scholarly journals 50SPATIAL EXPRESSION OF OCT4 IS NORMALLY REGULATED IN PREIMPLANTATION STAGE BOVINE SOMATIC CELL CLONES

2004 ◽  
Vol 16 (2) ◽  
pp. 147
Author(s):  
S. Kurosaka ◽  
S. Eckardt ◽  
M.K. Friez ◽  
N.A. Leu ◽  
R. Reinbold ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Inner Cell Mass ◽  
Marker Gene ◽  
Cell Mass ◽  
Calf Serum ◽  
Ectopic Expression ◽  
Cell Lineage ◽  
Embryonic Cells ◽  
Oct4 Protein

Oct4 is a member of the POU family of transcription factors and is expressed in blastomeres, pluripotent embryonic cells and the germ cell lineage. In the mouse blastocyst, expression of Oct4 becomes restricted to the inner cell mass (ICM). In bovine, Oct4 has not been considered to be a marker for pluripotency because Oct4 protein is expressed in both the ICM and the trophectoderm (TE) of bovine blastocysts (van Eijk MJT et al., 1999 Biol. Reprod. 60, 1093–1103; Kirchhof N et al., 2000 Biol. Reprod. 63, 1698–1705). Oct4 has been used as a marker gene for nuclear reprogramming in cloned embryos. Aberrant spatial distribution and levels of Oct4 have been observed in the majority of mouse clone blastocysts and blastocyst outgrowths (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), indicating reprogramming failure of mouse clones. Lack of or abnormal Oct4 protein expression was also observed in cloned monkey embryos (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159). The spatial distribution of Oct4 mRNA and protein in bovine clones has not been reported. Bovine oocytes were obtained from a commercial supplier (BOMED, Inc., Madison, WI, USA), and were matured in vitro. Enucleated oocytes were fused with fibroblasts from ear skin and then treated with 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D for 6h. Embryos were cultured in SOF supplemented with 1% fetal calf serum (FCS) at 39°C under 5% CO2, 5% O2 and 90% N2 for 2 days. At Day 2, embryos were transferred to SOF supplemented with 5% FCS and cultured under the same conditions until Day 7. Blastocysts were analyzed at Day 7. Oct4 mRNA expression was visualized by whole-mount in situ hybridization using a bovine Oct4-specific antisense riboprobe. Oct4 protein was detected by immunocytochemistry. Control embryos were produced by IVF and were cultured under the same conditions to the blastocyst stage (Day 7). We found that Oct4 mRNA signal was restricted to the ICM in bovine blastocysts. Bovine clones were not different from control embryos in that distribution of Oct4 mRNA signal was typically restricted to the ICM (14 of 16). In contrast to our previous report on mouse clones (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), ectopic expression of Oct4 mRNA in the TE was rarely detected in bovine clones (2 of 16). Distribution of Oct4 protein was also similar between clones and controls with distribution in both the ICM and TE (clones: 9 of 9; controls: 9 of 9). It is unclear why defects in Oct4 distribution should differ between bovine and other species tested including monkey (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159–160); however, the higher rate of normal Oct4 distribution is consistent with the generally higher rates of postimplantation development of bovine clones (Shi W et al., 2003 Differentiation 71, 91–113).

scholarly journals Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

2005 ◽  
Vol 17 (8) ◽  
pp. 799 ◽  
Author(s):  
Natalie I. Alexopoulos ◽  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Andrew J. French ◽  
Alan O. Trounson
Keyword(s):  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Collagen Gel ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Term Survival

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

Berberine alleviates LPS-induced apoptosis, oxidation, and skewed lineages during mouse preimplantation development

2022 ◽  
Author(s):  
Xiaosu Miao ◽  
Wei Cui
Keyword(s):  
Inner Cell Mass ◽  
Polycystic Ovary ◽  
Cell Mass ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Embryonic Cells ◽  
Preimplantation Embryo Development ◽  
New Treatment ◽  
Induced Apoptosis

Abstract Female infertility is a heterogeneous disorder with a variety of complex causes, including inflammation and oxidative stress, which are also closely associated with the pathogenesis of Polycystic Ovary Syndrome (PCOS). As a new treatment for PCOS, berberine (BER), a natural compound from Berberis, has been clinically applied recently. However, the mechanisms underlying the association between BER and embryogenesis are still largely unknown. In this study, effects of BER on preimplantation development was evaluated by using both normal and inflammatory culture conditions induced by lipopolysaccharide (LPS) in the mouse. Our data first suggest that BER itself (25 nM) does not affect embryo quality or future developmental potency, moreover, it can effectively alleviate LPS-induced embryonic damage by mitigating apoptosis via ROS−/caspase-3-dependent pathways and by suppressing pro-inflammatory cytokines via inhibition of NF-κB signaling pathway during preimplantation embryo development. In addition, skewed cell lineage specification in inner cell mass (ICM) and primitive endoderm (PE) caused by LPS can also be successfully rescued with BER. In summary, these findings for the first time demonstrate the non-toxicity of low doses of BER and its anti-apoptotic and anti-oxidative properties on embryonic cells during mammalian preimplantation development.

scholarly journals Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Young-Ho Choi ◽  
Pablo Ross ◽  
Isabel C Velez ◽  
B Macías-García ◽  
Fernando L Riera ◽  
...  
Keyword(s):  
High Glucose ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Effect Of Glucose

Equine embryos developin vitroin the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31–46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively forPOU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did.GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation inin vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

scholarly journals Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

2020 ◽  
Author(s):  
Giuliano Giuseppe Stirparo ◽  
Agata Kurowski ◽  
Stanley Eugene Strawbridge ◽  
Hannah Stuart ◽  
Thorsten Edwin Boroviak ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Specific Gene ◽  
List Type ◽  
Early Mouse

AbstractOCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism.Highlights and noveltyMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markersJAK/STAT signalling is defective in OCT4 null embryosOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identityOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

305 CONSTRUCTION OF TRANSCRIPTION FACTOR GENES FOR REPROGRAMMING OF ADULT GOAT FIBROBLAST CELLS FOR PRODUCTION OF INDUCED PLURIPOTENT STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 249
Author(s):  
D. Kumar ◽  
D. Malakar ◽  
R. Dutta ◽  
S. Garg ◽  
S. Sahu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Somatic Cells ◽  
Ectopic Expression ◽  
Fibroblast Cells ◽  
Induced Pluripotent

Embryonic stem cells (ESC) are derived from the inner cell mass of blastocysts and proliferate extensively while maintaining pluripotency. They can be used for the treatment of juvenile diabetes, Parkinson’s disease, heart failure, and spinal cord injury. However, the use of embryos and tissue rejection remain concerns for ESC transplantation. Reprogramming of somatic cells may be done by different methods such as somatic cell nuclear transfer (Wilmut et al. 1997), fusion of somatic cells (Cowen et al. 2005), treatment with the extract of the pluripotent stem cells (Johnson Rajasingh 2008), and by the stable ectopic expression of defined factors in the somatic cells (Takahashi and Yamanaka 2006). Several transcription factors, including Oct3/4 (Nichols et al. 1998; Niwa et al. 2000), Sox2 (Avilion et al. 2003), and Nanog (Chambers et al. 2003; Mitsui et al. 2003), function in the maintenance of pluripotency in both early embryos and ESC. Takahashi and Yamanaka reported reprogramming the fibroblast cells into stem cells by introducing Oct3/4, Sox2, c-Myc, and Klf4 in mouse embryonic and adult fibroblasts. Yu et al. (2007) demonstrated that four transcription factors (OCT-4, SOX2, NANOG, and LIN28) are sufficient to reprogramme human somatic cells to pluripotent stem cells that exhibit the essential characteristics of ESC. Nakagawa et al. (2008) used three factors (OCT3/4, SOX2, and KLF4) for human iPS cell production from somatic cells. We are trying to reprogramme the adult goat fibroblast cells in induced pluripotent stem cells by using ectopic expression of transcription factors such as Oct-4, Sox2, Nanog, and Lin28. We collected the ovaries from a slaughtered animal from Delhi and collected the oocytes from ovaries. Then after the collection, A and B grade oocytes were selected. Selected oocytes were processed and incubated in in vitro maturation media for 24 h. We collected semen from a male goat, and it was processed and capacitated in sperm TALP. Capacitated sperms were used for IVF of the in vitro matured oocytes in ferTALP. After 12 h sperm were washed from oocytes in embryo developing media (EDM), and oocytes were cultured (in vitro) in EDM. After 24 h cleavage occurred. The cleaved embryos were cultured for 6 to 7 days. At the 7th day, we got blastocysts. From these blastocysts, inner cell mass was isolated enzymatically and cultured to get ESC. The ESC were cultured for 7 passages and used for RNA isolation. The RNA was isolated from these stem cells by the Trizol method. Complementary DNA was prepared by RT-PCR. Using gene-specific primer for Oct-4, Nanog, and Sox2, DNA was amplified. The DNA for the Oct-4, Nanog, and Sox2 genes was cloned in pJET cloning vector and transformed in Top10 E. coli competence cells. After screening, plasmid was isolated and sent for sequencing. Sequences were analysed and the complete open reading frame was created for Oct-4, Nanog, and Sox2.

scholarly journals OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

2018 ◽  
Vol 115 (11) ◽  
pp. 2770-2775 ◽  
Author(s):  
Kilian Simmet ◽  
Valeri Zakhartchenko ◽  
Julia Philippou-Massier ◽  
Helmut Blum ◽  
Nikolai Klymiuk ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
Null Mouse ◽  
General Strategy ◽  
Vitro Fertilization ◽  
Domestic Species ◽  
Oct4 Protein ◽  
Salt And Pepper

Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 39-44 ◽  
Author(s):  
P. Chrenek ◽  
A.V. Makarevich
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Control Group ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Egfp Gene ◽  
Chimeric Blastocysts ◽  
Rabbit Embryos

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p<0.05), but significantly lower than in TR<>N chimeric blastocysts (p<0.001). Similarly, a higher proportion (p<0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

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