Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 675-685 ◽  
Author(s):  
Martin Morovic ◽  
Frantisek Strejcek ◽  
Shoma Nakagawa ◽  
Rahul S. Deshmukh ◽  
Matej Murin ◽  
...  
Keyword(s):  
Embryonic Development ◽  
De Novo ◽  
Blastocyst Stage ◽  
Rrna Synthesis ◽  
Control Group ◽  
Cell Stage ◽  
Nucleolar Proteins ◽  
Transmission Electron ◽  
Binding Factor ◽  
Upstream Binding Factor

SummaryIt is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

44 LIMITATIONS OF PORCINE OOPLASM TO REPROGRAM BOVINE SOMATIC CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 170
Author(s):  
F. Strejcek ◽  
O. Ostrup ◽  
M. Morovic ◽  
I. Petrovicova ◽  
A. Lucas-Hahn ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Stage ◽  
Tem Analysis ◽  
Time Points ◽  
Nucleolar Proteins ◽  
Transmission Electron ◽  
Binding Factor ◽  
Oocyte Cytoplasm ◽  
Upstream Binding Factor ◽  
Cell Genome

The oocyte cytoplasm (ooplasm) constitutes a unique mixture of factors that are critical for successful reprogramming of haploid maternal and paternal genomes at fertilization as well as the diploid somatic cell genome after somatic cell nuclear transfer (SCNT). The 1-cell-stage embryos (produced by transfer of bovine fibroblast into porcine enucleated ooplast; interspecies SCNT, iSCNT) were processed at different time points (2, 4, 8, and 12 h post-activation, hpa) for detailed nuclear and nucleolar analysis by transmission electron microscopy (TEM; 4 to 5 embryos per group), and immunofluorescence (4 and 12 hpa) for visualization of nucleolar proteins related to transcription (upstream binding factor, UBF) and processing (fibrillarin; 8 to 9 embryos per group). The parameters of interspecies embryos were compared with porcine parthenogenetic (PA) counterparts (4 to 5 embryos per group for TEM analysis; 8 to 9 embryos per group for immunofluorescence analysis). At all evaluated time points, embryos in both groups displayed pronucleus-like nuclei with abundant euchromatin and characteristic porcine nucleolus precursor body (NPB), indicating maternal origin of nucleolar components. Fibrillarin was in both groups localized into shell-like intranuclear entities surrounding NPB. On the contrary, UBF in PA embryos was at 12 hpa colocalized with fibrillarin, whereas in iSCNT embryos, UBF staining was absent at both time points. Despite the similar morphology and localization of processing factor fibrillarin, striking differences in localization of transcription factor UBF suggest limitation of porcine ooplasm to mediate initial phases of nucleolar remodelling. This work was supported by VEGA 1/0077/11.

Maternally inherited rRNA triggers de novo nucleolus formation in porcine embryos

Zygote ◽  
2018 ◽  
Vol 26 (5) ◽  
pp. 395-402
Author(s):  
Martin Morovic ◽  
Olga Østrup ◽  
Frantisek Strejcek ◽  
Michal Benc ◽  
Matej Murin ◽  
...  
Keyword(s):  
De Novo ◽  
Control Group ◽  
Cell Stage ◽  
Transcriptional Inhibition ◽  
Nucleolar Proteins ◽  
Transmission Electron ◽  
Embryonic Genome ◽  
Polymerase I ◽  
Genome Activation

SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.0 μg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 μg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription.

scholarly journals Recruitment of TATA-Binding Protein–TAFI Complex SL1 to the Human Ribosomal DNA Promoter Is Mediated by the Carboxy-Terminal Activation Domain of Upstream Binding Factor (UBF) and Is Regulated by UBF Phosphorylation

1999 ◽  
Vol 19 (4) ◽  
pp. 2872-2879 ◽  
Author(s):  
JoAnn C. Tuan ◽  
Weiguo Zhai ◽  
Lucio Comai
Keyword(s):  
Dna Binding ◽  
Ribosomal Dna ◽  
Transcriptional Activation ◽  
Direct Contact ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rrna Synthesis ◽  
Binding Factor ◽  
Upstream Binding Factor ◽  
Carboxy Terminal

ABSTRACT Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any sequence-specific DNA binding activity, and its recruitment to the promoter is mediated by specific protein interactions with UBF. Once on the promoter, the SL1 complex makes direct contact with the DNA promoter and directs promoter-specific initiation of transcription. To investigate the mechanism of UBF-dependent transcriptional activation, we first performed protein-protein interaction assays between SL1 and a series of UBF deletion mutants. This analysis indicated that the carboxy-terminal domain of UBF, which is necessary for transcriptional activation, makes direct contact with the TBP-TAFI complex SL1. Since this region of UBF can be phosphorylated, we then tested whether this modification plays a functional role in the interaction with SL1. Alkaline phosphatase treatment of UBF completely abolished the ability of UBF to interact with SL1; moreover, incubation of the dephosphorylated UBF with nuclear extracts from exponentially growing cells was able to restore the UBF-SL1 interaction. In addition, DNase I footprinting analysis and in vitro-reconstituted transcription assays with phosphatase-treated UBF provided further evidence that UBF phosphorylation plays a critical role in the regulation of the recruitment of SL1 to the ribosomal DNA promoter and stimulation of UBF-dependent transcription.

scholarly journals Baicalin improves the in vitro developmental capacity of pig embryos by inhibiting apoptosis, regulating mitochondrial activity and activating sonic hedgehog signaling

2019 ◽  
Vol 25 (9) ◽  
pp. 538-549 ◽  
Author(s):  
Qing Guo ◽  
Mei-Fu Xuan ◽  
Zhao-Bo Luo ◽  
Jun-Xia Wang ◽  
Sheng-Zhong Han ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cell Stage ◽  
Shh Signaling ◽  
Developmental Capacity

Abstract Baicalin, a traditional Chinese medicinal monomer whose chemical structure is known, can be used to treat female infertility. However, the effect of baicalin on embryonic development is unknown. This study investigated the effects of baicalin on in vitro development of parthenogenetically activated (PA) and in vitro fertilized (IVF) pig embryos and the underlying mechanisms involved. Treatment with 0.1 μg/ml baicalin significantly improved (P < 0.05) the in vitro developmental capacity of PA pig embryos by reducing the reactive oxygen species (ROS) levels and apoptosis and increasing the mitochondrial membrane potential (ΔΨm) and ATP level. mRNA and protein expression of sonic hedgehog (SHH) and GLI1, which are related to the SHH signaling pathway, in PA pig embryos at the 2-cell stage, were significantly higher in the baicalin-treated group than in the control group. To confirm that the SHH signaling pathway is involved in the mechanism by which baicalin improves embryonic development, we treated embryos with baicalin in the absence or presence of cyclopamine (Cy), an inhibitor of this pathway. Cy abolished the effects of baicalin on in vitro embryonic development. In conclusion, baicalin improves the in vitro developmental capacity of PA and IVF pig embryos by inhibiting ROS production and apoptosis, regulating mitochondrial activity and activating SHH signaling.

Early cleavage to formation of the unilaminar blastocyst in the marsupial Antechinus stuartii: ultrastructure

1997 ◽  
Vol 9 (2) ◽  
pp. 201 ◽  
Author(s):  
Henry Sathananthan ◽  
Lynne Selwood ◽  
Isabel Douglas ◽  
Kamani Nanayakkara
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Spindle Pole ◽  
Blastocyst Stage ◽  
Cell Junctions ◽  
Cell Stage ◽  
Transmission Electron ◽  
Embryonic Genome ◽  
Antechinus Stuartii ◽  
First Time

The development of Antechinus stuartiifrom the 2-cell stage to the blastocyst stage in vivo was examined by routine transmission electron microscopy. The 2–8-cell stages had a similar organization of organelles, whereas the 16- to 32-cell stages had pluriblast cells and trophoblast cells forming an epithelium closely apposed to the zona pellucida. Specialized cell–zona plugs were formed at the 8-cell stage, and primitive cell junctions appeared in later conceptuses. The cytoplasmic organelles included mitochondria, lysosomes, aggregates of smooth endoplasmic reticulum, lipid and protein yolk bodies and fibrillar arrays, possibly contractile in function. Nuclei had uniformly-dispersed dense chromatin. Nucleoli of 2–4-cell conceptuses were dense, compact and fibrillar, and those of 8-cell conceptuses and later conceptuses were finely granular and became progressively reticulated. The embryonic genome is probably not switched on before the 8-cell stage. Sperm tails were detected in cells in several early conceptuses. The yolk mass had the same organelles as cells. Centrioles were discovered for the first time in marsupial conceptuses. These were prominently situated at a spindle pole in a 32-cell blastomere and were associated with a nucleus and sperm tail at the 4-cell stage. It is very likely that the paternal centrosome is inherited at fertilization and perpetuated in Antechinus embryos during cleavage.

196 A NEW APPROACH TO PREDICT MOUSE EMBRYONIC DEVELOPMENTAL COMPETENCE USING A TIME-LAPSE SYSTEM AND THE 'SHORTEST HALF' analysis procedure

2007 ◽  
Vol 19 (1) ◽  
pp. 214 ◽  
Author(s):  
S. Yavin ◽  
A. Aroyo ◽  
Z. Roth ◽  
A. Arav
Keyword(s):  
Embryonic Development ◽  
Time Lapse ◽  
Time Frame ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Cell Stage ◽  
Developmental Potential ◽  
Additional Tool ◽  
Embryonic Cleavage

Embryonic development is a dynamic process in which embryo morphology may change immensely within several hours. Therefore, identifying and selecting embryos with the highest probability of developing and achieving a pregnancy is a major challenge. The timing of embryonic cleavage may serve as an additional indicator for the identification of quality embryos. The aim of this study was to characterize the cleavage timing of mouse embryos and to identify the stage that is most indicative of blastocyst formation. Mated mice (CB6F1) were sacrificed 20 h after hCG administration; putative zygotes were recovered and cultured (50 embryos in each 20-µL drop of M16) in a time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) inside the incubator. The time-lapse system was programmed to take photos at half-hour intervals such that culture dishes were not removed from the incubator. The ‘shortest half’ statistical procedure of JMPIN (SAS Institute, Inc., Cary, NC, USA) was utilized to evaluate the period during which at least 50% of the embryonic population cleaves within the shortest time frame. Captured images made it possible to search along the time axis for the densest 50% of cleavage observations. Developing embryos were categorized into 3 groups according to the time of cleavage after hCG administration: before, during, and after the ‘shortest half’ for each developmental stage. Two hundred thirty putative zygotes cleaved and created 2-cell-stage embryos, of which 55 arrested at various stages and 175 progressed to the blastocyst stage. During embryonic development, cleavage timing appeared to become less uniform and the ‘shortest half’ became longer for each successive cell division: Whereas the shortest period in which 50% of the 2-cell-stage embryos cleaved was a 2-h interval, cleavage into the 4-cell, 8-cell, and blastocyst stages took 2.5, 3.5, and 5 h, respectively. The ‘short half’ for the first cleavage appears to be a predictive time frame for subsequent embryonic development, because cleavage was closely synchronized with 80% of the embryos developing to the blastocyst stage. Note that only a small number of embryos were actually cleaving early, while the ‘shortest half’ consisted of 50% of the embryonic population. Moreover, late-cleaving embryos in the 2-cell stage expressed inferior developmental potential relative to those that cleaved within the ‘shortest half’ (see Table 1). In summary, 2-cell-stage embryos that cleaved within the ‘shortest half’ seemed to be better synchronized and consequently more competent than the rest of the embryonic population. Embryonic cleavage timing using the ‘shortest half’ parameter can be considered a biological indicator of embryo potential. It may be useful as an additional tool for selecting embryos for transfer and cryopreservation. Table 1. Cleavage timing distribution into the 2-cell stage according to the shortest half

190 EXPRESSION OF GENES ASSOCIATED WITH WINGLESS-RELATED MOUSE MAMMARY TUMOUR VIRUS SIGNALING IN THE PRE-IMPLANTATION BOVINE EMBRYO

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
P. Tribulo ◽  
J. I. Moss ◽  
P. J. Hansen
Keyword(s):  
Embryonic Development ◽  
Blastocyst Stage ◽  
Wnt Signalling ◽  
Mammary Tumour ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Mouse Mammary Tumour Virus ◽  
Ct Value ◽  
Morula Stage ◽  
Canonical Wnt

Wingless-related mouse mammary tumour virus (WNT) signalling participates in early embryonic development to maintain pluripotency, controls cell–cell communication, and modulates cell polarization and migration. To gain an understanding of the regulation of WNT signalling during embryonic development, expression patterns of a variety of molecules involved in WNT signal transduction were evaluated. Specific genes were DKK1, an endogenous inhibitor of canonical WNT signalling, the WNT co-receptors LRP5 and LRP6, WNT-responsive transcription factors, LEF1 and TCF7, and two repressors of WNT-regulated genes, the bovine orthologue of GROUCHO (LOC505120) and AES. Embryos were produced in vitro from oocytes obtained from ovaries collected at a local abattoir. Following oocyte maturation, fertilization was performed with sperm pooled from three randomly selected bulls; a different pool of bulls was used for each replicate. Groups of 30 matured oocytes or embryos at the 2-cell [28–32 h post-insemination (hpi)], 3–4 cell (44–48 hpi), 5–8 cell (50–55 hpi), 9–16 cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The zona pellucida was removed with proteinase, RNA was purified, cDNA synthesised using random hexamer primers and real-time qPCR performed. Data analysed were ΔCT values, which were calculated by subtracting the CT value of the geometric mean of the three housekeeping genes (GAPDH, YWHAZ, and SDHA) from the CT value of the sample. The relative transcript abundance was calculated as the 2ΔCT. Data were analysed by least-squares ANOVA using the Proc GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). A total of 5 replicates were analysed for each developmental stage. Results show significant effects of stage of development for each gene that ranged from P = 0.004 for LRP5 to P ≤ 0.0001 for AES, DKK1, LEF, LOC505120, LRP6, and TCF7. In all cases, expression declined as development advanced. Except for AES, lowest expression occurred at the blastocyst stage. Lowest expression for AES was at the morula stage; expression remained low at the blastocyst stage. For two genes, DKK1 and LEF1, there was no detectable expression at the blastocyst stage. The timing of decline in expression varied between genes, first occurring at the 9–16-cell stage (AES, LEF1, and LOC505120) or morula stage (DKK1, LRP5, LRP6, or TCF7). For DKK1, LEF1, and LRP6, there was also a slight increase in expression from the oocyte to two-cell stage. Results suggest that canonical WNT signalling is reduced at the morula and blastocyst stages relative to earlier stages in development. Research was supported by USDA-NIFA 2011-67015-30688.

53 Effect of anthocyanin supplementation in bovine pre-implantation embryonic development during invitro maturation of oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 133
Author(s):  
A. Zegarra ◽  
J. Rivas ◽  
A. Gallegos ◽  
E. Mellisho
Keyword(s):  
Embryonic Development ◽  
Decisive Role ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Maturation Medium ◽  
Advanced Stages ◽  
Commercial Medium ◽  
Antioxidant Effects

Oocyte protection against reactive oxygen species (ROS) during invitro maturation (IVM) may play a decisive role in pre-implantation embryonic development. For instance, anthocyanins have shown greater antioxidant effects than vitamins C and E. The objective of this study was to determine the anthocyanin supplementation level that influences quantity and quality of bovine blastocysts development during IVM. Cumulus–oocyte complexes (COC) were recovered from 185 abattoir ovaries in 6 sessions and classified (Grade 1 and 2) for maturation. Oocytes were in IVM in commercial medium (Vitrogen®) supplemented with anthocyanin (pelargonidin chloride) at different concentrations: 0 (control), 1, 10, 20, and 40μM, in droplets of 70μL with 12 to 15 COC at 38.5°C, 5% CO2 and 90% humidity for 22to 24h. Sperm selection was performed by Percoll gradient method (45/90%) with centrifugation at 600×g for 6min. The final concentration for IVF was 2×106 sperm mL−1. A total of 462 oocytes were used in the experiment (6 replicates). Presumptive zygotes were invitro cultured (IVC) in commercial medium (Vitrogen) in droplets of 70µL with 12–15 zygotes at 38°C, 5% CO2, and 90% humidity until the blastocyst stage (Day 7 of culture). The cleavage (Day 2), morulae (Day 4), and blastocyst (Day 7) rates were measured during IVC. The data were processed with non-parametric tests (Kruskal–Wallis test with independent samples, P&lt;0.05) using IBM SPSS Statistics 2.0 for Windows. The results in the control group of cleavage, morulae, and blastocyst rates were 67.3, 27.0, and 22.1%, respectively. Although, numerically, anthocyanin at 1μM resulted in a higher blastocyst rate (28.8%) and anthocyanin at 10μM resulted in a greater number of blastocysts of advanced stages (65.0%), anthocyanin supplementation during IVM did not influence the quantity and quality of bovine blastocyst development (P&gt;0.05). In conclusion, the supplementation of anthocyanin to the maturation medium did not affect invitro development of bovine embryos. Complementary studies at the cellular and gene expression level may be required.

scholarly journals K+ efflux through two-pore domain K+ channels is required for mouse embryonic development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Chang-Gi Hur ◽  
Eun-Jin Kim ◽  
Seong-Keun Cho ◽  
Young-Woo Cho ◽  
Sook-Young Yoon ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mammalian Cells ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Blastocyst Stage ◽  
Ruthenium Red ◽  
Channel Blockers ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Wide Range ◽  
Morula Stage

Numerous studies have suggested that K+ channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K+ channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K+ channel blockers to identify the functional role of K+ channels in mouse embryonic development. Voltage-dependent K+ channel blockers, such as tetraethylammonium and BaCl2, had no effect on embryonic development to the blastocyst stage, whereas K2P channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K2P channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ∼38% compared with scrambled siRNA injection (P<0.05). The blockade of K2P channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K2P channels could improve mouse embryonic development through the modulation of gating by activators.

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