scholarly journals Centrosome changes during meiosis in horse oocytes and first embryonic cell cycle organization following parthenogenesis, fertilization and nuclear transfer

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 661-667 ◽  
Author(s):  
Xihe Li ◽  
Y Qin ◽  
Sandra Wilsher ◽  
W R Allen
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Embryonic Cell ◽  
Immunofluorescent Staining ◽  
Sperm Chromatin ◽  
Germinal Vesicle Breakdown ◽  
Embryonic Cell Cycle

Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII stage oocytes to fertilization by intracytoplasmic sperm injection (ICSI), parthenogenetic treatment or nuclear transfer. Centrosome changes during meiosis and the first cell cycle organization were detected by indirect immunofluorescent staining, using a mouse anti-α-tubulin antibody for microtubules and a rabbit anti-γ-tubulin antibody for centrosomes. These examinations showed that the centrosomes of the horse oocyte reorganize themselves from the beginning of GV stage to leave only PCM of γ-tubulin surrounding both poles of the MI and MII stage spindles. These MII oocytes can organize the separation of metaphase chromosomes during the first embryonic cell cycle by parthenogenetic treatment. When the MII oocytes were subjected to ICSI or nuclear transfer, one or two red-stained centrosomes of γ-tubulin were introduced by the fertilising spermatozoon or the donor cell which associated with the sperm chromatin in the fertilized embryos and with the donor cell chromatin and microtubules in the cloned embryos. This finding suggests that centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development.

scholarly journals A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

1997 ◽  
Vol 137 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Xuequn Helen Hua ◽  
Hong Yan ◽  
John Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cyclin E ◽  
Embryonic Cell ◽  
S Phase ◽  
Negative Regulation ◽  
Sperm Chromatin ◽  
High Concentration ◽  
Low Concentrations ◽  
Embryonic Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.

scholarly journals Excess histone H3 is a Chk1 inhibitor that controls embryonic cell cycle progression

2020 ◽  
Author(s):  
Yuki Shindo ◽  
Amanda A. Amodeo
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Cell Cycle Progression ◽  
Histone H3 ◽  
Embryonic Cell ◽  
Cycle Progression ◽  
Cell Cycles ◽  
Embryonic Cell Cycle

AbstractThe early embryos of many species undergo a switch from rapid, reductive cleavage divisions to slower, cell fate-specific division patterns at the Mid-Blastula Transition (MBT). The maternally loaded histone pool is used to measure the increasing ratio of nuclei to cytoplasm (N/C ratio) to control MBT onset, but the molecular mechanism of how histones regulate the cell cycle has remained elusive. Here, we show that excess histone H3 inhibits the DNA damage checkpoint kinase Chk1 to promote cell cycle progression in the Drosophila embryo. We find that excess H3-tail that cannot be incorporated into chromatin is sufficient to shorten the embryonic cell cycle and reduce the activity of Chk1 in vitro and in vivo. Removal of the Chk1 phosphosite in H3 abolishes its ability to regulate the cell cycle. Mathematical modeling quantitatively supports a mechanism where changes in H3 nuclear concentrations over the final cell cycles leading up to the MBT regulate Chk1-dependent cell cycle slowing. We provide a novel mechanism for Chk1 regulation by H3, which is crucial for proper cell cycle remodeling during early embryogenesis.

Pronucleus development and organization of intranuclear bodies during the first bovine embryonic cell cycle in vitro

1997 ◽  
Vol 47 (1) ◽  
pp. 326 ◽  
Author(s):  
J. Laurinčík ◽  
P. Hyttel ◽  
V. Baran ◽  
A. Lucas-Hahn ◽  
J. Eckert ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Embryonic Cell ◽  
Embryonic Cell Cycle

scholarly journals The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation

Reproduction ◽  
2005 ◽  
Vol 130 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Luisa Gioia ◽  
Barbara Barboni ◽  
Maura Turriani ◽  
Giulia Capacchietti ◽  
Maria Gabriella Pistilli ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Hdac Inhibitors ◽  
Germinal Vesicle ◽  
Fertilization Rate ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Germinal Vesicle Breakdown

The present experiments compared the ability of pig oocytes matured eitherin vivoorin vitroto structurally reorganize the penetrated sperm chromatin into male pronucleus (PN) and to carry out, in parallel, the epigenetic processes of global chromatin methylation and acetylation, 12–14 h afterin vitrofertilization (IVF). In addition, PN distribution of histone deacetylase (HDAC), a major enzyme interfacing DNA methylation and histone acetylation, was investigated. The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The monospermic fertilization rate was significantly higher forin vivothan forin vitromatured (IVM) oocytes(P< 0.01) which, furthermore, showed a reduced ability to transform the chromatin of penetrated sperm into male PN(P< 0.01). Indirect immunofluorescence analysis of global DNA methylation, histone acetylation and HDAC distribution (HDAC-1, -2 and -3), carried out in monospermic zygotes that reached the late PN stage, showed that IVM oocytes also had a reduced epigenetic competence. In fact, while in about 80% ofin vivomatured and IVF oocytes the male PN underwent a process of active demethylation and showed a condition of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a similar PN remodelling asymmetry. Oocytes that carried out the first part of maturationin vivo(up to germinal vesicle breakdown; GVBD) and then completed the processin vitro, displayed the same PN asymmetry as oocytes matured entirelyin vivo. A crucial role of HDAC in the establishment of PN acetylation asymmetry seems to be confirmed by the use of HDAC inhibitors as well as by the abnormal distribution of the enzyme between the two PN in IVM zygotes. Collectively, these data demonstrated that some pig IVM oocytes fail to acquire full remodelling competence which is independent from their ooplasmic ability to morphologically reorganize the sperm nucleus into PN.

Sequence and regulation of morphological and molecular events during the first cell cycle of mouse embryogenesis

Development ◽  
1985 ◽  
Vol 87 (1) ◽  
pp. 175-206
Author(s):  
Sarah K. Howlett ◽  
Virginia N. Bolton
Keyword(s):  
Cell Cycle ◽  
Germinal Vesicle ◽  
In Vitro Translation ◽  
Careful Study ◽  
Germinal Vesicle Breakdown ◽  
Post Translational Modifications ◽  
Molecular Events ◽  
Terminal Events ◽  
Sperm Penetration

Mouse oocytes were fertilized in vitro and the precise timing and sequence of morphological and molecular events occurring during the first cell cycle were investigated. The timing of development through the first cell cycle was found to be initiated by an event associated with sperm penetration rather than with germinal vesicle breakdown. DNA replication is initiated randomly in either pronucleus of a given egg, beginning approximately 11 h post insemination (hpi), and S phase lasting 6–7 h in both. Careful study of polypeptide synthetic profiles revealed three classes of changes in polypeptide synthesis during the first few hours of development: fertilization-independent, fertilization-accelerated, and fertilization-dependent. Pulse-chase experiments and in vitro translation of extracted mRNA showed that the changes in polypeptide synthetic profile result from differential mRNA activation, differential polypeptide turnover and post-translational modifications. These results support the notion that following ovulation, development is controlled at two levels. An endogenous (oocyte) programme, set in train by the terminal events of oocyte maturation, may regulate the ‘housekeeping’ functions of the egg, while sperm penetration activates a further endogenous (fertilization) programme, which may serve to initiate subsequent embryogenesis.

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 165-171
Author(s):  
M. Kishi ◽  
R. Takakura ◽  
Y. Nagao ◽  
K. Saeki ◽  
Y. Takahashi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
Cell Cycle Stage

SummaryIn the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.

scholarly journals NuMA distribution and microtubule configuration in rabbit oocytes and cloned embryos

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 869-876 ◽  
Author(s):  
Li-Ying Yan ◽  
Jun-Cheng Huang ◽  
Zi-Yu Zhu ◽  
Zi-Li Lei ◽  
Li-Hong Shi ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Spindle Pole ◽  
Germinal Vesicle Breakdown ◽  
Spindle Poles ◽  
Oocyte Meiosis ◽  
Reconstructed Embryo ◽  
Oocyte Cytoplasm

The assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. α-Tubulin was localized around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around the introduced nucleus in most reconstructed embryos and formed a transient spindle 2–4 h post-fusion (hpf). A mass of microtubules surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMAwas detected in the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase. In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit oocyte meiosis and cloned embryo mitosis.

scholarly journals SUMOylation regulates germinal vesicle breakdown and the Akt/PKB pathway during mouse oocyte maturation

2018 ◽  
Vol 315 (1) ◽  
pp. C115-C121 ◽  
Author(s):  
Weber Beringui Feitosa ◽  
Patricia L. Morris
Keyword(s):  
Cell Cycle ◽  
Oocyte Maturation ◽  
Nuclear Translocation ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Serine Phosphorylation ◽  
Germinal Vesicle Breakdown ◽  
Akt Activation ◽  
Downstream Target

SUMOylation, a process of posttranslational modification of proteins by the small ubiquitin-related modifier (SUMO) family of proteins, is known to be involved in yeast and mammalian somatic cell-cycle regulation. However, the identities of the SUMO-modified oocyte targets are largely unknown and the functional role(s) for SUMOylation during mammalian oocyte maturation remains unclear. On the basis of studies in non-germline cells, protein kinase B/Akt is a potential SUMOylation target in the mouse oocyte, where it plays an essential role in cell-cycle resumption and progression during maturation. This study investigated the temporal patterns and prospective role(s) for interactions between SUMOylation and Akt serine-phosphorylation during oocyte meiotic resumption. Pharmacological inhibition of SUMOylation significantly decreased follicular fluid meiosis-activating sterol-induced cell-cycle resumption in oocytes matured in vitro and negatively affected the phosphorylation and nuclear translocation of Akt. Similarly, nuclear localization of cyclin D1, a downstream target of Akt activation, was significantly decreased following SUMOylation inhibition. Together these data show that SUMO and the posttranslational process of SUMOylation are involved in cell-cycle resumption during murine oocyte maturation and exert a regulatory influence on the Akt pathway during germinal vesicle breakdown.

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