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.

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.

Differential cytolocalization of prosomes in axolotl during oogenesis and meiotic maturation

1988 ◽  
Vol 90 (4) ◽  
pp. 543-553 ◽  
Author(s):  
J. Gautier ◽  
J.K. Pal ◽  
M.F. Grossi de Sa ◽  
J.C. Beetschen ◽  
K. Scherrer
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Immunoblot Analysis ◽  
Ambystoma Mexicanum ◽  
Meiotic Maturation ◽  
Crescent Formation ◽  
Oocyte Growth ◽  
Oocyte Cytoplasm

The prosomes, a novel type of small RNA-protein complex previously characterized in avian and mammalian cells, were isolated from axolotl (Ambystoma mexicanum) oocytes and identified by sedimentation analysis and protein composition. The prosomal nature of these particles was further ascertained by immunoblot analysis with anti-duck prosome monoclonal antibodies. By in vitro [35S]methionine labelling, de novo synthesis of prosomal proteins could be detected neither during oogenesis nor meiotic maturation. The results obtained by both indirect immunofluorescence and immunoblot analyses demonstrated a dramatic change in the localization of prosomal antigens during oocyte development. They were initially detected in the oocyte cytoplasm, during oocyte growth. At the end of vitellogenesis (stages V-VI), they entered the nucleus (germinal vesicle) and were accumulated there to the highest concentration. During oocyte maturation, after nuclear envelope breakdown, prosomal antigens were found to be localized again in the cytoplasm, until fertilization. No specific localization of prosomal antigens in mature oocytes, unfertilized and fertilized eggs was observed within the oocyte cytoplasm in relation to the cytoplasmic rearrangements leading to grey crescent formation.

scholarly journals Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

Zygote ◽  
2003 ◽  
Vol 11 (2) ◽  
pp. 119-129 ◽  
Author(s):  
R.C. Chian ◽  
J.T. Chung ◽  
K. Niwa ◽  
M.A. Sirard ◽  
B.R. Downey ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Two Dimensional Gel Electrophoresis ◽  
Germinal Vesicle Breakdown ◽  
One Dimensional ◽  
Cell Cycle Transition ◽  
Pronuclear Formation ◽  
Bovine Oocytes

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

scholarly journals Surubim-do-Paraíba oocytes viability after being exposed to different cryoprotectants

2018 ◽  
Vol 48 (6) ◽  
Author(s):  
Taís da Silva Lopes ◽  
Eduardo Antônio Sanches ◽  
Danilo Caneppele ◽  
Elizabeth Romagosa
Keyword(s):  
Dimethyl Sulfoxide ◽  
Propylene Glycol ◽  
Germinal Vesicle ◽  
Blue Staining ◽  
Mature Stage ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes ◽  
Stage Of Development ◽  
Oocyte Stage

ABSTRACT: To know the non-toxic cryoprotectants to fish oocytes is of extreme importance for tests that aim to increase oocyte resistance to cold, thus allowing more advanced studies in cryopreservation. Therefore, commonly used cryoprotectants such as methanol, dimethyl sulfoxide, ethylene glycol, propylene glycol, sucrose and fructose were studied. Immature oocytes from the initial to vitelogenic (diameter <1.7 mm) and mature (diameter >1.8 mm) stages of Steindachneridion parahybae were evaluated. Four distinct experiments were performed, three using immature oocytes and one using oocytes at the mature stage. For each oocyte stage, the best maintenance solution to be used: Hank or 50% L15 and; viability after baths for 30min (room temperature) at cryoprotectant concentrations ranging from 0.25 to 4M were evaluated. Different tests were used to evaluate oocyte viability: in vitro maturation followed by observation of germinal vesicle breakdown (only for immature oocytes), Trypan Blue staining (all stages) and fertilization and hatching rates (mature stage only). Results showed that the toxic effect of cryoprotectants on oocytes generally increases with increasing concentrations. Sensitivity of oocytes to cryoprotectants increases according to the stage of development, with mature oocytes being more sensitive. Sucrose, fructose, methanol, propylene glycol and dimethyl sulfoxide can be used as cryoprotectants for S. parahybae oocytes.

scholarly journals Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems

2015 ◽  
Vol 27 (7) ◽  
pp. 1082 ◽  
Author(s):  
Maricy Apparicio ◽  
Giuliano Q. Mostachio ◽  
Tathiana F. Motheo ◽  
Aracelle E. Alves ◽  
Luciana Padilha ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
System A ◽  
Cytoplasmic Maturation ◽  
Positive Effect

The aim of this study was to evaluate the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of oocytes obtained from bitches at different reproductive stages (follicular, luteal, anoestrous). In System A (control) oocytes were matured for 72 h in base medium (BM) with 10 IU mL–1 human chorionic gonadotrophin (hCG), 1 μg mL–1 progesterone (P4) and 1 μg mL–1 oestradiol (E2); in bi-phasic System B oocytes were matured for 48 h in BM with hCG and for 24 h in BM with P4; in bi-phasic System C oocytes were matured for 48 h in BM with hCG, P4 and E2, and for 24 h in BM with P4; in System D, oocytes were cultured in BM without hormonal supplementation. Data were analysed by ANOVA. There was a positive effect of the bi-phasic systems on germinal vesicle breakdown, metaphase I and metaphase II rates, irrespective of reproductive status (P < 0.05). Bi-phasic systems were also beneficial for cortical granule distribution (an indication of cytoplasmic maturation) and its relationship to nuclear status: 74.5% of the oocytes cultured in System B and 85.4% of those cultured in System C presented both nuclear and cytoplasmic maturation (P < 0.001). The stage of the oestrous cycle did not influence maturation rates.

scholarly journals Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos.

1990 ◽  
Vol 111 (2) ◽  
pp. 533-541 ◽  
Author(s):  
R S Freeman ◽  
J P Kanki ◽  
S M Ballantyne ◽  
K M Pickham ◽  
D J Donoghue
Keyword(s):  
Antisense Oligonucleotide ◽  
Xenopus Oocytes ◽  
Kinase Activity ◽  
Germinal Vesicle ◽  
Mitotic Arrest ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Transforming Activity ◽  
Cytostatic Factor

Previous work has demonstrated that the Xenopus protooncogene mosxe can induce the maturation of prophase-arrested Xenopus oocytes. Recently, we showed that mosxe can transform murine NIH3T3 fibroblasts, although it exhibited only 1-2% of the transforming activity of the v-mos oncogene. In this study we have investigated the ability of the v-mos protein to substitute for the mosxe protein in stimulating Xenopus oocytes to complete meiosis. Microinjection of in vitro synthesized RNAs encoding either the mosxe or v-mos proteins stimulates resting oocytes to undergo germinal vesicle breakdown. Microinjection of an antisense oligonucleotide spanning the initiation codon of the mosxe gene blocked progesterone-induced oocyte maturation. When oocytes were microinjected first with the mosxe antisense oligonucleotide, and subsequently with in vitro synthesized v-mos RNA, meiotic maturation was rescued as evidenced by germinal vesicle breakdown. The v-mos protein exhibited in vitro kinase activity when recovered by immunoprecipitation from either microinjected Xenopus oocytes or transfected monkey COS-1 cells; however, in parallel experiments, we were unable to detect in vitro kinase activity associated with the mosxe protein. Microinjection of in vitro synthesized v-mos RNA into cleaving Xenopus embryos resulted in mitotic arrest, demonstrating that the v-mos protein can function like the mosxe protein as a component of cytostatic factor. These results exemplify the apparently conflicting effects of the v-mos protein, namely, its ability to induce maturation of oocytes, its ability to arrest mitotic cleavage of Xenopus embryo, and its ability to transform mammalian fibroblasts.

Meiosis and embryo technology: renaissance of the nucleolus

2005 ◽  
Vol 17 (2) ◽  
pp. 3 ◽  
Author(s):  
Poul Maddox-Hyttel ◽  
Bolette Bjerregaard ◽  
Jozef Laurincik
Keyword(s):  
Topoisomerase I ◽  
De Novo ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Rdna Transcription ◽  
Morphological Marker ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Growth ◽  
Embryonic Genome

The nucleolus is the site of rRNA and ribosome production. This organelle presents an active fibrillogranular ultrastructure in the oocyte during the growth of the gamete but, at the end of the growth phase, the nucleolus is transformed into an inactive remnant that is dissolved when meiosis is resumed at germinal vesicle breakdown. Upon meiosis, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities harbour the development of fibrillogranular nucleoli and re-establishment of nucleolar function in conjunction with the major activation of the embryonic genome. This so-called nucleologenesis occurs at a species-specific time of development and can be classified into two different models: one where nucleolus development occurs inside the NPBs (e.g. cattle) and one where the nucleolus is formed on the surface of the NPBs (e.g. pigs). A panel of nucleolar proteins with functions during rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) are localised to specific compartments of the oocyte nucleolus and those engaged in late processing are, to some degree, re-used for nucleologenesis in the embryo, whereas the others require de novo embryonic transcription in order to be allocated to the developing nucleolus. In the oocyte, inactivation of the nucleolus coincides with the acquisition of full meiotic competence, a parameter that may be of importance in relation to in vitro oocyte maturation. In embryo, nucleologenesis may be affected by technological manipulations: in vitro embryo production apparently has no impact on this process in cattle, whereas in the pig this technology results in impaired nucleologenesis. In cattle, reconstruction of embryos by nuclear transfer results in profound disturbances in nucleologenesis. In conclusion, the nucleolus is an organelle of great importance for the developmental competence of oocytes and embryos and may serve as a morphological marker for the completion of oocyte growth and normality of activation of the embryonic genome.

scholarly journals NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuting Xiang ◽  
Chuanchuan Zhou ◽  
Yanyan Zeng ◽  
Qi Guo ◽  
Jiana Huang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
High Throughput Sequencing ◽  
Epigenetic Modification ◽  
Binding Protein ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Translation Efficiency ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p &lt; 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

161 USE OF FORSKOLIN TO DELAY MEIOSIS AND PRODUCE IN VITRO BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 194
Author(s):  
D. Paschoal ◽  
R. Maziero ◽  
M. Sudano ◽  
M. Guastali ◽  
L. Vergara ◽  
...  
Keyword(s):  
Rate Control ◽  
Germinal Vesicle ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Bovine Embryos ◽  
Hoechst 33342 ◽  
Germinal Vesicle Breakdown ◽  
Tunel Reaction ◽  
Level Of Significance ◽  
Bovine Oocytes

The maintenance of oocytes in germinal vesicle (GV) stage for a few hours could result in more competent oocytes for use in biotechnology. This study aimed to show if the use of forskolin is able to inhibit and reverse the maturation in bovine oocytes, producing a higher rate of in vitro embryos without apoptosis rates. Eight replicates in total were performed. Nellore oocytes were matured in TCM-199 and to delay meiosis, the oocytes (n = 584) were maintained for 6 h in medium in presence of 0.025, 0.05, or 0.1 mM Forskolin. Then, the oocytes were cultured for 18 h in agent-free medium to resume meiosis. After resumption of meiosis, the oocytes (n = 336) were stained with Hoechst 33342 to evaluate the state of the nucleus: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), or degenerated or unidentified (D/U). Then (Day 0) oocytes were fertilized in human tubal fluid (Irvine, New Zealand) and the presumed zygotes were culture in SOFaa + 0.6% BSA + 2.5% FCS until Day 7, when the blastocyst (n = 177) rate was evaluated. Apoptosis in blastocysts was assessed by terminal deoxynucleotidyl transferase uracil nick-end labeling (TUNEL) reaction. Data were analysed by ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. There were no statistical differences in state of the nucleus, only in MI (Control = GV: 0.0, GVBD: 0.8, MI: 8.3a, MII: 67.7, D/U: 7.3; F 0.025 mM = GV: 2.8, GVBD: 0.7, MI: 20.8ab, MII: 67.7, D/U: 8.9; F 0.05 mM = GV: 0.0, GVBD: 4.4, MI: 15.8ab, MII: 65.9, D/U: 13.7; and F 0.1 mM = GV: 0.0, GVBD: 1.0, MI: 34.1b, MII: 50.2, D/U: 14.6; P < 0.05). There were no statistical differences in blastocyst rate (Control: 36.7, F 0.025 mM: 32.6, F 0.05 mM: 29.2 and F 0.1 mM: 25.1 – P > 0.05). But when we analysed the apoptosis rate, differences were found among groups: (Control: 12.1a, F 0.025 mM: 12.9a, F 0.05 mM: 13.5a and F 0.1 mM: 30b; P < 0.05). Although Forskolin was able to inhibit meiosis and produce embryos at the same rates as controls, the higher dosage of this drug damaged the embryos. The authors acknowledge FAPESP 10/50410-2 for support.

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