scholarly journals 307CHARACTERIZATION OF PROTEIN PHOSPHORYLATIONS IN THE COURSE OF MEIOTIC MATURATION OF BOVINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 273
Author(s):  
M. Bhojwani ◽  
M. Marx ◽  
F. Melo-Sterza ◽  
W. Kanitz ◽  
C. Leiding ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
Meiotic Maturation ◽  
Detailed Knowledge ◽  
Phosphorylated Proteins ◽  
Stage Of Development ◽  
Peptide Mass ◽  
Fluorescence Dye ◽  
Bovine Oocytes

The importance of protein phosphorylations during meiotic maturation (transition from prophase I to metaphase II) of oocytes is documented by the fact that the inhibition of the M-phase kinases, cdc2k or MAPK, arrests the oocytes in the GV stage. A detailed knowledge of the targets of these kinases during this stage of development is still missing. Therefore, we have analyzed the proteome of bovine oocytes by high resolution 2D-gel electrophoresis to detect differences in the expression and phosphorylation state of proteins in the course of in vitro maturation (IVM). Bovine oocytes were matured for different times in TCM 199 containing 3% BSA and 300 oocytes each in GV stage (0-h maturation), in GVBD/M I (10-h maturation) or in M II stage (240h maturation) were separated on the gels. The proteins were visualized by staining them with silver or with the fluorescence dye Sypro Ruby, and phosphorylated proteins were detected by Western Blotting with Ser-, Thr-, or Tyr-phosphorylation specific antibodies or by staining with the phosphoprotein specific fluorescence dye Pro-Q Diamond. Gels made from oocytes at the above mentioned maturation stages were compared by a computerized gel-overlay software program (2D Decodon, Greitswald, Germany). The overall protein synthesis was statistically analysed by ANOVA (SigmaStat, Ekrath, Germany), pairwise multiple comparison procedure. Only distinct spots with a difference greater than 30% in their optical densities were considered to be differently expressed or phosphorylated. The results showed a three-fold increase in the rate of overall protein synthesis (p 0.05) during GVBD. Newly synthesized proteins were detected mainly in the higher molecular weight (MW) range (60–80kDa), and protein degradations were found mainly in the lower MW range (20–40kDa) after GVBD. Preliminary data obtained by analyzing the phosphorylation pattern showed that obviously no phosphorylated proteins could be detected in the GV-stage oocytes. Phosphorylation of different proteins was observed at the time of GVBD after 6 to 10h IVM, concomitantly with the activation of cdc2k and MAPK. A maximum of phosphorylated proteins was observed in metaphase II. The first results obtained by performing peptide mass fingerprinting using MALDI-Tof showed that members of the family of heat-shock proteins, ribosomal proteins and putative zinc finger proteins (transcription regulators) were differently expressed or phosphorylated during IVM. This work was supported by the DFG, To 178/1-1, 2 and by the Eibl-Stiftung.

scholarly journals Aspects of cytoplasmic maturation of bovine oocytes: Interplay between mapk, mRNA-cap binding complex and cytoplasmic mRNA metabolism in regulation of translation

2003 ◽  
Vol 19 (3-4) ◽  
pp. 1-8 ◽  
Author(s):  
Tatjana Smiljakovic ◽  
Melo Sterza ◽  
M. Kubelka ◽  
Z. Vohnikova ◽  
W. Tomek
Keyword(s):  
Binding Protein ◽  
Germinal Vesicle ◽  
Initiation Factor ◽  
Meiotic Maturation ◽  
Detailed Knowledge ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Growth ◽  
Stage Of Development ◽  
Bovine Oocytes

Bovine oocytes are arrested in the germinal vesicle stage (GV stage)and mature spontaneously when they are removed from their follicles and transferred to a suitable culture medium. This process, known as meiotic maturation is characterized among others, by germinal vesicle breakdown followed by metaphase I (MI) stage and further development to metaphase II (MII), where they become arrested again. During GVBD to MI transition, the overall protein synthesis reaches the highest level and it rapidly declines in MII. We have previously shown that transcription completely declines during meiotic maturation. Therefore we suppose that gene expression is exclusively regulated on translational level at this stage of development. This means that mRNAs, which were stored in repressed form during oocyte growth, were actively translated during meiotic maturation. Therefore we have investigated specific regulators of translation, namely the eukaryotic initiation factor of translation eIF4E (cap binding protein) and a specific repressor of eIF4E function, the 4E-binding protein 4E-BP1. Furthermore, we have elucidated pathways, which lead to eIF4E and 4E-BP1 phosphorylation by using specific M-phase kinase inhibitors, and we compare these results with transcription and cytoplasmic polyadenylation events during the course of meiotic maturation. The detailed knowledge of such regulatory processes can help to improve in vitro bio-techniques and to estimate the risk of these techniques.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

scholarly journals Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts fromIn VitroMaturation of Bovine Oocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jolanta Opiela ◽  
Joanna Romanek ◽  
Daniel Lipiński ◽  
Zdzisław Smorąg
Keyword(s):  
Dna Fragmentation ◽  
Oocyte Maturation ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
The Mean ◽  
Bovine Oocytes

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were maturedin vitroin control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higherBaxmRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

In Vitro Fertilization of Bovine Oocytes Matured In Vitro

1997 ◽  
Vol 1987 ◽  
pp. 28-28
Author(s):  
K.H. Lu ◽  
I. Gordon ◽  
M.P. Boland ◽  
T.F. Crosby
Keyword(s):  
Embryo Transfer ◽  
Normal Development ◽  
Present Report ◽  
Blastocyst Stage ◽  
Laboratory Procedure ◽  
Commercial Exploitation ◽  
Vitro Fertilization ◽  
Stage Of Development ◽  
Bovine Oocytes

The development of an efficient laboratory procedure which would enable cattle ovarian oocytes to be matured in vitro, fertilized and cultured in vitro to the blastocyst stage of development could have important practical and scientific implications. The commercial exploitation of certain embryo transfer techniques applicable in cattle (eg., twinning by embryo transfer) might be facilitated by the development of such a procedure and there would be many advantages to having a cheap source of embryos available for research purposes. The present report deals with some of the studies recently carried out in this laboratory aimed at utilising follicular oocytes recovered from the ovaries of cattle slaughtered for beef at the abattoir. Such studies have been undertaken over a period of almost twenty years, starting with the work of Sreenan (1968)* but it now realised that the oocytes of farm mammals are incapable of normal development until after the completion of complex changes during maturation.

scholarly journals THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

1972 ◽  
Vol 54 (1) ◽  
pp. 56-74 ◽  
Author(s):  
Paul M. Lizardi ◽  
David J. L. Luck
Keyword(s):  
Protein Synthesis ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Mitochondrial Protein ◽  
Selective Inhibitor ◽  
Mitochondrial Protein Synthesis ◽  
Ribosomal Subunits

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.

scholarly journals P11.36 Ribosome hydroxylase Mina53 is required for Glioblastoma and is involved in regulation of translation rateand fidelity by regulating ribosomal biogenesis

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii51-iii51
Author(s):  
D Pandey ◽  
F Mohammad ◽  
S Weissmann ◽  
P Hallenborg ◽  
B Blagoev ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
High Throughput Sequencing ◽  
Affinity Purification ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Ribosomal Biogenesis ◽  
Isolation And Identification ◽  
Knock Down

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive types of tumors with a poor response to standard treatment and a median 5-year survival of less than 5%. Therefore, there is an urgent need for new treatments. Recently, a large number of genome-wide studies have shown that the epigenetic modifiers are frequently deregulated in cancer. Using a mouse GBM model, we performed in vitro and in vivo shRNA screens to identify epigenetic regulators required for the tumorigenic process in GBM. Among these regulators is a ribosome hydroxylase Mina53 which hydroxylates His-39 of ribosomal protein, RPL27a. We have found that the knock-down (KD) of Mina53 reduces the in vitro proliferation and colony forming ability of mouse glioma initiating cells (mGIC) and this is dependent on the catalytic activity of Mina. Knock-down of Mina resulted into a small but significant reduction in the global protein synthesis rate. A tandem affinity purification experiment to identify proteins associated with Mina revealed that it is associated mainly with ribosomal proteins, including its substrate RPL27a. Global proteomic analyses revealed that final amounts and de novo protein synthesis of many ribosomal proteins were reduced upon Mina depletion. Isolation and identification of different polysome fraction bound mRNAs using high-throughput sequencing found that mRNAs encoding many ribosomal proteins have lower number of ribosomes loaded on them in the Mina depleted samples compared to the control. Taken together, this study has found that Mina53 is required for glioblastoma and it regulates translation through regulation of ribosomal biogenesis

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