scholarly journals Different co-culture systems have the same impact on bovine embryo transcriptome

Reproduction ◽  
2017 ◽  
Vol 154 (5) ◽  
pp. 695-710 ◽  
Author(s):  
A Vitorino Carvalho ◽  
E Canon ◽  
L Jouneau ◽  
C Archilla ◽  
L Laffont ◽  
...  
Keyword(s):  
Early Development ◽  
Embryo Development ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Cell Types ◽  
Culture Conditions ◽  
Bovine Embryo ◽  
Culture Systems ◽  
Beneficial Impact ◽  
Reduce Cell Survival

During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2. Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2. Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.

Quantitative expression analysis of blastocyst-derived gene transcripts in preimplantation developmental stages of in vitro-produced bovine embryos using real-time polymerase chain reaction technology

2004 ◽  
Vol 16 (8) ◽  
pp. 753 ◽  
Author(s):  
Nermin El-Halawany ◽  
Siriluck Ponsuksili ◽  
Klaus Wimmers ◽  
Markus Gilles ◽  
Dawit Tesfaye ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Expression Pattern ◽  
Embryo Development ◽  
Developmental Stages ◽  
Bovine Embryo ◽  
Chain Reaction ◽  
Quantitative Expression ◽  
Polymerase Chain

The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B–M) and used to establish a B–M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.

81 EFFECT OF IN VITRO CULTURE SYSTEM MODIFICATION USING CR1aa MEDIUM ON EMBRYO DEVELOPMENT AND PREGNANCY RATE IN CATTLE

2014 ◽  
Vol 26 (1) ◽  
pp. 154
Author(s):  
G. Singina ◽  
T. Taradajnic ◽  
N. Taradajnic ◽  
N. Zinovieva
Keyword(s):  
Pregnancy Rate ◽  
Embryo Development ◽  
Subsequent Pregnancy ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Developmental Potential ◽  
Culture Systems ◽  
System 2 ◽  
System 1

The culture of in vitro matured and fertilized oocytes is a critical step of in vitro production of bovine embryos. Generally, oocytes are co-incubated with sperm in TALP medium containing different additions and then zygotes are transferred to a medium with another composition. At the same time the effect of the medium alteration on the development of early embryos is unknown. Continual adjustment of fertilized oocytes to the changing culture environment may result in a reduction of their developmental potential. The aim of the present study was to compare effects of two different culture systems on the embryo development and subsequent pregnancy rate in cattle. Slaughterhouse-derived cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. Frozen/thawed sperm from different Russian Black Pied bulls were prepared in Sperm-TALP medium by swim-up procedure. In vitro matured oocytes were co-incubated for 18 h with prepared sperm in the modified Fert-TALP medium containing 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% MEM nonessential amino acids. The embryo culture was carried out using 2 systems. A total of 340 presumptive zygotes were incubated in CR1aa medium (Rosenkrans et al. 1994 J. Anim. Sci. 72, 434–437) up to Day 5 post-insemination (System 1) and a total of 442 presumptive zygotes were incubated for 24 h in a fresh Fert-TALP medium without PHE and heparin and then cleaved embryos were transferred to CR1aa medium and incubated until Day 5 post-insemination (System 2). Thereupon, the embryos were transferred to a fresh CR1aa medium supplemented with 5% FCS and cultured for 3 or 5 days. The embryo development was evaluated at Days 2, 8, and 10 for cleavage and blastocyst formation and hatching rates, respectively. A portion of blastocysts (of Grade 1 according to IETS classification) obtained at Day 8 were immediately transferred to recipients or were frozen in 1.5 M ethylene glycol and stored in liquid nitrogen until transplantation. The embryo development data (from 6–8 replicates) were analysed by ANOVA and the embryo transplantation data were analysed using the chi-squared test. The cleavage rates did not differ among Systems 1 and 2 and were 63.6–65.7%. On the other hand, the significant differences between culture Systems 1 and 2 were detected in rates of blastocysts (21.9 ± 1.4 v. 28.8 ± 2.8; P < 0.05) and hatched blastocysts (7.2 ± 1.2 v. 12.3 ± 1.6; P < 0.05). The pregnancy rate for frozen embryos was also higher (but not significantly) in System 2 than in System 1 [26.3% (9/34) v. 16.7% (2/12)], whereas for fresh embryos the similar values of the pregnancy rate were observed [on average 42.9% (6/14)]. Thus the additional 24-h culture of zygotes in Fert-TALP medium favourably affects bovine embryo development in vitro.

scholarly journals Unveiling the bovine embryo transcriptome during the maternal-to-embryonic transition

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 245-257 ◽  
Author(s):  
Christian Vigneault ◽  
Catherine Gravel ◽  
Maud Vallée ◽  
Serge McGraw ◽  
Marc-André Sirard
Keyword(s):  
Cdna Library ◽  
Developmental Stages ◽  
Protein Biosynthesis ◽  
Subtractive Hybridization ◽  
Culture Conditions ◽  
Bovine Embryo ◽  
Maternal Mrna ◽  
Activation Of Transcription ◽  
Early Embryos ◽  
Genome Activation

Bovine early embryos are transcriptionally inactive and subsist through the initial developmental stages by the consumption of the maternal supplies provided by the oocyte until its own genome activation. In bovine, the activation of transcription occurs during the 8- to 16-cell stages and is associated with a phase called the maternal-to-embryonic transition (MET) where maternal mRNA are replaced by embryonic ones. Although the importance of the MET is well accepted, since its inhibition blocks embryonic development, very little is known about the transcripts expressed at this crucial step in embryogenesis. In this study, we generated and characterized a cDNA library enriched in embryonic transcripts expressed at the MET in bovine. Suppression subtractive hybridization followed by microarray hybridization was used to isolate more than 300 different transcripts overexpressed in untreated late eight-cell embryos compared with those treated with the transcriptional inhibitor, α-amanitin. Validation by quantitative RT-PCR of 15 genes from this library revealed that they had remarkable consistency with the microarray data. The transcripts isolated in this cDNA library have an interesting composition in terms of molecular functions; the majority is involved in gene transcription, RNA processing, or protein biosynthesis, and some are potentially involved in the maintenance of pluripotency observed in embryos. This collection of genes associated with the MET is a novel and potent tool that will be helpful in the understanding of particular events such as the reprogramming of somatic cells by nuclear transfer or for the improvement of embryonic culture conditions.

scholarly journals Temporal expression of factors involved in chromatin remodeling and in gene regulation during early bovine in vitro embryo development

Reproduction ◽  
2007 ◽  
Vol 133 (3) ◽  
pp. 597-608 ◽  
Author(s):  
Serge McGraw ◽  
Christian Vigneault ◽  
Marc-André Sirard
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Epigenetic Modification ◽  
Chromatin Modification ◽  
Bovine Embryo ◽  
Mrna Levels ◽  
Temporal Expression ◽  
Regulate Gene Expression ◽  
Preimplantation Embryo Development

Distinct epigenetic modification events regulate gene expression and chromatin structure during the period between the immature oocyte and the blastocyst. Throughout this developmental period, important methylation fluctuations occur on genomic DNA and histones. Finding single orcombinations offactors, which are at work during this period is essential to understand the entire epigenetic process. With this in mind, we assessed the precise temporal expression profile, during preimplantation embryo development, of 15 key regulators involved in RNA, DNA or histone methylation, chromatin modification or silencing and transcription regulation. To achieve this, real-time RT-PCR was used to quantify the mRNA levels of ATF7IP, DMAP1, EHMT1, EHMT2, HELLS, JARID1A, JARID1B, JMJD1A, JMJD2A, LSD1, MeCP2, METTL3, PRMT2, PRMT5 and RCOR2, in the oocyte and throughout in vitro bovine embryo development. Our results demonstrate that all the 15 key regulators were present to different degrees in the developmental stages tested, and they can be divided into three different groups depending on their respective mRNA profile.

61 Extracellular vesicles from serum in culture media are internalized by bovine embryos produced in vitro

2019 ◽  
Vol 31 (1) ◽  
pp. 156 ◽  
Author(s):  
B. Melo-Baez ◽  
E. Mellisho ◽  
L. Rodriguez-Alvarez
Keyword(s):  
Early Development ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Control Treatment ◽  
Bovine Embryo ◽  
Embryonic Cells ◽  
Long Distance ◽  
Protein Sources

Extracellular vesicles (EV) are currently considered a mechanism of cell communication. These are secreted from different cell types, including embryos, to serve as mediators of short and long distance signals. EV can be identified in vivo in different biological fluids, as well as in vitro embryo culture medium. Usually, media used for embryo in vitro culture are supplemented with serum or other protein sources that favour cell proliferation and development. Serum and protein sources contain EV, including microvesicles and exosomes that in principle can be internalized by embryonic cells. The aim of this study was to evaluate if serum-derived EV are internalized by the embryo at different stages of the early development, and if EV from the serum are required for in vitro bovine embryo development. For that, it was first evaluated if EV depleted culture media affect embryo development up to the blastocyst stage; oocytes were in vitro matured for 22 to 23h and in vitro fertilized for 18h. Posteriorly, presumptive zygotes were in vitro cultured in groups (25 embryos/well in 4-well plates) in SOF or SOF depleted of EV for 8 days. To evaluate EV internalization, culture media was supplemented with labelled EV and confocal imaging was performed. The EV were obtained by ultrafiltration (centrifugal filter devices 100 kDa, Amicon; Millipore, Billerica, MA, USA) for 15min at 3000 rpm. Then, EV were stained with PKH67 dye and washed 3 times with PBS by ultrafiltration to remove excess dye. The EV labelled with PKH67 were resuspended in SOFaa depleted of EV (3×109 particles per 500µL) and supplemented for 24h at the 1-cell stage (Day 1 post IVF), 16 cells (Day 4 post IVF), and early blastocyst (Day 6 post IVF) in 5% CO2, 5% O2, and 90% N2. PBS with PKH67 dye was used as a control treatment. Hoechst 33343 was used to label the nuclei before washing with PBS and fixation with 0.4% paraformaldehyde. Images were acquired on a Zeiss (Zeiss, Jena, Germany) LSM 780 confocal microscope. There were no statistical differences on blastocyst rate at Day 8 between embryos cultured in SOF depleted of EV (19.5%) and control group (SOF; 22.7%; P&gt;0.05). We observed punctuated green fluorescence near the embryo nuclei in the 3 stages studied in embryos supplemented with EV but not in the control treatment, which indicates that EV from serum are uptaken by embryonic cells in early development. Therefore, we demonstrated uptake of EV from fetal calf serum added to culture media, although its absence does not affect embryo development. Research was supported by FONDECYT, Chile (1170310).

scholarly journals Early development of Hypogymnia physodes (L.) Nyl. in response to emissions from a copper smelter

2001 ◽  
Vol 33 (6) ◽  
pp. 527-538 ◽  
Author(s):  
Irina N. Mikhailova ◽  
Christoph Scheidegger
Keyword(s):  
Early Development ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Copper Smelter ◽  
Copper Smelting ◽  
Hypogymnia Physodes ◽  
Toxic Impact ◽  
Early Developmental ◽  
Desert Zone ◽  
Copper Smelting Plant

AbstractThe early development of Hypogymnia physodes from soredia to the formation of stratified lobes has been studied experimentally in the vicinity of a copper-smelting plant in theMiddle Urals. SEM investigations combined with life table analyses of early developmental stages revealed decreases in soredial survival and developmental rate in polluted localities. Non-stricatified pre-thallus stages without an epicortex were tolerant to toxic impact and were able to survive even in the zone with the highest pollution (lichen desert zone). The sensitivity of developmental stagesancreased after stratified lobes had developed.

scholarly journals Ceramide in Stem Cell Differentiation and Embryo Development: Novel Functions of a Topological Cell-Signaling Lipid and the Concept of Ceramide Compartments

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Erhard Bieberich
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Cell Signaling ◽  
Embryo Development ◽  
Knockout Mice ◽  
Lipid A ◽  
Developmental Stages ◽  
Stem Cell Differentiation ◽  
Cell Types

In the last two decades, the view on the function of ceramide as a sole metabolic precursor for other sphingolipids has completely changed. A plethora of studies has shown that ceramide is an important lipid cell-signaling factor regulating apoptosis in a variety of cell types. With the advent of new stem cell technologies and knockout mice for specific steps in ceramide biosynthesis, this view is about to change again. Recent studies suggest that ceramide is a critical cell-signaling factor for stem cell differentiation and cell polarity, two processes at the core of embryo development. This paper discusses studies on ceramide usingin vitrodifferentiated stem cells, embryo cultures, and knockout mice with the goal of linking specific developmental stages to exciting and novel functions of this lipid. Particular attention is devoted to the concept of ceramide as a topological cell-signaling lipid: a lipid that forms distinct structures (membrane domains and vesicles termed “sphingosome”), which confines ceramide-induced cell signaling pathways to localized and even polarized compartments.

Effect of embryo developmental stage and culture conditions on number and quality of ovine in vitro produced blastocysts

Zygote ◽  
2006 ◽  
Vol 14 (3) ◽  
pp. 181-187 ◽  
Author(s):  
R.M. Garcia-Garcia ◽  
V. Dominguez ◽  
A. Gonzalez-Bulnes ◽  
A. Veiga-Lopez ◽  
M.J. Cocero
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Defined Medium ◽  
Culture Conditions ◽  
Cell Groups ◽  
Early Developmental

SummaryThis study evaluated the final output and quality of in vitro produced blastocysts derived from in vivo recovered sheep embryos cultured at various early developmental stages to blastocyst. A total of 270 embryos were recovered from the oviduct, at different days of the early luteal phase, and were classified into three different developmental stages: 2- to 4-cell (n = 93); 5- to 8-cell (n = 92) and 9- to 12-cell (n = 85). The effect of culture conditions was studied, at the same time, by randomly allocating the embryos to one of four groups: three groups of culture with fresh oviduct monolayers (2, 4 and 5 days old) and a fourth group with 2-day monolayers derived from frozen-thawed oviduct cells. Two control groups were established: first, embryos cultured in semi-defined medium (n = 29) and, second, blastocysts obtained in vivo and cryopreserved (n = 43). Influence on blastocyst yield of embryo developmental stage at the start of culture was statistically significant (p < 0.001). Two- to four-cell embryos showed a significantly lower developmental rate (67.7%) than the 5- to 8-cell (83.6%; p < 0.001) and 9- to 12-cell groups (90.5%; p < 0.0001) and lower quality in terms of blastocyst cryotolerance (56.0 vs. 83.7%; p < 0.005). There were no detected effects relating to the age or handling of the monolayer on the embryo developmental rate, but the day of blastocyst appearance was different between embryos cultured on monolayers derived from fresh or frozen-thawed cells (p < 0.0001); the main influence was on the group of 9- to 12-cell embryos (p < 0.0001). Current results confirm the temporal sensitivities of sheep embryos to in vitro culture, regardless of the culture conditions.

Temporally differential protein expression of glycolytic and glycogenic enzymes during in vitro preimplantation bovine embryo development

2018 ◽  
Vol 30 (9) ◽  
pp. 1245 ◽  
Author(s):  
Manuel García-Herreros ◽  
Constantine A. Simintiras ◽  
Patrick Lonergan
Keyword(s):  
Embryo Development ◽  
Glucose Transporter ◽  
Developmental Stages ◽  
Glycogen Synthase ◽  
Differentially Expressed ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Glucose Transporter 1 ◽  
Glut 1

Proteomic analyses are useful for understanding the metabolic pathways governing embryo development. This study investigated the presence of enzymes involved in glycolysis and glycogenesis in in vitro-produced bovine embryos at five developmental stages leading up to blastocyst formation. The enzymes examined were: (1) glycolytic: hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase mutase 1/2 (PKM-1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (2) glycogenic: glycogen synthase kinase-3 isoforms α/ β (GSK-3α/β). Glucose transporter-1 (GLUT-1) was also analysed. The developmental stages examined were: (1) 2–4-cell, (2) 5–8-cell, (3) 16-cell, (4) morula and (5) expanded blastocyst. The enzymes HK-I, PFK-1, PKM-1/2, GAPDH and GLUT-1 were differentially expressed throughout all stages (P < 0.05). GSK-3α and β were also differentially expressed from the 2–4-cell to the expanded blastocyst stage (P < 0.05) and GLUT-1 was identified throughout. The general trend was that the abundance of PFK1, GAPDH and PKM-1/2 decreased whereas HK-I, phospho-GSK3α (P-GSK3α) and P-GSK3β levels increased as the embryo advanced. In contrast, GLUT-1 expression peaked at the 16-cell stage. These data combined suggest that in vitro bovine embryo metabolism switches from being glycolytic-centric to glycogenic-centric around the 16-cell stage, the developmental window also characterised by embryonic genome activation.

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