Somatic Cell Nuclei Research Articles

Various nuclear reprogramming systems using egg and oocyte materials.

Maternal factors stored in eggs and oocytes are necessary for reprogramming sperm for embryonic development. This reprogramming activity of maternal factors also works towards somatic cells, including terminally differentiated cells. Several different experimental systems utilizing egg and oocyte materials have been applied to study nuclear reprogramming by maternal factors. Among these systems, the most widely used is the transfer of a somatic cell nucleus to an oocyte arrested at the metaphase II stage, leading to the production of a cloned animal. Nuclear transfer to an unfertilized oocyte thus provides a unique opportunity to examine reprogramming processes involved in acquiring totipotency. Other experimental systems are also available to study maternal reprogramming, such as nuclear transfer to Xenopus laevis oocytes at the germinal vesicle stage, treatment with extracts obtained from eggs or oocytes, and induced pluripotency with overexpressed maternal factors. Each system can be used for answering different types of scientific questions. This review describes currently available reprogramming systems using egg and oocyte materials and discusses how we can deepen our understanding of reprogramming mechanisms by taking advantage of these various experimental systems.

Out-of-position telomeres in meiotic leptotene appear responsible for chiasmate pairing in an inversion heterozygote in wheat (Triticum aestivum L.).

Chromosome pairing in meiosis usually starts in the vicinity of the telomere attachment to the nuclear membrane and congregation of telomeres in the leptotene bouquet is believed responsible for bringing homologue pairs together. In a heterozygote for an inversion of a rye (Secale cereale L.) chromosome arm in wheat, a distal segment of the normal homologue is capable of chiasmate pairing with its counterpart in the inverted arm, located near the centromere. Using 3D imaging confocal microscopy, we observed that some telomeres failed to be incorporated into the bouquet and occupied various positions throughout the entire volume of the nucleus, including the centromere pole. Rye telomeres appeared ca. 21 times more likely to fail to be included in the telomere bouquet than wheat telomeres. The frequency of the out-of-bouquet rye telomere position in leptotene was virtually identical to the frequency of telomeres deviating from Rabl's orientation in the nuclei of somatic cells, and was similar to the frequency of synapsis of the normal and inverted chromosome arms, but lower than the MI pairing frequency of segments of these two arms normally positioned across the volume of the nucleus. Out-of-position placement of the rye telomeres may be responsible for reduced MI pairing of rye chromosomes in hybrids with wheat and their disproportionate contribution to aneuploidy, but appears responsible for initiating chiasmate pairing of distantly positioned segments of homology in an inversion heterozygote.

Analysis of stage-specific expression of the toll-like receptor family in the porcine endometrium throughout the estrous cycle and pregnancy

Toll-like receptors (TLRs) play critical roles in innate immunity by regulating antimicrobial responses in mucosal tissues. The expression and function of TLRs in female reproductive tissues have been studied in several species, but the expression and function of TLRs and MYD88, an adaptor molecule in the TLR signaling pathway, at the maternal-conceptus interface are not well understood in pigs. Thus, we determined the expression of TLR1 - TLR10 and MYD88 in the endometrium, conceptus, and chorioallantoic tissues of pigs. TLR1 - TLR10 and MYD88 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy in a stage-dependent manner. TLR and MYD88 mRNAs were also detected in early stage conceptuses and chorioallantoic tissues from Day 30 to term pregnancy. The expression of TLR2, TLR4, TLR5, and TLR7 was localized to epithelial and stromal cells in endometrial and chorioallantoic tissues. Increasing doses of P4, but not E2, induced the expression of TLR4, TLR5, TLR6, TLR7, and TLR8, while interferon-γ increased the expression of TLR2 and TLR7 in endometrial explant tissues. Expression of TLR3, TLR5, TLR6, TLR7, and MYD88 was higher in the endometrium with somatic cell nucleus transfer-derived conceptuses than conceptuses derived from natural mating on Day 12. These results indicate that the expression of TLR1 - TLR10 and MYD88 is dynamically regulated at the maternal-conceptus interface in pigs, suggesting that TLRs expressed in the endometrium and the placenta may play a critical role in regulating mucosal immune responses to support the establishment and maintenance of pregnancy.

Sequence Composition and Evolution of Mammalian B Chromosomes.

B chromosomes (Bs) revealed more than a hundred years ago remain to be some of the most mysterious elements of the eukaryotic genome. Their origin and evolution, DNA composition, transcriptional activity, impact on adaptiveness, behavior in meiosis, and transfer to the next generation require intensive investigations using modern methods. Over the past years, new experimental techniques have been applied and helped us gain a deeper insight into the nature of Bs. Here, we consider mammalian Bs, taking into account data on their DNA sequencing, transcriptional activity, positions in nuclei of somatic and meiotic cells, and impact on genome functioning. Comparative cytogenetics of Bs suggests the existence of different mechanisms of their formation and evolution. Due to the long and complicated evolvement of Bs, the similarity of their morphology could be explained by the similar mechanisms involved in their development while the difference between Bs even of the same origin could appear due to their positioning at different stages of their evolution. A complex analysis of their DNA composition and other features is required to clarify the origin and evolutionary history of Bs in the species studied. The intraspecific diversity of Bs makes this analysis a very important element of B chromosome studies.

DCAF13 promotes pluripotency by negatively regulating SUV39H1 stability during early embryonic development.

Mammalian oocytes and zygotes have the unique ability to reprogram a somatic cell nucleus into a totipotent state. SUV39H1/2-mediated histone H3 lysine-9 trimethylation (H3K9me3) is a major barrier to efficient reprogramming. How SUV39H1/2 activities are regulated in early embryos and during generation of induced pluripotent stem cells (iPSCs) remains unclear. Since expression of the CRL4 E3 ubiquitin ligase in oocytes is crucial for female fertility, we analyzed putative CRL4 adaptors (DCAFs) and identified DCAF13 as a novel CRL4 adaptor that is essential for preimplantation embryonic development. Dcaf13 is expressed from eight-cell to morula stages in both murine and human embryos, and Dcaf13 knockout in mice causes preimplantation-stage mortality. Dcaf13 knockout embryos are arrested at the eight- to sixteen-cell stage before compaction, and this arrest is accompanied by high levels of H3K9me3. Mechanistically, CRL4-DCAF13 targets SUV39H1 for polyubiquitination and proteasomal degradation and therefore facilitates H3K9me3 removal and zygotic gene expression. Taken together, CRL4-DCAF13-mediated SUV39H1 degradation is an essential step for progressive genome reprogramming during preimplantation embryonic development.

Can Reprogramming of Overall Epigenetic Memory and Specific Parental Genomic Imprinting Memory within Donor Cell-Inherited Nuclear Genome be a Major Hindrance for the Somatic Cell Cloning of Mammals? – A Review

Abstract Successful cloning of animals by somatic cell nuclear transfer (SCNT) requires epigenetic transcriptional reprogramming of the differentiated state of the donor cell nucleus to a totipotent embryonic ground state. It means that the donor nuclei must cease its own program of gene expression and restore a particular program of the embryonic genome expression regulation that is necessary for normal development. Transcriptional activity of somatic cell-derived nuclear genome during embryo pre- and postimplantation development as well as foetogenesis is correlated with the frequencies for spatial remodeling of chromatin architecture and reprogramming of cellular epigenetic memory. This former and this latter process include such covalent modifications as demethylation/re-methylation of DNA cytosine residues and acetylation/deacetylation as well as demethylation/re-methylation of lysine residues of nucleosomal core-derived histones H3 and H4. The main cause of low SCNT efficiency in mammals turns out to be an incomplete reprogramming of transcriptional activity for donor cell-descended genes. It has been ascertained that somatic cell nuclei should undergo the wide DNA cytosine residue demethylation changes throughout the early development of cloned embryos to reset their own overall epigenetic and parental genomic imprinting memories that have been established by re-methylation of the nuclear donor cell-inherited genome during specific pathways of somatic and germ cell lineage differentiation. A more extensive understanding of the molecular mechanisms and recognition of determinants for epigenetic transcriptional reprogrammability of somatic cell nuclear genome will be helpful to solve the problems resulting from unsatisfactory SCNT effectiveness and open new possibilities for common application of this technology in transgenic research focused on human biomedicine.

CDK-11-Cyclin L is required for gametogenesis and fertility in C. elegans

CDK11, a member of the cyclin-dependent kinase family, has been implicated in a diverse array of functions including transcription, RNA processing, sister chromatid cohesion, spindle assembly, centriole duplication and apoptosis. Despite its involvement in many essential functions, little is known about the requirements for CDK11 and its partner Cyclin L in a developing multicellular organism. Here we investigate the function of CDK11 and Cyclin L during development of the nematode Caenorhabditis elegans. Worms express two CDK11 proteins encoded by distinct loci: CDK-11.1 is essential for normal male and female fertility and is broadly expressed in the nuclei of somatic and germ line cells, while CDK-11.2 is nonessential and is enriched in hermaphrodite germ line nuclei beginning in mid pachytene. Hermaphrodites lacking CDK-11.1 develop normally but possess fewer mature sperm and oocytes and do not fully activate the RAS-ERK pathway that is required for oocyte production in response to environmental cues. Most of the sperm and eggs that are produced in cdk-11.1 null animals appear to complete development normally but fail to engage in sperm-oocyte signaling suggesting that CDK-11.1 is needed at multiple points in gametogenesis. Finally, we find that CDK-11.1 and CDK-11.2 function redundantly during embryonic and postembryonic development and likely do so in association with Cyclin L. Our results thus define multiple requirements for CDK-11-Cyclin L during animal development.

Intrinsic and extrinsic molecular determinants or modulators for epigenetic remodeling and reprogramming of somatic cell-derived genome in mammalian nuclear-transferred oocytes and resultant embryos

The efficiency of somatic cell cloning in mammals remains disappointingly low. Incomplete and aberrant reprogramming of epigenetic memory of somatic cell nuclei in preimplantation nuclear- transferred (NT) embryos is one of the most important factors that limit the cloning effectiveness. The extent of epigenetic genome-wide alterations, involving histone or DNA methylation and histone deacetylation, that are mediated by histone-lysine methyltransferases (HMTs) or DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be modulated/reversed via exogenous inhibitors of these enzymes throughout in vitro culture of nuclear donor cells, nuclear recipient oocytes and/or cloned embryos. The use of the artificial modifiers of epigenomically-conditioned gene expression leads to inhibition of both chromatin condensation and transcriptional silencing the genomic DNA of somatic cells that provide a source of nuclear donors for reconstruction of enucleated oocytes and generation of cloned embryos. The onset of chromatin decondensation and gene transcriptional activity is evoked both through specific/selective inactivating HMTs by BIX-01294 and through non-specific/non-selective blocking the activity of either DNMTs by 5-aza-2'-deoxycytidine, zebularine, S-adenosylhomocysteine or HDACs by trichostatin A, valproic acid, scriptaid, oxamflatin, sodium butyrate, m-carboxycinnamic acid bishydroxamide, panobinostat, abexinostat, quisinostat, dacinostat, belinostat and psammaplin A. Epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or cloned embryos may facilitate and accelerate the reprogrammability for gene expression of donor cell nuclei that have been transplanted into a host ooplasm and subsequently underwent dedifferentiating and re-establishing the epigenetically dependent status of their transcriptional activity during pre- and postimplantation development of NT embryos. Nevertheless, a comprehensive additional work is necessary to determine whether failures in the early-stage reprogramming of somatic cell-inherited genome are magnified downstream in development of cloned conceptuses and neonates.

PH Change in Culture Medium Induces Reorganization of Intranuclear Actin in Two-Cell Mouse Embryos

Actin is a permanent component of the cell nucleus involved in many nuclear processes. However, some nuclear functions of actin remain insufficiently explored. The role played by various extracellular stimuli in regulation of nuclear actin still remains enigmatic. Deviation of basic parameters of culture medium from optimal values is a member of the group of extracellular stimuli that are very important for mammalian embryos cultured in vitro. Change in culture medium pH from the level optimal for embryo homeostasis is one such signals. The purpose of this study was to investigate the intranuclear actin distribution in nuclei of two-cell mouse embryos under stress conditions induced by changes in extracellular pH. The pattern of actin localization has been tracked after short-term culturing of the embryos at optimal (pH 7.2), increased (pH 7.8), or decreased (pH 6.5) pH conditions. Analysis was carried out with confocal microscopy using methods of direct fluorescent and indirect immunofluorescent identification of actin. It has been shown that the change of culture medium pH from the optimum value is the signal that alters intranuclear actin distribution in nuclei of the embryonic cells. Culture of two-cell mouse embryos in suboptimal pH conditions (pH 6.5 and pH 7.8) induced alterations in the intranuclear actin localization, which, in particular, were expressed in accumulation of monomeric actin and the appearance of phalloidin-stainable actin in the nuclei. These changes, in our opinion, show some signs of similarity with stress-induced changes in nuclear-actin distribution, which, as has been reported earlier by a number of researchers, have been observed in the nuclei of somatic cells.

The miR-302-Mediated Induction of Pluripotent Stem Cells (iPSC): Multiple Synergistic Reprogramming Mechanisms.

Pluripotency represents a unique feature of embryonic stem cells (ESCs). To generate ESC-like-induced pluripotent stem cells (iPSCs) derived from somatic cells, the cell genome needs to be reset and reprogrammed to express the ESC-specific transcriptome. Numerous studies have shown that genomic DNA demethylation is required for epigenetic reprogramming of somatic cell nuclei to form iPSCs; yet, the mechanism remains largely unclear. In ESCs, the reprogramming process goes through two critical stages: germline and zygotic demethylation, both of which erase genomic DNA methylation sites and hence allow for different gene expression patterns to be reset into a pluripotent state. Recently, miR-302, an ESC-specific microRNA (miRNA), was found to play an essential role in four aspects of this reprogramming mechanism-(1) initiating global genomic DNA demethylation, (2) activating ESC-specific gene expression, (3) inhibiting developmental signaling, and (4) preventing stem cell tumorigenicity. In this review, we will summarize miR-302 functions in all four reprogramming aspects and further discuss how these findings may improve the efficiency and safety of the current iPSC technology.

Oocyte Cytoplasmic Gas6 and Heparan Sulfate (HS) are Required to Establish the Open Chromatin State in Nuclei During Remodeling and Reprogramming

Background/Aims: Previously, we found that silencing of growth arrest-specific gene 6 (Gas6) in oocytes impaired cytoplasmic maturation, resulting in failure of sperm chromatin decondensation (SCD) and pronuclear (PN) formation after fertilization. Thus, we conducted this study to determine the effect of Gas6 RNAi on downstream genes and to elucidate the working mechanism of Gas6 on oocyte cytoplasmic maturation and SCD. Methods: Using RT-PCR, Western blot and immunofluorescence, the expression levels of various target genes and the localization of heparan sulfate (HS) were analyzed after Gas6 RNAi. The roles of Gas6 in HS biosynthesis, production of ATP and GSH, ROS generation and ΔΨm were also investigated. SCD and micrococcal nuclease (MNase) analyses were used to examine the effects of HS on the open chromatin state in sperm and somatic cell nuclei, respectively. Results: Disruption of Gas6 expression led to the inhibition of HS biosynthesis through the reduction of several HS biosynthetic enzymes. The rescue experiment, HS treatment in vitro, significantly recovered SCD and PN formation, confirming that HS had the ability to induce sperm head remodeling during fertilization. Interestingly, excessive mitochondrial activation in Gas6-depleted MII oocytes caused ROS generation and glutathione (GSH) degradation via mitochondrial activation, such as elevated ΔΨm and ATP production. Indeed, HS-treated NIH3T3 cell nuclei showed an open chromatin state, as determined by diffuse DAPI staining and increased sensitivity to MNase. Conclusion: We propose that the addition of HS to sperm and/or oocyte maturation would improve the efficiency of in vitro fertilization and somatic cell nuclear transfer (SCNT) reprogramming.

31 Overall Goat Cloning Efficiency Under Suboptimal Conditions — A 6-Year Experience

Animal cloning involves a combination of several simple steps that need to be carried out at the highest efficacy to provide acceptable yet low overall cloning efficiency. Oocyte competence is key for proper somatic cell nucleus reprogramming, and technical elements must be refined to minimize interference in the overall outcome. The aim of this study was to compare the progress of a goat-cloning program over a 6-year period in which oocyte donors’ protocols and oocyte and embryo manipulation were continuously refined to cope with success under Brazilian semi-arid conditions. The cloning dataset was divided in 3 periods (P1, 2011-2012; P2, 2013-2014; P3, 2015-2016), using either in vivo- or in vitro-matured goat oocytes for cloning by nuclear transfer (NT) with different cell lines for subsequent transfer to synchronized recipients to produce transgenic liveborn kids. Over time, protocols for recovery of competent oocytes were adapted to existing conditions, also optimizing animal well being and nutritional status to attain better success. Data on total and viable oocytes, oocyte maturation, pregnancy, and cloning efficiency were analysed by the Chi-squared or t-test (P < 0.05). After 111 replicates, viable and matured oocyte rates were improved during the time, but mean number of total oocytes/donor were higher in the second period (25.4 ± 8.9 v. 18.5 ± 10.8 for P1 and 19.5 ± 6.1 for P3), whereas the mean of viable and matured oocytes were similar between P2 (19.4 ± 6.0 and 9.8 ± 4.6) and P3 (18.8 ± 7.0 and 9.6 ± 3.7), but higher than in P1 (12.8 ± 7.6 and 6.0 ± 4.1), respectively. Higher pregnancy rate was achieved in P1 and lower in P2, with both being similar to P3. However, the efficiency based on transferred embryos was similar between periods (Table 1). When compared with total oocytes, birth rate improved from the first to the last period (P1, 0.01%; P2, 0.02%; and P3, 0.09%). Cloning efficiency was measured by the number of transferred embryos compared with matured structures used for cloning, which showed an improvement over time (P1, 34.5%; P2, 43.9%; P3, 60.1%). A 5-fold enhancement was observed in the number of matured oocytes needed to produce a liveborn animal (P1, 2,779/1; P2, 1,892/1; P3, 545/1). Also, the number of donors (P1, 432; P2, 193.5; P3, 56.6) and recipients (P1, 114; P2, 57; P3, 27) needed to produce 1 animal was reduced by 7.6- and 4.2-fold between periods. In conclusion, complex and inefficient procedures such as cloning by NT require technical refinement and adjustments to existing conditions, including animal nutrition and welfare, and cumulative gain of expertise to attain successful results. Table 1.Overall goat cloning efficiency over time.

A 100-Year Review: Reproductive technologies in dairy science

Reproductive technology revolutionized dairy production during the past century. Artificial insemination was first successfully applied to cattle in the early 1900s. The next major developments involved semen extenders, invention of the electroejaculator, progeny testing, addition of antibiotics to semen during the 1930s and 1940s, and the major discovery of sperm cryopreservation with glycerol in 1949. The 1950s and 1960s were particularly productive with the development of protocols for the superovulation of cattle with both pregnant mare serum gonadotrophin/equine chorionic gonadotrophin and FSH, the first successful bovine embryo transfer, the discovery of sperm capacitation, the birth of rabbits after in vitro fertilization, and the development of insulated liquid nitrogen tanks. Improved semen extenders and the replacement of glass ampules with plastic semen straws followed. Some of the most noteworthy developments in the 1970s included the initial successes with in vitro culture of embryos, calves born after chromosomal sexing as embryos, embryo splitting resulting in the birth of twins, and development of computer-assisted semen analysis. The 1980s brought flow cytometric separation of X- and Y-bearing sperm, in vitro fertilization leading to the birth of live calves, clones produced by nuclear transfer from embryonic cells, and ovum pick-up via ultrasound-guided follicular aspiration. The 20th century ended with the birth of calves produced from AI with sexed semen, sheep and cattle clones produced by nuclear transfer from adult somatic cell nuclei, and the birth of transgenic cloned calves. The 21st century has seen the introduction of perhaps the most powerful biotechnology since the development of artificial insemination and cryopreservation. Quick, inexpensive genomic analysis via the use of single nucleotide polymorphism genotyping chips is revolutionizing the cattle breeding industry. Now, with the introduction of genome editing technology, the changes are becoming almost too rapid to fully digest.

Production of cloned mice using oocytes derived from ICR-outbred strain.

Recently, it has become possible to generate cloned mice using a somatic cell nucleus derived from not only F1 strains but also inbred strains. However, to date, all cloned mice have been generated using F1 mouse oocytes as the recipient cytoplasm. Here, we attempted to generate cloned mice from oocytes derived from the ICR-outbred mouse strain. Cumulus cell nuclei derived from BDF1 and ICR mouse strains were injected into enucleated oocytes of both strains to create four groups. Subsequently, the quality and developmental potential of the cloned embryos were examined. ICR oocytes were more susceptible to damage associated with nuclear injection than BDF1 oocytes, but their activation rate and several epigenetic markers of reconstructed cloned oocytes/embryos were similar to those of BDF1 oocytes. When cloned embryos were cultured for up to 4 days, those derived from ICR oocytes demonstrated a significantly decreased rate of development to the blastocyst stage, irrespective of the nuclear donor mouse strain. However, when cloned embryos derived from ICR oocytes were transferred to female recipients at the two-cell stage, healthy cloned offspring were obtained at a success rate similar to that using BDF1 oocytes. The ICR mouse strain is very popular for biological research and less expensive to establish than most other strains. Thus, the results of this study should promote the study of nuclear reprogramming not only by reducing the cost of experiments but also by allowing us to study the effect of oocyte cytoplasm by comparing it between strains.

Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa).

ObjectiveInvestigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT).MethodsPorcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR.ResultsAscorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid.ConclusionAscorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

Remodeling somatic nuclei via exogenous expression of protamine 1 to create spermatid-like structures for somatic nuclear transfer.

This protocol describes how to convert the chromatin structure of sheep and mouse somatic cells into spermatid-like nuclei through the heterologous expression of the protamine 1 gene (Prm1). Furthermore, we also provide step-by-step instructions for somatic cell nuclear transfer (SCNT) of Prm1-remodeled somatic nuclei in sheep oocytes. There is evidence that changing the organization of a somatic cell nucleus with that which mirrors the spermatozoon nucleus leads to better nuclear reprogramming. The protocol may have further potential application in determining the protamine and histone footprints of the whole genome; obtaining 'gametes' from somatic cells; and furthering understanding of the molecular mechanisms regulating the maintenance of DNA methylation in imprinted control regions during male gametogenesis. The protocol is straightforward, and it requires 4 weeks from the establishment of the cell lines to their transfection and the production of cloned blastocysts. It is necessary for researchers to have experience in cell biology and embryology, with basic skills in molecular biology, to carry out the protocol.

The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

Abstract One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

Effect of Long-Term Exposure of Donor Nuclei to the Oocyte Cytoplasm on Production of Cloned Mice Using Serial Nuclear Transfer.

Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT.

Parp1 Deficiency Confers Defects in Chromatin Surveillance and Remodeling During Reprogramming by Nuclear Transfer.

Accumulating evidence suggests that cloned mice production by the injection of a somatic cell nucleus into an enucleated oocyte is inefficient. DNA damage and chromatin remodeling failures that occur during embryogenesis following nuclear transfer (NT) might explain the poor development of cloned embryos. To avoid these problems, it is important to elucidate somatic chromatin remodeling after NT. Because polyADP-ribosylation, which is catalyzed mainly by poly(ADP-ribose) polymerase 1 (Parp1), is a major post-translational modification that facilitates DNA repair and chromatin remodeling, we examined the effects of Parp1 deficiency in developing NT embryos. Parp1 was located within the pseudo-pronuclei (PPN) of NT eggs. We observed that NT eggs, after activation by Sr2+, formed PPN with significantly more efficiency in Parp1-null embryos than in wild-type NT embryos. However, most the Parp1-null embryos stopped developing by the four-cell stage. Immunostaining for γH2AX foci, a marker of DNA double strand breaks, showed longer retention in the PPN of Parp1-/- donor NT embryos than in wild-type NT embryos, suggesting that, in the absence of Parp1, DNA breaks are slowly repaired and consequently, entry into the S phase might be delayed. Furthermore, decreases in histone H3 acetylation, H3 monomethylation at lysine 4, and H3 trimethylation at lysine 27 after the Sr2+ activation step were observed in the PPN of Parp1-/- donor embryos. Taken together, our data suggest that Parp1 is involved in the plastic remodeling of chromatin structure after NT by supporting DNA repair and specific histone code modifications.

Phenotypes of Aging Postovulatory Oocytes After Somatic Cell Nuclear Transfer in Mice.

Oocytes rapidly lose their developmental potential after ovulation, termed postovulatory oocyte aging, and often exhibit characteristic phenotypes, such as cytofragmentation, abnormal spindle shapes, and chromosome misalignments. Here, we reconstructed mouse oocytes using somatic cell nuclear transfer (SCNT) to reveal the effect of somatic cell-derived nuclei on oocyte physiology during aging. Normal oocytes started undergoing cytofragmentation 24 hours after oocyte collection; however, this occurred earlier in SCNT oocytes and was more severe at 48 hours, suggesting that the transferred somatic cell nuclei affected oocyte physiology. We found no difference in the status of acetylated α-tubulin (Ac-Tub) and α-tubulin (Tub) between normal and SCNT aging oocytes, but unlike normal oocytes, aging SCNT oocytes did not have astral microtubules. Interestingly, aging SCNT oocytes displayed more severely scattered chromosomes or irregularly shaped spindles. Observations of the microfilaments showed that, in normal oocytes, there was a clear actin ring beneath the plasma membrane and condensed microfilaments around the spindle (the actin cap) at 0 hours, and the actin filaments started degenerating at 1 hour, becoming completely disrupted and distributed to the cytoplasm at 24 hours. By contrast, in SCNT oocytes, an actin cap formed around the transplanted nuclei within 1 hour of SCNT, which was still present at 24 hours. Thus, SCNT oocytes age in a similar but distinct way, suggesting that they not only contain nuclei with abnormal epigenetics but are also physiologically different.