Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals

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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Methylation levels of maternal and paternal genomes during preimplantation development

Development ◽

10.1242/dev.113.1.119 ◽

1991 ◽

Vol 113 (1) ◽

pp. 119-127 ◽

Cited By ~ 8

Author(s):

S.K. Howlett ◽

W. Reik

Keyword(s):

Methylation Status ◽

Germinal Vesicle ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Cell Stage ◽

Global Methylation ◽

Cell Cycles ◽

Dna Methylase ◽

Parthenogenetic Embryos ◽

Metaphase Ii Oocytes

The methylation status of three highly repeated sequences was studied in sperm, eggs and preimplantation embryos with different combinations of parental chromosomes. High levels of methylation of the IAP and MUP sequence families were found in sperm and in eggs, whereas the L1 repeat was found to be highly methylated in sperm but only about 42% methylated in eggs. To assess how the two parental genomes behaved during preimplantation development, normal, fertilised embryos were compared with parthenogenetic embryos where the chromosomes are exclusively of maternal origin. It was observed that the high levels of methylation at the IAP and MUP sequences were retained through early development, with the first signs of demethylation at the IAP sequences apparent on both parental chromosomes in the blastocyst. Methylation at the sperm-derived L1 sequences dropped to about the same level as that of the egg-derived sequences by the late 2-cell stage, both then remain at this intermediate level until around the time of cavitation when levels fell to about 10% in the blastocyst. High levels of DNA methylase were detected in germinal vesicle and metaphase II oocytes; these high levels were maintained in fertilised and parthenogenetic embryos through into the morula and then declined to be undetectable in the blastocyst. Our comparison of maternal and paternal genomes suggests that methylation levels at repeat sequences are remarkably similar at the time of fertilisation or, as in the case of the L1 sequences, they become so during the first few cell cycles. Hence, there do not appear to be global methylation differences between the genomes that are retained through preimplantation development.(ABSTRACT TRUNCATED AT 250 WORDS)

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Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

Reproduction ◽

10.1530/rep-16-0277 ◽

2016 ◽

Vol 152 (5) ◽

pp. 417-430 ◽

Cited By ~ 10

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.

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336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab336 ◽

2010 ◽

Vol 22 (1) ◽

pp. 324 ◽

Cited By ~ 3

Author(s):

M. De los Reyes ◽

D. Luna ◽

J. Palomino

Keyword(s):

In Vitro Maturation ◽

Germinal Vesicle ◽

Fluorescein Isothiocyanate ◽

Cumulus Cells ◽

Homogeneous Distribution ◽

Cortical Granules ◽

Dapi Staining ◽

Nuclear Maturation ◽

Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

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

Zygote ◽

10.1017/s0967199410000705 ◽

2011 ◽

Vol 20 (2) ◽

pp. 147-158 ◽

Cited By ~ 6

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab115 ◽

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.

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

Development ◽

10.1242/dev.105.2.317 ◽

1989 ◽

Vol 105 (2) ◽

pp. 317-322 ◽

Cited By ~ 1

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.

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Metabolic behaviour and cleavage capacity in the amphibian egg

Development ◽

10.1242/dev.47.1.161 ◽

1978 ◽

Vol 47 (1) ◽

pp. 161-168

Author(s):

Arnaldo H. Legname ◽

Marta I. Buhler

Keyword(s):

Winter Season ◽

Germinal Vesicle ◽

Fundamental Aspect ◽

Embryonic Cells ◽

Germinal Vesicle Breakdown ◽

Nuclear Maturation ◽

Ovarian Metabolism ◽

Cytoplasmic Maturation ◽

Short Time ◽

Metabolic Behaviour

During the winter season the full grown Bufo arenarum oocyte shows the metabolic behaviour characteristic of differentiated tissues of the same species. Due to seasonal variations, uring the amplexus period, it acquires the metabolic behaviour of the segmenting egg. Short-time-induced ovulations (5–6 h) determine germinal vesicle breakdown immediately before the expulsion of the oocyte, without modifying the ovarian metabolism of the same. The incidence of the operative type of metabolism upon their capacity to cleave after insemination and needle pricking, has been studied in coelomic oocytes, which have attained nuclear maturation and have not experienced oviducal secretion effects. The results obtained indicate that the segmenting capacity of the egg is attained only when, through biochemical modifications, the oocyte acquires the metabolic behaviour characterizing embryonic cells. It is postulated that the metabolic changes observed in the oocyte constitute a fundamental aspect of cytoplasmic maturation.

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