162 EVALUATION OF THE DEMI-EMBRYOS AGGREGATION TECHNIQUE EFFICACY ON THE PRODUCTION OF CHIMERAS WITH IN VIVO PRODUCED MURINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 239
Author(s):  
M. P. M. Mancini ◽  
B. C. S. Campanha ◽  
D. M. Souza ◽  
C. P. Godoi ◽  
F. Frei ◽  
...  
Keyword(s):  
Advanced Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Stage Of Development ◽  
Embryo Cells ◽  
Genotype Composition ◽  
23 Gauge ◽  
Aggregation Technique

Mixing embryo cells coming from different fertilizations (i.e. embryonic chimera) have been used as a tool to understand embryogenesis, organo- genesis, and pluripotency, as well as a source to obtain transgenic mammals. The objectives of this work were to evaluate the potential of mice demi-embryos, in advanced stage of the development (morulae and blastocysts) to aggregate in chimeras; to compare the chimerism rate of those embryos with the rate of whole 8- to 16-cell-stage embryos; and to measure the genotype composition of the resultant chimera. One-month-old transgenic (C57/BL6/EGFP strain, GFP) or non-transgenic (Swiss Webster strain, SW) mice weighing approximately 35 g were superstimulated with 5 or 10IU of eCG (for GFP or SW mice, respectively) followed with hCG injection of 5 or 10IU (GFP or SW mice, respectively) 48 h later. Embryos were harvested at different stages of development and allocated in 3 groups for aggregation technique. Blastocysts and morulae were bisected (microblade mounted on TransferMan NK-2, Eppendorf), whereas 8- to 16-cell-stage embryos had their zona pellucida mechanically removed (23-gauge needle). Embryos were manipulated in M2 culture medium at room temperature, and aggregation groups consisted of G1 (2 demi-blastocysts, n = 28), G2 (demi-blastocyst and demi-morula, n = 20), and G3 (2 whole 8- to 16-cell-stage embryos, n = 25). All embryos were placed in wells (Embryo GPS dish, SunIVF) containing KSOMaa medium (EmbryoMax, Millipore) under oil (Sigma, St. Louis, MO, USA) and were incubated at 37°C, 5% CO2 in air saturated with humidity. After 24 h of incubation, the presence of chimera was verified, and the percentage of area (square pixel) occupied by each embryonic type (GFP or SW) from both G2 (n = 3) and G3 (n = 3) were measured by the ImageJ program (v. 1.42i, USA). General results of the chimerism rate were 3.6%, 15.0%, and 60.0% (G1, G2, and G3, respectively; P < 0.001, chi-square). The G3 group differed from others (G1, P < 0.001 and G2, P = 0.003), which appeared similar (P = 0.294; Fisher’s exact test). The mean percentage (±SD) of GFP cells in the resultant chimera were 51.3 ± 4.1% and 50.6 ± 10.0% (for G2 and G3, respectively; P = 0.91, t-test). Moreover, the percentages of GFP cells within the same group of G2 or G3 at 0 v. 24 h of culture were not statistically different (data not shown). It was concluded that in our conditions, the embryonic chimerism by aggregation of murine demi-embryos is a feasible procedure, even for embryos in an advanced stage of development (morulae and blastocysts). Nevertheless, the chimerism rate with whole pre-compaction embryos (G3) was higher than that of G1 and G2 groups. Furthermore, the phenotype of embryonic chimera was equally composed, with no effect of strain (GFP or SW cells) or culture (0 or 24 h) on its composition. Supported by FAPESP, Brazil: 2006/06491-2 and 2007/07705-9 (MFGN) and 2007/04291-9 (MPMM).

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
C. Pontes Godoi ◽  
P. D. Moço ◽  
B. Cazari ◽  
P. T. Mihara ◽  
P. V. Silva ◽  
...  
Keyword(s):  
Cell Mass ◽  
Farm Animals ◽  
Control Group ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Chi Square Test ◽  
Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 175
Author(s):  
E. M. Razza ◽  
I. P. Emanuelli ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Aggregation Rate ◽  
Stage Of Development

Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

9 SINGLE FIXED-TIME LAPAROSCOPIC INTRAUTERINE INSEMINATION IN PIGS TO PRODUCE LOW-DIVERSE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 152
Author(s):  
K.-P. Brüssow ◽  
H. Torner ◽  
J. Rátky
Keyword(s):  
Intrauterine Insemination ◽  
Fixed Time ◽  
Uterine Wall ◽  
Sperm Cells ◽  
Chi Square ◽  
Cell Stage ◽  
Stage Of Development ◽  
Sperm Migration ◽  
Chi Square Test

In vivo-derived embryos at a defined stage of development are often a necessary requirement for ongoing biotechnological applications. Because double fixed-time insemination after ovulation induction is commonly used in pigs to produce embryos, variations in the time of ovulation and fertilization of the ovulated oocytes by spermatozoa mainly of 1 of the 2 inseminations can cause, however, diversities in embryo development. To moderate embryo diversity and to realize a uniform outcome of porcine embryo stages, single laparoscopic fixed-time insemination can be used to minimize embryo diversity. The potential of laparoscopic intrauterine insemination (LIUI) has been demonstrated in sperm-mediated gene transfer (Fantinati et al. 2005) and evaluation of sperm migration (Brüssow et al. 2006, 2011). The aim of the present study was to analyze the development and possible diversity of embryos after LIUI. Forty-eight puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day Regu-Mate® (Intervet, Millsboro, DE, USA) feeding and follicle development was stimulated with 850 IU of eCG 24 h after Regu-Mate® treatment. Ovulation was induced by 500 IU of hCG 80 h after eCG treatment. The LIUI was performed 31 h after hCG treatment. To that, ketamine/azaperone-anaesthetized gilts were fixed in a dorsal position, a pneumoperitoneum was produced and 3 trocar cannulas were inserted into the abdomen for optics and instruments. Laparoscopic handling was observed on a television monitor. Each uterine horn was carefully fixed with an atraumatic forceps 10 to 15 cm caudal from the utero-tubal junction and the uterine wall was punctured with a 2.5-mm diameter trocar. A 2.2-mm catheter connected to a syringe was inserted about 3 cm into the uterine lumen and 20 mL of extended, fresh boar semen (32.2 × 106 sperm cells mL–1; 65% motility) was deposited in the lumen. Embryos were surgically flushed from the genital tract on Day 2 and 3, respectively. Altogether, 778 oocytes were recovered (recovery rate 68 ± 17%); 45 of 48 gilts (93.8%) revealed fertilization and 76.1% of the recovered embryos (n = 592) were at the 2- and 4-cell stage. On Day 2 (n = 22 gilts), a higher percentage of gilts displayed only 2-cell embryos compared with both 2- and 4-cell, and only 4-cell embryos (72.2 v. 22.7 and 4.6%, P < 0.05; chi-square test). On Day 3 (n = 23 gilts), there was a shift regarding the embryo stage. The proportion of gilts with 2-cell, 2- and 4-cell, and only 4-cell embryos was 4.3, 0, and 95.7%, respectively (P < 0.05). Results of the present study demonstrate high rates of fertilization and of non-diverse developed embryos after single fixed-time LIUI in gilts. Additionally, these results were achieved after inseminating a 75% lower number of sperm cells per insemination dose. Laparoscopic intrauterine insemination can be suggested as an alternative for insemination of sex-sorted semen where the number of available sperm cells after the sorting procedure is restricted.

Effect of GnRH injection timing in the production of pronuclear-stage zygotes used for DNA microinjection

Zygote ◽  
2004 ◽  
Vol 12 (3) ◽  
pp. 257-261 ◽  
Author(s):  
Hernan Baldassarre ◽  
Bin Wang ◽  
Melanie Gauthier ◽  
Nathalie Neveu ◽  
Anthoula Lazaris ◽  
...  
Keyword(s):  
Medroxyprogesterone Acetate ◽  
Treatment Protocol ◽  
Hormonal Treatment ◽  
Injection Timing ◽  
Chi Square ◽  
Cell Stage ◽  
Embryo Recovery ◽  
Stage Of Development ◽  
Production Programme ◽  
Dna Microinjection

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE® and 47 adult standard goats (1–5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 μg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 μg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p<0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p=0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.

Nutrient uptake and culture of Sminthopsis macroura (stripe-faced dunnart) embryos

1996 ◽  
Vol 8 (4) ◽  
pp. 685 ◽  
Author(s):  
DK Gardner ◽  
L Selwood ◽  
M Lane
Keyword(s):  
Glucose Uptake ◽  
Nutrient Uptake ◽  
Culture System ◽  
Energy Demand ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Stage Of Development ◽  
Order Of Magnitude ◽  
Sminthopsis Macroura

Glucose and pyruvate uptake by individual embryos were measured in a marsupial species (stripe-faced dunnart) and a eutherian species (mouse). At each stage of development, nutrient uptake by the dunnart embryo was around an order of magnitude greater than that of the mouse embryo. The pattern of glucose uptake by the dunnart embryo was not like that for any eutherian embryo, all of which have a low glucose uptake before the blastocyst stage. Rather, in the dunnart embryo there was a significant increase in glucose uptake after the third cleavage division, increasing from 13.6 pmol embryo h-1 at the 4-cell stage to 34.9 pmol embryo h-1 by the 8-cell stage. This increase in glucose uptake before blastocyst formation may be attributed to an increased energy demand associated with the movement of cells within the dunnart embryo. Using a new culture system, it was possible to culture 66% of dunnart embryos at the 2-4-cell stage and 80% of those at the 8-16-cell stage to the unilaminar blastocyst stage. Embryos cultured from the 2-cell to the 4-cell stage were retarded by around 12 h when they reached the blastocyst stage. Developmental retardation was also reflected in the pattern of nutrient uptake, which lagged behind that of embryos developed in vivo. The present study has shown that it is possible to culture the early marsupial embryo to the blastocyst stage in a serum-free culture system, while concomitantly quantifying embryonic nutrient requirements. Such an approach is essential for species where there is a paucity of material for study.

Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos

2015 ◽  
Vol 27 (2) ◽  
pp. 385 ◽  
Author(s):  
Yanfang Li ◽  
Ki-Eun Park ◽  
Ryan A. Cabot
Keyword(s):  
Nuclear Import ◽  
In Vitro Assays ◽  
Cell Stage ◽  
Nuclear Trafficking ◽  
Nuclear Localisation ◽  
Cellular Events ◽  
Stage Of Development ◽  
Importin Α

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/β (importin α/β) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/β heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.

25 THE EFFECTS OF OOCYTE SOURCES AND ACTIVATION PROTOCOLS ON THE DEVELOPMENT OF CLONED GOAT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 131
Author(s):  
M. Apimeteeumrong ◽  
A. Thuangsanthia ◽  
N. Leingchaloen ◽  
V. Yiengvisavakul ◽  
A. Harintharanon ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Cytochalasin B ◽  
Vero Cells ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates ◽  
Matured Oocyte

The objective of this study was to compare the development to the morula and blastocyst stages, after either cycloheximide (CHX) or ethanol (ETOH) activation, in somatic nuclear transfer (NT) goat embryos derived from 2 sources of oocytes. In vivo- and in vitro-matured oocytes were obtained from FSH-stimulated goats (Native, Saanen, and Native-Saanen crossbred goats). Gonadotropin treatment was performed with a modified program of a previous report (Reggio et al. 2001 Biol. Reprod. 64, 849-856). In vivo-matured oocytes were flushed from the oviduct of donor goats by exposing the reproductive tract via a small ventral laparotomy incision. In vitro-matured oocytes were aspirated and cultured in maturation medium (M199 + 10% FCS, 10 �g mL-1 FSH, 10 �g mL-1 LH, and 1 �g mL-1 17�-estradiol) for 22 h, at 38.5�C in 5% CO2 and air. Donor cells were prepared from ear skin fibroblasts of a female goat (Native breed). Cells, at passage 3-9, starved by culturing in 0.5% FCS for 4-8 days, were used for NT. Matured oocytes were enucleated, and cell-cytoplast couplets (n = 162 in vivo-, and n = 190 in vitro-matured oocyte groups, respectively) were fused by applying 2 DC pulses of 2.2 kV cm-1 for 30 �s. One to 2 h after fusion, fused embryos were either incubated in 10 �g mL-1 cycloheximide plus 5 �g mL-1 cytochalasin B for 5 h (CHX treatment) or in 7% ethanol for 5 min followed by a 4-h incubation in 2 mM 6-dimethylaminopurine plus 5 �g mL-1 cytochalasin B (ETOH treatment). NT embryos were then cultured in B2 medium supplemented with 5% FCS and Vero cells for 9 days. At the end of the culture period, the NT embryos were fixed and stained with Hoechst 33342 (Begin et al. 2003 Theriogenology 59, 1839-1850). The numbers of nuclei were counted under ultraviolet light. Fusion, cleavage, and development rates were compared using chi-square test or Fisher&apos;s exact test. For the in vivo-matured oocyte group, there were no significant differences in fusion rates (78.1% vs. 68.7%), cleavage rates (87.7% vs. 87.0%, based on the numbers of embryos fused) between the CHX and ETOH treatment groups, respectively (P &gt; 0.05). However, the development rates to morula and blastocyst stages of NT embryos derived from either in vivo- or in vitro-matured oocytes were significantly higher in the ETOH group than in the CHX group (in vivo: 15.2% vs. 0%, and in vitro: 7.1% vs. 0%, for ETOH and CHX groups, respectively; P &lt; 0.05). For the in vitro-matured oocyte group, no significant differences were found between the CHX and ETOH groups in fusion rates (78.6% vs. 83.6%; P &gt; 0.05), cleavage rates (80.5% vs. 83.9%: P &gt; 0.05, based on the numbers of embryos fused). NT embryos from the CHX treatment group derived from in vivo- or in vitro-matured oocytes did not develop beyond the 16-cell stage. These results demonstrate that activation with CHX plus cytochalasin B treatment affects the development to the blastocyst stage of cloned goat embryos whether derived from in vivo- or in vitro-matured oocytes. This work was supported by the RGJ PhD program, Thailand Research Fund, and the Bureau of Biotechnology in Animal Production, Department of Livestock Development.

Transcription profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat

2020 ◽  
Vol 32 (7) ◽  
pp. 714
Author(s):  
Yunsheng Li ◽  
Jiangwen Sun ◽  
Yinghui Ling ◽  
Hao Ming ◽  
Zhen Chen ◽  
...  
Keyword(s):  
Embryo Development ◽  
Mammalian Species ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Transcriptional Networks ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Transcription Profiles ◽  
Stage Of Development

RNA sequencing performed on goat matured oocytes and preimplantation embryos generated invivo enabled us to define the transcriptome for goat preimplantation embryo development. The largest proportion of changes in gene expression in goat was found at the 16-cell stage, not as previously defined at the 8-cell stage, and is later than in other mammalian species. In all, 6482 genes were identified to be significantly differentially expressed across all consecutive developmental stage comparisons, and the important signalling pathways involved in each development transition were determined. In addition, we identified genes that appear to be transcribed only at a specific stage of development. Using weighted gene coexpression network analysis, we found nine stage-specific modules of coexpressed genes that represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the goat transcriptional networks. Their association with other embryo genes suggests that they may have important regulatory roles in embryo development. Our cross-mammalian species transcriptomic comparisons demonstrate both conserved and goat-specific features of preimplantation development.

scholarly journals 252A COMPARATIVE EXPRESSION ANALYSIS OF GENES IN PREIMPLANTATION DEVELOPMENTAL STAGES OF BOVINE EMBRYOS PRODUCED IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
D. Tesfaye ◽  
K. Wimmers ◽  
M. Gilles ◽  
S. Ponsuksili ◽  
K. Schellander
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
The Novel ◽  
Cell Stage ◽  
Novel Transcript ◽  
Stage Of Development ◽  
Significant Difference

A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P&lt;0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P&lt;0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P&lt;0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P&lt;0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.

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