Arrowheads show the chromosomes distributed throughout the cytoplasm. Well-organized spindle and chromosomes aligned on the metaphase plate are visible in both types of cytoplasm.
The spindle remained intact and some scattered chromosomes could be detected in the cytoplasm 1 h after activation Fig. Chromosomes separated into two groups and moved toward their respective poles 3 h after activation Fig.
The two groups of chromosomes remained condensed and a spindle continued to be assembled 5 h after activation, with no pseudopronuclei formation observed Fig. Chromosomes were moving toward their respective poles and the reconstructed oocyte was at the anaphase stage 1 h after onset of activation Fig. Two sets of separated chromosomes were starting to decondense 3 h after activation Fig.
Two large decondensed pseudopronuclei formed 5 h after activation, and the spindle remained visible Fig. The reconstructed oocyte was at the telophase stage and two groups of chromosomes were at their respective poles 1 h after activation Fig. Cytoskeleton organization and DNA configuration in reconstructed oocytes during activation. No pronuclei formation is visible, and chromosomes are segregated into two groups.
MII reconstructed oocytes showed more microfilament distributed in the cortex than do the other two types of reconstructed oocytes. However, the survival rate using MII cytoplasm was slightly higher than that for the other two types of cytoplasm. No reconstructed oocytes from GV cytoplasm were activated after strontium treatment. There were no differences in pseudopronuclei formation between MII and pro-MI reconstructed oocytes after activation.
Among the embryos that underwent the first cleavage, MII reconstructed embryos showed a much higher subsequent development rate than did pro-MI reconstructed embryos Table 1. After further culture, only 8. After embryo transfer, six pups were recovered from the surrogate mothers at Development of embryos reconstructed by injection of R1 ES cell nuclei into mature cytoplasm enucleated at different stages.
In this study, GV material was essential for nuclear remodeling after nuclear transfer. When mature GV stage enucleated oocytes were used as recipients for nuclear transfer, reconstructed oocytes could not form pseudopronuclei after activation treatment, and chromosomes remained condensed.
Although reconstructed embryos from mature pro-MI stage enucleated oocytes could form pseudopronuclei normally, very few embryos cleaved and developed to morulae. In animal cloning experiments, nuclear transfer has been performed mostly with enucleated MII oocytes that were subsequently activated to initiate embryo development. The transferred nucleus undergoes disassembly and reassembly in the MII cytoplasm [ 28 ].
Disassembly involves nuclear envelope and lamina breakdown and chromatin condensation [ 29 — 33 ]. Following activation of the reconstructed oocyte, nuclear reassembly involves decondensation of the chromatin, formation of a new nuclear envelope, polymerization of a new lamina, formation of nucleoli and expansion of the remodeled nucleus [ 34 ].
In the present study, when a mature GV stage enucleated oocyte was used as a recipient for nuclear transfer the transferred ES cell nucleus could be disassembled, as indicated by chromatin condensation. However, following activation the nucleus could not be fully remodeled; no nucleolus formation and no expansion of the nucleus was observed in the reconstructed oocyte.
Disassembly of the transferred nucleus is related to the level of MPF activity, and others have demonstrated that the anucleate oocyte can undergo MPF activation [ 12 — 16 ]. Our results also indicate that GV material is not required for MPF activation because the spindle can organize and the nucleus can condense into chromosomes 1—3 h after injection of the ES cell nucleus into a mature GV stage enucleated oocyte. However, following activation treatment no pseudopronuclei formation was observed in the reconstructed oocyte.
These results suggest that the GV material is responsible for the reassembly of the transferred nucleus. Both of these molecules can mediate the transfer of the core histones to DNA and the assembly of nucleosomes [ 39 ].
Nucleoplasmin is important in removing sperm protamines and decondensing the sperm chromatin to allow the assembly of the paternal pronucleus [ 40 , 41 ]. To elucidate the mechanism of reprogramming, we must determine which molecules, such as nucleoplasmin and others, are released from the GV at GVBD in mammal oocytes.
In contrast to GV stage enucleated oocytes, the transferred nucleus can undergo disassembly and reassembly in the mature pro-MI stage enucleated oocyte. Similar to the MII enucleated oocyte, a bipolar spindle formed and condensed chromosomes aligned on the metaphase plate 1—3 h after nuclear transfer. Only a large cell G2 nucleus could condense to form an orderly chromosome array resembling an MII plate. A different staining method used in this study showed the association of chromosomes and spindle much more directly.
Compared with MII reconstructed embryos, the developmental potential of reconstructed embryos with mature pro-MI cytoplasm is very limited. Microfilament distribution in the pro-MI reconstructed embryos was very different from that of MII reconstructed embryos, and less microfilament distribution was observed in pro-MI reconstructed embryos.
Actin is a major component of the cytoskeleton. Early studies suggested that actin filaments were responsible for maintenance of the meiotic spindle, spindle rotation, polar body release, pronuclei migration, and mitotic cleavage [ 42 — 46 ].
The decreased microfilament distribution may be one important reason for the poor development of reconstructed embryos with mature pro-MI cytoplasm. In previous studies, embryos produced by in vitro fertilization of in vitro-matured oocytes showed very different microfilament distribution and limited developmental ability compared with in vivo-produced embryos [ 47 , 48 ]. Another possible reason for the poor development in pro-MI reconstructed embryos could be that enucleated oocytes were matured without nuclei.
Nuclear-cytoplasm coordinated maturation is believed to be necessary for full maturation of oocytes [ 49 ]. Cumulus cell could also be a factor in the poor development of pro-MI reconstructed embryos; the pro-MI cytoplasts were matured in vitro without cumulus cells. In previous studies, cumulus cells were important for oocyte maturation and further development after fertilization [ 50 — 53 ].
The results of the present study demonstrated that GV material is essential for nucleus remodeling after nuclear transfer. More work is needed to determine what factors are released from the GV during GVBD, as this information will enable us to better understand the molecular basis of nuclear reprogramming.
We thank Miss Heather A. Warnock and Louise V. Rahr from the small animal unit of Roslin Institute for assistance with cesarean sections and pup care. Viable offspring derived from fetal and adult mammalian cells. Nature ; : — Google Scholar. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Eight calves cloned from somatic cells of a single adult. Science ; : — Production of goats by somatic nuclear transfer. Nat Biotechnol ; 17 : — Pig cloning by microinjection of fetal fibroblast nuclei.
Cloned pigs produced by nuclear transfer from adult somatic cells. Mice cloned from embryonic stem cells. Thus, the NSN-to-SN transition is one of the most important aspects in oocyte cytoplasmic maturation. Thus, the signaling pathways leading to the NSN-to-SN transition must be studied in order to better understand the epigenetic mechanisms of oocyte gene expression and to select oocytes that are more competent for in vitro maturation.
Furthermore, such research will also provide new insights into the molecular regulation of somatic cell reprogramming. The aim of the present study was to explore the signaling pathways leading to oocyte NSN-to-SN transition by using pig oocytes from small antral follicles.
The data not only will contribute to our understanding of the epigenetic mechanisms for oocyte maturation but also will provide important models for research on regulation of DNA transcription and the epigenetics and reprogramming in somatic cells.
The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on disappearance of nucleolus and nuclear membrane Fig. The GV0 configuration was characterized by a distinct nucleolus and a diffuse, filamentous pattern of chromatin in the whole GV area.
In GV1, the nucleolus was surrounded by a complete heterochromatin ring and heterochromatin was not obvious in the nucleoplasm. In GV2 and GV3, the heterochromatin ring around the nucleolus was often incomplete or forming a horseshoe, and clumps and strands of heterochromatin were observed in the GV.
In GV4, the heterochromatin clumps or strands remained but the nuclear membrane was less distinct and the nucleolus disappeared completely. Gene activities in oocytes with different chromatin configurations were determined by observing global RNA transcription after 5-ethynyl uridine EU labeling. Photographs in the top and middle rows for each chromatin configuration are the same oocyte observed with phase contrast and fluorescence, respectively, after Hoechst staining.
The nucleolus is indicated with arrows in the phase contrast images. Photographs in the bottom row are laser confocal merged images showing global RNA transcription of porcine oocytes with different GV chromatin configurations. Each treatment was repeated 3 times with each replicate containing about 30 oocytes. Each treatment was repeated 3 times with each replicate containing cumulus-free oocytes for p-MAPK and 40 oocytes for MPF activity assays.
Each treatment was repeated 4—5 times with each replicate containing about 25 oocytes. Each treatment was repeated 3 times with each replicate containing cumulus-free oocytes. G Intra-oocyte cAMP levels in oocytes from follicles of different diameters. Each treatment was repeated 3—4 times with each replicate including 25 oocytes. Each treatment was repeated 4—5 times with each replicate including 25 oocytes.
Data in Fig. DOs were cultured with or without dispersed CCs. In the pig, FSH secretion is relatively constant throughout the estrous cycle 20 and the growth of 1. To observe chromatin configuration, each treatment was repeated 4—5 times with each replicate containing about 25 oocytes.
For FSH assays in follicular fluid, each treatment was repeated 3 times with each replicate including 50 follicles. Each treatment was repeated 3 times with each replicate containing follicular fluid from 50 follicles.
Each treatment was repeated 3 times with each replicate containing 25 COCs. By manipulating enzyme activities in vitro and by measuring related molecules both in vivo and in vitro , this study has established a pivotal role for MAPK in the regulation of the NSN-to-SN transition. Furthermore, by knocking out Erk1 and Erk2 in mouse oocytes, Zhang et al. Studies on histone acetylation also indicated different mechanisms for the GV chromatin condensation and the post-GVBD chromosome condensation.
For example, fully grown mouse oocytes mostly of SN configuration were fully acetylated at all the lysine residues on H3 and H4, but underwent deacetylation after GVBD 1 , Thus, the different mechanisms between the GV chromatin condensation and the post-GVBD chromosome condensation will be an very interesting topic in future studies.
The present results indicated that in CCs of the small porcine follicles, a series of molecular events resulted in reduced production and delivery of cGMP into the oocyte, leading to a decline in intra-oocyte cAMP that inhibited the NSN-to-SN transition Fig.
It is known that the dominant follicles produce a large quantity of estrogen, which turns down the pituitary secretion of FSH Thus, it was expected that most of the medium subordinate follicles would suffer FSH insufficiency and undergo atresia at this stage of follicle selection In both cows 38 and sows 39 , the concentration of E2 increased significantly with increasing follicular sizes.
Studies in the human suggested that GDF9 levels in the follicular fluid were highly correlated with oocyte nuclear maturation and embryo quality Furthermore, a significant positive correlation was observed between BMP and E2 levels in the same human follicle LH-dependent activation of MAPK has been observed in granulosa cells of different species including the pig However, in the summary by Conti et al.
As a result, oocytes remain at NSN. The data not only will contribute to our understanding of the epigenetic mechanisms for oocyte maturation but also will provide important models for research on the epigenetics and reprogramming in somatic cells. The experimental procedures used for animal care and handling were approved by the Animal Care and Use Committee of the Shandong Agricultural University P. The methods were carried out in accordance with the approved guidelines. Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich Corp.
Only COCs with uniform ooplasm and compact cumulus were chosen for further treatment. Whereas some of the COCs were cultured directly after collection, others were mechanically denuded of cumulus cells CCs before culture as denuded oocytes DOs. Pellets were resuspended, and cells were counted on a hemocytometer. Oocytes were then placed on glass slides and compressed with coverslips to visualize GV. Oocytes were first examined with phase contrast to visualize morphology of nucleoli and nuclear envelope, and then observed with fluorescence optics.
After EU labeling, oocytes were 1 fixed using 3. The relative quantities of proteins were determined with an Image-Pro Plus software by analyzing the sum density of each protein band image. Immediately after amplification, PCR products were analyzed by sequencing, dissociation-curve analysis and gel electrophoresis to determine specificity of the reaction. The minimum detectable amounts of estradiol and FSH were 1.
We conducted at least three replicate trials for each treatment. How to cite this article : Sun, M. De La Fuente, R. Chromatin modifications in the germinal vesicle GV of mammalian oocytes.
Dev Biol , 1—12 Mattson, B. Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol Reprod Dev 25 , — Large-scale chromatin remodeling in germinal vesicle bovine oocytes: interplay with gap junction functionality and developmental competence. Oocyte morphology and transcriptional silencing in relation to chromatin remodeling during the final phases of bovine oocyte growth.
Hypoxanthine HX inhibition of in vitro meiotic resumption in goat oocytes. Chromatin configuration and transcriptional control in human and mouse oocytes. Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro. RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study. Meiotic competence in vitro of pig oocytes isolated from early antral follicles.
Germinal vesicle stages in pig follicular oocytes collected by different methods. Follicular oocytes of the man cytological and physiological characteristics. Meiotic competence and acetylation pattern of UV light classified mouse antral oocytes after meiotic arrest with isobutylmethylxanthine.
Human antral follicles: oocyte nucleus and the karyosphere formation electron microscopic and autoradiographic data. Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology.
Cytogenetic analysis of human oocytes and their maturation process outside the body. Involvement of apoptosis in disruption of developmental competence of bovine oocytes by heat shock during maturation. Modifications in chromatin morphology and organization during sheep oogenesis. Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys.
Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocytes. Configurations of germinal vesicle GV chromatin in the goat differ from those of other species. Changes in germinal vesicle GV chromatin configurations during growth and maturation of porcine oocytes. Ultrastructural cytochemistry of the nucleus and nucleolus in growing rabbit oocytes.
Dynamic changes of germinal vesicle chromatin configuration and transcriptional activity during maturation of rabbit follicles correlate with oocyte potential for in vitro maturation and developmental competence.
Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes. Changes in expression of connexin 43 gap junction messenger ribonucleic acid and protein during ovarian follicular growth.
A growth-maturation system that enhances the meiotic and developmental competence of porcine oocytes isolated from small follicles. Studies of the cell cycle of in vitro cultured skin fibroblasts in goats: work in progress. Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts.
A study on chromatin configuration in the germinal vesicle of rat oocytes. The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst.
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Proceedings of the Royal Society of London Biology — Molecular Reproduction and Development 25 — Molecular Reproduction and Development 64 — Journal of Cell Biology 60 — Gamete Research 22 — Biology of Reproduction 69 — Molecular Reproduction and Development 43 36 — Current Biology 10 — Reproduction, Fertility, and Development 19 1 — Whittingham DG Culture of mouse ova.
Journal of Reproduction and Fertility 14 7 — Developmental Biology 62 — Molecular Reproduction and Development 41 — Biology of Reproduction 58 — Zygote 10 73 — BMC Developmental Biology 8 Sign in Create account. Author guidelines Submit now Reasons to publish Ethical policy Open-access policy Publication charges Author resource centre. Advanced Search Help. The position of the germinal vesicle and the chromatin organization together provide a marker of the developmental competence of mouse antral oocytes in Reproduction.
Free access. Download PDF. Check for updates. Get Permissions. Abstract Based on their chromatin organization, antral oocytes can be classified into two classes, namely surrounded nucleolus SN, chromatin forms a ring around the nucleolus , and not surrounded nucleolus NSN, chromatin has a diffuse pattern.
Introduction A number of morphological and molecular markers have been suggested to predict the meiotic and developmental competence of antral oocytes Albertini et al. Discussion A major problem in the use of assisted reproductive technologies is the difficulty to make predictions on the quality of the available oocytes.
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