scholarly journals Bovine embryo-oviduct interaction in vitro reveals an early cross talk mediated by BMP signaling

Reproduction ◽  
2017 ◽  
Vol 153 (5) ◽  
pp. 631-643 ◽  
Author(s):  
Elina V García ◽  
Meriem Hamdi ◽  
Antonio D Barrera ◽  
María J Sánchez-Calabuig ◽  
Alfonso Gutiérrez-Adán ◽  
...  
Keyword(s):  
Bone Morphogenetic Proteins ◽  
Relative Abundance ◽  
Cross Talk ◽  
Early Embryo ◽  
Bmp Signaling ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Signaling Components

Signaling components of bone morphogenetic proteins (BMPs) are expressed in an anatomically and temporally regulated fashion in bovine oviduct. However, a local response of this signaling to the presence of the embryo has yet to be elucidated. The aim of the present study was to evaluate if early embryo-oviduct interaction induces changes in the gene expression of BMP signaling components. For this purpose, we used an in vitro co-culture system to investigate the local interaction between bovine oviductal epithelial cells (BOEC) from the isthmus region with early embryos during two developmental periods: before (from the 2-cell to 8-cell stage) or during (from the 8-cell to 16-cell stage) the main phase of embryonic genome activation (EGA). Exposure to embryos, irrespective of the period, significantly reduced the relative abundance of BMPR1B, BMPR2, SMAD1, SMAD6 and ID2 mRNAs in BOEC. In contrast, embryos that interacted with BOEC before EGA showed a significant increase in the relative abundance of SMAD1 mRNA at the 8-cell stage compared to embryos cultured without BOEC. Moreover, embryos at the 16-cell stage that interacted with BOEC during EGA showed a significant increase in BMPR1B, BMPR2 and ID2 mRNA. These results demonstrate that embryo-oviduct interaction in vitro induces specific changes in the transcriptional levels of BMP signaling, causing a bidirectional response that reduces the expression levels of this signaling in the oviductal cells while increases them in the early embryo. This suggests that BMP signaling pathway could be involved in an early cross talk between the bovine embryo and the oviduct during the first stages of development.

scholarly journals Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo

Reproduction ◽  
2019 ◽  
Vol 158 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Meriem Hamdi ◽  
María J Sánchez-Calabuig ◽  
Beatriz Rodríguez-Alonso ◽  
Sandra Bagés Arnal ◽  
Kalliopi Roussi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Metabolic Response ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Transcriptomic Response ◽  
Early Embryos ◽  
Oviduct Epithelial Cells

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

83 BONE MORPHOGENETIC PROTEIN SIGNALING DURING INTERACTION OF THE BOVINE EMBRYO WITH OVIDUCTAL EPITHELIAL CELLS IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 149
Author(s):  
E. V. García ◽  
M. Hamdi ◽  
A. D. Barrera ◽  
M. J. Sánchez-Calabuig ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mrna Expression ◽  
Relative Abundance ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Expression Levels ◽  
Bmp Receptors ◽  
Bmp Signalling ◽  
Mrna Expression Levels

In previous studies, we have demonstrated that different signalling components of bone morphogenetic proteins (BMP) are expressed in an anatomically and temporally regulated fashion in the bovine oviduct. However, a local response of this signalling to the embryo presence has not been elucidated yet. The aim of the present study was to evaluate whether the interaction of the embryo with the oviduct can induce changes in the gene expression of BMP signalling components. For this purpose, we used an in vitro co-culture system of a bovine oviducal epithelial cell (BOEC) monolayer with pre-implantation embryos in 2 developmental time points: before and during the main phase of embryonic genome activation (EGA). Isthmus epithelial cells from post-ovulatory stage oviducts (Day 2–4) were cultured in 500 μL of SOF + 10% FCS in 4-well plates at 38.5°C, 5% CO2, 5% O2, and 90% N2. On Day 6 of culture, medium was replaced with SOF + 5% FCS, and 24 h later BOEC monolayer was cultured in the absence or presence of in vitro-produced embryos from 2- to 8-cell stage [G1 BOEC; 33–54 h post-insemination (hpi)] or from 8- to 16-cell stage (G2 BOEC; 54–98 hpi) in the same conditions. In both groups, a polyester mesh was used to define a local co-culture area, and 30 embryos per well were placed in a 6 × 5 grid over the monolayer. In addition, as control groups, embryos in both developmental stages were cultured either in SOF + 5% FCS (G1 FCS and G2 FCS) or in SOF + 3 mg mL−1 BSA (G1 BSA and G2 BSA). At 54 hpi (G1 BOEC/BSA/FCS) or 98 hpi (G2 BOEC/BSA/FCS), embryos that reached 8- or 16-cell stage, respectively, were transferred to SOF + BSA and cultured until Day 9. The mRNA expression levels of 3 BMP receptors (BMPRIA/IB/II), 2 signalling proteins (SMAD1/5), 1 inhibitor (SMAD6), and 1 target gene (ID2) were analysed by qPCR in 5 samples of BOEC cultured with or without embryos before or during EGA, and in 3 pools of 10 embryos at 8 (54 hpi), 16 (98 hpi), and blastocyst stage (Day 7–8) from all groups. Genes H2A.Z and ACTG1 were used as housekeeping genes, and statistical differences were assessed by ANOVA. The presence of the embryo, irrespective the stage, significantly reduced the expression levels of BMPRIB, BMPRII, SMAD1, SMAD6, and ID2 in BOEC. Embryos that interacted with BOEC before EGA (G1 BOEC) showed a significant increase in the relative abundance of SMAD1 at the 8-cell stage compared with controls. Moreover, embryos that interacted with BOEC during EGA (G2 BOEC) showed a significant increase in the relative abundance of BMPRIB, BMPRII, and ID2 at the 16-cell stage when compared with controls. However, no differences were observed in the mRNA expression levels of BMP signalling components in the blastocysts between groups. In conclusion, local embryo-oviduct interaction in vitro induces changes in the transcriptional levels of BMP signalling, causing a bidirectional response that reduces the expression levels of this signalling in the oviducal cells while increases them in the embryo at early stages. This suggests that BMP signalling pathway could be involved in an early cross-talk between the bovine embryo and the oviduct during first stages of development.

Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro

2021 ◽  
Author(s):  
Yulia N Cajas ◽  
Karina Cañón-Beltrán ◽  
Carolina Núñez-Puente ◽  
Alfonso Gutierrez-Adán ◽  
Encina M González ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Developmental Effect ◽  
Promote Cell Survival ◽  
Development In Vitro

Abstract During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2 to 8-cell stage, MNEGA) or major (8 to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 μM AKT-InhIII; 10 μM AKT-InhIV; 10 μM nobiletin; nobiletin+AKT-InhIII; nobiletin+AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors which infers that nobiletin probably uses another signalling cascade that PI3K/AKT during early embryo development in bovine.

scholarly journals Profile and regulation of annexin II expression during early embryogenesis in cattle

2007 ◽  
Vol 59 (6) ◽  
pp. 1493-1499
Author(s):  
L.F.S. Costa ◽  
M.S.N Machado ◽  
J.F.C. Oliveira ◽  
J.C. Silva ◽  
R.S. Loguercio ◽  
...  
Keyword(s):  
Embryo Development ◽  
Early Stage ◽  
Early Embryo ◽  
Annexin Ii ◽  
Control Group ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Igf I ◽  
Mrna Extraction

The presence of annexin II (Ann-II) during the initial stages of bovine embryo development and the regulation of Ann-II expression by retinol and insulin-like growth factor I (IGF-I) were studied. Bovine embryos at different stages of development were produced in vitro on Synthetic Oviductal Fluid (SOF) medium (control group), SOF supplemented with retinol (retinol group; 0.1ng/ml), or IGF-I (IGF-I group; 10ng/ml). The embryos were processed for mRNA extraction, cDNA production and polymerase chain reaction (PCR) using Ann-II-specific oligonucleotides. Ann-II was detected in all stages of early embryo development, except for the 16-cell stage. The blastocyst rates were significantly higher (P<0.05) in the group supplemented with retinol (37.8%, 45/119) during in vitro embryo culture (IVC) than in those cultured in SOF (20.5%, 24/117) or SOF with IGF-I (25.8%, 24/93). Semiquantitative analysis of Ann-II expression in embryos produced in medium supplemented with IGF-I or retinol revealed a lower expression of this gene when compared with embryos cultured in SOF (P<0.05). The Ann-II expression was not different in embryos cultured in the presence of retinol and IGF-I. The presence of retinol increased the production of embryos in vitro by decreasing the expression of Ann-II in early-stage of bovine embryo.

scholarly journals Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage

Reproduction ◽  
2012 ◽  
Vol 144 (3) ◽  
pp. 349-359 ◽  
Author(s):  
Tereza Toralová ◽  
Veronika Benešová ◽  
Kateřina Vodičková Kepková ◽  
Petr Vodička ◽  
Andrej Šušor ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genome Activation

This study was conducted to investigate the effect of silencing nucleophosmin in the development of in vitro-produced bovine embryos. Nucleophosmin is an abundant multifunctional nucleolar phosphoprotein that participates, for example, in ribosome biogenesis or centrosome duplication control. We showed that although the transcription of embryonic nucleophosmin started already at late eight-cell stage, maternal protein was stored throughout the whole preimplantation development and was sufficient for the progression to the blastocyst stage. At the beginning of embryogenesis, translation occurs on maternally derived ribosomes, the functionally active nucleoli emerge during the fourth cell cycle in bovines. We found that nucleophosmin localisation reflected the nucleolar formation during bovine preimplantation development. The protein was detectable from the beginning of embryonic development. Before embryonic genome activation, it was dispersed throughout the nucleoplasm. The typical nucleolar localisation emerged with the formation of active nucleoli. At the blastocyst stage, nucleophosmin tended to localise especially to the trophectoderm. To see for how long is maternal nucleophosmin preserved, we silenced the nucleophosmin mRNA using RNA interference approach. Although a large portion of nucleophosmin was degraded in embryos with silenced nucleophosmin mRNA, an amount sufficient for normal development was preserved and we detected only a temporal delay in nucleophosmin relocalisation to nucleoli. Moreover, we observed no defects in nuclear shape or cytoskeleton previously found in somatic cells and only a non-significant decrease in embryonic developmental competence. Thus, our results show that the preserved amount of maternal nucleophosmin is sufficient for preimplantation development of bovine embryo.

Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription

Development ◽  
1995 ◽  
Vol 121 (1) ◽  
pp. 113-122 ◽  
Author(s):  
E. Christians ◽  
E. Campion ◽  
E.M. Thompson ◽  
J.P. Renard
Keyword(s):  
Dna Replication ◽  
Mouse Embryo ◽  
Culture Conditions ◽  
Hsp 70 ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genome Activation ◽  
Alpha Amanitin ◽  
Zygotic Genome

Activation of the mouse embryonic genome at the 2-cell stage is characterized by the synthesis of several alpha-amanitin-sensitive polypeptides, some of which belong to the multigenic hsp 70 family. In the present work we show that a member of this family, the HSP 70.1 gene, is highly transcribed at the onset of zygotic genome activation. Transcription of this gene began as early as the 1-cell stage. Expression of the gene continued through the early 2-cell stage but was repressed before the completion of the second round of DNA replication. During this period we observed that the level of transcription was modulated by in vitro culture conditions. The coincidence of repression of HSP70.1 transcription with the second round of DNA replication was not found for other transcription-dependent polypeptides synthesized at the 2-cell stage.

The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Embryo Development ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Early Embryo ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

193 DIFFERENTIAL EFFECTS OF CULTURE ON APOPTOTIC GENE EXPRESSION IN THE PRE-IMPLANTATION CLONED EMBRYOS OF MINIATURE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 213
Author(s):  
M. R. Park ◽  
I. S. Hwang ◽  
H. J. Moon ◽  
J. H. Shim ◽  
D. H. Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Total Concentration ◽  
Control Group ◽  
Specific Cell ◽  
Miniature Pig ◽  
Cell Stage ◽  
Apoptotic Gene ◽  
Early Embryos

Manipulations of early embryos require that the embryos be placed in vitro. The ability to reproduce in vivo conditions in vitro would greatly facilitate studies on the development of early embryos. A variety of different conditions have been described that result in development of pig embryos from the 1-cell stage to the blastocyst stage in vitro. There is a species-specific cell stage at which the early embryo is very sensitive to in vitro conditions, which generally corresponds to the stage at which the embryo begins producing significant amounts of RNA. The present study was conducted to investigate the relative amounts of apoptotic gene expression in miniature pig NT embryos under culture conditions of different osmolarity. Oocytes were cultured in TCM-199 for 40–44 h at 38.5�C under 5% CO2 in air. Miniature pig ear fibroblast cells were cultured to reach confluency, and the culture was continued for an additional 5–6 days. The NaCl group of embryos was cultured in PZM-3 supplemented with 138 mM NaCl in total concentration (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for a further 4 days. The control group of embryos was cultured in the PZM-3 for the entire period of in vitro culture. Total RNA samples were prepared from 2 blastocysts using the Roche 1st strand cDNA synthesis kit. Bax and Bcl-xl gene expression of blastocysts was analyzed by real-time RT-PCR. Developemntal rates were analyzed by a GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Relative gene expression was compared by Student&apos;s t-test. Blastocyst formation rate in the NaCl group was not different from that in the control group (25.4% and 23.2%, respectively), but the apoptosis rate was significantly lower (P &lt; 0.05) in the NaCl group (1.6%) than in the control (7.1%). The relative abundance of Bax mRNA expression was significantly higher (P &lt; 0.05) in the control group (n = 32) than in the NaCl group (n = 33). However, the relative abundance of Bcl-xl mRNA was significantly higher (P &lt; 0.05) in NaCl group. The relative abundance of Bax/Bcl-xl was significantly higher in the control group than in the NaCl group (P &lt; 0.05). These results indicate that the hypertonic culture condition at the early embryonic stage of miniature pig NT embryos could reduce the frequency of apoptosis through regulating Bax and Bcl-xl gene expression.

83 STAGE-SPECIFIC PROTEOME SIGNATURES IN EARLY BOVINE EMBRYO DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 134
Author(s):  
D. R. Deutsch ◽  
T. Fröhlich ◽  
K. A. Otte ◽  
A. Beck ◽  
F. A. Habermann ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Principal Component ◽  
Proteome Analysis ◽  
Early Embryogenesis ◽  
Bovine Embryo ◽  
Protein Abundance ◽  
Absolute Quantitation ◽  
Cell Stage ◽  
Proteolytic Activation

Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome and adequate activation, deactivation, and relocation of proteins. To establish protein function, several posttranslational events (e.g. proteolytic activation, phosphorylation, or secretion) are frequently essential and thereby prevent prediction of protein abundance from transcript abundance. Consequently, proteomic studies are indispensable to characterise the molecular processes governing early embryonic development and to establish corresponding regulatory networks. Here, we present a quantitative proteome analysis of bovine zygotes and embryos at the 2-cell and 4-cell stage. Cumulus-oocyte complexes (COC) were prepared from bovine ovaries obtained from a local abattoir and selected for a compact layer of cumulus cells. In vitro maturation, fertilization, and embryo production were performed according to standard procedures. For quantitative isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, protein from batches of 50 MII oocytes (serving as a reference), zygotes, 2-cell and 4-cell stage embryos, respectively, was extracted. Quantitative proteome analysis of iTRAQ-labelled tryptic peptides was performed on an Orbitrap XL instrument (Thermo Fisher, Waltham, MA, USA) coupled to an Eksigent nano-liquid chromatography system (AB Sciex, Framingham, MA, USA). The tandem MS data were analysed by MASCOT and filtered for a false discovery rate (FDR) of <1%. Quantification of iTRAQ signals was accomplished with the Q+ module of the Scaffold software (Proteome Software Inc., Portland, OR, USA). t-Tests, ANOVA and principal component analysis (PCA) analysis were performed using R (R Core Development Team, Vienna, Austria). From 4 biological replicates, 1072 proteins were identified and quantified. Eighty-seven differed significantly in abundance between the 4 stages (log2 fold change ≥ |0.6|, P ≤ 0.05). The proteomes of 2-cell and 4-cell embryos differed most from the reference MII oocyte, and a considerable fraction of proteins continuously increases in abundance during the stages analysed. Bioinformatic analysis of abundance altered proteins provided evidence that the proteins RPS14 and HNRNPK involved in the p53 pathway play a major role during early development, as well as proteins of the lipid metabolism, in particular APOA1. Furthermore, a group of proteins (e.g. SPTBN1, PPP1CC, RABGAP1, STMN1, and WEE2) is engaged in mitosis. In addition, we detected relevant differences between transcript and protein abundance levels; for example, for WEE2. In conclusion, this study identified and quantified numerous proteins important for early embryogenesis so far not described in the mammalian system, and contributed protein profiles for key players previously described. Our results highlight the importance of innovative proteomic tools and workflows to complement transcriptome data of early embryogenesis.

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