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.

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Mohammad Salehi ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Control Group ◽  
Developmental Potential ◽  
Significant Difference ◽  
Mouse Somatic Cell

SummaryThe beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

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.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

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

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.

282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 256
Author(s):  
W. J. Son ◽  
M. K. B. ◽  
Y. J. Jeong ◽  
S. Balasubramanian ◽  
S. Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Patterns ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Development ◽  
Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P &lt; 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P &lt; 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P &lt; 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P &lt; 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P &lt; 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P &lt; 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

5 TRANSCRIPTIONAL PROFILING OF PIG IN IN VIVO, IN VITRO-FERTILIZED, AND NUCLEAR TRANSFER-DERIVED BLASTOCYSTS AND THE DONOR SOMATIC CELL LINE

2008 ◽  
Vol 20 (1) ◽  
pp. 83
Author(s):  
K. M. Whitworth ◽  
L. D. Spate ◽  
R. Li ◽  
A. Rieke ◽  
D. M. Wax ◽  
...  
Keyword(s):  
Cell Line ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Reference Sample ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Electrical Activation ◽  
Shock Proteins

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix� 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

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

29 PRODUCTION OF BOVINE CLONED EMBRYOS BY NUCLEAR TRANSFER USING VITRIFIED IMMATURE OOCYTES AS RECIPIENT CYTOPLASTS

2009 ◽  
Vol 21 (1) ◽  
pp. 115
Author(s):  
F. Forell ◽  
C. Feltrin ◽  
L. C. Santos ◽  
A. D. Vieira ◽  
U. M. Costa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Technological Development ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
National Council ◽  
Immature Oocytes ◽  
Oocyte Recovery

The cryopreservation of immature oocytes is a logistic alternative to make cytoplasts available throughout the year for cloning by somatic cell nuclear transfer (SCNT). Oocyte cryopreservation will help to overcome hurdles related to oocyte availability, seasonality, or sanitary constraints. The objective of this experiment was to determine the efficiency of vitrification of bovine immature oocytes for use as cytoplasts to produce clone embryos. Cumulus–oocyte complexes (COCs) obtained from bovine ovaries by slicing from a local abattoir were selected and vitrified prior to maturation. Vitrification and warming solutions and exposure times were as previously described (Vieira AD et al. 2008 Rep. Dom. Anim. 43, 314–318) with minor modifications. Groups of 15 COCs were loaded in a 5-μL vitrification solution microdrop in beveled-cut straws (0.5 mL), which were plunged into N2L. Following warming, vitrified and control (non-vitrified) oocytes were in vitro-matured for 22 h and 17 h, respectively (Oliveira ATD et al. 2005 Theriogenology 64, 1559–1572). After maturation, cumulus cells were removed and oocytes were selected by the presence of a polar body. Embryo reconstruction by SCNT, carried out by standard micromanipulation procedures using fibroblast cells from adult origin, and in vitro culture to the blastocyst stage (Day 7) were based on our established procedures (Forell F et al. 2008 Acta Sci. Vet. 36, 141–148). Data regarding oocyte recovery following cumulus cell removal, oocyte survival after micromanipulation, and maturation, fusion, cleavage (Day 2), and blastocyst (Day 7) rates were analyzed by the chi-square test. Oocyte recovery (73.0%, n = 558/764 v. 91.4%, n = 529/579), maturation (46.8%, n = 261/558 v. 65.8%, n = 348/529) and cleavage (47.2%, n = 60/127 v. 60.2%, n = 77/128) rates were lower in the vitrified than in the non-vitrified group, respectively (P < 0.05). Conversely, oocyte survival after micromanipulation (77.8% and 78.4%) and fusion (82.1% and 82.3%) and blastocyst (16.7%, 10/60 v. 23.4%, n = 18/77) rates were similar between vitrified and non-vitrified groups. However, the overall efficiency (blastocysts produced from selected COCs) was 3.4-fold lower for vitrified oocytes than controls. In conclusion, the vitrification of immature bovine oocytes was proven as a valuable procedure for the production of blastocysts by SCNT, providing that a strict selection is made following warming, being an alternative resource either for the use of large numbers of oocytes obtained from slaughterhouse ovaries or to overcome seasonal variations in oocyte supply for use in animal cloning. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).

scholarly journals Characterization of mitochondrial replication and transcription control during rat early development in vivo and in vitro

Reproduction ◽  
2007 ◽  
Vol 133 (2) ◽  
pp. 423-432 ◽  
Author(s):  
Yuichi Kameyama ◽  
France Filion ◽  
Jae Gyu Yoo ◽  
Lawrence C Smith
Keyword(s):  
Early Development ◽  
Copy Number ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Mtdna Copy Number ◽  
Mitochondrial Transcription ◽  
Mtdna Replication

In vitroculture (IVC), used in assisted reproductive technologies, is a major environmental stress on the embryo. To evaluate the effect of IVC on mitochondrial transcription and the control of mtDNA replication, we measured the mtDNA copy number and relative amount of mRNA for mitochondrial-related genes in individual rat oocytes, zygotes and embryos using real-time PCR. The average mtDNA copy number was 147 600 (±3000) in metaphase II oocytes. The mtDNA copy number was stable throughoutin vivoearly development and IVC induced an increase in mtDNA copy number from the 8-cell stage onwards.GapdmRNA levels vary during early development and IVC did not change the patterns of these housekeeping gene transcripts.PolrmtmRNA levels did not vary during early development up to the morula stage but increased at the blastocyst stage. IVC induced the up-regulation ofPolrmtmRNA, one of the key genes regulating mtDNA transcription and replication, at the blastocyst stage. An increase inmt-Nd4mRNA preceded the blastocyst-related event observed in nuclear-encodedGapdandPolrmt, suggesting that the expression of mitochondrial encoded genes is controlled differently from nuclear encoded genes. We conclude that the IVC system can perturb mitochondrial transcription and the control of mtDNA replication in rat embryos. This perturbation of mtDNA regulation may be responsible for the abnormal physiology, metabolism and viability ofin vitro-derived embryos.

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