ABSTRACT
The fertilizing spermatozoa induce a Ca2+ oscillatory pattern, the universal hallmark of oocyte activation, in all sexually reproducing animals. Assisted reproductive technologies (ARTs) like intracytoplasmic sperm injection (ICSI) bypass the physiological pathway; however, while a normal Ca2+ release pattern occurs in some species, particularly humans, artificial activation is compulsory for ICSI-fertilized oocytes to develop in most farm animals. Unlike the normal oscillatory pattern, most artificial activation protocols induce a single Ca2+ spike, undermining proper ICSI-derived embryo development in these species. Curiously, diploid parthenogenetic embryos activated by the same treatments develop normally at high frequencies and implant upon transfer in the uterus. We hypothesized that, at least in ruminant embryos, the oscillatory calcium waves late in the first cell cycle target preferentially the paternal pronucleus and are fundamentally important for paternal nuclear remodeling. We believe that Ca2+ signaling is central to full totipotency deployment of the paternal genome. Research in this area could highlight the asymmetry between the parental genome reprogramming timing/mechanisms in early development and impact ARTs like ICSI and cloning.
Subject(s)
Calcium , Semen , Animals , Female , Male , Humans , Calcium/metabolism , Semen/metabolism , Cytoplasm/metabolism , Fertilization , Spermatozoa/metabolism , Oocytes/metabolismABSTRACT
Human pre-mRNA processing relies on multi-subunit macromolecular complexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD) histone pre-mRNA processing requires association with a stem-loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3' processing and polyadenylation.
Subject(s)
Histones , RNA Precursors , Humans , RNA Precursors/genetics , RNA Precursors/metabolism , Histones/genetics , Histones/metabolism , Polyadenylation , mRNA Cleavage and Polyadenylation Factors/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Nucleoside diphosphate (NDP) kinases 1 and 2 (NME1/2) are well-characterized enzymes known for their NDP kinase activity. Recently, these enzymes have been shown by independent studies to bind coenzyme A (CoA) or acyl-CoA. These findings suggest a hitherto unknown role for NME1/2 in the regulation of CoA/acyl-CoA-dependent metabolic pathways, in tight correlation with the cellular NTP/NDP ratio. Accordingly, the regulation of NME1/2 functions by CoA/acyl-CoA binding has been described, and additionally, NME1/2 have been shown to control the cellular pathways consuming acetyl-CoA, such as histone acetylation and fatty acid synthesis. NME1/2-controlled histone acetylation in turn mediates an important transcriptional response to metabolic changes, such as those induced following a high-fat diet (HFD). This review discusses the CoA/acyl-CoA-dependent NME1/2 activities and proposes that these enzymes be considered as the first identified carriers of CoA/short-chain acyl-CoAs.
Subject(s)
Adenosine Triphosphate , Humans , Animals , Adenosine Triphosphate/metabolism , Acyl Coenzyme A/metabolism , NM23 Nucleoside Diphosphate Kinases/metabolism , NM23 Nucleoside Diphosphate Kinases/genetics , Nucleoside-Diphosphate Kinase/metabolism , Nucleoside-Diphosphate Kinase/genetics , AcetylationABSTRACT
Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis of transcriptomic data from lung samples of patients who died from COVID-19 demonstrates an increased expression of the gene encoding AKR1B10. Measurements of the AKR1B10 protein in sera from hospitalised COVID-19 patients suggests a significant link between AKR1B10 levels and the severity of the disease. In macrophages and lung cells, the over-expression of AKR1B10 induces the expression of the pro-inflammatory cytokines Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor a (TNFα), supporting the biological plausibility of an AKR1B10 involvement in the COVID-19-related cytokine storm. When macrophages were stressed by lipopolysaccharides (LPS) exposure and treated by Zopolrestat, an AKR1B10 inhibitor, the LPS-induced production of IL-6, IL-1ß, and TNFα is significantly reduced, reinforcing the hypothesis that the pro-inflammatory expression of cytokines is AKR1B10-dependant. Finally, we also show that AKR1B10 can be secreted and transferred via extracellular vesicles between different cell types, suggesting that this protein may also contribute to the multi-organ systemic impact of COVID-19. These experiments highlight a relationship between AKR1B10 production and severe forms of COVID-19. Our data indicate that AKR1B10 participates in the activation of cytokines production and suggest that modulation of AKR1B10 activity might be an actionable pharmacological target in COVID-19 management.
Subject(s)
Aldo-Keto Reductases/physiology , COVID-19/genetics , Cytokine Release Syndrome/genetics , Respiratory Distress Syndrome/genetics , Aldo-Keto Reductases/antagonists & inhibitors , Aldo-Keto Reductases/genetics , Animals , COVID-19/complications , COVID-19/metabolism , COVID-19/pathology , Case-Control Studies , Cells, Cultured , Cytokine Release Syndrome/metabolism , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/virology , Cytokines/metabolism , Enzyme Inhibitors/pharmacology , Humans , Macrophages/drug effects , Macrophages/metabolism , Mice , Patient Acuity , RAW 264.7 Cells , Respiratory Distress Syndrome/metabolism , Respiratory Distress Syndrome/pathology , Respiratory Distress Syndrome/virology , SARS-CoV-2/physiology , TranscriptomeABSTRACT
PURPOSE: This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm injection (ICSI). METHODS: Prior to in vitro fertilization (IVF) or ICSI, the oocytes were subjected to in vitro maturation (IVM) for 24 h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation. Next, spermatozoa were mechanically (by piezo-electrical pulses) and/or chemically (by ionomycin/Triton X-100) treated to break membranes and acrosomes and were injected into oocytes, grouped as follows: (i) piezo-pulsed spermatozoa (PPS), (ii) PPS pre-treated with ionomycin (PPS-I), (iii) PPS pre-treated with Triton X-100 (PPS-T), and (iv) intact and untreated spermatozoa as a control (CTR-IS). RESULTS: No differences were observed in the zygote/cleavage/blastocyst rate between chemically activated and non-activated oocytes (50 vs. 45 %, 11.6 vs. 10.1 %; 1.8 vs. 1.1 %, respectively), after ICSI with CTR-IS. Injection of PPS compared to CTR-IS increased the proportion of zygotes and blastocysts (84.6 vs. 45 %, p < 0.01; 15.5 vs. 1.1 %, p < 0.0001, respectively). Moreover, the percentage of PPS-derived blastocysts was not significantly different from that obtained by conventional IVF (15.5 vs. 20.2 %). The ICSI blastocysts' development was also improved with PPS pre-treated with ionomycin (15.6 %), but was completely impeded with PPS pre-treated with Triton X-100 (0 %). CONCLUSION: Our findings confirm that ICSI with spermatozoa whose plasma membrane and acrosome have been mechanically damaged substantially improves embryonic development until the blastocyst stage.
Subject(s)
Acrosome Reaction , Cell Membrane/ultrastructure , Embryonic Development , Sheep/embryology , Sperm Injections, Intracytoplasmic/veterinary , Animals , Embryo Culture Techniques/veterinary , In Vitro Oocyte Maturation Techniques/veterinary , Male , Sperm Injections, Intracytoplasmic/methods , Sperm-Ovum Interactions , Spermatozoa/ultrastructureABSTRACT
The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.
Subject(s)
Nucleoside-Diphosphate Kinase , Animals , Mice , Nucleoside-Diphosphate Kinase/genetics , Diet, High-Fat/adverse effects , Epigenesis, Genetic , Histones , Liver , Fatty Acids , Mice, KnockoutABSTRACT
PURPOSE: To create an accurate 3D reconstruction of the vascular trees, it is necessary to know the exact geometrical parameters of the angiographic imaging system. Many previous studies used vascular structures to estimate the system's exact geometry. However, utilizing interventional devices and their relative features may be less challenging, as they are unique in different views. We present a semi-automatic self-calibration approach considering the markers attached to the interventional instruments to estimate the accurate geometry of a biplane X-ray angiography system for neuroradiologic use. METHODS: A novel approach is proposed to detect and segment the markers using machine learning classification, a combination of support vector machine and boosted tree. Then, these markers are considered as reference points to optimize the acquisition geometry iteratively. RESULTS: The method is evaluated on four clinical datasets and three pairs of phantom angiograms. The mean and standard deviation of backprojection error for the catheter or guidewire before and after self-calibration are [Formula: see text] mm and [Formula: see text] mm, respectively. The mean and standard deviation of the 3D root-mean-square error (RMSE) for some markers in the phantom reduced from [Formula: see text] to [Formula: see text] mm. CONCLUSION: A semi-automatic approach to estimate the accurate geometry of the C-arm system was presented. Results show the reduction in the 2D backprojection error as well as the 3D RMSE after using our proposed self-calibration technique. This approach is essential for 3D reconstruction of the vascular trees or post-processing techniques of angiography systems that rely on accurate geometry parameters.
Subject(s)
Angiography , Imaging, Three-Dimensional , Angiography/methods , Calibration , Humans , Imaging, Three-Dimensional/methods , Phantoms, ImagingABSTRACT
Dynamic MRI is a technique of acquiring a series of images continuously to follow the physiological changes over time. However, such fast imaging results in low resolution images. In this work, abdominal deformation model computed from dynamic low resolution images have been applied to high resolution image, acquired previously, to generate dynamic high resolution MRI. Dynamic low resolution images were simulated into different breathing phases (inhale and exhale). Then, the image registration between breathing time points was performed using the B-spline SyN deformable model and using cross-correlation as a similarity metric. The deformation model between different breathing phases were estimated from highly undersampled data. This deformation model was then applied to the high resolution images to obtain high resolution images of different breathing phases. The results indicated that the deformation model could be computed from relatively very low resolution images.
Subject(s)
Magnetic Resonance Imaging , Abdomen , Algorithms , RespirationABSTRACT
Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitously detectable in the environment and in the food chain. Prenatal exposure to PCBs negatively affects fetal development and produces long-term detrimental effects on child health. The present study sought to evaluate the cytotoxic and genotoxic effects of chronic PCB exposure on fetal cells during pregnancy. To this aim, sheep embryonic fibroblasts (SEF) and amniocytes (SA) were cultured in vitro in the presence of low doses of PCBs for a period of 120days, comparable to the full term of ovine pregnancy. Cellular proliferation rates, global DNA methylation, chromosome integrity, and markers of DNA damage were evaluated at different time points. Moreover, SEF treated with PCBs for 60days were left untreated for one further month and then examined in order to evaluate the reversibility of PCB-induced epigenetic defects. PCB-treated SEF were more sensitive than SA treated with PCBs, in terms of low cell proliferation, and increased DNA damage and global DNA methylation, which were still detectable after interruption of PCB treatment. These data indicate that chronic exposure of fetal cells to PCBs causes permanent genomic and epigenetic instability, which may influence both prenatal and post-natal growth up to adulthood. Our in vitro model offer a simple and controlled means of studying the effects of different contaminants on fetal cells - one that could set the stage for targeted in vivo studies.
Subject(s)
Embryo, Mammalian/cytology , Environmental Pollutants/toxicity , Fibroblasts/drug effects , Polychlorinated Biphenyls/toxicity , Sheep/embryology , Animals , DNA Methylation , Female , Models, Biological , Pregnancy , Pregnancy Complications , Toxicity TestsABSTRACT
Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM ("starved"-ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer.
Subject(s)
Fibroblasts/metabolism , Histones/metabolism , Protamines/metabolism , Sheep/metabolism , Acetylation/drug effects , Animals , Biomarkers/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Cellular Reprogramming/drug effects , DEAD-box RNA Helicases/metabolism , Female , Fibroblasts/drug effects , Gene Expression/drug effects , Hydroxamic Acids/pharmacology , Male , Mice , Nuclear Transfer Techniques , Ribonuclease III/metabolism , Spermatids/metabolismABSTRACT
In addition to the iconic wild species, such as the pandas and Siberian tigers, an ever-increasing number of domestic species are also threatened with extinction. Biobanking of spermatozoa could preserve genetic heritages of extinct species, and maintain biodiversity of existing species. Because lyophilized spermatozoa retain fertilizing capacity, the aim was to assess whether freeze-dried spermatozoa are an alternative option to save endangered sheep breeds. To achieve this objective, semen was collected from an Italian endangered sheep breed (Pagliarola), and a biobank of cryopreserved and freeze-dried spermatozoa was established, and evaluated using IVF (for frozen spermatozoa) and ICSI procedures (for frozen and freeze-dried spermatozoa). As expected, the fertilizing capacity of cryopreserved Pagliarola's spermatozoa was comparable to commercial semen stocks. To evaluate the activating capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI, and allocated to two groups: 56 oocytes were activated by incubation with ionomycin (ICSI-FDSa) and 52 were not activated (ICSI-FDSna). Pronuclear formation (2PN) was investigated at 14-16â¯h after ICSI in fixed presumptive zygotes. Only artificially activated oocytes developed into blastocysts after ICSI. In the present study, freeze-dried ram spermatozoa induced blastocyst development following ICSI at a relatively high proportion, providing evidence that sperm lyophilization is an alternative, low cost storage option for biodiversity preservation of domestic species.
Subject(s)
Biological Specimen Banks , Endangered Species , Sheep/physiology , Spermatozoa/physiology , Animals , Cryopreservation/veterinary , Embryo Culture Techniques/veterinary , Fertilization in Vitro/veterinary , Freeze Drying , In Vitro Oocyte Maturation Techniques/veterinary , Male , Semen Preservation , Sheep/genetics , Sperm Injections, Intracytoplasmic/veterinaryABSTRACT
In various animal species, the main cause of pregnancy loss in conceptuses obtained by somatic cell nuclear transfer (SCNT) are placental abnormalities. Most abnormalities described in SCNT pregnancies (such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium) suggest that placental cell degeneration may be triggered by mitochondrial failure. We hypothesized that placental abnormalities of clones obtained by SCNT are related to mitochondrial dysfunction. To test this, early SCNT and control (CTR, from pregnancies obtained by in vitro fertilization) placentae were collected from pregnant ewes (at day 20 and 22 of gestation) and subjected to morphological, mRNA and protein analysis. Here, we demonstrated swollen and fragmented mitochondria and low expression of mitofusin 2 (Mfn2), the protein which plays a crucial role in mitochondrial functionality, in SCNT early placentae. Furthermore, reduced expression of the Bcnl3L/Nix protein, which plays a crucial role in selective elimination of damaged mitochondria, was observed and reflected by the accumulation of numerous damaged mitochondria in SCNT placental cells. Likely, this accumulation of damaged organelles led to uncontrolled apoptosis in SCNT placentae, as demonstrated by the high number of apoptotic bodies, fragmented cytoplasm, condensed chromatin, lack of integrity of the nuclear membrane and the perturbed mRNA expression of apoptotic genes (BCL2 and BAX). In conclusion, our data indicate that deregulated expression of Mfn2 and Bcnl3L is responsible for placental abnormalities in SCNT conceptuses. Our results suggest that some nuclear genes, that are involved in the regulation of mitochondrial function, do not work well and consequently this influence the function of mitochondria.
Subject(s)
GTP Phosphohydrolases/genetics , Mitochondrial Proteins/metabolism , Nuclear Transfer Techniques/adverse effects , Placenta/metabolism , Proto-Oncogene Proteins/genetics , Animals , Apoptosis , Female , GTP Phosphohydrolases/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/genetics , Placenta/pathology , Pregnancy , Proto-Oncogene Proteins/metabolism , SheepABSTRACT
Cloning animals by somatic cell nuclear transfer (SCNT) has remained an uncontrollable process for many years. High rates of embryonic losses, stillbirths, and postnatal mortality have been typical outcomes. These developmental problems arise from abnormal genomic reprogramming: the capacity of the oocyte to reset the differentiated memory of a somatic cell. However, effective reprogramming strategies are now available. These target the whole genome or single domains such as the Xist gene, and their effectiveness has been validated with the ability of experimental animals to develop to term. Thus, SCNT has become a controllable process that can be used to 'rescue' endangered species, and for biomedical research such as therapeutic cloning and the isolation of induced pluripotent stem cells (iPSCs).
Subject(s)
Cloning, Organism , Nuclear Transfer Techniques , Animals , Humans , Mice , RNA, Long Noncoding/genetics , SheepABSTRACT
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.
Subject(s)
Nuclear Transfer Techniques , Protamines/genetics , Spermatids/cytology , Animals , Gene Expression , Male , Mice , NIH 3T3 Cells , Plasmids/genetics , SheepABSTRACT
Protamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer.
Subject(s)
Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Protamines/metabolism , Acetylation , Animals , Cell Nucleus/chemistry , Cells, Cultured , Chromatin/metabolism , DNA/chemistry , DNA/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Histones/metabolism , Humans , Male , Methylation , Microscopy, Electron, Transmission , Oocytes/metabolism , Protamines/genetics , Sheep , Spermatids/chemistry , Spermatids/metabolismABSTRACT
Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.
Subject(s)
Cloning, Organism , Oocytes/ultrastructure , Animals , Blastocyst , Culture Media , Demecolcine/administration & dosage , Female , SheepABSTRACT
The scope of this article is not to provide an exhaustive review of nuclear transfer research, because many authoritative reviews exist on the biological issues related to somatic and embryonic cell nuclear transfer. We shall instead provide an overview on the work done specifically on sheep and the value of this work on the greater nuclear transfer landscape.
Subject(s)
Cloning, Organism , Models, Animal , Nuclear Transfer Techniques , Sheep/genetics , AnimalsABSTRACT
The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.
Subject(s)
Cloning, Organism/veterinary , DNA/genetics , Freeze Drying/methods , Genomic Instability , Nuclear Transfer Techniques/veterinary , Sheep/genetics , Animals , Cell Nucleus/genetics , Cells, Cultured , Cloning, Organism/methods , DNA Damage , Embryo Culture Techniques/methods , Embryo Culture Techniques/veterinary , Female , Lymphocytes/metabolism , Oocytes/cytology , Oocytes/metabolism , Sheep/embryologyABSTRACT
The production of androgenetic embryos in large animals is a complex procedure. Androgenetic embryos have been produced so far only in cattle and sheep using pronuclear transfer (PT) between zygotes derived from in vitro fertilization (IVF) of previously enucleated oocytes. PT is required due to the poor developmental potential of androgenotes derived from IVF of enucleated oocytes. Here we compare the developemt to blastocyst of androgenetic embryos produced by the standard pronuclear transfer and by fertilization of oocytes enucleated in Ca2+/Mg2+-free medium, without pronuclear transfer. The enucleation in Ca2+/Mg2+-free medium abolished almost completely the manipulation-induced activation, significantly improving the development to blastocyst of the androgenetic embryos (IVF followed by PT; 18.6%: IVF only; 17.7%, respectively). Karyotype analysis of IVF revealed a similar proportion of diploid embryos in androgenetic and control blastocysts (35% and 36%, respectively), although mixoploid blastocysts were frequently observed in both groups (64%). Androgenotes had lower total cell numbers than control and parthenogenetic embryos, but more cells in ICM cells comparing to parthenogenotes (30.42 vs. 17.15%). Higher expression of the pluripotency-associated gene NANOG, and trophoblastic-specific gene CDX2, were also observed in androgenotes compared to parthenogenotes and controls. The global methytion profile of androgenetic embryos was comparable to controls, but was lower than parthenogenetic embryos. The cell composition and methylation pattern we have detected in monoparental sheep monoparental embryos are unprecedented, and differ considerably from the standard reference mouse embryos. Altogether, these finding indicate significant differences across species in the molecular mechanisms regulating early development of monoparental embryos, and highlights the need to study postimplantation development of androgenetic embryos in sheep.