scholarly journals 229 EFFECT OF CULTURE CONDITIONS ON AQUAPORIN mRNA ABUNDANCE IN MOUSE BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 265
Author(s):  
H. Offenberg ◽  
P.D. Thomsen
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Mrna Abundance ◽  
Control Group ◽  
Mrna Levels ◽  
Blastocyst Formation ◽  
Gapdh Mrna ◽  
The Media

It is known that culture conditions can alter gene expression of the pre-implantation embryo. We have previously shown that aquaporins (AQPs) are expressed in the mouse embryo and that they are involved in the passage of water across the trophoblast cells during blastocyst formation. This study was conducted to investigate whether AQP mRNA abundance is altered by culturing embryos in vitro compared to in vivo developed embryos. Furthermore we wanted to investigate if AQP mRNA abundance was influenced by the osmolality of the media. It is possible to compare the effect of hyperosmolality that the embryo may be able to compensate for by adding glycerol which can cross some AQPs, compared to the addition of sucrose which can not cross the membranes. Mouse embryos were obtained by superovulating B6D2F1 mice followed by culture of the flushed presumptive zygotes in KSOM to the blastocyst stage (in vitro) or by flushing blastocysts from the uterus (in vivo). For the study of the influence of osmolality on AQP mRNA abundance, zygotes were flushed and cultured to the compacted 8-cell stage and then placed in media of increasing osmolality, using either glycerol or sucrose. The osmolalities of the media were 243 (control), 300, 350, and 400 mOsm. Embryos were cultured to the blastocyst stage and frozen in liquid nitrogen. Embryonic RNA was extracted using a Dynabeads mRNA Capture kit (Dynal, Oslo, Norway). Real time PCR was performed on embryonic cDNA on a Lightcycler (Roche Diagnostics, 2650 Hvidovre, Denmark) using aquaporin-specific primers and primers for β-actin and GAPDH. The results of the quantitative RT-PCR analysis showed that in vitro-cultured embryos had a lower mRNA abundance for AQP 8, 9, and 11 compared to the in vivo-developed embryos but that the AQP 3 mRNA abundance was unaltered. Analysis of the housekeeping genes showed that GAPDH mRNA levels were unchanged in vitro, whereas β-actin was up-regulated in vitro. The osmotically challenged embryos showed the following blastocyst rates compared to the controls: glycerol 300: 100%; glycerol 350: 100%; glycerol 400: 100%; sucrose 300: 100%; sucrose 350: 78%; and sucrose 400: 0%. Thus, glycerol up to 400 mOsm had no effect on blastocyst rates, whereas addition of sucrose reduced blastocyst formation, with a total inhibition at 400 mOsm. Analysis of the mRNA abundance showed a reduction of AQP 8 in the glycerol solutions. The level was reduced to 30% of the control group at 300 mOsm, to 27% at 350 mOsm and to 8% at 400 mOsm. There was no corresponding reduction of AQP 8 mRNA abundance in sucrose solutions. Further, AQP 3, 7, 9, and 11 mRNA levels as well as β-actin and GAPDH mRNA levels were unaltered in the osmotically challenged embryos. In conclusion, this study shows that embryonic culture affects the abundance of several AQPs and that compensation of a glycerol-induced osmotical challenge induces down-regulation of AQP 8 expression. Embryos tolerate high glycerol concentrations better than high sucrose concentrations but the possible role of AQP 8 in this process is unclear at present.

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

scholarly journals 245RELATIVE ABUNDANCE OF HSP 70 MRNA IN BOVINE EMBRYOS PRODUCED IN VITRO USING DIFFERENT EMBRYO/VOLUME RATIOS IN CULTURE

2004 ◽  
Vol 16 (2) ◽  
pp. 243
Author(s):  
A.T.D. Oliveira ◽  
C. Gebert ◽  
R.F.F. Lopes ◽  
H. Niemann ◽  
J.L. Rodrigues
Keyword(s):  
Relative Abundance ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Gene Transcripts

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P<0.05 were considered significant. Results show that no significative difference in hatching rate per blastocyst produced was detected among the four groups. Cleavage rate and blastocyst formation were significantly higher in groups with 5, 10 and 20 embryos compared with drops containing 30 embryos. Hsp transcripts were detected in morula and blastocyst stages in all groups. In morula stage, no differences were observed in the RA of Hsp 70.1mRNA among groups with 5, 10, 20 and 30 embryos cultured per drop. However, in blastocyst stage, the RA was significantly increased in the group with 20 embryos per drop as compared to the group with 5 embryos. The results show that different embryo/volume ratios in culture influence not only cleavage rate, blastocyst formation and hatching rate, but also expression of Hsp 70.1 gene. Further studies changing other culture conditions and using in vivo-derived bovine embryos will aid in elucidating which culture systems are ideal to produce bovine embryos in vitro. This research was supported by CAPES/DAAD program and CNPq.

Growth and viability of human blastocysts in vitro

2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Early Cleavage ◽  
Uterine Endometrium ◽  
Stage Of Development ◽  
Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 81-87 ◽  
Author(s):  
P.N. Moreira ◽  
R. Fernández-Gonzalez ◽  
M.A. Ramirez ◽  
M. Pérez-Crespo ◽  
D. Rizos ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Differential Effects ◽  
Glut 1 ◽  
Mouse Somatic Cell

It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMα, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMα, as well as on in vivo cultured and MEMα cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMα cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 136
Author(s):  
M. Hoelker ◽  
A. Kassens ◽  
E. Held ◽  
C. Wrenzycki ◽  
U. Besenfelder ◽  
...  
Keyword(s):  
Lipid Content ◽  
Ex Vivo ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
In Vitro Production ◽  
Negative Effects ◽  
Uterine Flushing

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses

1999 ◽  
Vol 11 (8) ◽  
pp. 457 ◽  
Author(s):  
Christopher G. Grupen ◽  
Paul J. Verma ◽  
Zhong Tao Du ◽  
Stephen M. McIlfatrick ◽  
Rodney J. Ashman ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Single Treatment ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
Electrical Pulses ◽  
Number Of Cells

The current protocols used to activate pig nuclear transfer embryos are less efficient than those used for other species. To address this problem, the effect of multiple sets of electrical pulses on the parthenogenetic development of in vivo- and in vitro-derived porcine oocytes was examined. Each set of pulses consisted of two 1.5 kV cm–1 DC pulses of 60 s duration each, administered 1 s apart. For in vivo-derived oocytes, application of a second set of pulses 30 min after the first set increased the proportion of oocytes that developed to the blastocyst stage compared with a single treatment (51 v. 34%). Application of a third set of pulses 30 min after the second set reduced the rate of blastocyst formation compared with two sets of pulses. In contrast, the rate of blastocyst formation was greater with one set of pulses compared with two sets for in vitro matured oocytes (31 v. 16%). Additional sets of electrical pulses did not affect the number of cells in blastocysts obtained from either group of oocytes compared with a single treatment. In summary, the study demonstrates that the application of a second set of activating pulses 30 min after the first set is beneficial to in vivo-derived oocytes, but detrimental to in vitro matured oocytes, in terms of their ability to develop parthenogenetically to the blastocyst stage.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

132 DIFFERENCES IN RESPIRATION RATES BETWEEN IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 174
Author(s):  
A. S. Lopes ◽  
S. E. Madsen ◽  
N. B. Ramsing ◽  
L. H. Larsen ◽  
T. Greve ◽  
...  
Keyword(s):  
Metabolic Stress ◽  
Average Diameter ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Outer Diameter ◽  
Bovine Embryos ◽  
Physiological Level ◽  
Respiration Rates

In vitro-produced (IVP) bovine embryos differ (e.g. morphology and physiology) from their in vivo counterparts. Oxygen consumption is an indicator of the overall metabolic activity of a single embryo. Therefore, the aim of this study was to determine and compare respiration rates of in vivo- and in vitro-produced bovine day 7 embryos. Diameters of these two embryo types were also compared. In vivo embryos (n = 28) were recovered from 8 superovulated Holstein Frisian cows on day 7 following AI, while IVP embryos (n = 160; Holm et al. 1999 Theriogenology 52, 683-700) were used on day 7 after fertilization. Embryos were measured (outer diameter) and morphologically evaluated (Quality 1 to 4, IETS Manual, 1998). Only transferable in vivo embryos were used (i.e. excluding Quality 4). Respiration rates were measured on each embryo by Nanorespirometer technology (Lopes et al. 2005 Reprod. Fertil. Develop. 17, 151). Data were analyzed using Proc Mixed, and values are presented as mean � SEM. Values with different superscripts differ significantly (P < 0.05). The average respiration rates were 0.82 � 0.06a nL/h for in vivo vs. 1.37 � 0.06b nL/h for IVP embryos. The average respiration rates for the different morphological qualities were as follows (nL/h, numbers in brackets): IVP: 2.1 � 0.08a (38), 1.37 � 0.07b (55), 1.08 � 0.07c (48) and 0.62 � 0.11d (19) for Quality 1, 2, 3, and 4, respectively. In vivo: 1.17 � 0.21b,c,e (6), 0.80 � 0.15c,d,e (12), and 0.64 � 0.16d,f (10) for Quality 1, 2, and 3, respectively. The average diameter (mm) of in vivo and IVP embryos was 0.157 � 0.002a and 0.176 � 0.002b, respectively. Respiration rates were directly related to embryo diameter; larger embryos were associated with higher respiration rates (y = 17.55 � 1.32 nL/h � mm, n = 188). Respiration rates of in vivo embryos were significantly lower than those of IVP embryos, regardless of quality. This difference could reflect an effect of the culture conditions on IVP embryos because media components affect embryo metabolism. Moreover, the different ages (day 7 for IVP vs. approximately Day 6.5 for in vivo embryos, because in vivo embryos are less than 7 days after fertilization at recovery) and stages (IVP: up to expanded blastocyst stage; in vivo: morula or early blastocyst stage) could have influenced the results and also partly explain the smaller diameter of the in vivo embryos. Finally, respiration rates decreased proportionately to the morphological quality within embryo type, indicating that morphological differences are reflected at the physiological level. In conclusion, this study further outlines metabolic differences between in vivo and IVP bovine embryos. Whether such differences are a manifestation of metabolic stress associated to the separation from the natural environment or reflect suboptimal culture conditions is yet to be determined. ASL is supported by FCT, Portugal.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

215 INVESTIGATION OF A PREFERENTIALLY UPREGULATED GENE CLUSTER IN DAY 7 BOVINE EMBRYOS DERIVED FROM RNA SEQUENCING DATA

2013 ◽  
Vol 25 (1) ◽  
pp. 256 ◽  
Author(s):  
A. Al Naib ◽  
S. Mamo ◽  
P. Lonergan
Keyword(s):  
Rna Sequencing ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Sequencing Data ◽  
Cell Stage ◽  
Uterine Endometrium ◽  
Embryonic Origin

Successful establishment and maintenance of pregnancy requires optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. A recent RNA sequencing study from our group (Mamo et al. 2011 Biol. Reprod. 85, 1143–1151) described the global transcriptome profile of the bovine conceptus at 5 key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using RNA sequencing techniques. One cluster of genes (n = 1680 transcripts) was preferentially upregulated at Day 7 and subsequently downregulated, suggesting that these genes might be markers of blastocyst formation. The objective of this study was to characterise the pattern of expression of these genes before Day 7 (i.e. from the zygote to blastocyst stage). The list of genes was submitted to DAVID (Database for Annotation, Visualisation, and Integrated Discovery) to take advantage of available ontology information contained therein. The expression of 9 genes belonging to ontologies specifically related to embryo developmental (GINS1, TAF8, ESRRB, NCAPG2, SP1, XAB2, CDC2L1, MSX1, and AQP3) plus Na/K ATPase, a gene previously known to be involved in blastocoe formation, was studied by quantitative real-time PCR (QPCR) in 6 replicate pools of 5 embryos produced by maturation, fertilization, and embryo culture in vitro. Stages studies included immature and mature oocyte, zygote, 2- cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, and hatched blastocyst. In addition, in vivo derived Day 13 and Day 16 embryos were included as controls to confirm down-regulation after Day 7. Data were analysed using the GLM procedure of SAS. The QPCR expression data supported the RNA Seq data in that expression of all transcripts was downregulated after the blastocyst stage. Expression before the blastocyst stage was characterised by 1 of 3 broad patterns: (1) the expression was of maternal origin where the expression was very high up to 8-cell stage and decreased subsequently (MSX1), (2) the expression was of embryonic origin being low up to the 8-cell stage and increasing thereafter (TAF8, ESRRB, AQP3, and Na/K ATPase), or (3) static or decreased expression from oocyte to the maternal-zygotic transition followed by increased expression from the 16-cell stage (GINS1, NCAPG2, SP1, XAB2, and CDC2L1). In conclusion, the genes identified in this cluster, despite having different patterns of expression before the blastocyst stage, may represent markers of blastocyst formation in cattle given their downregulation subsequently. Supported by Science Foundation Ireland (07/SRC/B1156).

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