228. Proteasomal activity during mouse preimplantation development

2008 ◽  
Vol 20 (9) ◽  
pp. 28 ◽  
Author(s):  
K. McCue ◽  
M. Pantaleon ◽  
P. L. Kaye
Keyword(s):  
Cell Proliferation ◽  
Protein Degradation ◽  
Specific Treatment ◽  
Preimplantation Development ◽  
26S Proteasome ◽  
Preimplantation Embryos ◽  
Cell Cycle Regulators ◽  
Loss Of Function ◽  
Cell Stage ◽  
Proteasomal Activity

Function of the 26S proteasome, a proteolytic organelle directed at proteins targeted for turnover by polyubiquitination, in preimplantation embryos is unclear. But it is well known to play a role in regulating meiosis. This paper reports the distribution of the proteasome and assessment of its functional importance in preimplantation development. Embryos from superovulated mice were either paraformaldehyde fixed for immunolabelling with a rabbit polyclonal antibody against the 20S proteasome core or cultured in KSOM medium with and without reversible (MG132) or irreversible (β-lactone) proteasomal inhibitors. Morphology, cell number, apoptosis and proteolysis were measured. Although diffuse throughout embryonic cytoplasm, there were distinct proteasomal concentrations in pronuclei, nuclei and cortical cytoplasm. When β-lactone was used to block blastocyst proteasomal proteolysis, ~25% of protein degradation was found to be proteasome-specific. Treatment of 2-cell embryos for more than 3 h with MG132 blocked blastocyst formation completely, even after washout, whilst both inhibitors reduced cell proliferation over the ensuing 48 h. Two hours exposure to MG132 tripled the proportion of apoptotic cells in expanded blastocysts 96 h post hCG. The nuclear concentration of proteasomes suggests a particular role in nuclear protein degradation possibly including the timed destruction of cell-cycle regulators and anti-apoptotic factors. This is supported by the loss-of-function studies which show that cell proliferation as well as morphogenesis require proteasomal activity at the late 2-cell stage and that without it apoptosis is dramatically increased. The mechanisms involved in the activation of apoptosis as a result of proteasomal inhibition in the early embryo are unknown but may include JNK signalling although this is controversial. More intriguing however is the identity of the proteasomal targets in the 2-cell embryo that must be degraded to permit continued morphogenesis.

scholarly journals Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

Reproduction ◽  
2016 ◽  
Vol 152 (5) ◽  
pp. 417-430 ◽  
Author(s):  
Atsushi Fukuda ◽  
Atsushi Mitani ◽  
Toshiyuki Miyashita ◽  
Hisato Kobayashi ◽  
Akihiro Umezawa ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Splice Variants ◽  
Protein Localization ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Sequencing Analysis ◽  
Dependent Manner ◽  
Cell Stage ◽  
Protein Levels ◽  
Oct4 Protein

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8–16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3–5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.

scholarly journals Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage

Reproduction ◽  
2009 ◽  
Vol 138 (5) ◽  
pp. 783-791 ◽  
Author(s):  
Tereza Toralová ◽  
Andrej Šušor ◽  
Lucie Němcová ◽  
Kateřina Kepková ◽  
Jiří Kaňka
Keyword(s):  
Fluorescent Protein ◽  
Staining Intensity ◽  
Preimplantation Development ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Average Decrease ◽  
Complex Protein ◽  
Green Fluorescent

Identification of genes that are important for normal preimplantation development is essential for understanding the basics of early mammalian embryogenesis. In our previous study, we have shown that CENPF (mitosin) is differentially expressed during preimplantation development of bovine embryos. CENPF is a centromere–kinetochore complex protein that plays a crucial role in the cell division of somatic cells. To our best knowledge, no study has yet been done on either bovine model, or oocytes and preimplantation embryos. In this study, we focused on the fate of bovine embryos after injection of CENPF double-stranded RNA (dsRNA) into the zygotes. An average decrease of CENPF mRNA abundance by 94.9% or more and an extensive decline in immunofluorescence staining intensity was detected relative to controls. There was no disparity between individual groups in the developmental competence before the 8-cell stage. However, the developmental competence rapidly decreased then and only 28.1% of CENPF dsRNA injected 8-cell embryos were able to develop further (uninjected control: 71.8%; green fluorescent protein dsRNA injected control: 72.0%). In conclusion, these results show that depletion of CENPF mRNA in preimplantation bovine embryos leads to dramatic decrease of developmental competence after embryonic genome activation.

Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals

1996 ◽  
Vol 8 (4) ◽  
pp. 485 ◽  
Author(s):  
JJ Eppig
Keyword(s):  
Growth Phase ◽  
Germinal Vesicle ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Maternal Factors ◽  
Cell Stage ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation ◽  
Developmental Programme ◽  
Critical Aspects

As oocytes near the end of their growth phase, they become competent to undergo two aspects of maturation, cytoplasmic and nuclear. Both are essential for the formation of an egg having the capacity for fertilization and development to live offspring. Nuclear maturation encompasses the processes reversing meiotic arrest at prophase I and driving the progression of meiosis to metaphase II. Cytoplasmic maturation refers to the processes that prepare the egg for activation and preimplantation development. This review focuses on the developmental programmes whereby oocytes at the germinal vesicle (GV) stage acquire competence to undergo nuclear and cytoplasmic maturation, the coordination of programmes regulating the acquisition of these competencies in GV-stage oocytes, and the coordination of the maturational processes themselves. Although the developmental programme of the GV-stage oocyte for acquiring competence to complete preimplantation development does not appear to be tightly linked to the acquisition of competence to complete nuclear maturation, GV breakdown (GVB) is probably essential for activating some critical aspects of cytoplasmic maturation, particularly those related to fertilization and activation. Nuclear and cytoplasmic maturation are normally coordinated by this mechanism requiring the mixing of the GV contents with the cytoplasm at the time of GVB, but some processes of cytoplasmic maturation related to successful preimplantation development probably still occur without coordination with nuclear maturation. Thus, continued differentiation of GV-stage oocytes is necessary after the acquisition of competence to undergo nuclear maturation, to allow for the deposition of the maternal factors required for the development of preimplantation embryos beyond the 2-cell stage.

Imprinting of phosphoribosyltransferases during preimplantation development of the mouse mutant, Hprtb-m3

Development ◽  
1992 ◽  
Vol 115 (4) ◽  
pp. 1011-1016 ◽  
Author(s):  
T.F. Moore ◽  
D.G. Whittingham
Keyword(s):  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Parental Origin ◽  
Mouse Mutant ◽  
Wild Type ◽  
Cell Stage ◽  
Unequivocal Identification ◽  
Deficient Mice

The measurement of the activity of the X-linked enzyme HPRT has been widely used as an indicator of X-chromosome activity during preimplantation development in the mouse. More recently, the concomitant measurement of the activity of the autosomally-encoded enzyme APRT has been used in an attempt to decrease the variability inherent in the measurement of enzyme activity from minute samples such as preimplantation embryos. In this study the use of the HPRT-deficient mouse mutant, Hprtb-m3, allowed the unequivocal identification of the parental origin of HPRT activity measured in embryos derived from crosses between wild-type mice, and mice which were homozygous or hemizygous for the Hprtb-m3 allele. Results were similar to those of a previous study, where oocyte-encoded HPRT activity accounted for about 10% of total HPRT activity at 76 hours post human chorionic gonadotrophin injection and the paternally-derived Hprt allele was shown to be transcriptionally active by the late 2-cell stage. In contrast to other studies, differential expression of the two Hprt alleles was detected during the preimplantation period, in embryos derived from crosses between wild-type and HPRT-deficient mice. Evidence was also found for the existence of an X-linked locus which influences the amount of APRT activity in the unfertilized oocyte. We propose that the expression pattern of this locus may be influenced by its parental origin.

Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 147-158 ◽  
Author(s):  
Shangdan Xu ◽  
Jibak Lee ◽  
Masashi Miyake
Keyword(s):  
Western Blotting ◽  
Contact Region ◽  
Relative Concentration ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Homogeneous Distribution ◽  
Mrna Levels ◽  
Cell Stage ◽  
Specific Expression ◽  
Morula Stage

SummaryExpression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α− was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α− throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

115 APOPTOSIS EVENT OF PREIMPLANTATION DEVELOPMENT STAGES IN PORCINE IN VITRO-FERTILIZED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 157
Author(s):  
S. M. Hong ◽  
S. H. Jeong ◽  
S. H. Hyun
Keyword(s):  
Cell Death ◽  
Actinomycin D ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Development Stages

Little is known about apoptosis events in porcine preimplantation embryos. In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro-fertilized (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22 h postinsemination were treated at different concentrations of actinomycin D (0, 5, 50 and 500 ng mL–1 in NCSU medium). Four groups were incubated at 37°C in 5% CO2, 5%O2 for 8 h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2 days and BL development rate at 7 days after in vitro culture (IVC). A significantly less rate of cleavage was found in the 500 ng mL–1 group compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 15.4% v. 48.6%, 40%, 32%). In the results of BL formation rate, there was a significantly less BL formation rate in 500 ng mL–1 compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 0% v. 10%, 8.8%, 9%). In experiment 2, to evaluate apoptotic cells at different stage (2-cell, 4-cell, 8-cell and BL stage) of all groups, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng mL–1 actinomycin D). A high number of the BL derived control group contained at least one apoptotic cell. Actinomycin D treated BL responded to the presence of apoptotic inductor by a significant decrease in the average number of blastomeres and a significant increase in the incidence of apoptotic cell death. In the 500 ng mL–1 group, the incidence of apoptosis increased at the 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is a useful tool for the apoptosis study of porcine preimplantation embryos. This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.

Involvement of oocyte-coded message in cell differentiation control of early human embryos

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 317-322 ◽  
Author(s):  
J. Tesarik
Keyword(s):  
Plasma Membrane ◽  
Cell Mass ◽  
Preimplantation Development ◽  
Gene Activity ◽  
Cell Junctions ◽  
Preimplantation Embryos ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Continuous Increase ◽  
Embryonic Genome

Considerable evidence indicates that the first phenotypical diversification of embryonic cells during mammalian preimplantation development is achieved in two successive steps: (i) generation of cell asymmetry and (ii) unequal cell division. This paper shows that ultrastructural signs of blastomere surface regionalization in human preimplantation embryos are evident as early as the 2-cell stage when modifications of the plasma membrane (loss of microvilli and endocytotic activity, formation of cell junctions) are induced in places of blastomere contact. The capacity of the plasma membrane to undergo these cell-contact-dependent changes precedes any detectable activity of the embryonic genome. The area of the modified plasma membrane shows a continuous increase during the first three cleavage stages. The progression of these membrane modifications is the same in embryos that have properly enhanced their transcriptional activity at the 8-cell stage and in those that have not. In spite of the failure of this early-cleavage-progressed-cleavage transition of gene activity, the formation of zonula adherens and gap junctions goes on apparently normally in the respective embryos and morphologically distinct inner cell mass and trophectoderm cell lineages are subsequently segregated in 16-cell morulae. However, tight junctions do not develop under these conditions. The occurrence of the progressed-cleavage pattern of gene activity in the majority of embryonic cells is a necessary prerequisite for the appearance of the blastocyst cavity. Thus, oocyte-coded message is apparently involved in the control of relatively late stages of human preimplantation development including the differentiation of the first two embryonic tissues, but the embryonic genome is required for the full achievement of this early differentiative event.

scholarly journals ZFAND5/ZNF216 is an activator of the 26S proteasome that stimulates overall protein degradation

2018 ◽  
Vol 115 (41) ◽  
pp. E9550-E9559 ◽  
Author(s):  
Donghoon Lee ◽  
Shinichi Takayama ◽  
Alfred L. Goldberg
Keyword(s):  
Protein Degradation ◽  
Zinc Finger ◽  
Mouse Embryonic Fibroblast ◽  
Cellular Protein ◽  
26S Proteasome ◽  
Peptidase Activity ◽  
Protein Breakdown ◽  
Proteasomal Activity ◽  
Ubiquitinated Proteins ◽  
Proteasome Regulators

ZFAND5/ZNF216, a member of the zinc finger AN1-type domain family, is abundant in heart and brain, but is induced in skeletal muscle during atrophy (although not in proteotoxic stress). Because mice lacking ZFAND5 exhibit decreased atrophy, a role in stimulating protein breakdown seemed likely. Addition of recombinant ZFAND5 to purified 26S proteasomes stimulated hydrolysis of ubiquitinated proteins, short peptides, and ATP. Mutating its C-terminal AN1 domain abolished the stimulation of proteasomal peptidase activity. Mutating its N-terminal zinc finger A20 domain, which binds ubiquitin chains, prevented the enhanced degradation of ubiquitinated proteins without affecting peptidase activity. Mouse embryonic fibroblast (MEF) cells lacking ZFAND5 had lower rates of protein degradation and proteasomal activity than WT MEFs. ZFAND5 addition to cell lysates stimulated proteasomal activity and protein degradation. Unlike other proteasome regulators, ZFAND5 enhances multiple 26S activities and overall cellular protein breakdown.

scholarly journals Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 181-191 ◽  
Author(s):  
Satoko Kanzaki ◽  
Shiori Tamura ◽  
Toshiaki Ito ◽  
Mizuki Wakabayashi ◽  
Koji Saito ◽  
...  
Keyword(s):  
Asymmetric Cell Division ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Blastocyst Development ◽  
Triple Mutant ◽  
Cell Stage

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

Transcription profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat

2020 ◽  
Vol 32 (7) ◽  
pp. 714
Author(s):  
Yunsheng Li ◽  
Jiangwen Sun ◽  
Yinghui Ling ◽  
Hao Ming ◽  
Zhen Chen ◽  
...  
Keyword(s):  
Embryo Development ◽  
Mammalian Species ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Transcriptional Networks ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Transcription Profiles ◽  
Stage Of Development

RNA sequencing performed on goat matured oocytes and preimplantation embryos generated invivo enabled us to define the transcriptome for goat preimplantation embryo development. The largest proportion of changes in gene expression in goat was found at the 16-cell stage, not as previously defined at the 8-cell stage, and is later than in other mammalian species. In all, 6482 genes were identified to be significantly differentially expressed across all consecutive developmental stage comparisons, and the important signalling pathways involved in each development transition were determined. In addition, we identified genes that appear to be transcribed only at a specific stage of development. Using weighted gene coexpression network analysis, we found nine stage-specific modules of coexpressed genes that represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the goat transcriptional networks. Their association with other embryo genes suggests that they may have important regulatory roles in embryo development. Our cross-mammalian species transcriptomic comparisons demonstrate both conserved and goat-specific features of preimplantation development.

Sign in / Sign up

Export Citation Format

Share Document