Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues

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.

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Comparative analysis of different nuclear transfer techniques to prevent the transmission of mitochondrial DNA variants

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz062 ◽

2019 ◽

Author(s):

M Tang ◽

R R Guggilla ◽

Y Gansemans ◽

M Van der Jeught ◽

A Boel ◽

...

Keyword(s):

Mitochondrial Dna ◽

Disease Transmission ◽

Nuclear Transfer ◽

Smooth Endoplasmic Reticulum ◽

Scientific Evidence ◽

Polar Body ◽

Comparable Rate ◽

Carry Over

Abstract Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates amongst PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregates (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.

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199 EVIDENCE FOR A NOVEL PERTURBATION IN CLONED FETUSES: MITOCHONDRIAL DNA DEPLETION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab199 ◽

2007 ◽

Vol 19 (1) ◽

pp. 216

Author(s):

S. Hiendleder ◽

D. Bebbere ◽

S. E. Ulbrich ◽

V. Zakhartchenko ◽

M. Weppert ◽

...

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Nuclear Gene ◽

Complement C3 ◽

Mtdna Copy Number ◽

Vitro Fertilization ◽

Mitochondrial Transcription Factor ◽

Mitochondrial Dna Depletion

The reported mtDNA turnover and plasticity of mtDNA copy number in mammalian zygotes and early embryos (McConnel and Petrie 2004 Reprod. Biomed. Online 9, 418–424) have revealed a potential for adverse effects of in vitro embryo techniques on mtDNA and mitochondrial function. We explored the effects of in vitro fertilization (IVF) and somatic cell nuclear transfer cloning (NT) on relative mtDNA amount and phenotype in viable bovine fetuses recovered 80 days after the initiation of embryonic development (Hiendleder et al. 2004 Biol. Reprod. 71, 217–223). We sampled brain, liver, and skeletal muscle to represent all 3 embryonic germ layers, and compared IVF-fetuses (n = 24), NT-fetuses (n = 23), and fetuses generated by in vivo insemination (controls, n = 24). This experimental approach allowed us to distinguish abnormalities specific to cloning from more general consequences of in vitro embryo manipulation. We analyzed relative mtDNA amounts by real-time quantitative PCR (qPCR) and amplified a segment of the mtDNA control region that was normalized against the nuclear gene complement C3. ANOVA (SPSS 13.0) of qPCR data and phenotypic parameters revealed significant effects of fetus group on mtDNA amount in liver (P < 0.05) and muscle (P < 0.01), and on fetus (P < 0.001), heart (P < 0.001), and liver (P < 0.001) weights. The mtDNA amount in all tissues from IVF-fetuses was normal, but mtDNA levels in liver (-23%; P < 0.05) and muscle (-24%; P < 0.01) of NT-fetuses were significantly lower than in controls. Fetuses derived from IVF- or NT-embryos were similar in weight and displayed fetal overgrowth (+19% and +22%; P < 0.001), but only the NT-fetuses were affected by disproportionate hepatomegaly and cardiomegaly with 31% and 49% increases (ANCOVA; P < 0.001) in their respective organ weights. This further partitioned NT-fetuses from IVF-fetuses and identified symptoms that are also encountered in mitochondrial DNA depletion syndromes (MDDS): a phenotypically heterogeneous group of human disorders characterized by loss of mtDNA from various tissues during development and associated respiratory chain dysfunction. The MDDS phenotypes have mainly been classified into a hepatocerebral (MIM 251880) or myopathic (MIM 609560) form, and neonates and infants display a spectrum of abnormalities, including hepatomegaly and cardiomegaly, that are similar or identical to phenotypic abnormalities commonly encountered in cloned mammals. Reduced mtDNA amounts in NT-fetuses could stem from perturbation of mtDNA during the reported turnover period, or be a secondary effect of epigenetic change in nuclear-encoded genes involved in mtDNA replication and stability. Mitochondrial transcription factor A (TFAM) is regulated by CpG methylation in vitro, but our real-time RT-PCR quantification of TFAM transcript in liver and muscle of a subset of NT- and control fetuses failed to detect significant differences (P > 0.10). In conclusion, our observed reduction of mtDNA amount in cloned fetuses provides the molecular basis for a mitochondrial perspective on pathological phenotypes of cloned mammals, and may explain similarities to mitochondrial disease in human.

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44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab44 ◽

2011 ◽

Vol 23 (1) ◽

pp. 128

Author(s):

J. Lee ◽

J. Park ◽

Y. Chun ◽

W. Lee ◽

K. Song

Keyword(s):

Nuclear Transfer ◽

Polar Body ◽

Donor Cell ◽

Skin Fibroblasts ◽

Developmental Competence ◽

Cleavage Rate ◽

Cell Stage ◽

Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

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

10.32469/10355/66732 ◽

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.

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5 TRANSCRIPTIONAL PROFILING OF PIG IN IN VIVO, IN VITRO-FERTILIZED, AND NUCLEAR TRANSFER-DERIVED BLASTOCYSTS AND THE DONOR SOMATIC CELL LINE

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab5 ◽

2008 ◽

Vol 20 (1) ◽

pp. 83

Author(s):

K. M. Whitworth ◽

L. D. Spate ◽

R. Li ◽

A. Rieke ◽

D. M. Wax ◽

...

Keyword(s):

Cell Line ◽

Nuclear Transfer ◽

Transcriptional Profiling ◽

Reference Sample ◽

Donor Cell ◽

Blastocyst Stage ◽

Electrical Activation ◽

Shock Proteins

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix� 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

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37 Healthy Foals Produced Using Bone Marrow-Mesenchymal Stem Cells as Nuclear Donors in Horse Cloning

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab37 ◽

2018 ◽

Vol 30 (1) ◽

pp. 158

Author(s):

R. Olivera ◽

L. Moro ◽

R. Jordan ◽

C. Luzzani ◽

S. Miriuka ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Umbilical Cord ◽

Donor Cell ◽

Control Group ◽

Nuclear Reprogramming ◽

Marrow Mesenchymal Stem Cells

Somatic cell nuclear transfer efficiency is based on the capacity of the donor cell to be reset and reprogrammed to an embryonic state. So, the less differentiated the donor cells are, the more easily they could be reprogrammed by a recipient cytoplasm. Failures on appropriate nuclear reprogramming frequently lead to abnormalities associated with the placenta, umbilical cord, birthweight, and limbs. In the present study, we evaluated the efficiency of bone marrow mesenchymal stem cells (BM-MSC) compared with adult fibroblasts (AF) as nuclear donors in horse cloning and evaluated both in vitro and in vivo development of the embryos generated. Moreover, we focused on comparing the health of the foals generated and on the presence of anatomical abnormalities in foals produced from the different treatments. Embryos produced by AI, recovered by uterine flushing, and transferred to recipient mares were used as controls. All variables were analysed by Fisher test (P < 0.05). The cloning procedure was performed according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Both cleavage and blastocyst rates were higher when MSC were used as nuclear donors (P < 0.05). Cleavage rates were 85.6% (3875/4527) v. 90.2% (3095/3432) and blastocyst rates were 10.9% (492/4527) and 18.1% (622/3432) for AF and MSC groups, respectively. In the AF group, 476 blastocysts were transferred to recipient mares (232 transfers), and in the MSC group, 594 blastocysts were transferred 297 transfers). In the AI control group, 88 embryos were transferred. Pregnancies were diagnosed by transrectal ultrasonography 15 days after embryo transfer in all the groups. Pregnancy rates were similar between both cloning groups (41/232, 17.7% and 37/297, 12.5%for AF and MSC, respectively), but higher in the AI group (71/88, 80.7%). However, significant differences were observed in the birth of viable offsprings among the cloning groups. Despite similar rates of foal delivery (AF, 17/41, 41.5%; MSC, 21/37, 56.7%), a higher proportion of viable foals were obtained from the MSC group (20/37, 54.1%) compared with the AF group (9/41, 22%; P < 0.05). Surprisingly, as in the AI group (63/63, 100%), all of the viable foals obtained using MSC (20/20, 100%) were considered normal and did not show abnormalities associated with cloning. In contrast, in the AF group, only 4/9 (44.4%) were considered normal foals. The defects present in the other 5 foals were related to flexural and angular limb deformities and umbilical cord malformations. These were corrected rapidly with standard treatments or, in the case of the umbilical cords, minor surgery. This study shows for the first time that BM-MSC can be used as nuclear donors in horse cloning and that the foals obtained are as healthy as those produced by AI, showing no abnormalities related to deficiencies in nuclear reprogramming.

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Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽

10.1530/rep.0.1220801 ◽

2001 ◽

pp. 801-808 ◽

Cited By ~ 5

Author(s):

JL Liu ◽

MK Wang ◽

QY Sun ◽

XR Zhang ◽

LK Jiang ◽

...

Keyword(s):

Nuclear Transfer ◽

Cultured Cells ◽

Donor Cell ◽

Serum Starvation ◽

Fresh Tissue ◽

Reconstructed Embryos ◽

Donor Cells ◽

Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

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Chromatin Modifying Agents in theIn VitroProduction of Bovine Embryos

Veterinary Medicine International ◽

10.4061/2011/694817 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 4

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.

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No differences in sheep somatic cell nuclear transfer outcomes using serum-starved or actively growing donor granulosa cells

Reproduction Fertility and Development ◽

10.1071/rd02092 ◽

2003 ◽

Vol 15 (3) ◽

pp. 157 ◽

Cited By ~ 17

Author(s):

T. T. Peura ◽

K. M. Hartwich ◽

H. M. Hamilton ◽

S. K. Walker

Keyword(s):

Somatic Cell ◽

Granulosa Cells ◽

Nuclear Transfer ◽

Survival Rates ◽

Fetal Loss ◽

Donor Cell ◽

Perinatal Period ◽

Offspring Mortality

The aim of this study was to compare serum-starved and non-starved donor cells in sheep nuclear transfer with a special emphasis on cloning outcomes. Sheep oocytes, derived either in vivo or in vitro, were fused with cultured serum-starved or actively growing adult granulosa cells. Resulting blastocysts were transferred to recipients fresh or after vitrification, and subsequent pregnancies followed to term. Donor cell treatment did not significantly affect preimplantation development, pregnancy rates, fetal loss or neonate survival rates. Of 22 lambs born, ten survived the immediate perinatal period but all succumbed at various timepoints within the first few weeks of life. The results of the study suggest that the use of serum-starved cells offers no advantages or disadvantages to cloning outcomes. Neither were significant differences in outcomes observed when using either in vivo- or in vitro-derived oocytes or embryos transferred fresh or after vitrification. Yet, these results continue to highlight problems associated with somatic cell cloning as indicated by offspring mortality. It remains unclear whether the high offspring mortality in the current study was related to species, associated with the cell lines used or the result of other causes.

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66 IN VITRO DEVELOPMENT OF YAK (POEPHAGUS MUTUS) CLONED EMBRYOS BY INTERSPECIES SOMATIC NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab66 ◽

2005 ◽

Vol 17 (2) ◽

pp. 183

Author(s):

L. Su ◽

F.L. Du ◽

L.Y. Sung ◽

S. Yang ◽

B.S. Jeong ◽

...

Keyword(s):

Nuclear Transfer ◽

Bos Taurus ◽

Donor Cell ◽

Cumulus Cells ◽

Fusion Rate ◽

Cell Number ◽

Developmental Potential ◽

Somatic Nuclear Transfer ◽

Humidified Air

Interspecies nuclear transfer (NT) is an important tool for preservation of endangered animal species. This study was carried out to clone Yak (Poephagus mutus) embryos by using Yak skin fibroblasts and bovine (Bos taurus) recipient cytoplasts, and to compare the efficiency of YAK interspecies NT (bovine cytoplast-Yak donor cell) and bovine somatic NT (bovine cytoplast-bovine donor cell). Recipient oocytes were extracted from antral follicles of bovine ovaries, and subsequently cultured in maturation medium for 18–20 h in 5% CO2 and 95% humidified air at 39°C. Cumulus cells were removed from the oocytes by vortexing also facilitated further enucleation. Yak skin fibroblast cells were prepared from cultured ear explants of an adult 5-year-old female. Fibroblasts were cultured at passage 6–9 in 10% FBS DMEM at 37°C in 5% CO2 humidified air. The donor cell at a diameter of 19–20 μm was inserted into the perivitelline space of an enucleated oocyte. A bovine female cell line at similar passage number was used for bovine somatic NT as control. Somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0 kV/cm for a duration of 6–10 μs/pulse. Fused embryos were activated in 10 μg/mL cycloheximide and 2.5 μg/mL cytochalasin D in M199 plus 7.5% FBS for 5 h. Reconstructed Yak embryos were cultured in CR1aa plus 6 mg/mL BSA for 2 days (initiation of activation = Day 0) at 39°C, 5% CO2, 5% O2, and 90% N2, and then in 7.5% FBS CR1aa medium for 5 successive days on bovine cumulus monolayers. Expanding and hatching blastocysts on Day 7 were recorded and cryopreserved for further embryo transfer trials. The percentage of cleavage and the development to morulae and blastocysts were statistically analyzed using a General Linear Model (GLM, Univariate, SPSS 9.0, SPSS Inc, Chicago, IL, USA). As indicated in Table 1, the results demonstrated that the efficiencies of fusion rate as well as developmental potential in vitro were significantly higher in the bovine somatic NT group compared to those of the Yak interspecies NT group. However, the morphology and cell number per embryo of interspecies Yak cloned embryos were indistinguishable from those of bovine NT embryos. Our data suggest that bovine oocytes possess the capability of reprogramming/reactivation of the genome from differentiated somatic Yak nuclei. Table 1. Comparison of yak interspecies and bovine somatic nuclear transfer

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