scholarly journals Scientific and technological approaches to improve SCNT efficiency in farm animals and pets

Reproduction ◽  
2021 ◽  
Author(s):  
Pasqualino Loi ◽  
Luca Palazzese ◽  
Pier Augusto Scapolo ◽  
Josef Fulka ◽  
Helena Fulka ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Large Scale ◽  
Epigenetic Modifications ◽  
Farm Animals ◽  
Nuclear Reprogramming ◽  
Nuclear Maturation ◽  
Offspring Production ◽  
Almost All ◽  
Main Message

The birth of Dolly through somatic cell nuclear transfer (SCNT) was a major scientific breakthrough of the last century. Yet, while significant progress has been achieved across the technics required to reconstruct and in vitro culture nuclear transfer embryos, SCNT outcomes in terms of offspring production rates are still limited. Here we provide a snapshot of the practical application of SCNT in farm animals and pets. Moreover, we suggest a path to improve SCNT through alternative strategies inspired by the physiological reprogramming in male and female gametes in preparation for the totipotency required after fertilization. Almost all papers on SCNT focused on nuclear reprogramming in the somatic cells after nuclear transfer. We believe that this is misleading, and even if it works sometimes, it does so in an uncontrolled way. Physiologically, the oocyte cytoplasm deploys nuclear reprogramming machinery specifically designed to address the male chromosome, the maternal alleles are prepared for totipotency earlier, during oocyte nuclear maturation. Significant advances have been made in remodeling somatic nuclei in vitro through the expression of protamines, thanks to a plethora of data available on spermatozoa epigenetic modifications. Missing are the data on large-scale nuclear reprogramming of the oocyte chromosomes. The main message our article conveys is that the next generation nuclear reprogramming strategies should be guided by insights from in-depth studies on epigenetic modifications in the gametes in preparation for fertilization.

scholarly journals Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniela Bebbere ◽  
Susanne E. Ulbrich ◽  
Katrin Giller ◽  
Valeri Zakhartchenko ◽  
Horst-Dieter Reichenbach ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Donor Cell ◽  
Farm Animals ◽  
Nuclear Reprogramming ◽  
Mtdna Depletion ◽  
Mitochondrial Dna Depletion

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

scholarly journals TP53Mutations Promote Immunogenic Activity in Breast Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-19 ◽  
Author(s):  
Zhixian Liu ◽  
Zehang Jiang ◽  
Yingsheng Gao ◽  
Lirui Wang ◽  
Cai Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Large Scale ◽  
Bioinformatics Analysis ◽  
Therapeutic Option ◽  
Survival Prognosis ◽  
Mutation Status ◽  
Immune Signatures ◽  
Immunotherapeutic Strategy ◽  
Almost All

Background. Although immunotherapy has recently achieved clinical successes in a variety of cancers, thus far there is no immunotherapeutic strategy for breast cancer (BC). Thus, it is important to discover biomarkers for identifying BC patients responsive to immunotherapy.TP53mutations were often associated with worse clinical outcomes in BC whose triple-negative subtype has a highTP53mutation rate (approximately 80%). To explore a potentially promising therapeutic option for theTP53-mutated BC subtype, we studied the association betweenTP53mutations and immunogenic activity in BC.Methods. We compared the enrichment levels of 26 immune signatures that indicated activities of diverse immune cells, functions, and pathways betweenTP53-mutated andTP53-wildtype BCs based on two large-scale BC multiomics datasets. Moreover, we explored the molecular cues associated with the differences in immunogenic activity betweenTP53-mutated andTP53-wildtype BCs. Furthermore, we performed experimental validation of the findings from bioinformatics analysis.Results. Bioinformatics analysis showed that almost all analyzed immune signatures showed significantly higher enrichment levels inTP53-mutated BCs than inTP53-wildtype BCs. Moreover,in vitroexperiments confirmed that mutant p53 could increase BC immunogenicity. Both computational and experimental results demonstrated thatTP53mutations could promote BC immunogenicityviaregulation of the p53-mediated pathways including cell cycle, apoptosis, Wnt, Jak-STAT, NOD-like receptor, and glycolysis. Furthermore, we found that elevated immune activity was likely associated with a better survival prognosis inTP53-mutated BCs, but not necessarily inTP53-wildtype BCs.Conclusions.TP53mutations may promote immunogenic activity in BC, suggesting that theTP53mutation status could be a useful biomarker for stratifying BC patients responsive to immunotherapy.

scholarly journals Bradyrhizobia Nodulating the Acacia mangium × A. auriculiformis Interspecific Hybrid Are Specific and Differ from Those Associated with Both Parental Species

2009 ◽  
Vol 75 (24) ◽  
pp. 7752-7759 ◽  
Author(s):  
Christine Le Roux ◽  
Diana Tentchev ◽  
Yves Prin ◽  
Doreen Goh ◽  
Yani Japarudin ◽  
...  
Keyword(s):  
Interspecific Hybrid ◽  
Large Scale ◽  
Parental Species ◽  
Root Nodules ◽  
Acacia Mangium ◽  
Rhizobium Strains ◽  
Homologous Strain ◽  
Yeast Extract Mannitol ◽  
Almost All

ABSTRACT In the context of an increasing utilization of the interspecific hybrid Acacia mangium × A. auriculiformis as a plantation tree in the tropical humid zone, its symbiotic characterization was carried out in comparison with that of its two parental species. Rhizobium strains of diverse geographical origins were isolated from root nodules of the hybrid and its parents. Almost all Acacia hybrid isolates were fast growing on yeast extract-mannitol medium, in contrast to those isolated from both parental species, which were mostly slow growing. The rhizobium strains were characterized through partial sequencing of the rRNA operon. In the phylogenetic tree, almost all strains isolated from the hybrid were grouped together in a clade close to Bradyrhizobium japonicum, while all strains isolated from both parental species were close to Bradyrhizobium elkanii. Inoculation experiments performed under in vitro or greenhouse conditions showed that all strains were infective with their original hosts but exhibited very variable degrees of effectivity according to the host plant tested. Thus, homologous strain-host associations were more effective than heterologous ones. This shows that there is still a high potential for isolating and testing new strains from hybrids to be used as inoculants in the context of large-scale afforestation programs.

Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Yongjin Lee ◽  
Hanna Lee ◽  
Joohyeong Lee ◽  
Seung Tae Lee ◽  
Geun-Shik Lee ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Glucose Supplementation

Summary This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

scholarly journals Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

2017 ◽  
Author(s):  
◽  
Bethany Rae Mordhorst
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Pharmaceutical Agents ◽  
Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

Scriptaid Improves In Vitro Development and Nuclear Reprogramming of Somatic Cell Nuclear Transfer Bovine Embryos

2011 ◽  
Vol 13 (5) ◽  
pp. 431-439 ◽  
Author(s):  
Li-Jun Wang ◽  
Hui Zhang ◽  
Yong-Sheng Wang ◽  
Wen-Bing Xu ◽  
Xian-Rong Xiong ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Nuclear Reprogramming ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development

scholarly journals Chromatin Modifying Agents in theIn VitroProduction of Bovine Embryos

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Fabio Morato Monteiro ◽  
Clara Slade Oliveira ◽  
Letícia Zoccolaro Oliveira ◽  
Naiara Zoccal Saraiva ◽  
Maria Eugênia Zerlotti Mercadante ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Epigenetic Reprogramming ◽  
Nuclear Reprogramming ◽  
Bovine Embryos ◽  
Aberrant Gene Expression ◽  
Low Efficiency ◽  
Aberrant Gene

The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjustin vitromanipulations in order to better mimicin vivoconditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use ofin vitrofertilization and cloning technologies in cattle and other species.

scholarly journals Nuclear Reprogramming in Mouse Primordial Germ Cells: Epigenetic Contribution

2011 ◽  
Vol 2011 ◽  
pp. 1-15 ◽  
Author(s):  
Massimo De Felici
Keyword(s):  
Germ Cells ◽  
Germ Line ◽  
Primordial Germ Cells ◽  
Germ Cell Tumors ◽  
Cell Lineage ◽  
Epigenetic Modifications ◽  
Epigenetic Reprogramming ◽  
Nuclear Reprogramming

The unique capability of germ cells to give rise to a new organism, allowing the transmission of primary genetic information from generation to generation, depends on their epigenetic reprogramming ability and underlying genomic totipotency. Recent studies have shown that genome-wide epigenetic modifications, referred to as “epigenetic reprogramming”, occur during the development of the gamete precursors termed primordial germ cells (PGCs) in the embryo. This reprogramming is likely to be critical for the germ line development itself and necessary to erase the parental imprinting and setting the base for totipotency intrinsic to this cell lineage. The status of genome acquired during reprogramming and the associated expression of key pluripotency genes render PGCs susceptible to transform into pluripotent stem cells. This may occurin vivounder still undefined condition, and it is likely at the origin of the formation of germ cell tumors. The phenomenon appears to be reproduced under partly definedin vitroculture conditions, when PGCs are transformed into embryonic germ (EG) cells. In the present paper, I will try to summarize the contribution that epigenetic modifications give to nuclear reprogramming in mouse PGCs.

Laboratory production of buffalo (Bubalus bubalis) embryos

1998 ◽  
Vol 10 (5) ◽  
pp. 379 ◽  
Author(s):  
P. Palta ◽  
M. S. Chauhan
Keyword(s):  
In Vitro Maturation ◽  
Large Scale ◽  
Follicle Stimulating Hormone ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Nuclear Maturation ◽  
Fertilization Rates ◽  
Vitro Production

There is an increasing interest in large-scale in vitro production (IVP) of buffalo embryos through in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes for faster multiplication of superior germplasm. The recovery of total and usable quality oocytes from slaughterhouse ovaries is low in this species. The nuclear maturation rates of buffalo oocytes matured in the presence of follicular fluid or serum and hormones like luteinizing hormone, follicle-stimulating hormone and oestradiol vary from 70 to 80% and are comparable to those reported for cattle oocytes. However, with fertilization rates of 40–55%, and the yield of blastocysts at around 10–15%, the efficiency of IVP is much lower than that in cattle. The in vitro sperm preparation procedures and the systems employed for performing IVF and culture of zygotes up to blastocyst stage are suboptimal and need substantial improvements. The quality and viability of blastocysts produced need to be checked by cell count, and after transfer to synchronized recipients, for development of quality control standards.

scholarly journals Enhancing Oocyte Competence With Milrinone as a Phosphodiesterase 3A Inhibitor to Improve the Development of Porcine Cloned Embryos

2021 ◽  
Vol 9 ◽  
Author(s):  
Pantu Kumar Roy ◽  
Ahmad Yar Qamar ◽  
Bereket Molla Tanga ◽  
Xun Fang ◽  
Ghangyong Kim ◽  
...  
Keyword(s):  
Formation Rate ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Nuclear Reprogramming ◽  
Blastocyst Development ◽  
Expression Levels ◽  
Oocyte Competence ◽  
Cresyl Blue ◽  
Nuclear Maturation

The objective of this study was to investigate the effect of milrinone supplementation as a phosphodiesterase 3A inhibitor during in vitro maturation (IVM) to coordinate the cytoplasmic and nuclear maturation of porcine oocytes and subsequent development of porcine cloned embryos. Brilliant cresyl blue (BCB)-stained (BCB +) oocytes, classified as well-developed, and BCB− oocytes were used in parthenogenesis (PA) and cloning, and their preimplantation development was compared. In PA embryos, BCB + oocytes had significantly higher rates of development than BCB− oocytes in terms of maturation (87.5 vs. 71.3%), cleavage (88.6 vs. 76.3%), and blastocyst development (34.3 vs. 25.3%) and also had higher cell numbers (46.9 vs. 38.9%), respectively (p < 0.05). In cloned embryos, the BCB + group also had a significantly higher blastocyst formation rate than the BCB− group (30.6 vs. 20.1%; p < 0.05). Supplementation with 75 μM milrinone during IVM of BCB− oocytes showed improvement in maturation and blastocyst development rates, which may be due to the coordinated maturation of the cytoplasm with the nucleus as an effect of milrinone. Moreover, the analysis of nuclear reprogramming via the examination of the expression levels of the reprogramming-related genes POU5F1, DPPA2, and NDP52IL in milrinone-supplemented BCB− oocytes showed higher expression levels than that in non-treated BCB− oocytes. These findings demonstrate that milrinone is useful in improving developmental competence in less competent oocytes during IVM and for proper nuclear reprogramming in the production of porcine cloned embryos by coordinating cytoplasmic and nucleus maturation.

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