59 Selected Reaction Monitoring-Based Absolute Quantification of Developmentally Relevant Proteins in Early Bovine Embryos Reveals Differences Between In Vitro and In Vivo Embryo Culture and Between Different Maternal Metabolic Stages

2018 ◽  
Vol 30 (1) ◽  
pp. 168
Author(s):  
G. J. Arnold ◽  
K. Gegenfurtner ◽  
T. Frohlich ◽  
D. R. Deutsch ◽  
P. Salvetti ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Developmental Process ◽  
Absolute Quantification ◽  
Early Embryogenesis ◽  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Cell Stage ◽  
Cell Potential

Early embryogenesis is a highly complex developmental process, accompanied by a plethora of changes at the morphological and molecular level. Particularly at the level of proteins, these changes are still poorly characterised and understood. During the first cleavages, the embryo depends mainly on maternal transcripts and proteins that were accumulated and stored during oogenesis until embryonic genome activation (EGA) occurs. In the bovine system, the major EGA takes place at the 8- to 16-cell stage. However, we recently demonstrated by liquid chormatography-tandem mass spectrometry (LC-MS/MS)-based holistic proteome approaches that despite transcriptional and translational silencing, the proteome of the early embryo is highly dynamic (Deutsch et al. 2014; Demant et al. 2015). Based on these findings, we established a targeted LC-MS/MS approach based on multiplexed selected reaction monitoring (mSRM), which facilitates an absolute quantification of 27 proteins relevant in early embryogenesis. Each protein is targeted by 2 independent peptides to facilitate highly reliable quantifications. Nine characteristic developmental stages from germinal vesicle oocyte to hatched blastocyst were analysed (n = 6 per stage), and absolute protein contents are reported as femtomole per embryo, with limits of quantification (LOQ) down to 100 attomoles per embryo. Based on their abundance profiles during maturation, zygote formation, and embryonic development, the 27 proteins could be grouped into 6 SOTA clusters. By principal component analysis (PCA), absolute SRM quantifications of only 9 selected proteins were shown to discriminate between all 9 developmental stages analysed, thus providing molecular fingerprints significant for each developmental stage. We used the 27-plex SRM assay as a powerful readout tool and demonstrated substantial quantitative differences between embryos derived from a well-established in vitro culture system and embryos transferred into the oviduct of living animals for 2 days (in vivo culture). Furthermore, in vivo development of embryos in animals differing in their metabolic stress levels led to significant alterations in the 27-plex SRM profiles. This work was supported by a grant to GJA from Deutsche Forschungsgemeinschaft DFG FOR1041 ‘Germ Cell Potential’ AR 362/7-1 and European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 312097 - FECUND.

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

scholarly journals A uterus-inspired 3D niche drives embryo development beyond implantation

2020 ◽  
Author(s):  
Zhen Gu ◽  
Jia Guo ◽  
Jinglei Zhai ◽  
Guihai Feng ◽  
Xianning Wang ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Developmental Process ◽  
High Rate ◽  
Mammalian Embryo ◽  
Uterine Endometrium ◽  
Experimental Systems ◽  
In Vitro Systems ◽  
Development In Vitro

Abstract The mammalian embryo must undergo dramatic morphogenetic changes to invade the uterine endometrium and achieve implantation. Thus, recapitulation of implantation using in vitro systems is crucial for revealing the mechanisms controlling early development and the main problems compromising human fertility. Experimental systems based on two-dimensional (2D) platforms cannot fully recapitulate the in vivo 3D microenvironments of the embryo. Therefore, here we use collagen grafted onto polydimethylsiloxane (PDMS) based on the uterine mechanics and microstructure to establish a uterus-inspired 3D niche (U3N). Our U3N enables mouse embryos to form egg cylinders at high rate and reach the developmental stages of heartbeat. Moreover, a unique interface forms between the embryo and collagen, showing the invasion of trophoblasts into collagen fires, which simulate the developmental process of implantation. Our findings highlight embryo-substrate interaction as a key characteristic of post-implantation development in vitro and as an important design parameter of 3D conditions for embryo culture.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

2008 ◽  
Vol 56 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Chang-Liang Yan ◽  
Qi-En Yang ◽  
Guang-Bin Zhou ◽  
Yun-Peng Hou ◽  
Xue-Ming Zhao ◽  
...  
Keyword(s):  
Survival Rate ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Significant Loss ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Fresh Embryos

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3–100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8–89.5%) and hatched blastocyst rates (61.1–69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3–30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

246 FUNCTIONAL ANALYSIS OF Sebox DURING OOCYTE MATURATION AND EARLY EMBRYOGENESIS BY RNAi

2008 ◽  
Vol 20 (1) ◽  
pp. 202
Author(s):  
K. H. Kim ◽  
E. Y. Kim ◽  
H. S. Lee ◽  
K. A. Lee
Keyword(s):  
Oocyte Maturation ◽  
Developmental Stages ◽  
Homeobox Gene ◽  
Germinal Vesicle ◽  
Basic Research ◽  
Early Embryogenesis ◽  
Rnai Phenotype ◽  
Dot Blot ◽  
Cell Stage

Previously, we found that the skin-embryo-brain-oocyte homeobox (Sebox) gene was highly expressed in germinal vesicle (GV)-stage oocytes. The function of Sebox is unknown as yet; thus the objective of this study was to determine the role(s) of Sebox during oocyte maturation and early embryogenesis using RNA interference (RNAi). Cumulus-free GV oocytes were collected from 4-week-old ICR mice 48 h after pregnant mare serum gonadotropin (PMSG) injection. The expression pattern of Sebox mRNA was evaluated in various tissues, including gonads, oocytes, and embryos at different developmental stages. To determine the role of Sebox in oocyte maturation and preimplantation embryonic development, Sebox dsRNA was microinjected into the cytoplasm of GV oocytes and pronuclear (PN)-stage embryos, respectively. After Sebox RNAi, phenotype and maturation rates were recorded prior to measuring changes in Sebox mRNA and protein concentration. RNA content was determined by RT-PCR and protein content was determined by oocyte dot blot. Anti-Sebox antibody was produced against a 14 amino acid synthetic peptide corresponding to residues 67–80. Additionally, chromosomal status of the oocytes was confirmed by orcein staining, while spindle shape was confirmed by immunofluorescence staining. Sebox mRNA was ubiquitous in adult mice tissues, with relatively high expression in the brain and ovary. Expression of Sebox was detected in oocytes, granulosa cells, and theca cells. Sebox mRNA was highly expressed from GV up to 2-cell-stage embryos, but dramatically decreased afterward in later developmental stages. Sebox dsRNA microinjection into GV oocytes resulted in a markedly decreased Sebox mRNA (80%) and protein (70%) expression. However, Sebox RNAi resulted in rates in MII oocytes (82%) similar to that of control (87%) and the buffer injection (80%) group during in vitro maturation. Sebox RNAi did not affect the spindle and chromosomal organizations of the MII oocytes. But microinjection of Sebox dsRNA into PN embryos inhibited preimplantational embryo development and blocked at the 2-cell stage (79%). Results suggest that the Sebox gene is not related to the regulation of oocyte nuclear maturation in mice. However, we concluded that Sebox is a new entry of maternally expressed homeobox gene that is involved in the regulation of early embryogenesis, especially at the 2-cell block. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-311-E00067).

Consumption of amino acids by bovine preimplantation embryos

1996 ◽  
Vol 8 (6) ◽  
pp. 945 ◽  
Author(s):  
RJ Partridge ◽  
HJ Leese
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Amino Acid Requirement ◽  
Zygote Stage

Bovine embryos produced in vitro from the putative zygote stage to the blastocyst stage, and blastocysts freshly flushed from the uterus, were cultured in a physiological mixture of amino acids. Depletion of amino acids from the medium and, in a few cases, their appearance, was measured by high performance liquid chromatography. Amino acids were depleted at widely differing rates. The depletion of amino acids was higher when embryos at later developmental stages were cultured, implying an increase in amino acid requirement with development. Threonine was the only amino acid to be depleted at all stages of development; depletion increased from 0.18 +/- 0.07 pmol embryo-1 h-1 at the putative zygote stage to 1.96 +/- 0.49 pmol embryo-1 h-1 at the blastocyst stage. Glutamine was depleted at the putative zygote stage and the 4-cell stage (0.76 +/- 0.05 and 0.94 +/- 0.10 pmol embryo-1 h-1 respectively), but was not significantly depleted at the later stages. Alanine was the only amino acid that appeared consistently in the medium and its production increased progressively throughout development. Aspartate, glutamate, threonine and lysine were depleted significantly by blastocysts derived both in vitro and in vivo; the embryos in vivo also depleted arginine, phenylalanine, isoleucine and tyrosine. These results indicate that individual amino acids are depleted at different rates by bovine preimplantation embryos and suggest that amino acid requirements change during development.

scholarly journals 252A COMPARATIVE EXPRESSION ANALYSIS OF GENES IN PREIMPLANTATION DEVELOPMENTAL STAGES OF BOVINE EMBRYOS PRODUCED IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
D. Tesfaye ◽  
K. Wimmers ◽  
M. Gilles ◽  
S. Ponsuksili ◽  
K. Schellander
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
The Novel ◽  
Cell Stage ◽  
Novel Transcript ◽  
Stage Of Development ◽  
Significant Difference

A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P&lt;0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P&lt;0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P&lt;0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P&lt;0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.

scholarly journals Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Shipra Kumari ◽  
Bashistha Kumar Kanth ◽  
Ju young Ahn ◽  
Jong Hwa Kim ◽  
Geung-Joo Lee
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Lilium Longiflorum ◽  
Biotic And Abiotic Stress ◽  
Biotic Stresses ◽  
Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

Glucose utilization by sheep embryos derived in vivo and in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 571 ◽  
Author(s):  
JG Thompson ◽  
AC Simpson ◽  
PA Pugh ◽  
RW Wright ◽  
HR Tervit
Keyword(s):  
Glucose Oxidation ◽  
Glucose Utilization ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stage ◽  
Glycolytic Activity ◽  
Total Utilization ◽  
Oxidation Of Glucose

Embryos were collected from superovulated donors at various intervals from onset of oestrus, ranging from Day 1.5 to Day 6. In addition, blastocysts obtained from the culture of 1-cell embryos collected in vivo or of oocytes matured and fertilized in vitro were used to assess the effects of in vitro manipulation and culture on glucose utilization. Glycolytic activity was determined by the conversion of [5-3H]glucose to 3H2O, and oxidation of glucose was determined by the conversion of [U-14C]glucose to 14CO2. Glucose utilization increases significantly from the 8-cell stage and during compaction and blastulation. Glucose oxidation was at a relatively low level (5-12% of total utilization) compared with glycolysis. No difference was observed between the glycolytic activity of blastocysts derived from in vivo or in vitro sources. However, glucose oxidation was lower (P less than 0.05) in blastocysts derived from the culture of 1-cell embryos or from oocytes matured and fertilized in vitro. Exogenous tricarboxylic acid cycle substrates (i.e. pyruvate and lactate supplied in the medium) affected the level of glucose oxidation.

scholarly journals Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Stefanie Schmitteckert ◽  
Cornelia Ziegler ◽  
Liane Kartes ◽  
Alexandra Rolletschek
Keyword(s):  
Transcription Factor ◽  
Developmental Stages ◽  
Troponin T ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cardiac Cells ◽  
Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

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