277 PLASMID MEDIATED EXPERIMENTAL TELOMERE EXTENSION IN BOVINE EMBRYOS BY ECTOPIC EXPRESSION OF HUMAN TERT

2009 ◽  
Vol 21 (1) ◽  
pp. 235 ◽  
Author(s):  
K. Iqbal ◽  
W. A. Kues ◽  
H. Niemann
Keyword(s):  
Telomere Length ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Fluid Medium ◽  
Gfp Fluorescence ◽  
Morula Stage

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situ hybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells. Supported by DFG and Goyaike SAACIYF.

103 ECTOPIC EXPRESSION OF TELOMERASE IN BOVINE PREIMPLANTATION EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 132
Author(s):  
K. Iqbal ◽  
W. A. Kues ◽  
H. Niemann
Keyword(s):  
Telomere Length ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Fluid Medium ◽  
Gfp Fluorescence ◽  
Morula Stage

Recently, we have demonstrated a stage-specific increase of telomere length at morula-blastocyst transition in bovine and murine embryos (Schaetzlein et al. 2004 PNAS 101, 8034–8038). Telomeres are composed of repetitive hexanucleotide sequences (TTAGGG) encompassing several kilobasepairs and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage up to the morula stage, telomerase is not active or only found at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) was selected as the target molecule due to its 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from local abattoir followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. In preliminary tests, it was shown that co-injection of green fluorescent protein (GFP) and red fluorescent protein (dsRed) constructs resulted in the simultaneous expression of the 2 proteins. The onset of fluorescent protein expression was recorded 30 to 40 hours after injection by fluorescence microscopy. Because all cDNA were driven by the cytomegalovirus (CMV) promoter, it was assumed that hTERT is expressed in parallel. In the main experiment, 2 constructs encoding human TERT and GFP were co-injected to allow live separation of embryos. A total of 400 bovine embryos were injected, 209 (53%) of the treated embryos showed specific GFP fluorescence; out of a total of 104 blastocysts (26%), 55 showed GFP fluorescence (53%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic human TERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. Surprisingly, expression pattern of the endogenous TERT revealed a transient increase at the 2–4 cell stage and a major increase at the blastocyst stage. The mRNA level of the ectopic hTERT started to rise from 4 to 8 cell stage and remained high up to the morula stage. Currently, qFISH and TRAP techniques are being employed to assess enzymatic activity of hTERT and to quantitatively determine telomere length. In conclusion, this study demonstrates the ectopic expression of TERT and fluorescent proteins in early embryos; overexpression of TERT may facilitate the derivation of bovine ES cells. Supported by DFG and Goyaike S.A.A.C.I. Y.F.

X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Development ◽  
2000 ◽  
Vol 127 (19) ◽  
pp. 4137-4145 ◽  
Author(s):  
I. Okamoto ◽  
S. Tan ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Ectopic Expression ◽  
Blastocyst Stage ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Current View ◽  
Replication Banding ◽  
Xist Rna ◽  
Morula Stage

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.

166 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS IN IN VITRO-PRODUCED BUFFALO EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 163
Author(s):  
T. Anand ◽  
D. Kumar ◽  
M. K. Singh ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
...  
Keyword(s):  
Cell Surface ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Tumor Rejection ◽  
Cell Surface Expression ◽  
Human Embryos ◽  
Strong Expression

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. These are pluripotent cells that retain the ability to differentiate into all cell types. Various cell surface antigens, the expressions of which have been widely used as markers to monitor the pluripotency of ESCs, include Oct-4, stage-specific embryonic antigens (SSEAs) such as SSEA-1, SSEA-3, and SSEA-4, and tumor rejection antigens (TRAs) such as TRA-1-60 and TRA-1-81. In this study, the cell surface expression patterns of these markers were examined in in vitro-produced buffalo embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages using immunofluorescence microscopy. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF, following which the cleaved embryos were cultured for 9 days for production of embryos at different stages (n = 246). The embryos were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 30 min, permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min, and incubated first with the blocking solution (4% normal goat serum) for 30 min and then with the primary antibody (Oct-4: clone 9E3; SSEA-1: MC-480; SSEA-3: MC-631; SSEA-4: MC-813-70; TRA-1-60: clone TRA-1-60; and TRA-1-81: clone TRA-1-81, Chemicon� Inc., Temecula, CA, USA) at a dilution of 1:10 to 1:20 for 1 h. After being washed with DPBS, the embryos were incubated with appropriate FITC-labeled second antibody (anti-rat IgM or anti-mouse IgG or IgM, diluted 1:100 to 1:200) for 1 h and then examined under a fluorescence microscope. Oct-4 expression was detected at all embryonic stages starting from the 2-cell to the blastocyst stage, in which ICM, but not trophectoderm cells, exhibited a strong expression. SSEA-4 signal was found to be strongest at the 2-cell stage, with continued expression at all intermediate stages until the blastocyst stage in which there was a strong expression in ICM cells. In contrast, all of the embryonic stages were found to be negative for SSEA-3 expression. The SSEA-1 signal was present at all of the embryonic stages but was very weak. Expression of TRA-1-60 and TRA-1-81, which was detected only on the inner surface of the zona pellucida and in the perivitelline space in early embryonic stages, was absent in morulae and blastocysts. The results of this study indicate that the pluripotency-determining markers are differentially expressed in buffalo embryos and that the pattern of their expression is distinct from that of murine and human embryos but resembles to some extent that of goat embryos. Comparison of the expression pattern of these markers needs to be done between embryonic cells and ESCs for a better understanding of their developmental regulation.

161 MODULATION OF TELOMERASE ACTIVITY IN BOVINE EMBRYOS USING CYTOPLASMATIC PLASMID INJECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 239
Author(s):  
W. Garrels ◽  
W. Kues ◽  
U. Baulain ◽  
H. Niemann
Keyword(s):  
Telomere Length ◽  
Telomerase Activity ◽  
Control Group ◽  
Activity Measurement ◽  
Bovine Embryos ◽  
Gfp Fluorescence ◽  
Plasmid Injection ◽  
Significant Extension ◽  
Human Telomerase ◽  
The Impact

Telomeres are repetitive, noncoding sequences at the ends of linear chromosomes that shorten with each cell division. They play an important role in aging and affect the regenerative capacity of cells. The holoenzyme telomerase rebuilds telomeres and is composed of 2 components, i.e. the catalytic protein component telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). In mammals, telomerase is active during embryogenesis, in germ cells and a subset of stem and progenitor cells. In the present study, we set out to express the TERC component alone and then in combination with TERT, the human telomerase complex, in bovine embryos. The human telomerase components are highly homologous to bovine telomerase genes. Here, 3 different expression constructs encoding hTERC, hTERT, and a green fluorescent protein (GFP) reporter were co-injected into bovine zygotes cytoplasm, and three groups of 528, 1865, and 110 zygotes were constituted; hTERC/GFP (Group 1), hTERT/hTERC/GFP (Group 2), and GFP alone (Group 3), respectively. GFP fluorescence was used to identify successfully injected embryos. This method has recently been established in our laboratory. Injected and control embryos were cultured for 7 days to the blastocyst stage in vitro and the impact on early embryonic development and the physiological consequences of an ectopic overexpression of telomerase in early bovine embryos were assayed. We obtained 45 blastocysts with green fluorescence in the first, 192 in the second, and 28 in the third group. Embryos with GFP fluorescence were frozen for subsequent PCR analysis and telomerase activity measurement. Some blastocyts were analyzed using quantitative fluoresence in situ hybridization to monitor telomere length. Control groups were analyzed for the endogenous levels of TERC and TERT. Results indicate that endogenous TERC and TERT are up-regulated in morulae and blastocyts. In this study, we show that human TERC and TERT can be expressed in blastocysts by cytoplasmic plasmid injection in bovine zygotes. Statistical analyses were performed using the JMP 7.0.1 for Windows software (SAS Institute Inc., Cary, NC, USA). The Tukey-Kramer test was applied to compare the group means. The expression of hTERC alone resulted in a significant extension of telomere length of 280 telomere fluorescence units. Expression of both components also resulted in a significant extension of telomere length. In conclusion, TERC component alone is sufficient to elongate telomere length. The activity measurement showed that telomerase activity in the hTERT and hTERC injected group is 1.77 times higher than in the control group. Findings from this study will allow a comprehensive analysis of the functions of TERT and TERC in early embryogenesis. The ectopic expression of telomerase components in bovine embryos could pave new avenues for generating stem cells and for the development of novel regenerative therapies. Funded by DFG.

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).

scholarly journals Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Reproduction ◽  
2013 ◽  
Vol 145 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Shahin Eghbalsaied ◽  
Kamran Ghaedi ◽  
Götz Laible ◽  
Sayed Morteza Hosseini ◽  
Mohsen Forouzanfar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Gfp Expression ◽  
Exogenous Dna ◽  
Bovine Sperm ◽  
Gfp Fluorescence ◽  
Gfp Reporter ◽  
Green Fluorescent

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

scholarly journals Lysophosphatidic Acid Signaling in Late Cleavage and Blastocyst Stage Bovine Embryos

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Ana Catarina Torres ◽  
Dorota Boruszewska ◽  
Mariana Batista ◽  
Ilona Kowalczyk-Zieba ◽  
Patricia Diniz ◽  
...  
Keyword(s):  
Lysophosphatidic Acid ◽  
Culture Medium ◽  
Marker Gene ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Human Embryos ◽  
Lipid Mediator ◽  
Bovine Embryos ◽  
Pluripotency Marker

Lysophosphatidic acid (LPA) is a known cell signaling lipid mediator in reproductive tissues. In the cow, LPA is involved in luteal and early pregnancy maintenance. Here, we evaluated the presence and role of LPA in bovine early embryonic development. In relevant aspects, bovine embryos reflect more closely the scenario occurring in human embryos than the mouse model. Transcription of mRNA and protein expression of enzymes involved in LPA synthesis (ATX andcPLA2) and of LPA receptors (LPAR1–4) were detected in Days 5 and 8in vitroproduced embryos. Embryonic LPA production into culture medium was also detected at both stages of development. Supplementation of culture medium with LPA (10−5 M) between Days 2 and 8 had no effect on embryo yield and quality and on blastocyst relative mRNA abundance of genes involved in prostaglandin synthesis (PTGS2,PGES, andPGFS) and steroidogenesis (3βHSD). However, LPA treatment affected transcription levels of embryo quality markers, decreasingBAX(apoptotic) and increasingBCL2(antiapoptotic) andIGF2R(growth marker) gene transcription levels. Blastocyst transcription ofOCT4(pluripotency marker) was not affected by LPA stimulation. In conclusion, LPA is an early bovine embryonic autocrine/paracrine signaling mediator, and LPA action may be relevant in early embryo-maternal interactions leading to embryonic survival.

182 EXPRESSION PATTERN OF EMBRYONIC STEM CELL-SPECIFIC microRNAs IN MOUSE PREIMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 190
Author(s):  
T.-Y. Fu ◽  
P.-C. Tang
Keyword(s):  
Expression Pattern ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Cell Stage ◽  
Morula Stage

The endogenous non-coding microRNAs (miRNAs) of 18–25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by repression of translation or cleavage of mRNAs. In mammals, there are approximately 250 miRNAs that have been identified, and the cluster of miRNA-290 s (miR-290 s) has been demonstrated to express dramatically from the 2-cell to the 4-cell stage in mouse embryos examined from oocytes to the 8-cell stage. The association of miR-290 to 295 with pluripotency has been reported according to their specific expression in embryonic stem (ES) cells. It is interesting to explore the roles of these ES cell-specific miRNAs during the preimplantation stages and early differentiation at the blastocyst stage. Therefore, the objective of this study was to profile the expression pattern of ES cell-specific miRNAs (miR-291-5p, miR-293-3p, and miR-294-3p) from the 4-cell, 8- to 16-cell, morula, and blastocyst stages of mouse embryos. CD-1 F1 embryos at various developmental stages were collected from superovulated and naturally mated CD-1 mice. Total miRNAs of each stage analyzed were collected from 3 embryos for every replicate. Real-time RT-PCR was performed by using the specific stem-loop primers and the embryo lysate as template, which was prepared by heating in 4 μL of PBS at 95°C. Additionally, the in situ expressions of miR-291-5p, miR-293-3p, and miR-294-3p in mouse preimplantation embryos were confirmed by LNA™ probes specific for individual miRNAs. The embryo was fixed with 4% paraformaldehyde for 2 h at room temperature, followed by 3 times wash in PBST (0.1% TritonX-100 in PBS). After hybridization with individual 5′-fluorescein-labeled LNA™ probe, the embryo was washed with 0.1 × SSC, 2 × SSC, and TN buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl) subsequently. The in situ expressions of miRNAs were detected by immunocytochemical reaction. The results indicated that the expressions of miR-291-5p, miR-293-3p, and miR-294-3p were up-regulated from the 4-cell to the morula stage and then down-regulated afterwards. It was found that the signals of miR-293-5p in an expanded blastocyst were weaker than those at the early blastocyst stage. However, it showed that the intensity of expression at the morula stage was 2 to 4 folds higher compared to that at the 4-cell stage in each miRNA analyzed. Also, the result showed that the ES cell-specific miRNAs examined were expressed in all cells in a blastocyst, i.e. tropectoderm and inner cell mass. In conclusion, we have established the expression profile of ES cell-specific miRNAs during preimplantation stages in mouse embryos. The specific roles of these miRNAs would be further investigated in the short future.

СРАВНИТЕЛЬНАЯ ОЦЕНКА ЭФФЕКТИВНОСТИ ПРИЖИВЛЯЕМОСТИ ИНТАКТНЫХ И БИСЕКЦИОННЫХ ЭМБРИОНОВ У ТЕЛОК-РЕЦИПИЕНТОВ

2020 ◽  
pp. 32-36
Author(s):  
A.V. BRIGIDA ◽  
О.А. SKACHKOVA
Keyword(s):  
Blastocyst Stage ◽  
Uterine Horn ◽  
Bisection Method ◽  
Initial Number ◽  
Bovine Embryos ◽  
Experimental Conditions ◽  
Group I ◽  
Morula Stage ◽  
Group Ii ◽  
Donor Cows

Рассмотрены результаты исследований по оценке эффективности приживляемости интактных и бисекционных эмбрионов крупного рогатого скота у телок-реципиентов. Эмбрионы, полученные от коров-доноров швицкой породы и отобранные в процессе морфологической оценки на стадии развития поздней морулы (n244), были разделены на 2 группы. В I группе эмбрионы (n135) оставили интактными, во II произвели деление интактных эмбрионов (n109) пополам (бисекция) и получили бисекционные эмбрионы (n182), пригодные для пересадки. В обеих группах для обеспечения аналогичности исходных условий эксперимента эмбрионы были культивированы до стадии экспандированной бластоцисты. Затем интактные эмбрионы из I группы и бисекционные эмбрионы из II группы были пересажены телкам-реципиентам. Пересадку эмбрионов осуществляли в верхнюю часть рога матки с использованием катетера для аппликации эмбрионов, по одному каждому реципиенту. Результат приживляемости бисекционных эмбрионов из II группы (53,133,97 ) был ниже на 15,76 в сравнении с уровнем приживляемости интактных эмбрионов из I группы (68,894,01). Несмотря на увеличение исходного количества эмбрионов на 66,97 за счет применения метода бисекции во II группе, количество стельных животных увеличено не было. Таким образом, в процессе исследования определено, что метод бисекции позволяет значительно увеличить число эмбрионов, пригодных к пересадке. Необходимы дальнейшие исследования, направленные на изучение вопроса повышения уровня приживляемости бисекционных эмбрионов у телок-реципиентов.The paper presents the results on evaluating the engraftment of intact and bisected bovine embryos to recipient heifers. Embryos of Swiss breed donor cows were selected at the late morula stage (n244) and divided into two groups. In the group I (n135) the embryos were remained intact, in the group II (n109) the intact embryos were divided in half (bisection) and 182 bisected embryos suitable for transplantation were obtained. In both groups, to provide the similar initial experimental conditions, the embryos were cultured to the expanded blastocyst stage. Then, the intact embryos from the group I and bisected embryos from the group II were transferred to recipient heifers. Embryo transplantation (one embryo per a recipient) was carried out in the upper part of the uterine horn of recipients using a catheter for application of embryos The engraftment rate of bisected embryos from the group II (53.133.97) was 15.76 lower than the engraftment rate of intact embryos from the group I (68.894.01). As can be seen from this data, despite the increase in the initial number of embryos by 66.97 due to the application of the bisection method in group II, the number of pregnant animals was not increased. Thus, it was determined that the bisection method can significantly increase the number of embryos suitable for transplantation, but further studies are needed to study the issue of increasing the engraftment of bisection embryos to recipient heifers.

scholarly journals Embryonic Stem Cell Research and Religion: The Ban on Federal Funding as a Violation of the Establishment Clause

2006 ◽  
Vol 68 (1) ◽  
Author(s):  
Larry J. Pittman
Keyword(s):  
Stem Cells ◽  
Medical Condition ◽  
Embryonic Stem ◽  
Petri Dish ◽  
Medical Personnel ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Vitro Fertilization ◽  
New Treatments

Many Americans die each day from diseases affecting the heart, liver, kidneys, brain and a whole host of other bodily organs. Scientific researchers are constantly trying to develop new treatments for such medical conditions. Presently, the research community is working hard to develop medical treatments using stem cells from human embryos. That process involves extracting stem cells from either excess embryos that are no longer needed for in vitro fertilization or from embryos that are created through therapeutic cloning. At the blastocyst stage, about five days after the beginning of an embryo, researchers extract stem cells from the embryo and place them in a petri dish where the cells divide to produce a line of millions of stem cells. These stem cells are undifferentiated, meaning that they are still capable of transforming themselves into many different types of cells that exist in the human body. The hope is that physicians and other medical personnel will one day be able to inject these stem cells into a patient’s diseased heart, kidney, brain, liver, spinal cord or other organ, and the stem cells willtransform themselves into the same type of cells that comprise the host organ. The expectation is that the stem cells will repair the patient’s heart or other organs by curing diseases and otherwise improving the patient’s medical condition and life expectancy.

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