Gene silencing in bovine zygotes: siRNA transfection versus microinjection

2011 ◽  
Vol 23 (4) ◽  
pp. 534 ◽  
Author(s):  
Ciara M. O'Meara ◽  
James D. Murray ◽  
Solomon Mamo ◽  
Emma Gallagher ◽  
James Roche ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Relative Abundance ◽  
Messenger Rna ◽  
Blastocyst Stage ◽  
Small Interfering Rnas ◽  
Sirna Transfection ◽  
Cell Stage ◽  
Transfection Medium ◽  
E Cadherin

The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1 h at 39°C and then cultured to Day 7. 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% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P < 0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P < 0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P < 0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05).

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.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

scholarly journals 243TEMPORAL DIVERGENCE IN THE PATTERN OF MRNA EXPRESSION IN BOVINE EMBRYOS CULTURED FROM THE ZYGOTE TO BLASTOCYST STAGE IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
P. Lonergan ◽  
D. Rizos ◽  
A. Gutierrez-Adan ◽  
P.M. Moreira ◽  
B. Pintado ◽  
...  
Keyword(s):  
Growth Factor ◽  
Relative Abundance ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Interferon Tau ◽  
Cell Stage

The objective of this study was to examine the time during the post-fertilization culture period that gene expression patterns of in vitro cultured bovine embryos diverge from those of their in vivo cultured counterparts. Presumptive bovine zygotes were produced by IVM/IVF of immature oocytes collected from the ovaries of slaughtered animals. At approximately 20h post-insemination (hpi), presumptive zygotes were randomly divided into two culture groups, either in vitro in synthetic oviduct fluid or in vivo, and transferred into the ewe oviduct. Embryos were recovered from both systems at approximately 30hpi (2-cell), two (4-cell), three (8-cell), four (16-cell), five (early morula), six (compact morula) or seven (blastocyst) days pi and snap-frozen for the analysis of transcript abundance using real-time PCR. The transcripts studied were interferon-tau, apoptosis regulator box-a (Bax), connexin 43, sarcosine oxidase, glucose transporter 5, mitochondrial Mn-superoxide dismutase, insulin-like growth factor II, and insulin-like growth factor-I receptor, most of which are known from our previous work to be differentially transcribed in blastocysts derived from culture in vitro or in vivo. Analysis was done on pools of 10 embryos. Data were analyzed using one-way repeated measures ANOVA. The relative abundance of the transcripts studied varied throughout the preimplantation period and was strongly influenced by the culture environment. For example, transcripts for interferon-tau were detected from the 8-cell stage onwards in in vitro-cultured embryos but not until the early morula stage in those cultured in vivo. Levels of this transcript increased significantly at the compact morula and blastocyst stages in both groups but were significantly higher (P&lt;0.05) in in vitro-cultured embryos at both stages. mRNA for Bax was not detected before the 8-cell stage in in vitro cultured embryos and not until the 16-cell stage in in vivo cultured embryos. The abundance of this transcript increased significantly thereafter up to the blastocyst stage in both groups. The level of expression was significantly higher (P&lt;0.05) at all stages of development in in vitro-cultured embryos than those cultured in vivo. The relative abundance of Cx43 transcripts decreased in both in vitro- and in vivo-cultured embryos at the 8- to 16-cell stage. Levels remained low thereafter in the in vitro-cultured embryos but significantly increased in those cultured in vivo. Transcript abundance was significantly higher in in vivo cultured embryos from Day 4 onwards with a ten-fold difference presence at the blastocyst stage. Differences also existed for the other transcripts studied. These data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident in as short as 10h of culture.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

193 DIFFERENTIAL EFFECTS OF CULTURE ON APOPTOTIC GENE EXPRESSION IN THE PRE-IMPLANTATION CLONED EMBRYOS OF MINIATURE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 213
Author(s):  
M. R. Park ◽  
I. S. Hwang ◽  
H. J. Moon ◽  
J. H. Shim ◽  
D. H. Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Total Concentration ◽  
Control Group ◽  
Specific Cell ◽  
Miniature Pig ◽  
Cell Stage ◽  
Apoptotic Gene ◽  
Early Embryos

Manipulations of early embryos require that the embryos be placed in vitro. The ability to reproduce in vivo conditions in vitro would greatly facilitate studies on the development of early embryos. A variety of different conditions have been described that result in development of pig embryos from the 1-cell stage to the blastocyst stage in vitro. There is a species-specific cell stage at which the early embryo is very sensitive to in vitro conditions, which generally corresponds to the stage at which the embryo begins producing significant amounts of RNA. The present study was conducted to investigate the relative amounts of apoptotic gene expression in miniature pig NT embryos under culture conditions of different osmolarity. Oocytes were cultured in TCM-199 for 40–44 h at 38.5�C under 5% CO2 in air. Miniature pig ear fibroblast cells were cultured to reach confluency, and the culture was continued for an additional 5–6 days. The NaCl group of embryos was cultured in PZM-3 supplemented with 138 mM NaCl in total concentration (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for a further 4 days. The control group of embryos was cultured in the PZM-3 for the entire period of in vitro culture. Total RNA samples were prepared from 2 blastocysts using the Roche 1st strand cDNA synthesis kit. Bax and Bcl-xl gene expression of blastocysts was analyzed by real-time RT-PCR. Developemntal rates were analyzed by a GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Relative gene expression was compared by Student&apos;s t-test. Blastocyst formation rate in the NaCl group was not different from that in the control group (25.4% and 23.2%, respectively), but the apoptosis rate was significantly lower (P &lt; 0.05) in the NaCl group (1.6%) than in the control (7.1%). The relative abundance of Bax mRNA expression was significantly higher (P &lt; 0.05) in the control group (n = 32) than in the NaCl group (n = 33). However, the relative abundance of Bcl-xl mRNA was significantly higher (P &lt; 0.05) in NaCl group. The relative abundance of Bax/Bcl-xl was significantly higher in the control group than in the NaCl group (P &lt; 0.05). These results indicate that the hypertonic culture condition at the early embryonic stage of miniature pig NT embryos could reduce the frequency of apoptosis through regulating Bax and Bcl-xl gene expression.

175 BOVINE EMBRYO DEVELOPMENT AND MRNA EXPRESSION IN BLASTOCYSTS CULTURED IN ISOLATED MOUSE OVIDUCTS MAINTAINED IN SOF OR KSOM

2006 ◽  
Vol 18 (2) ◽  
pp. 195
Author(s):  
D. Rizos ◽  
B. Pintado ◽  
J. de la Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan
Keyword(s):  
Embryo Development ◽  
Relative Abundance ◽  
Culture Medium ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Glut 1 ◽  
Gene Transcripts ◽  
Culture Environment ◽  
Chi Square Analysis

It is well known that modification of the post-fertilization culture environment of mammalian pre-attachment embryos can affect blastocyst quality, manifested in terms of morphology, cryotolerance, and relative abundance of certain gene transcripts. Culture of in vitro-produced bovine zygotes in the ewe oviduct leads to the development of blastocysts of a quality similar to those derived totally in vitro (Rizos et al. 2002 Biol. Reprod. 66, 589-595). However, such a system has disadvantages from a practical and animal welfare point of view. The isolated mouse oviduct (IMO) culture system is a potential alternative and has been successfully used in the in vitro culture of mouse, rat, hamster, and pig embryos from the one-cell stage to the morula/blastocyst stage. The aim of this study was to examine (1) the development of bovine zygotes in the IMO maintained in two different media (SOF and KSOM) in organ culture, and (2) the quality of the resultant blastocysts assessed in terms of the relative abundance of transcripts for several genes that have been previously implicated in embryo quality. Mouse oviducts were isolated from adult Swiss females (CD1, Harlan) the day after mating with an intact male. Approximately 10-15 presumptive bovine zygotes, produced by in vitro oocyte maturation and fertilization, were transferred to the ampullae of the isolated oviducts and were cultured in Transwell plates (Costar, Corning, NY, USA) over 1.1 mL of culture medium (SOF, n = 241 or KSOM, n = 320) at 39�C in an atmosphere of 5% CO2 in air at maximum humidity. A control group of embryos was cultured in droplets (25 �L) of the same culture medium and conditions in parallel (SOF, n = 278, KSOM, n = 225). Five replicates (=days of bovine ovary collection) were carried out. Following 6 days of culture, embryos were recovered from the oviducts/culture drops and blastocysts were snap-frozen in liquid nitrogen. Quantification of all gene transcripts was carried out by real time quantitative RT-PCR. Data on embryo development were analyzed by chi-square analysis and differences in transcript abundance by ANOVA. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 21.0 vs. 21.9%; KSOM: 22.0 vs. 22.2%). Culture in the IMO in SOF resulted in an increase (P d 0.05) in the abundance of transcripts for Oct-4 and SOX and reduced abundance of Glut-1, Na/K transporter, Cx43, and survivin, compared to control embryos. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and a reduced expression of Na/K transporter and SOX. Transcripts for G6PDH, IFN, and E-Cad were unaffected by culture environment. In conclusion, culture in the IMO leads to alterations in the relative abundance of transcripts that have been previously associated with embryo quality following culture in the ewe oviduct. However, the effect is dependent on the basal medium used.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

135 EXPRESSION OF APOPTOSIS-RELATED GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED THROUGH IN VITRO FERTILIZATION AND PARTHENOGENETIC ACTIVATION

2011 ◽  
Vol 23 (1) ◽  
pp. 172
Author(s):  
S. Saw ◽  
K. P. Singh ◽  
R. Kaushik ◽  
M. Muzaffar ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Culture Medium ◽  
Mammalian Species ◽  
Control Cell ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Immature Oocytes ◽  
Tissue Size

Apoptosis, a highly conserved evolutionary mechanism that allows an organism to tightly control cell numbers, tissue size, and protect itself from dangerous cells and unfavourable environments that threaten homeostasis, is generally directed by specific genes involved in the regulation of a series of pro-apoptotic (BAX) and anti-apoptotic (BCL-XL) proteins that are expressed during early development. All mammalian species show the highest level of spontaneous apoptotic processes at the blastocyst stage. These proteins prevent apoptosis by maintaining the cell survival by interfering with the release of cytochrome-C from mitochondria. In this study, immature oocytes were obtained from buffalo slaughterhouse ovaries and were subjected to in vitro maturation (IVM) in TCM-199 + 10% FBS + 5 μg mL–1 porcine FSH for 24 h in a CO2 incubator (5% CO2, 90 to 95% relative humidity) at 38.5°C. The mature oocytes were used for IVF, and the cleaved embryos were cultured for 8 days in culture medium (CR2 medium containing 0.6% BSA and 10% FBS) for production of embryos at different stages. The parthenotes were produced with exposure of 7% ethanol, 6-dimethyl aminopurine and cultured for 8 days in culture medium. The total RNA was isolated from oocytes and embryos and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA), according to manufacturer protocol. The PCR cycle included heating to 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60 (BAX) and 62°C (BCL) for 30 s, and 72°C for 45 s with a final extension at 72°C for 10 min. The amplified product of both genes were separated on agarose gel and densitometry data for band intensities were generated using AlphaDigiDocTM AD-1201 software under a WindowsTM environment and data analysed with the help of SYSTAT software. Relative abundance of BCL-XL transcripts in immature, mature oocytes and embryos produced through IVF (i.e. 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stage) were 25.33 ± 0.90, 12.67 ± 1.20, 37.67 ± 0.90, 30.67 ± 0.30, 23.67 ± 0.90, 18.33 ± 0.90, and 27.00 ± 1.20, respectively, whereas in parthenogenesis these values were 23.67 ± 0.88, 13.67 ± 1.20, 23.67 ± 1.20, 22.34 ± 0.88, 24.34 ± 0.88, 33.67 ± 0.88, and 45.34 ± 1.20, respectively. Relative abundance of BAX transcripts by IVF were 23.0 ± 0.60, 0.33 ± 0.10, 4.00 ± 0.60, 5.00 ± 0.60, 0.37 ± 0.06, 13.0 ± 0.66, and 56.7 ± 0.90; and by parthenonenesis were 22.3 ± 0.90, 0.13 ± 0.03, 13.67 ± 0.90, 14.0 ± 0.60, 15.33 ± 0.90, 64.67 ± 2.20, and 55.0 ± 2.10, respectively. In conclusion, the expression pattern of the apoptosis-related genes revealed that the incidence of apoptosis was significantly higher in IVF and parthenogenetically produced buffalo embryos at stages such as immature oocytes, morula, and blastocyst than the early cleavage stage embryos.

14 EQUINE CLONING AND EMBRYO AGGREGATION: EFFECT OF BOVINE, PORCINE, FELINE AND EQUINE OOPLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 118
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlanian ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Aggregation Effect ◽  
Group I

The low number of horse slaughterhouses is one of the reasons for the limited availability of horse oocytes for research in cloning. The aim of our study was to assess the capability of equine, bovine, porcine, or feline ooplast to produce cloned embryos when equine cells are used as donor nuclei and to evaluate if embryo aggregation improves their development. Oocytes from mentioned species were collected from ovaries derived from slaughterhouses, except for cat ovaries that were obtained from ovariectomized queens. Oocytes were matured in TCM199 supplemented following standard protocols for each species. After maturation, cumulus and zona pellucida were removed. Enucleation was performed by aspiration of the metaphase plate under ultraviolet light. Donor cell and ooplast were attached by phytohemagglutinin treatment and then electrofused. Activation protocols were ionomycin for 4 min, except for porcine, which were electrically activated, followed by culture in 1.9 mM 6-DMAP for bovine, feline and porcine, except for equine: 1 mM 6-DMAP with 5 mg mL–1 of cycloheximide. Reconstructed embryos (RE) were cultured in SOF in the well of well system in 2 different groups: only one RE per well (1X) and three RE per well (3X, aggregated embryos, AE). Blastocysts derived from homospecific clones were transferred to synchronized mares. Cleavage and maximum development stage achieved of all experimental groups were assessed. In vitro development was compared using the chi-square test. In group 1X, a total of 64, 49, 38 and 145 RE were performed for porcine, bovine, feline and equine, respectively and in group 3X, 88, 48, 48 and 195 RE. Cleavage of cloned embryos ranged from 67 to 87%. Aggregated of homospecific equine clones showed the highest blastocyst rates (1X: 5.5%, 3X: 34%) and after embryo transfer (4 recipients for each group), an ongoing pregnancy (day 300, at the time of submission) was only achieved with aggregated embryo confirming the positive effect of embryo aggregation in these clones. The stages with higher developmental arrest of heterospecific nonaggregated embryos were 2 to 4 cells for porcine ooplast (23/64, 36%) and 4 to 8 cells for bovine and feline ooplast (37/49, 75% and 18/38, 47%, respectively). Blastocyst stage was only reached using feline ooplast (group I: 2/38, 5.26% and group II: 2/16, 12.5%). Heterospecific aggregated clones were able to achieve 16-cell stage, showing statistic differences compared with group 1X. As we reported previously, embryo aggregation shows benefits for homospecific equine clones, although more studies are needed to clarify if aggregation of heterospecific clones has the same effect. All heterospecific ooplasm was able to support embryo development. The stage of major developmental arrests was similar to embryonic genomic activation stage. Our results suggest that cat oocyte seems to be the best receptor to support equine cloned embryo development.

178 RELATIVE ABUNDANCE OF Oct-4, Nanog, AND Sox-2 TRANSCRIPTS IN PORCINE OOCYTES AND CLEAVAGE-STAGE EMBRYOS PRODUCED VIA FERTILIZATION IN VITRO OR PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 168
Author(s):  
L. Magnani ◽  
R. Cabot
Keyword(s):  
Gene Expression ◽  
Similar Pattern ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Developmental Expression ◽  
Fertilization In Vitro ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Parthenogenetic Embryos

Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.

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