatory Networks ◽  
Preimplantation Embryo ◽  
Cumulus Cells ◽  
Transcriptional Regulators ◽  
Genetic Regulatory Networks ◽  
Molecular Regulation ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Knock Out ◽  
Functional Studies

<p>In recent times, cattle embryology has been under the spotlight of investigation due to its apparent economic values. This is especially relevant in the case of New Zealand, owing to its high percentage of livestock export. Specifically, the period of peri-implantation development has been of particular relevance. During this stage, the developing zygote will establish 3 key lineages – epiblast, hypoblast and trophoblast. Previous studies have elucidated that a significant number of embryos die prior to implantation, therefore highlighting the importance of correctly establishing these 3 lineages to overall embryonic survival. However, while embryological stages of the preimplantation embryo have been extensively studied in their eutherian cousin, mice, the molecular regulation of that of cattle remains much less addressed. Whereas the regulation of bovine embryo development is orchestrated by many transcriptional regulators, or genetic regulatory networks (GNP), we aimed to focus our studies on 2 key transcriptional regulators, GATA4 and GATA6. During early embryogenesis, both these transcriptional factors are known molecular regulators that drive the establishment of the hypoblast lineage in mice. By and large, while their respective expression has been documented in cattle embryos, functional studies towards these markers have not yet been performed. Latest advances in molecular biology have given us novel methods to study the mechanism of bovine embryogenesis. To this end, the continuing perfection of CRISPR technologies in the last decade - in particular its delivery through lentiviral vectors, has established an ability to generate stable, targeted knock-out mutants. Therefore, it is aimed in this thesis to design and test lentiviral particles that induce knock-out mutants of GATA4 and GATAT6, to test their efficacy in primary cell cultures (bovine cumulus cells) and to functionally analyse the effect of GATA4 and GATA6 knockdowns in early bovine embryos.</p>

scholarly journals Adapting CRISPR/Cas9 Lentivirus Technology for Functional Studies of GATA6 & GATA4 during Bovine Preimplantation Embryogenesis

2021 ◽  
Author(s):  
◽  
Shicheng Ni
Keyword(s):  
Regulatory Networks ◽  
Preimplantation Embryo ◽  
Cumulus Cells ◽  
Transcriptional Regulators ◽  
Genetic Regulatory Networks ◽  
Molecular Regulation ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Knock Out ◽  
Functional Studies

<p>In recent times, cattle embryology has been under the spotlight of investigation due to its apparent economic values. This is especially relevant in the case of New Zealand, owing to its high percentage of livestock export. Specifically, the period of peri-implantation development has been of particular relevance. During this stage, the developing zygote will establish 3 key lineages – epiblast, hypoblast and trophoblast. Previous studies have elucidated that a significant number of embryos die prior to implantation, therefore highlighting the importance of correctly establishing these 3 lineages to overall embryonic survival. However, while embryological stages of the preimplantation embryo have been extensively studied in their eutherian cousin, mice, the molecular regulation of that of cattle remains much less addressed. Whereas the regulation of bovine embryo development is orchestrated by many transcriptional regulators, or genetic regulatory networks (GNP), we aimed to focus our studies on 2 key transcriptional regulators, GATA4 and GATA6. During early embryogenesis, both these transcriptional factors are known molecular regulators that drive the establishment of the hypoblast lineage in mice. By and large, while their respective expression has been documented in cattle embryos, functional studies towards these markers have not yet been performed. Latest advances in molecular biology have given us novel methods to study the mechanism of bovine embryogenesis. To this end, the continuing perfection of CRISPR technologies in the last decade - in particular its delivery through lentiviral vectors, has established an ability to generate stable, targeted knock-out mutants. Therefore, it is aimed in this thesis to design and test lentiviral particles that induce knock-out mutants of GATA4 and GATAT6, to test their efficacy in primary cell cultures (bovine cumulus cells) and to functionally analyse the effect of GATA4 and GATA6 knockdowns in early bovine embryos.</p>

95 ROLE OF POLYAMINES IN BOVINE PRE-IMPLANTATION DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 177
Author(s):  
J. Herrick ◽  
A. Greene ◽  
W. Schoolcraft ◽  
R. Krisher
Keyword(s):  
Amino Acids ◽  
Inner Cell Mass ◽  
Gradient Centrifugation ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Human Albumin ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation

Polyamines are involved in trophectoderm attachment and outgrowth, but little is known about their role in earlier stages of development. The objective of this study was to evaluate the effects of an inhibitor of polyamine synthesis (difluoromethylornithine, DFMO) on development (blastocyst formation and hatching) and cell allocation to the trophectoderm (TE, CDX2-positive) and inner cell mass (ICM, SOX2-positive) in the bovine embryo. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries and matured for 24 h in a defined maturation medium (5.0 mM glucose, 0.6 mM cysteine, 0.5 mM cysteamine, 0.1 IU mL–1 FSH, 50 ng mL–1 EGF, and 2.5 mg mL–1 recombinant human albumin). Frozen-thawed spermatozoa were processed by gradient centrifugation and co-incubated (2 × 106 mL–1) with COC [10 COC/50 µL; 7.5 µg mL–1 heparin, 2 mM caffeine, and 8.0 mg mL–1 fatty-acid free (FAF) BSA] for 20 to 22 h. After removing cumulus cells, zygotes were cultured (10 embryos/20 µL) in a medium for cleavage stage bovine embryos (0.5 mM glucose, 0.3 mM pyruvate, 6.0 mM lactate, 0.25 mM citrate, 1.0 mM alanyl-glutamine, 0.25 × MEM nonessential and essential amino acids, 5 µM EDTA, and 8.0 mg mL–1 FAF BSA). After 72 h, embryos with >4 cells were randomly allocated (5 embryos/20 µL) to a culture medium for compaction and blastocyst formation (3.0 mM fructose, 0.1 mM pyruvate, 6.0 mM lactate, 0.5 mM citrate, 1.0 mM alanyl-glutamine, 1× MEM nonessential amino acids, 0.5× MEM essential amino acids, 0.075 mM myo-inositol, and 8.0 mg mL–1 FAF BSA) containing 0 (control), 5, or 10 mM DFMO. Embryonic development was evaluated at 192 h post-insemination (96 h in the second medium containing DFMO treatments), and hatching or hatched blastocysts were fixed for analysis of cell allocation. All data were analysed by ANOVA and P < 0.05 was considered significant. Blastocyst formation and hatching (% of embryos cultured in the presence of treatments) were both inhibited (P < 0.05) when embryos (n = 157/treatment) were cultured with 5 (39.5 ± 3.9%, 14.6 ± 2.8%) or 10 (39.5 ± 3.9%, 14.0 ± 2.8%) mM DFMO compared with embryos cultured without DFMO (53.5 ± 4.0%, 26.1 ± 3.5%). The number of TE cells was also reduced (P < 0.05) in the presence of 5 (121.4 ± 7.2) and 10 (123.6 ± 6.7) mM DFMO compared with embryos cultured without DFMO (152.4 ± 9.7), but the number of ICM cells (45.2 to 54.0) and the total number of cells (TE+ICM, 168.8 to 201.1) were not affected (P > 0.05). In a second experiment (n = 163 to 165/treatment), the negative effects of DFMO on hatching (17.0 ± 2.9%; P < 0.05, v. control, 30.7 ± 3.6%) could be partially reversed when embryos were cultured with both 10 mM DFMO and an exogenous polyamine (100 µM putrescine, 23.0 ± 3.3% DFMO+Put; P > 0.05 v. control). The number of TE cells for embryos cultured with DFMO+Put (153.9 ± 8.7) was intermediate between embryos cultured with (138.0 ± 6.9) or without DFMO (control, 161.6 ± 8.7), but these differences were not significant (P > 0.05). These results provide the first evidence of a role for polyamines during blastocyst formation and hatching of bovine embryos, with specific effects on trophectoderm proliferation and hatching.

scholarly journals 127 LETHAL EFFECT OF HIGH CONCENTRATIONS OF PARECOXIB AND FLUNIXIN MEGLUMINE ON THE IN VITRO CULTURE OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 163 ◽  
Author(s):  
E. M. Razza ◽  
R. A. Satrapa ◽  
C. F. Silva ◽  
R. A. L. Simões ◽  
T. Nabhan ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Lethal Effect ◽  
Control Group ◽  
Published Data ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Flunixin Meglumine ◽  
Plastic Bags ◽  
Drug Concentrations

The aim of this experiment was to evaluate the effects of cycloxigenase inhibitor drugs, i.e. flunixin meglumine (FM) and parecoxib (P), on the development of bovine embryos cultured in vitro until the blastocyst and hatched blastocyst stages. Immature oocytes were aspirated from slaughterhouse ovaries and morphologically selected for IVM (Monteiro FM et al. 2007 Anim. Reprod. 4, 51–58). Twenty hours after maturation (39°C and 5% CO2 in air), matured oocytes were transferred to fertilization media, inseminated with frozen–thawed semen, and incubated for 10 to 12 h. Presumptive zygotes (PZ) were then transferred to TCM 199 HEPES medium, vortexed to remove cumulus cells and finally to drops of IVC media (SOFaaci plus 5% BFS [Gibco] with 13 mm sodium pyruvate). Each drop of IVC medium had appropriate concentrations of FM (0.14/n = 123; 1.4/n = 122; 14/n = 117; 140/n = 44 or 1400 μg mL–1/n = 44 PZ) or P (0.09/n = 134; 0.9/n = 109; 9/n = 118; 90/n = 113 or 900 μg mL–1/n = 45 PZ), besides extra drops as control groups (CFM; n = 124 and CP; n = 149 PZ). Based on published data from bovine (FM) and human (P) administered concentrations, it was calculated the blood concentration to a bovine weighing 450 kg (FM = 14 and P = 9 μg mL–1). Both drugs were used from available commercial preparations, and in a pilot test, there were no deleterious effects of the solvent itself on the blastocyst and hatched blastocyst rates. During culture, petri dishes containing PZ/embryos were kept into plastic bags, under controlled atmosphere of 5% O2, 5% CO2 and 90% N2 at 39°C. There were 11 replicates for each treatment. In all drops (both drug concentrations and control group) the blastocyst and hatched blastocyst rates (BR and HBR, respectively) were evaluated at 144 and 192 h after fertilization, respectively. Statistical analysis was performed with ANOVA on ranks (Dunn’s test a posteriori and significance being considered when P < 0.05; BioEstat version 5.0). According to the results, FM (1400 and 140 μg mL–1) and P (900 μg mL–1) concentrations were toxic enough for a complete inhibition of in vitro bovine embryo development. There were no significant differences among the other drug concentrations and their respective control group, on the BR (27.7 ± 3.9; 29.6 ± 3.4 and 29.8% ± 4.8) and HBR (13.5 ± 4.4; 15.6 ± 3.8 and 22.1% ± 5.1), respectively to 0.14; 1.4 and 14 μg mL–1 for FM; on BR (26.0 ± 2.6; 18.2 ± 4.6; 25.8 ± 5.9 and 23.2% ± 4.8) and HBR (14.1 ± 3.3; 10.2 ± 3.3; 16.8 ± 3.8 and 12.0% ± 3.4), respectively to 0.09; 0.9; 9 and 90 μg mL–1 for P; and on BR (35.3 ± 5.2 and 36.5% ± 3.4) and HBR (26.6 ± 4.5 and 19.8% ± 3.6), respectively for CFM and CP. The results suggest that, during in vitro bovine embryo culture, there was no significant toxicity of either drug, with exception of the complete lethal concentrations of 140 and 1400 μg mL–1 (flunixin meglumine) and 900 μg mL–1 (parecoxib) on blastocyst production. Supported by FAPESP – Brazil (MFGN 06/06491-2 and 07/07705-9; EMR 07/04284-2; RAS; CFS and RALS) and CAPES – Brazil.

Perifusion culture system for bovine embryos: improvement of embryo development by use of bovine oviduct epithelial cells, an antioxidant and polyvinyl alcohol

1997 ◽  
Vol 9 (4) ◽  
pp. 411 ◽  
Author(s):  
J. M. Lim ◽  
B. C. Reggio ◽  
R. A. Godke ◽  
W. Hansel
Keyword(s):  
Epithelial Cells ◽  
Polyvinyl Alcohol ◽  
Embryo Development ◽  
Culture Medium ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Static System ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Oviduct Epithelial Cells

Three experiments were conducted in an attempt to improve a continuous flow-perifusion system capable of maintaining embryo development for long periods of time. Bovine embryos (8–16 cells) obtained from static co-culture with cumulus cells in a serum-free medium were perifused in an ACUSYST-S cell culture incubator. Culture chambers of the incubator consisted of a 0·2-mL unit (Chamber 1) connected to a 1·5-mL-1 unit (Chamber 2), with the outflow from Chamber 1 routed to the inlet to Chamber 2. A bovine embryo culture medium supplemented with 3 mg mL 1 bovine serum albumin (BSA) and 25 mM HEPES was used as a perifusion culture medium (PCM). Embryos were perifused in Chamber 2 for 24, 48 and 72 h and further co-cultured in a static system up to 216 h after insemination. In Experiment 1, conditioning PCM with frozen–thawed bovine oviduct epithelial cells (BOEC) placed in Chamber 1 enhanced (P < 0·05) blastocyst formation of embryos in Chamber 2, after 24, 48 and 72 h of perifusion culture. The proportion of blastocysts was not further increased by placing BOEC in Chamber 2 along with the embryos. In Experiment 2, embryos were perifused with PCM conditioned with BOEC in Chamber 1 for 48 h or 72 h. A higher proportion of perifused embryos developed to the blastocyst stage after addition of 25 U mL-1 or 50 U mL-1 of superoxide dismutase (SOD) to PCM than in its absence. However, blastocyst formation of embryos perifused for 72 h was not increased after addition of 50 U mL-1 SOD compared with its absence. In Experiment 3, the proportions of morulae and blastocysts were not decreased by replacement of 3 mg mL-1 BSA with 1 mg mL-1 polyvinyl alcohol (PVA) in a BOEC-conditioned medium containing 50 U mL-1 SOD after perifusion for 48 h. In conclusion, PCM conditioning with BOEC and addition of an antioxidant to the perifusion medium improved the developmental capacity of perifused embryos. PVA is an adequate replacement for BSA in the perifusion medium.

140 THE EFFECTS OF INTERVALS OF MECHANICAL VIBRATION DURING IN VITRO CULTURE ON THE DEVELOPMENT OF BOVINE EMBRYO DERIVED FROM LOW-QUALITY OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 183
Author(s):  
M. Takehisa ◽  
S. Kondo ◽  
K. Imai ◽  
O. Dochi ◽  
H. Koyama
Keyword(s):  
In Vitro Culture ◽  
Vibration Control ◽  
Mechanical Vibration ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate

Mechanical vibration enhances the cytoplyasmic maturation of in vitro-matured (IVM) pig oocytes (Mizobe et al. 2010 J. Reprod. Dev. 56, 285–290), as well as the development of in vitro-cultured (IVC) bovine embryos (Fujita et al. 2010 Rakuno Gakuen University Graduation thesis,1–36). In this study, the effects of intervals of mechanical vibration during in vitro culture, after IVF, on the development of embryos derived from low-quality oocytes were examined. Cumulus-oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter = 2 to 6 mm) obtained from a local abattoir. In this experiment, only grade 3 oocytes (i.e. those with one layer or partially remaining cumulus cells and normal cytoplasm) were used. Groups of 20 COC were matured in 100-μL droplets of in vitro TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. Matured COC were inseminated with 5 × 106 sperms mL–1 for 18 h. After 18 h of gamete co-culture, the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). Presumptive zygotes were cultured in vitro without mechanical vibration (control; n = 467) and with mechanical vibration for 5 s at 5 min (n = 180), 10 min (n = 180), 15 min (n = 180), and 60 min (n = 200) for 9 days. Embryo development was evaluated for cleavage and blastocyst rates, on Days 3 and 7 to 9 after IVF, respectively. The cleavage and blastocyst formation rates were analysed by the chi-squared test. Vibration at 15-min intervals increased (P < 0.05) cleavage rate compared to 5 min, 60 min, and control (control: 66.2 ± 22.1%; 5 min: 49.4 ± 10.2%; 10 min: 70.0 ± 7.7%; 15 min: 86.2 ± 6.6%; and 60 min: 64.0 ± 8.5%).The highest (P < 0.05) blastocyst rate among the experimental groups was found with 15-min intervals for vibration (control: 21.6 ± 9.2%; 5 min: 15.0 ± 5.3%; 10 min: 22.8 ± 1.8%; 60 min: 21.5 ± 5.0%). These results indicated that the cleavage and blastocyst formation rates of IVM-IVF-IVC bovine embryos derived from low-quality oocytes can be improved by physical stimulus during IVC. In addition, it was shown that 15-min intervals of mechanical vibration elicited the highest benefit for the development of embryos.

226 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING CHEMICAL PACKETS THAT REGULATE CO2 AND O2

2015 ◽  
Vol 27 (1) ◽  
pp. 202
Author(s):  
K. Saeki ◽  
Y. Fujiki
Keyword(s):  
Gas Phase ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Catalog Number ◽  
Co2 Level ◽  
Vitro Production

Bovine embryos are now routinely produced with oocytes collected from slaughterhouse ovaries or by transvaginal ovum pickup. The oocytes are matured, fertilized, and cultured in a water-jacketed CO2/O2 incubator. Gas phase in incubators is usually maintained at 5% CO2 in air for in vitro maturation (IVM) and IVF of oocytes and at 5% CO2, 5% O2, and 90% N2 for in vitro culture (IVC) of embryos. Here we investigated whether two chemical packets that regulate CO2 and O2 for culturing bacteria (Mitsubishi Gas Chemical, Tokyo, Japan) could be used to control the gas phase in vitro production (IVP) of cattle embryos. One packet (Anaero Pack-CO2) was maintained at a CO2 level of ~5% in a 2.5-L container and the other (Anaero Pack-MicroAnaero) was maintained at a CO2 level of 5–8% and an O2 level of 6 to 12%. Bovine cumulus-oocyte complexes (COC, n = 970) were collected from slaughterhouse ovaries, matured in HEPES-buffered TCM-199 (catalog number 12340–030, Invitrogen) supplemented with 10% FCS, 0.02 Armour unit mL–1 FSH and 1 µg mL–1 E2 for 22 h, and fertilized in medium IVF100 [Research Institute for the Functional Peptides Co. Ltd. (IFP), Yamagata, Japan] with frozen-thawed sperm (4 × 106 cells mL–1) for 6 h. Sperm and cumulus cells were removed from the oocytes. The denuded oocytes were cultured in IVD101 (IFP, 20 to 30 embryos/50 μL) for 8 days (Day 0 = IVF). Culture was carried out at 39°C with maximum humidity. Five different combinations of gas conditions were used for incubation (Table 1). Experiments were repeated 3 times. Cleavage and blastocyst rates were assessed on Day 8. Data were analysed by ANOVA followed by Fisher's PLSD test. In the five conditions, rates of matured oocytes (oocytes at MII, n = 210) were 70 to 73% and rates of normal fertilized oocytes (oocytes with 2 pronuclei, n = 310) were 67 to 75%. Cleavage rates of embryos after 8 days of culture (n = 450) were 68 to 75%, and rates of blastocysts from cleaved embryos were 25 to 40%. None of the above measures were significantly different among the 5 conditions (P > 0.05). These results indicate that gas phase control is not needed for IVM and IVF of bovine oocytes for their subsequent development. Anaero Pack-MicroAnaero (5–8% CO2, 6–12% O2) can be used for IVC of bovine embryos. The CO2-generating and deoxidizing packets can be successfully used to control the gas phase during bovine embryo production. Table 1.Five different combinations of gas conditions used for incubation

scholarly journals Evaluation of Bovine Embryo Biopsy Techniques according to Their Ability to Preserve Embryo Viability

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
M. Cenariu ◽  
E. Pall ◽  
C. Cernea ◽  
I. Groza
Keyword(s):  
Pregnancy Rate ◽  
Genetic Diagnosis ◽  
Needle Aspiration ◽  
Embryo Biopsy ◽  
Embryo Cryopreservation ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Biopsy Methods ◽  
Biopsy Method

The purpose of this research was to evaluate three embryo biopsy techniques used for preimplantation genetic diagnosis (PGD) in cattle and to recommend the least invasive one for current use, especially when PGD is followed by embryo cryopreservation. Three hundred bovine embryos were biopsied by either one of the needle, aspiration or microblade method, and then checked for viability by freezing/thawing and transplantation to recipient cows. The number of pregnancies obtained after the transfer of biopsied frozen/thawed embryos was assessed 30 days later using ultrasounds. The results were significantly different between the three biopsy methods: the pregnancy rate was of 57% in cows that received embryos biopsied by needle, 43% in cows that received embryos biopsied by aspiration, and 31% in cows that received embryos biopsied by microblade. Choosing an adequate biopsy method is therefore of great importance in embryos that will undergo subsequent cryopreservation, as it significantly influences their viability after thawing.

Sign in / Sign up

Export Citation Format

Share Document