scholarly journals Function of JARID2 in bovines during early embryonic development

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4189 ◽  
Author(s):  
Yao Fu ◽  
Jia-Jun Xu ◽  
Xu-Lei Sun ◽  
Hao Jiang ◽  
Dong-Xu Han ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Embryonic Stem ◽  
Early Embryonic Development ◽  
Mouse Development ◽  
Expression Levels ◽  
Relative Expression ◽  
Bovine Oocytes

Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC) differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV), MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05), and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc) after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the efficiency ofin vitroembryo culture as well as a theoretical basis for further studying the regulatory mechanisms involved in early embryonic development.

144 Oct-4 EXPRESSION PATTERN IN THE EQUINE EMBRYO

2007 ◽  
Vol 19 (1) ◽  
pp. 189
Author(s):  
Y. H. Choi ◽  
H. D. Harding ◽  
A. D. Obermiller ◽  
K. Hinrichs
Keyword(s):  
Embryonic Development ◽  
Expression Pattern ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Cell Mass ◽  
Population Variation ◽  
Early Embryonic Development

Oct-4 is a key transcription factor in the control of early embryonic development and maintenance of a pluripotent cell population. Variation in Oct-4 expression patterns during embryo development have been reported among species, and have been related to the time of placental development in those species. This study was conducted to investigate Oct-4 expression pattern during early embryonic development in the horse, a species with relatively delayed placentation. In vitro-produced embryos were obtained from in vitro-matured oocytes via fertilization by intracytoplasmic sperm injection. Ex vivo blastocysts were recovered from mares that had been artificially inseminated. Oct-4 status was determined by immunocytochemistry; photomicrographs were taken at 4 standardized settings to aid in qualitative comparison of the amount of fluorescence. A total of 106 oocytes and embryos were evaluated. Immature oocytes showed Oct-4 expression in the nucleus and cytoplasm, as did early-cleaved embryos (2 to 5 cells, 1 to 2 days). Oct-4 expression in embryos at 3 to 4 days (6 to 12 cells) decreased and was restricted to the cytoplasm. From 5 to 6 days (15 cells to morulae), Oct-4 intensity increased and was exclusively found in the nuclei. In vitro-produced blastocysts (7 to 8 days) expressed Oct-4 equivalently in the trophectoderm and inner cell mass nuclei; culture for 2 to 3 more days (10 to 11 days) did not alter Oct-4 expression. However, when in vitro-produced blastocysts were transferred to the uteri of mares and recovered after 2 to 3 days (IVP-ET), the embryos showed strong expression of Oct-4 within the inner cell mass and limited expression in the trophectoderm, and a similar pattern was seen for ex vivo-recovered embryos. In bigger embryos (such as a 1779-�m ex vivo embryo and a 1121-�m IVP-ET embryo), the trophectoderm lost staining completely. These results suggest that Oct-4 expression is present in both nucleus and cytoplasm in equine oocytes and early-cleaved embryos as a result of maternal mRNA accumulation. Oct-4 protein decreases over the first few days of embryonic development as these stores are used. The shift to greater expression, in the nucleus only, during further embryo development suggests embryonic genome activation. Oct-4 expression in the trophectoderm of in vitro-produced blastocysts was different from that in blastocysts that had been exposed to the uterus (both ex vivo and IVP-ET); this indicates that differentiation of the trophectoderm is dependent upon factors present in the uterine environment. The Oct-4 expression in the trophectoderm of in vitro-produced equine blastocysts thus appears to be an artifact due to in vitro culture; this finding may be applicable to the reported patterns of Oct-4 expression in embryos of other species. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

MicroRNAs in Normal Human Terminal Granulocytic Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1463-1463
Author(s):  
Su Ming Sun ◽  
Menno K Dijkstra ◽  
André C Bijkerk ◽  
Rik Brooijmans ◽  
Peter J Valk ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Mirna Expression ◽  
Expression Patterns ◽  
Granulocytic Differentiation ◽  
Expression Levels ◽  
Mirna Expression Pattern ◽  
Normal Human

Abstract Abstract 1463 Poster Board I-486 Normal human myelopoiesis is a complex biological process, where the balance between cell proliferation, differentiation and apoptosis is tightly regulated by a transcriptional program that results in the production of appropriate numbers of circulating mature myeloid cells. MicroRNAs (miRNAs) are small non-coding RNAs of 18∼25 nt that can affect cellular protein levels. Several studies show specific miRNA expression patterns in different subtypes of myeloid malignancies, however only limited data is available on miRNA expression patterns during normal myeloid differentiation of primary human cells. We set out to characterize miRNA expression patterns in the different stages of granulocytic differentiation in two models. First myeloblast, promyelocytes, metamyelocytes and granulocytes from normal human bone marrow were cell-sorted with flow cytometry using the markers CD10, CD11, CD34, CD36, CD45 and CD117. Second, CD34+ cells from primary human fetal livers were differentiated in vitro towards neutrophils. MiRNA expression levels were determined at different time points (day 0, 3 and 10), representing different stages of granulocytic differentiation. MiRNA expression was measured using the qPCR platform, containing 365 miRNAs, from Applied Biosystems. To identify potential miRNA target genes, we performed mRNA expression profiling in the latter in vitro differentiation. The negative correlations between miRNA and mRNA expression were identified and integrated with a target prediction database (Targetscan). The miRNA profiling showed that approximately 70% of the 365 miRNAs analyzed, were expressed during granulocytic differentiation and that the miRNA expression pattern during this process change significantly in both models. Principal component analysis showed clear separation of the different subsets of granulopoiesis based on the miRNA expression. We determined the differentially expressed miRNAs between the various subsets using ANOVA with a P value <0.05, after correction for multiple testing. We found 24 miRNAs to be differentially upregulated in the both models. The top 5 upregulated miRNA, with the highest fold change in granulocytes as compared to myeloblasts, were miR-223, miR-145, miR-148, miR-24 and miR-23a. We identified 27 miRNAs that were downregulated, the top 5 were of miR-10a, miR-196a, miR-130a, miR-135a and miR-125b. Concomitant miRNA and mRNA expression analysis of the in vitro model with the Targetscan database, demonstrates a potential regulatory role for these miRNAs in various processes, such as cell proliferation, apoptosis and cell cycle regulation. For example, miR-130a, miR-20b and miR-191, miR-301 expression levels were negatively correlated with E2F2 and SOX4 respectively. Furthermore, MAPK1 levels correlated inversely with miR-17-5p, miR-130a, miR-181b, miR-181d and miR-20b. We observed potential regulation of BCL2L11 by miR-10a, miR-10b and CDK6 by miR-148a, miR-148b, miR-191 and miR-21, as well as CHEK1 by the miR-15a and miR-16, LATS2 by miR-142-3p and CCND3 by miR-133a. In addition we also identified myeloid specific genes to be potentially regulated by miRNAs such as CEBPA by miR-181b, KIT by miR-148a, miR-148b and miR-301 and RUNX3 by miR-301. This is the first comprehensive study of miRNA expression in normal human granulocytic differentiation. We show in two models that the miRNA expression pattern changes during granulocytic differentiation. miRNA-mRNA analyses suggest involvement of miRNAs in regulation of important cellular processes during granulocytic differentiation. Experimental validations of several candidate targets as well as functional studies are currently ongoing. Disclosures No relevant conflicts of interest to declare.

267 CLASSICAL AND NON-CLASSICAL MAJOR HISTOCOMPATABILITY COMPLEX CLASS I GENE EXPRESSION IN IN VITRO-DERIVED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 250
Author(s):  
J. Doyle ◽  
S. A. Ellis ◽  
P. Lonergan ◽  
T. Fair
Keyword(s):  
Gene Expression ◽  
Sequence Analysis ◽  
Mrna Expression ◽  
Complex Class ◽  
Mhc I ◽  
Expression Levels ◽  
Pcr Products ◽  
Bovine Oocytes ◽  
I Gene

The major histocompatibility complex class I (MHC-I) genes have been shown to play a role in the establishment of pregnancy in humans and mice. In particular, the secretion of the soluble isoform of the human non-classical HLA-G gene during in vitro embryo culture has been linked to higher pregnancy rates following embryo transfer. In contrast to the situation in humans, knowledge of the bovine MHC-I is quite limited. The objective of the current study was to characterize MHC-I gene expression during bovine pre-implantation development in vitro. Immature bovine oocytes were matured, fertilized, and cultured in vitro. Three replicate pools of 20 samples were collected at immature and mature oocyte, presumptive zygote, 2–4 cell, 8–16 cell, morula, blastocyst, and hatched blastocyst stages. Relative MHC-I mRNA expression levels were quantified using quantitative real-time PCR across pre-implantation development with a generic primer pair for global classical and non-classical MHC-I gene expression and primer pairs for two known non-classical MHC-I genes, N*50001 and N*50101 (NCBI accession numbers: X80936 and AY188807), cDNA sequencing, and wholemount immunocytochemistry with a Pan MHC-I-protein antibody (ILA88). MHC-I mRNA transcripts amplified by the generic primers were detected in greatest abundance in the oocyte and cumulus cells of immature oocytes (P &lt; 0.05); following maturation, the expression levels decreased by 5-fold but remained stable until the 2- to 4-cell stage. Levels remained low until the morula and blastocyst stages when a small increase in relative expression was detected. Sequence analysis of oocyte and blastocyst PCR products amplified by the generic primers was carried out. The generic primers amplified a total of 6 discrete sequences, which are listed on the bovine MHC database (http://www.ebi.ac.uk/ipd/mhc/bola): (i) N*02601, (ii) N*02301, (iii) N*01301, (iv) N*04001, (v) N*01601, and (vi) non-classical N*5003. The mRNA expression pattern of Gene X differed from the global MHC-I mRNA profile in that mRNA abundance was highest in the matured oocytes (P &lt; 0.05), after which abundance decreased 5-fold in the zygote and was not detected in the subsequent embryo stages until the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were Gene X. Relative HD15 mRNA expression was highest in immature oocytes (P &lt; 0.05); however, there was a steep decrease in abundance following oocyte maturation (P &lt; 0.05), followed by a transient increase at the presumptive zygote stage (P &lt; 0.05). Thereafter, expression levels remained low up to the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were HD15. MHC-I protein expression was detected in immature and mature oocytes and their surrounding granulosa cells, in 2- and 4-cell embryos, and in blastocysts. In conclusion, these data demonstrate that classical and non-classical MHC-I genes are expressed in bovine oocytes and embryos at both the mRNA and the protein levels, and the expression pattern is gene- and stage-specific.

Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 383 ◽  
Author(s):  
Jin-Tae Chung ◽  
Bruce R. Downey ◽  
Robert F. Casper ◽  
Ri-Cheng Chian
Keyword(s):  
Embryonic Development ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Early Embryonic Development ◽  
Ionophore A23187 ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation ◽  
Treatment Groups ◽  
Bovine Oocytes

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL–1 FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen–thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 M calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v. 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (10.8% v. 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca2+-ionophore. However, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10 000g and stimulation with calcium ionophore A23187 than in the control (18.4% v. 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be ≤7000g to enhance the visibility of nuclear elements for further micromanipulation.

scholarly journals The roles of ERAS during cell lineage specification of mouse early embryonic development

Open Biology ◽  
2015 ◽  
Vol 5 (8) ◽  
pp. 150092 ◽  
Author(s):  
Zhen-Ao Zhao ◽  
Yang Yu ◽  
Huai-Xiao Ma ◽  
Xiao-Xiao Wang ◽  
Xukun Lu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Embryonic Stem Cells ◽  
Embryonic Development ◽  
Expression Pattern ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Primitive Streak ◽  
Early Embryonic Development ◽  
Lineage Specification

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β -catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.

scholarly journals MLL3/MLL4 methyltransferase activities regulate embryonic stem cell differentiation independent of enhancer H3K4me1

2020 ◽  
Author(s):  
Guojia Xie ◽  
Ji-Eun Lee ◽  
Kaitlin McKernan ◽  
Young-Kwon Park ◽  
Younghoon Jang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Embryonic Development ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Enzymatic Activities ◽  
Early Embryonic Development ◽  
Specific Gene ◽  
Cell Type ◽  
Mouse Development ◽  
Cell Type Specific

Enhancers drive cell-type-specific gene transcription and are marked by H3K4me1. MLL4 (KMT2D), a major H3K4me1 methyltransferase with partial functional redundancy with MLL3 (KMT2C), is critical for enhancer activation and cell-type-specific gene induction during cell differentiation and development. However, the roles of MLL3/4-mediated enhancer H3K4me1 and MLL3/4 enzymatic activities in general in these processes remain unclear. Here, we report that MLL3/4 enzymatic activities are partially redundant during mouse development. Simultaneous elimination of both leads to embryonic lethality around E8.5. Using embryoid body (EB) differentiation as an in vitro model for early embryonic development, we show that Mll3 knockout MLL4 enzyme-dead embryonic stem cells (ESCs) are capable of differentiating towards the three germ layers but display severe cavitation defects, likely due to impaired induction of visceral endoderm. Importantly, MLL3/4-catalyzed H3K4me1 is dispensable for enhancer activation during early EB differentiation and lineage-specific neural differentiation. Together, these results suggest a critical, but enhancer H3K4me1-independent, role of MLL3/4 enzymatic activities in early embryonic development and ESC differentiation.

200 STAGE-SPECIFIC EXPRESSION PATTERNS OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 191 ◽  
Author(s):  
F. Poppicht ◽  
H. Stinshoff ◽  
C. Wrenzycki
Keyword(s):  
Growth Factor ◽  
Embryonic Development ◽  
Protein Expression ◽  
Mrna Expression ◽  
Early Embryonic Development ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Igf1r Expression

Insulin-like growth factor 1 (IGF1) is a key regulator in early embryonic development, influencing physiological processes and stimulating growth and development (Fowden et al. 2003). Supplementing IGF1 during in vitro culture of bovine embryos improved cleavage and developmental rates while it reduced apoptosis (Byrne et al. 2002). The signal transduction of IGF1 is performed by its binding to the insulin-like growth factor 1 receptor (IGF1R). At the mRNA level, IGF1R is expressed throughout pre-implantation embryonic development and was identified as a potential marker of good quality embryos (Yaseen et al. 2001). However, information on protein level is rare. Therefore, protein expression of the IGF1R during early embryonic development in vitro was analysed in the present study by immunofluorescence staining. Furthermore, the mRNA expression of the IGF1R was investigated by RT-qPCR. In vitro derived embryos of different stages (2-cell, 4-cell, 8-cell, 16-cell stage, morula, blastocyst, and expanded blastocyst) were either directly subjected to immunofluorescence staining or frozen at –80°C for use in RT-qPCR. Staining was performed with a peptide antibody against two peptide sequences of the bovine IGF1R α unit, which was specifically produced. Pixel intensity of immunofluorescence was measured and a mean grey value was calculated using the cellsens® software (Olympus, Hamburg, Germany). Data were analysed by one-way ANOVA followed by a Tukey's test using SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany). The detection of the IGF1R mRNA and protein was possible in all stages of embryonic development beginning at the 2-cell stage up to the expanded blastocyst. The maximal mRNA expression could be observed in 2- and 4-cell embryos. It significantly decreased to the 8-cell stage, followed by an increase up to the expanded blastocyst. The IGF1R protein was mainly localised in the plasma membrane of single blastomeres and also weakly in the cytoplasm. Mean grey values are highest in the 2-cell stage, showing a significant decline up to the 16-cell stage and an increase again until the expanded blastocyst. The mRNA and protein expression showed similar patterns during early embryonic development. IGF1R expression started to increase at the 8-cell stage (mRNA) and 16-cell stage (protein) indicating a link to the maternal-embryonic transition. For the first time, these results show that in bovine embryos, the IGF1R expression is related to the activation of the embryonic genome. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).

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