scholarly journals Nuclear–cytoplasmic incompatibility and inefficient development of pig–mouse cytoplasmic hybrid embryos

Reproduction ◽  
2011 ◽  
Vol 142 (2) ◽  
pp. 295-307 ◽  
Author(s):  
Dasari Amarnath ◽  
Inchul Choi ◽  
Adel R Moawad ◽  
Teruhiko Wakayama ◽  
Keith H S Campbell
Keyword(s):  
Nuclear Transfer ◽  
Cytoplasmic Incompatibility ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Meiotic Spindle ◽  
Copy Numbers ◽  
Mouse Zygotes ◽  
Mouse Zygote ◽  
Dna Copy Numbers ◽  
Cytoplasmic Hybrids

Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development. We hypothesized that nuclear–cytoplasmic incompatibility could be another reason for failure of embryonic development from iSCNT. We used pig–mouse cytoplasmic hybrids as a model to address nuclear–cytoplasmic incompatibility in iSCNT embryos. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/10 (small) and 1/5 (large) total volume of mouse zygote). The presence of pig cytoplasm significantly reduced the development of mouse zygotes to the blastocyst stage compared with control embryos at 120 h post-human chorionic gondotropin (41 vs 6 vs 94%, P<0.05; 1/10, 1/5, control respectively). While mitochondrial DNA copy numbers remained relatively unchanged, expression of several important genes namely Tfam, Polg, Polg2, Mfn2, Slc2a3 (Glut3), Slc2a1 (Glut1), Bcl2, Hspb1, Pou5f1 (Oct4), Nanog, Cdx2, Gata3, Tcfap2c, mt-Cox1 and mt-Cox2 was significantly reduced in cytoplasmic hybrids compared with control embryos. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of inter-species nuclear transfer embryos.

scholarly journals Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

2017 ◽  
Vol 7 (7) ◽  
pp. 2065-2080 ◽  
Author(s):  
Kanokwan Srirattana ◽  
Justin C St. John
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Trichostatin A ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Genetic Integrity ◽  
Rna Seq ◽  
Mtdna Copy Number ◽  
Donor Cells

Abstract The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.

81 COMPARISON OF DEVELOPMENTAL ABILITY AMONG CLONED EMBRYOS WITH VARIOUS INDIVIDUAL RECIPIENT CYTOPLASMS IN BOVINE

2006 ◽  
Vol 18 (2) ◽  
pp. 148
Author(s):  
J. G. Zhao ◽  
X. Y. Yang ◽  
H. F. Liu ◽  
H. Li ◽  
S. Z. Huang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Vero Cells ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Chi Square ◽  
Reconstructed Embryos ◽  
Maturation Rate ◽  
Chi Square Test

Faithful reprogramming ensures the proper activation of genes during embryonic development of the somatic cell nuclear transfer (NT) in bovine. It is unambiguous that all these remodeling factors are presented in the oocyte cytoplasm (Du et al. 2002 Mol. Reprod. Dev. 63, 183–191). It will be interesting to determine if the recipient cytoplasms derived from individuals have different development ability and reprogramming competence during NT. Oocytes recovered by Ovum pickup from five Holstein heifers at 14 months of age were used as recipient cytoplasms. Cultured granulosa cells of the same origin were used as donor cells. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were cultured in B2 medium (Laboratoire CCD, Paris, France) on a monolayer of Vero cells for 7 days. The oocyte number, development ability, and NT efficiency of recipient cytoplasm derived from each individual were compared (Table 1). Differences among individuals were verified using a chi-square test, SAS 6.12 version (SAS Institute, Cary, NC, USA). There were significant differences of survival after fusion and the rate of development to the blastocyst stage for embryos reconstructed with recipient cytoplasm from five different individual heifers (P < 0.05). However, maturation rate, fusion rate and cleavage rate of embryos reconstructed with recipient cytoplasm from five different individual heifers presented no significant differences (P > 0.05). Reconstructed embryos with recipient cytoplasm from one heifer (03025) showed a lower survival after fusion (61% vs. 80%, 86%, 77%, 91%) but a higher ability to develop to blastocyst stage (61% vs. 24%, 31%, 52%, 31%) than the embryos from the other four heifers. The current study showed that recipient cytoplasm from various individuals may present great differences in developmental ability in nuclear transfer. This may result from different compatibility between nucleus and mitochondria or the content of maternal RNA as well as proteins in the oocyte. Further studies are needed to elucidate the genetic factors that affect the reprogramming in nuclear transfer. Table 1. Nuclear transfer efficiency with various individual recipient cytoplasms

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

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.

65 SCRIPTAID CORRECTS GENE EXPRESSION OF A FEW ABERRANTLY REPROGRAMMED TRANSCRIPTS IN NUCLEAR TRANSFER PIG BLASTOCYST STAGE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 138
Author(s):  
K. M. Whitworth ◽  
J. Zhao ◽  
L. D. Spate ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Histone Deacetylase ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Hdac Activity

Scriptaid is a histone deacetylase inhibitor (HDACi) that can increase cloning efficiency. The objective of this study was to identify aberrantly reprogrammed transcripts by performing transcriptional profiling between in vivo (IVV), nuclear transfer (NT) blastocyst stage embryos and the donor cell line (cells). This was followed by measuring HDAC activity (Epigentek) in zygotes and by real-time PCR on a selected subset of genes at the blastocyst stage to determine if Scriptaid treatment (NTS) corrected the aberrant gene expression. NTS embryos were treated with 500 nM Scriptaid for 14 h after activation. NT and NTS embryos were transferred into gilts on Day 0 or 1 of oestrus and collected 6 days later by uterine flush. IVV samples were collected on Day 8 of gestation. 3 pools of 10 to 15 embryos and cells were collected for each treatment and analysed twice. For transcriptional profiling, total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA), amplified by using an Ovation Ribo-SPIA linear amplification kit (Nugen), labelled with Cy5 and compared to reference labelled with Cy3. Lowess normalization and analysis was performed in Genespring 7.3.1. ANOVA was performed with the Benjamini and Hochberg False Discovery Rate. Transcripts that were different between IVV and NT (P ≤ 0.20) and significantly different from the donor cell line (P ≤ 0.05) were classified as being aberrantly reprogrammed. This comparison resulted in 119 under- and 60 over-compensated transcripts. Functional annotation classification was performed in DAVID and identified under-compensated pathways (oxidative phosphorylation and protein biosynthesis) and over-compensated pathways (chromatin packaging/remodelling and protein complex assembly). Fourteen transcripts were chosen for real-time PCR validation and evaluation of the effect of Scriptaid. Relative gene expression was compared between IVV, NT, NTS, and cells by the comparative Ct method with SYBR Green Supermix (Bio-Rad) and statistical analysis was performed in SAS 9.1 (SAS Institute Inc., Cary, NC, USA) by using a least significant difference test (P ≤ 0.05). NTS embryos had 3 transcripts returning to the same level as IVV (H3F3A, CAPG, and SEPT7). The level of the majority of the transcripts (8/14) was not affected by NTS treatment, e.g. histone deacetylase SIRT1 and H1 histone, member 0 (H1F0). However, Scriptaid treatment caused COX5A to be further over compensated in NTS with expression levels higher than IVV and NT. 2 transcripts had expression levels that were lower in NTS compared to both IVV and NT including GPD1L and EIF3E. Scriptaid treatment significantly affected gene expression in 6 of the 14 transcripts evaluated. Scriptaid treatment of the reconstructed zygotes did not affect the majority of the transcripts when measured at the blastocyst stage. HDAC activity was significantly reduced in NTS compared to NT 1-cell stage embryos (P ≤ 0.038). While Scriptaid reduced HDAC activity, it returned only a few genes to normal IVV levels. This project was supported in part by the USDA NRI (2006-35203-17282) and Food for the 21st Century.

scholarly journals Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 245-256 ◽  
Author(s):  
Hung-Fu Liao ◽  
Chu-Fan Mo ◽  
Shinn-Chih Wu ◽  
Dai-Han Cheng ◽  
Chih-Yun Yu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Trichostatin A ◽  
Cell Mass ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Oct4 Expression ◽  
Donor Cells

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.

19 CHANGES IN PLASMA STEROID CONCENTRATIONS DURING GESTATION IN COWS WITH SPONTANEOUS ABORTION OF SOMATIC CELL CLONED FETUSES

2012 ◽  
Vol 24 (1) ◽  
pp. 121
Author(s):  
M. Hirako ◽  
H. Takahashi ◽  
K. Kimura ◽  
N. Adachi ◽  
S. Akagi
Keyword(s):  
Sex Steroids ◽  
Repeated Measures ◽  
Nuclear Transfer ◽  
Plasma Concentrations ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Steroid Synthesis ◽  
Estrone Sulfate

Cloning of mammals by nuclear transfer frequently results in gestational failure with a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Monitoring the placental function during gestation is important to clarify the cause of abnormalities in cloned animals. Sex steroids are produced in the bovine placenta and their levels in maternal peripheral blood are a useful measure of placentation. The objective of this study was to investigate changes in plasma concentrations of sex steroids during gestation in cows aborting cloned fetuses. Donor cells for nuclear transfer were obtained from subculture of cumulus cells retrieved from ovarian follicles of a Japanese Black cow. Recipient oocytes were derived from ovaries obtained at an abattoir and matured in vitro. Metaphase II oocytes were enucleated and each fused with a donor cell by DC pulses. Nuclear-transferred oocytes were activated and cultured for 7 days. Embryos developed to the blastocyst stage were each transferred into the uterine horn ipsilateral to the ovary bearing the CL of 39 multiparous Japanese Black and Holstein crossbred cows at 7 to 8 days after the day of standing oestrus (day 0). Fourteen recipient cows were diagnosed pregnant on Day 40 by ultrasonography and 7 cows delivered at full term. The other seven miscarried on Day 66, 81, 85, 89, 97, 104 and 211. Blood was collected from these cows at least once a week following the pregnancy diagnosis. Progesterone, estrone, oestradiol-17β and estrone sulfate in the blood plasma were measured by RIA and were compared with those in pregnant AI cows. Statistical differences at stages of gestation were analysed with repeated-measures ANOVA. In all miscarried cows, progesterone concentrations were similar to those in AI cows until several days before abortion and then rapidly decreased to the basal level. Concentrations of all estrogens stayed low until abortion in six cows aborting by day 104, whereas estrone and oestradiol-17β started to increase around Day 80 and estrone sulfate gradually increased from around Day 50 and started to increase drastically around Day 80 in AI cows. In another cow aborting on Day 211, profiles of estrone and oestradiol-17β were similar to those in AI cows until around Day 150. Thereafter, concentrations of these estrogens gradually decreased to the basal levels by Day 160 and stayed low until abortion. In this cow, gradual increase in estrone sulfate during Day 50 to 80 was not observed, but the difference in the concentration was not statistically significant from AI cows. The following profile of estrone sulfate was similar to those in active estrogens. The fetus was still alive on day 160 and fetal death was confirmed on day 180 by ultrasonography. These results suggest the possibility that developmental or functional failure of placenta associated with steroid synthesis may be a cause of mid-term miscarriage of a cloned fetus.

65 NUCLEAR REPROGRAMMING OF PORCINE CELLS AND THEIR USE AS DONOR CELL FOR NUCLEAR TRANSFER AFTER TREATMENT IN XENOPUS EGG EXTRACTS

2007 ◽  
Vol 19 (1) ◽  
pp. 150 ◽  
Author(s):  
K. Miyamoto ◽  
M. Ohnuki ◽  
N. Minami ◽  
M. Yamada ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Nuclear Reprogramming ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Revealing an adequate cell state for nuclear reprogramming is essential to achieve efficient production of cloned embryos and animals. Previous reports suggest that nuclei from undifferentiated cells such as blastomeres or embryonic stem cells can support efficient development of cloned embryos to term. In recent years, differentiated somatic cells are frequently used for donor cells because of ease of preparation and application for genetic modification. The efficiency of the somatic cell nuclear transfer (SCNT) is still extremely low. We hypothesized that somatic cells that had been reprogrammed to dedifferentiated states before SCNT might support higher developmental ability of SCNT embryos. To test this hypothesis, porcine fibroblast cells were treated with Xenopus egg extracts, and the extract-treated cells (ETCs) were used as donor cell for SCNT to examine their ability to support early embryonic development. Xenopus egg extracts were prepared from activated S-phase eggs. Porcine fibroblast cells (106/mL) were permeabilized by 500 ng mL-1 of Streptolysin O and were incubated in the egg extracts with the energy-regenerating system for 2 hours at 23�C. After the extract treatment, permeabilized membranes were resealed in DMEM containing 2 mM CaCl2. The ETCs were fused with porcine enucleated oocytes and simultaneously activated. The reconstructed embryos were cultured in PZM-3 medium for 7 days. All statistical differences were analyzed by ANOVA. Reprogramming of ETCs was evaluated on changes of chromatin states and gene expression. Chromatin-binding proteins of ETCs were separated and analyzed on SDS-PAGE. Some proteins were incorporated onto and/or released from chromatins after the extract treatment. Especially, Xenopus egg-specific linker histone B4 was assembled on chromatins. Non-permeabilized control cells did not show these protein exchanges. Deacetylation of histone H3 lysine9 was detected in half number of ETCs in an ATP-dependent manner. In contrast, a high population of histone H3-acetylated cells was observed in buffer-treated cells as well as cells before the extract treatment. The pluripotent marker gene expression, such as OCT4 and SOX2, was also observed in ETCs after culture. The gene expression of these genes was not detected in non-treated cells. These results indicate that the extract treatment induces or triggers a part of dedifferentiation of somatic cells. These ETCs were used as donor cell for SCNT, and reconstructed cloned embryos were cultured. SCNT embryos showed no significant difference in cleavage rates and developmental rates to the blastocyst stage (25%) compared with non-treated control cells (26%). However, the total cell number of embryos at the blastocyst stage was significantly higher in SCNT embryos from ETCs compared with those of control cells (62 � 7 vs. 43 � 2, respectively; P &lt; 0.05). These results indicate that the extract treatment before nuclear transfer may stimulate cell proliferation of SCNT embryos but not improve early development. More studies, however, are needed to investigate their developmental ability to term.

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 134
Author(s):  
P. Q. Cong ◽  
E. S. Song ◽  
E. S. Kim ◽  
Z. H. Li ◽  
Y. J. Yi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pulse Number ◽  
Donor Cells ◽  
First Polar Body ◽  
Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

scholarly journals 55IN VITRO DEVELOPMENT OF ENUCLEATED DOMESTIC CAT OOCYTES RECONSTRUCTED WITH SKIN FIBROBLASTS OF DOMESTIC AND LEOPARD CATS

2004 ◽  
Vol 16 (2) ◽  
pp. 149 ◽  
Author(s):  
C. Lorthongpanich ◽  
C. Laowtammathron ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
M. Ketudat-Cairns ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Skin Fibroblasts ◽  
Domestic Cat ◽  
Developmental Potential ◽  
A Cell ◽  
Vitro Development

The domestic cat is a valuable model for studies in assisted reproductive technology in felid species. Therefore, in this experiment we evaluated the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with somatic cells from domestic and leopard cats. Skin fibroblasts were isolated from female domestic and leopard cats. The oocytes were collected by aspiration of follicles from ovaries that were superovulated with 200IU PMSG. In vitro-matured oocytes were enucleated and individual donor cells (diameter 14–16μm) were inserted into the perivitelline space of the enucleated oocyte. Fusion was performed at 26–27h post-maturation by placing a cell-oocyte couplet between both tips of the needle electrode and electrostimulating with a 2-DC pulse (30V, 30μs) in fusion medium containing 0.3M Mannitol+0.1mM MgCl2. Activation was performed 1 to 2h post-fusion by incubation in 7% ethanol at room temperature for 5min followed by cultured in 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D at 38°C in 5% O2, 5% CO2, 90% N2 conditions. After activation, the reconstructed embryos were cultured in 100-μL droplets of Tyrode’s medium (Gomez et al., 2003 Theriogenology 60, 239–251.) supplemented with 0.3% BSA at 38°C in a 5% O2, 5% CO2, 90% N2 environment for 2d. Then, 8-cell embryos were cultured in 100-μL droplets of Tyrode’s medium supplemented with 10% FCS at 38°C in a 5% O2, 5% CO2, 90% N2environment for 5d. The cleavage rates of oocytes reconstructed with either donor cell types were not different. The percentages of blastocyst formation from parthenogenotes and nuclear transfer embryos derived from domestic cat fibroblasts (8/56, 14.3% and 7/51, 13.7%, respectively) were significantly higher than that for nuclear transfer embryos constructed with leopard cat fibroblasts (3/45, 6.7%). These results indicate that enucleated domestic cat oocytes reconstructed with skin fibroblasts of leopard cats can develop to the blastocyst stage. This experiment was supported by Suranaree University of Technology. Table 1 In vitro development of domestic cat oocytes reconstructed with domestic and leopard skin fibroblasts and parthenogenetic activation

scholarly journals Complete Replacement of the Mitochondrial Genotype in a Bos indicus Calf Reconstructed by Nuclear Transfer to a Bos taurus Oocyte

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 351-356 ◽  
Author(s):  
Flávio V Meirelles ◽  
Vilceu Bordignon ◽  
Yeda Watanabe ◽  
Michelle Watanabe ◽  
André Dayan ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Donor Cell ◽  
Female Offspring ◽  
Blastocyst Stage ◽  
Complete Replacement ◽  
Animal Cloning ◽  
Mitochondrial Genotype ◽  
Viable Approach

Abstract Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.

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