Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

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.

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Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽

10.1530/rep-19-0516 ◽

2020 ◽

Vol 160 (2) ◽

pp. 181-191 ◽

Cited By ~ 2

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.

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Regulation of desmocollin transcription in mouse preimplantation embryos

Development ◽

10.1242/dev.121.3.743 ◽

1995 ◽

Vol 121 (3) ◽

pp. 743-753 ◽

Cited By ~ 9

Author(s):

J.E. Collins ◽

J.E. Lorimer ◽

D.R. Garrod ◽

S.C. Pidsley ◽

R.S. Buxton ◽

...

Keyword(s):

Protein Expression ◽

Molecular Mechanisms ◽

Inner Cell Mass ◽

Splice Variants ◽

Cell Mass ◽

Cumulus Cells ◽

Early Embryo ◽

Preimplantation Embryos ◽

Cell Stage ◽

Cleavage Stages

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527–539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

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228. Proteasomal activity during mouse preimplantation development

Reproduction Fertility and Development ◽

10.1071/srb08abs228 ◽

2008 ◽

Vol 20 (9) ◽

pp. 28 ◽

Cited By ~ 1

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.

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

Reproduction ◽

10.1530/rep-09-0234 ◽

2009 ◽

Vol 138 (5) ◽

pp. 783-791 ◽

Cited By ~ 14

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.

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CAR expression in human embryos and hESC illustrates its role in pluripotency and tight junctions

Reproduction ◽

10.1530/rep-14-0253 ◽

2014 ◽

Vol 148 (5) ◽

pp. 531-544 ◽

Cited By ~ 14

Author(s):

M Krivega ◽

M Geens ◽

H Van de Velde

Keyword(s):

Outer Layer ◽

Transmembrane Domain ◽

Splice Variants ◽

Embryonic Stem ◽

Cell Types ◽

Preimplantation Development ◽

Coxsackie Virus ◽

Preimplantation Embryos ◽

Human Embryos ◽

Adenovirus Receptor

Coxsackie virus and adenovirus receptor,CXADR(CAR), is present during embryogenesis and is involved in tissue regeneration, cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells, from the cleavage stage up to the precursor epiblast, and corresponded with the presence of solubleCXADR3/7splice variant. CAR was displayed on the membrane, involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells, and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts, CAR was reduced in the membrane and concentrated in the nucleus, which correlated with the switch in RNA expression to theCXADR4/7andCXADR2/7splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus, corresponding with the transmembraneCXADRandCXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage, the expression ofCXADRwas downregulated. We concluded thatCXADRis differentially expressed during human preimplantation development. We described various CAR expressions: i) solubleCXADRmarking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types, such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated.

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Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals

Reproduction Fertility and Development ◽

10.1071/rd9960485 ◽

1996 ◽

Vol 8 (4) ◽

pp. 485 ◽

Cited By ~ 255

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.

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

Development ◽

10.1242/dev.115.4.1011 ◽

1992 ◽

Vol 115 (4) ◽

pp. 1011-1016 ◽

Cited By ~ 1

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.

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Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4694-4703.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4694-4703

Author(s):

E M Thompson ◽

E Christians ◽

M G Stinnakre ◽

J P Renard

Keyword(s):

Transgene Expression ◽

Developmental Stages ◽

Preimplantation Embryos ◽

Dependent Manner ◽

Cell Stage ◽

Position Effects ◽

Adult Mice ◽

Preimplantation Mouse ◽

Transgenic Lines

Eukaryotic interphase chromatin is thought to be organized into topologically discrete, independent domains acting as units upon which differential patterns of gene expression are established. Sequences which attach chromatin to in vitro preparations of a nucleoprotein matrix (scaffold attachment regions [SARs]) may act as domain boundaries, but their role remains poorly defined compared with those of other elements such as locus control regions. We have produced mice homozygous for a transgene which is transcribed as early as the activation of the embryonic genome at the two-cell stage and which is expressed ubiquitously in a number of differentiated tissues. Transgenic lines were generated in the presence or absence of flanking SAR sequences, creating an original model which enabled us to examine the effects of these elements at different developmental stages. In the preimplantation mouse embryo, flanking SARs stimulated transgene expression in a copy-dependent manner. In contrast, in the differentiated tissues of newborn and adult mice, no significant SAR-dependent increase in transgene expression was found, correlation with copy number was lost, and position effects were observed. These results suggest a limited capacity of SARs to act as insulating elements but are consistent with a proposed model of SAR-mediated chromatin opening and closing.

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De novo transcription of thyroid hormone receptors is essential for early bovine embryo development in vitro

Reproduction Fertility and Development ◽

10.1071/rd17165 ◽

2018 ◽

Vol 30 (5) ◽

pp. 779 ◽

Cited By ~ 1

Author(s):

N.-Y. Rho ◽

F. A. Ashkar ◽

T. Revay ◽

P. Madan ◽

G.-J. Rho ◽

...

Keyword(s):

Thyroid Hormone ◽

Embryo Development ◽

De Novo ◽

Preimplantation Embryos ◽

Embryo Morphology ◽

Bovine Embryo ◽

Cell Stage ◽

Protein Levels ◽

Development In Vitro

Thyroid hormone receptor (THR) α and THRβ mediate the genomic action of thyroid hormones (THs) that affect bovine embryo development. However, little is known about THRs in the preimplantation embryo. The aim of the present study was to investigate the importance of THRs in in vitro preimplantation bovine embryos. THR transcripts and protein levels were detected in developing preimplantation embryos up to the blastocyst stage. Embryonic transcription of THRs was inhibited by α-amanitin supplementation, and both maternal and embryonic transcription were knocked down by short interference (si) RNA microinjection. In the control group, mRNA and protein levels of THRs increased after fertilisation. In contrast, in both the transcription inhibition and knockdown groups there were significant (P < 0.05) decreases in mRNA expression of THRs from the 2-cell stage onwards. However, protein levels of THRs were not altered at 2-cell stage, although they did exhibit a significant (P < 0.05) decrease from the 4-cell stage. Moreover, inhibition of de novo transcripts of THRs using siRNA led to a significant (P < 0.01) decrease in the developmental rate and cell number, as well as inducing a change in embryo morphology. In conclusion, THRs are transcribed soon after fertilisation, before major activation of the embryonic genome, and they are essential for bovine embryo development in vitro.

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timrit Lengthens Circadian Period in a Temperature-Dependent Manner through Suppression of PERIOD Protein Cycling and Nuclear Localization

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4343 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4343-4354 ◽

Cited By ~ 35

Author(s):

Akira Matsumoto ◽

Kenji Tomioka ◽

Yoshihiko Chiba ◽

Teiichi Tanimura

Keyword(s):

Nuclear Localization ◽

Temperature Compensation ◽

Feedback Loop ◽

Gene Dosage ◽

Dependent Manner ◽

Temperature Dependent ◽

Protein Levels ◽

Free Running ◽

Free Running Period ◽

Posttranscriptional Level

ABSTRACT A fundamental feature of circadian clocks is temperature compensation of period. The free-running period of ritsu(timrit ) (a novel allele oftimeless [tim]) mutants is drastically lengthened in a temperature-dependent manner. PER and TIM protein levels become lower in timrit mutants as temperature becomes higher. This mutation reduces per mRNA but not tim mRNA abundance. PER constitutively driven by the rhodopsin1 promoter is lowered in ritmutants, indicating that timrit mainly affects the per feedback loop at a posttranscriptional level. An excess of per + gene dosage can ameliorate allrit phenotypes, including the weak nuclear localization of PER, suggesting that timrit affects circadian rhythms by reducing PER abundance and its subsequent transportation into nuclei as temperature increases.

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