scholarly journals CPEB2 Is Necessary for Proper Porcine Meiotic Maturation and Embryonic Development

2018 ◽  
Vol 19 (10) ◽  
pp. 3138 ◽  
Author(s):  
Barbora Prochazkova ◽  
Pavla Komrskova ◽  
Michal Kubelka
Keyword(s):  
Embryonic Development ◽  
Translational Control ◽  
Translational Regulation ◽  
Basic Mechanism ◽  
Meiotic Maturation ◽  
Cytoplasmic Polyadenylation ◽  
Ribonucleic Acids ◽  
Element Binding Protein ◽  
Oocyte Meiotic Maturation ◽  
Post Transcriptional Regulation

Oocyte meiotic maturation and embryogenesis are some of the most important physiological processes that occur in organisms, playing crucial roles in the preservation of life in all species. The post-transcriptional regulation of maternal messenger ribonucleic acids (mRNAs) and the post-translational regulation of proteins are critical in the control of oocyte maturation and early embryogenesis. Translational control affects the basic mechanism of protein synthesis, thus, knowledge of the key components included in this machinery is required in order to understand its regulation. Cytoplasmic polyadenylation element binding proteins (CPEBs) bind to the 3′-end of mRNAs to regulate their localization and translation and are necessary for proper development. In this study we examined the expression pattern of cytoplasmic polyadenylation element binding protein 2 (CPEB2) both on the mRNA (by real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR) and protein (by Western blotting, WB) level, as well as its localization during the meiotic maturation of porcine oocytes and early embryonic development by immunocytochemistry (ICC). For the elucidation of its functions, CPEB2 knockdown by double-strand RNA (dsRNA) was used. We discovered that CPEB2 is expressed during all stages of porcine meiotic maturation and embryonic development. Moreover, we found that it is necessary to enable a high percentage of oocytes to reach the metaphase II (MII) stage, as well as for the production of good-quality parthenogenetic blastocysts.

scholarly journals Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

1990 ◽  
Vol 1 (13) ◽  
pp. 1057-1067 ◽  
Author(s):  
Z Xu ◽  
M B Hille
Keyword(s):  
Translational Control ◽  
Translational Regulation ◽  
High Capacity ◽  
Meiotic Maturation ◽  
Immature Oocytes ◽  
Initiation Factors ◽  
Initiation Of Translation ◽  
Free Translation ◽  
Translation Systems

Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimental application of the hormone 1-methyladenine induces their maturation, synchronizing meiosis. We have prepared such systems from both immature and mature oocytes of starfish. Changes in protein synthesis rates and the specificity of proteins synthesized in these cell-free translation supernatants mimic those seen in vivo. Supernatants both from immature and mature oocytes have a high capacity to initiate new translation because 90% of the proteins made are newly initiated from mRNAs. Cell-free supernatants from mature oocytes have a much higher rate of initiation of translation than those from immature oocytes and use the 43S preinitiation complexes more efficiently in initiation of translation. Similarly, we have shown that mRNAs and initiation factors are rate limiting in cell-free translation systems prepared from immature oocytes. In addition, cell-free translation systems prepared from immature oocytes are only slightly, if at all, inhibitory to cell-free translation systems from mature oocytes. Thus, soluble inhibitors, if they exist, are rapidly converted by cell-free supernatants from mature oocytes. The similarities between translation in our starfish cell-free translation systems and in intact oocytes suggests that the cell-free translation systems will be useful tools for further studies of maturation events and translational control during meiosis.

scholarly journals Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster

2021 ◽  
Author(s):  
Lisa Shao ◽  
Jaclyn M. Fingerhut ◽  
Brook L. Falk ◽  
Hong Han ◽  
Giovanna Maldonado ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cytoplasmic Polyadenylation ◽  
Eukaryotic Translation ◽  
Eukaryotic Initiation Factor 4E ◽  
Sperm Development ◽  
Element Binding Protein ◽  
Eukaryotic Translation Initiation ◽  
Post Transcriptional Regulation

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. Drosophila has multiple paralogs of eIF4E, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Other than eIF4E-3, none of these has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 mutants exhibit defects during post-meiotic stages, including a fully penetrant defect in individualization, resulting in failure to produce mature sperm. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, where it regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. eIF4E-5 mutants also have mild defects in spermatid cyst polarization, similar to mutants affecting the cytoplasmic polyadenylation-element binding protein Orb2 and atypical protein kinase C (aPKC). Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

Molecular characterization of cyclin B and its expression profile during oogenesis in the red swamp crayfish, Procambarus clarkii (Girard, 1852) (Decapoda, Astacidea)

Crustaceana ◽  
2016 ◽  
Vol 89 (8) ◽  
pp. 915-931 ◽  
Author(s):  
Y. Shui ◽  
Z. H. Xu ◽  
Y. H. Shi ◽  
X. Zhou
Keyword(s):  
Procambarus Clarkii ◽  
Cyclin B ◽  
Meiotic Maturation ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Cytoplasmic Polyadenylation ◽  
Calculated Molecular Mass ◽  
Red Swamp Crayfish ◽  
M Phase ◽  
Oocyte Meiotic Maturation

The maturation promoting factor (MPF), a complex of CDC2 (CDK1) and cyclin B, is a key regulator of controlling the G2/M phase transition in the meiotic maturation of the oocyte in multi-cellular organisms. In this study, full-length cDNA of cyclin B (Pc-cyclin B) from the red swamp crayfish,Procambarus clarkii, was cloned using the degenerate RT-PCR and RACE methods. The cDNA of Pc-cyclin B is 2595 bp in length and encoding a protein of 402 amino acids, with a calculated molecular mass of 45.75 kDa. Six potential cytoplasmic polyadenylation elements (CPE) as well as one signal sequences (AATAAA) were found in the 3′-UTR location. Semi-quantitative RT-PCR analysis revealed that the amount of cyclin B mRNA was highest in the ovary, followed by the heart (); and also significantly higher in the pre-vitellogenesis (pre-Vt) and primary-vitellogenesis (pVt) stages, while low in the tertiary-vitellogenesis and GVBD stages (), suggesting that differential expression of Pc-cyclin B is closely related to oogonial proliferation (mitosis) and oocyte meiotic maturation in this species of crayfish.

200 SYNCHRONIZATION OF OOCYTE MEIOTIC MATURATION IN SUPEROVULATED MICE IMPROVES IN VITRO FERTILIZATION RATE

2012 ◽  
Vol 24 (1) ◽  
pp. 212
Author(s):  
A. M. Taiyeb Ridha ◽  
D. C. Kraemer
Keyword(s):  
Embryonic Development ◽  
Fertilization Rate ◽  
Early Embryonic Development ◽  
Meiotic Maturation ◽  
Control Group ◽  
Vitro Fertilization ◽  
Development Rates ◽  
Oocyte Meiotic Maturation

In vitro synchronization of oocyte nuclear and cytoplasmic maturation has been found to improve the IVF rate of ovarian oocytes in several species, including humans, in comparison with nonsynchronized in vitro-matured oocytes. Here, we tested the hypothesis that synchronization of oocyte meiotic maturation by an in vivo system in superovulated mice would increase the oocyte fertilization rate when compared to that of conventional superovulated oocytes. Recently, we observed that cilostazol (CZL), a PDE3-I, was able to inhibit mouse oocyte meiotic maturation in both in vitro and in vivo systems. Administering CZL at 7.5 mg, 4 or 7 h pre-hCG allowed retrieval of ovulated oocytes of which >95% were at MI stage, scored by Nikon stereo microscope (SMZ 1500). A conventional superovulation program was adapted in all treated and their control groups, in which mice were injected with eCG and after 48 h with hCG (7.5 IU for each hormone). On the second morning, 13 to 14 h post-hCG, mice were killed and oocytes were collected from oviducts and in vitro fertilized (control). For the treated groups, CZL was administered in a single 7.5 mg oral dose (gavage) 4 or 7 h before the hCG injection. On the second morning, CZL-treated animals were killed at the same timing as control animals and oocytes were retrieved from the oviduct and in vitro matured for 6 h (for those gavaged with CZL, 4 h pre-hCG) or 3 h (for those gavaged with CZL, 7 h pre-hCG) to MII oocytes before IVF. These groups were designated as in vivo-in vitro synchronized/matured oocytes. In other groups treated with CZL, 4 or 7 h pre-hCG, the ovulated oocytes were allowed to mature in the oviduct (full in vivo synchronization and maturation) and oocytes were retrieved and fertilized with the same fertilization timings as the in vivo-in vitro synchronized/matured oocytes. Oocytes were cultured for 1 day after IVF and examined for cleavage. Statistical differences were analyzed by cross-tabulated chi-square test. The full in vivo synchronization and maturation (for both CZL dose timings of 4 and 7 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [89% (n = 219) and 92.2% (n = 374) vs 81.8% (n = 198); P = 0.034 and P < 0.0001, respectively]. The in vivo-in vitro synchronized/matured oocytes (CZL dose timing at 7 h, but not 4 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [88.5% (n = 339) vs 83.4% (n = 458), respectively; P = 0.043]. However, the increase of the IVF rate of the oocytes from mice treated with CZL, 4 h pre-hCG, in the in vivo-in vitro synchronized/matured group was not significantly different from the control group [88.5% (n = 399) vs 83.4% (n = 458), respectively; P = 0.43]. It is concluded from the present study that synchronization of oocyte meiotic maturation by the in vivo and in vivo-in-vitro protocols can increase the IVF rate of oocytes in superovulated mice.

scholarly journals Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1α mRNA 3′-UTR and modulate HIF-1α protein expression

2008 ◽  
Vol 417 (1) ◽  
pp. 235-246 ◽  
Author(s):  
Sonja Hägele ◽  
Uwe Kühn ◽  
Melanie Böning ◽  
Dörthe M. Katschinski
Keyword(s):  
Translational Control ◽  
Binding Protein ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Insulin Stimulation ◽  
Luciferase Reporter Gene ◽  
Cytoplasmic Polyadenylation ◽  
Protein Levels ◽  
Hypoxia Inducible Factor 1 ◽  
Element Binding Protein

The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1α subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1α mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3′-UTR (untranslated region) of HIF-1α mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1α protein levels mediated by the 3′-UTR of HIF-1α mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3′-UTR of HIF-1α as well as endogenous HIF-1α protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1α cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1α following insulin stimulation.

scholarly journals Detecting translational regulation by change point analysis of ribosome profiling datasets

2014 ◽  
Author(s):  
Anze Zupanic ◽  
Catherine Meplan ◽  
Sushma N Grellscheid ◽  
John C Mathers ◽  
Tom BL Kirkwood ◽  
...  
Keyword(s):  
Translational Control ◽  
Change Point ◽  
Translational Regulation ◽  
Embryonic Stem ◽  
Ribosome Profiling ◽  
Change Point Analysis ◽  
Coding Region ◽  
Ribosome Density ◽  
Point Analysis ◽  
Post Transcriptional Regulation

Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While ribosome density is constant along the length of the mRNA coding region, it can be altered by translational regulatory events. In this study, we developed a method to detect translational regulation of individual mRNAs from their ribosome profiles, utilizing changes in ribosome density. We used mathematical modelling to show that changes in ribosome density should occur along the mRNA at the point of regulation. We analyzed a Ribo-Seq dataset obtained for mouse embryonic stem cells and showed that normalization by corresponding RNA-Seq can be used to improve the Ribo-Seq quality by removing bias introduced by deep-sequencing and alignment artefacts. After normalization, we applied a change point algorithm to detect changes in ribosome density present in individual mRNA ribosome profiles. Additional sequence and gene isoform information obtained from the UCSC Genome Browser allowed us to further categorize the detected changes into different mechanisms of regulation. In particular, we detected several mRNAs with known post-transcriptional regulation, e.g. premature termination for selenoprotein mRNAs and translational control of Atf4, but also several more mRNAs with hitherto unknown translational regulation. Additionally, our approach proved useful for identification of new gene isoforms.

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