17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

2019 ◽  
Vol 31 (1) ◽  
pp. 134
Author(s):  
D. Veraguas ◽  
C. Aguilera ◽  
D. Echeverry ◽  
D. Saez-Ruiz ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Relative Expression ◽  
Standard Curve ◽  
Morula Stage ◽  
Leopardus Guigna

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P<0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P<0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P<0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P>0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P>0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P>0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1061 ◽  
Author(s):  
RD Schramm ◽  
BD Bavister
Keyword(s):  
Growth Hormone ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Morula Stage

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 24 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Shuang Liang ◽  
Zheng-Wen Nie ◽  
Jing Guo ◽  
Ying-Jie Niu ◽  
Kyung-Tae Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Dna Methyltransferases ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

212 INTERSPECIFIC CLONING AND EMBRYO AGGREGATION INFLUENCE THE EXPRESSION OF oct4, nanog, sox2, AND cdx2 IN CHEETAH AND DOMESTIC CAT BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 196
Author(s):  
L. N. Moro ◽  
D. Veraguas ◽  
L. Rodriguez-Alvarez ◽  
M. I. Hiriart ◽  
C. Buemo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Internal Control ◽  
Gene Expression Analysis ◽  
Early Embryo ◽  
Domestic Cat ◽  
Nuclear Reprogramming ◽  
Acinonyx Jubatus ◽  
Standard Curve

The cheetah (Ch, Acinonyx jubatus) is a species considered globally endangered and cloning is one of the assisted reproductive techniques that can help to preserve it and to study early embryo development. However, the production of cloned felid embryos remains inefficient, probably because of the difficulty to control the process of nuclear reprogramming and obtain adequate gene expression. Embryo aggregation has been demonstrated to improve the cloning efficiency in several species and to normalise cdx2 in the mouse by lowering its expression (Balbach et al. 2010), but it has not been evaluated in felids before. To better understand the effect of interspecific somatic-cell nuclear transfer (iSCNT) and embryo aggregation in nuclear reprogramming, we analysed the expression of oct4, sox2, nanog, and cdx2 in cheetah blastocysts generated by iSCNT, domestic cat blastocysts (Dc) generated by SCNT, and IVF blastocysts as control. To achieve this, domestic cat oocytes were in vitro matured and zona-free SCNT or iSCNT was performed, as previously described (Moro et al. 2014, Reprod. Fertil. Dev.). Zona-free reconstructed embryos were then cultured individually (1X) or two embryo were cultured together (2X) in microwells, in synthetic oviductal fluid (SOF) medium. The experimental groups were Dc1X, Dc2X, Ch1X, Ch2X, and IVF. After 8 days of in vitro culture the blastocysts obtained were stored in RNA-later at –20°C. For gene expression analysis, blastocysts were pooled as follows: Dc1X, 4 replicates of 3 blastocysts each; Dc2X, 4 replicates of 3 blastocysts each; Ch1X, 2 replicates of 2 blastocysts and 1 replicate of 1 blastocyst; Ch2X, 4 replicates of 3 blastocysts each; IVF 3 replicates of 3 blastocysts each. Embryos were treated with a Cells-to-cDNA TM II kit (Life Technologies, Carlsbad, CA, USA) lyses buffer and treated with DNase I (0.04 U μL–1) for genomic DNA digestion. Gene expression analysis was performed by real-time qPCR using the standard curve method. In all qPCRs, GAPDH was used as an internal control. The statistical analysis was performed using a non-parametric Kruskal–Wallis test (P < 0.05). We observed that Dc1X blastocysts overexpressed the 4 genes evaluated respect to the IVF control. However, the gene expression of the aggregated group (Dc2X) was lower for all the genes, achieving the same levels of nanog and sox2 as the IVF blastocysts. The expression of oct4 and cdx2 were also closer to the expression levels of the control in the Dc2X group than in the Dc1X group. With respect to interspecific embryos, the amount of oct4 and cdx2 was also significantly reduced in the Ch2X blastocysts respect to Ch1X blastocysts. Both cheetah groups showed significantly lower expression of oct4, cdx2, and nanog than the IVF control. In conclusion, transcription of pluripotent and early differentiation factors in cheetah embryos was not as efficient as in the domestic cat embryos, probably caused by interspecific transfer. Our study demonstrated for the first time that defects in gene expression of domestic cat embryos can be corrected by embryo aggregation, providing a simple strategy to improve felid cloning.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

25 THE DEVELOPMENTAL COMPETENCE AND PLURIPOTENT GENE EXPRESSION OF PORCINE SOMATIC CELL NUCLEUS TRANSFER EMBRYOS IMPROVED BY m-CARBOXYCINNAMIC ACID BISHYDROXAMIDE

2012 ◽  
Vol 24 (1) ◽  
pp. 124
Author(s):  
S.-H. Park ◽  
S.-Y. Kim ◽  
M.-R. Lee ◽  
H.-J. Eun ◽  
S.-K. Baik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Nucleus ◽  
Histone Deacetylase Inhibitor ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Deacetylase Inhibitor ◽  
Somatic Cell Nucleus ◽  
Nucleus Transfer ◽  
Somatic Cell Nucleus Transfer

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic state after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Miyamoto et al. 2010 JBC), use of m-carboxycinnamic acid bishydroxamide (CBHA), a histone deacetylase inhibitor, during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA on the in vitro developmental competence and pluripotent gene expression of porcine SCNT embryos. The porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h) and then were assessed to cleavage rates and development to the blastocyst stage. In addition, pluripotent gene expression of SCNT embryos was analysed by RT-PCR. All data were analysed by chi-square. Following 4 replicates (207 and 124 for NT and CBHA-treated NT embryos, respectively), no significant difference was observed between NT and CBHA-treated NT embryos for cleavage rate (83.9 vs 82.2%). However, the developmental competence to the blastocyst stage differed significantly between NT and CBHA-treated NT embryos (6.8 vs 18.6%; P < 0.05). In addition, pluripotent transcription factors including Oct4, Nanog and Sox2 were higher expressed in the cloned embryos treated with CBHA (P < 0.05). The results of the present study suggest that treatment with CBHA as a histone deacetylase inhibitor significantly increased the developmental competence as well as the pluripotent gene expression of porcine SCNT embryos. This work was partly supported by a grant from the NRF (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.

27 Quisinostat, a Potent Histone Deacetylase Inhibitor, Regulates the Expression of Pluripotency- and Reprogramming-Related Genes on Somatic Cell Nuclear Transferred Porcine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 153
Author(s):  
A. Taweechaipaisankul ◽  
J.-X. Jin ◽  
S. Lee ◽  
G. A. Kim ◽  
B. C. Lee
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Acetylation ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Formation Rate ◽  
Developmental Competence ◽  
Limited Information ◽  
Relative Expression

The low efficiency of somatic cell nuclear transfer (SCNT) has been attributed mostly to inefficient epigenetic reprogramming. Recently, various histone deacetylase inhibitors (HDACi) were used to improve developmental competence of SCNT embryos in several species. However, limited information is available on the effects of quisinostat (JNJ-26481585, JNJ), a second-generation HDACi with high cellular potency towards Class I and II histone deacetylases. Based on our previous study, among various concentrations, treatment with 100 nM JNJ could improve embryo development into blastocysts compared with the control (23.50 ± 1.30 v. 13.97 ± 1.37; P < 0.05). Thus, in the present study, treatment with 100 nM JNJ was used for further investigation into the relative expression of genes related to pluripotency and reprogramming in order to assess the quality of pre-implantation embryos cultured in media with JNJ using quantitative real-time PCR. Porcine fibroblasts isolated from kidney of adult pigs from passage 6 to 8 were used as nuclear donor cells for SCNT. After SCNT, embryos were cultured with or without 100 nM JNJ during the first 24 h of in vitro culture, and blastocysts from each experimental group were collected and kept at –80°C until analysis. Total RNAs were extracted, and transcribed into cDNA before amplification. Then, the relative expression of development-related (Oct4, Sox2, and Nanog), histone acetylation-related (HDAC1, HDAC2, and HDAC3) and DNA methylation-related (DNMT1, DNMT3a, and DNMT3b) genes between the control and 100 nM JNJ groups were compared. All experiments were repeated 4 times and results were analysed by independent t-test using SPSS 17.0K (SPSS Inc., Chicago, IL, USA). Treatment with 100 nM JNJ showed significant increases in the expression Oct4, Sox2, and Nanog compared with the control (P < 0.05). Moreover, there was significantly lower expression of HDAC1, HDAC2, HDAC3, DNMT1, DNMT3a, and DNMT3b in the 100 nM JNJ treatment than in the control (P < 0.05). These expression results moderately illustrated more active transcriptional factors, stable maintenance of embryonic pluripotency, and lesser activity of histone acetylation and DNA methylation enzymes, enhancing the blastocyst formation rate in the treatment group. In conclusion, we suggest that improvement of the in vitro developmental competence of porcine SCNT embryos might be related to positive regulations of JNJ on the expression levels of genes related to pluripotency and reprogramming. This study was supported by the NRF (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

Production of somatic cell nuclear transfer embryos using in vitro-grown and in vitro-matured oocytes in rabbits

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 494-500 ◽  
Author(s):  
Hironobu Sugimoto ◽  
Yuta Kida ◽  
Noriyoshi Oh ◽  
Kensaku Kitada ◽  
Kazuya Matsumoto ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase Inhibitor ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Deacetylase Inhibitor ◽  
Number Of Cells

SummaryWe examined growing oocytes collected from follicles remaining in superovulated rabbit ovaries, that were grown (in vitro growth, IVG) and matured (in vitro maturation, IVM) in vitro. We produced somatic cell nuclear transfer (SCNT) embryos using the mature oocytes and examined whether these embryos have the ability to develop to the blastocyst stage. In addition, we examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), on the developmental competence of SCNT embryos derived from IVG–IVM oocytes. After growth for 7 days and maturation for 14–16 h in vitro, the growing oocytes reached the metaphase II stage (51.4%). After SCNT, these reconstructed embryos reached the blastocyst stage (20%). Furthermore, the rate of development to the blastocyst stage and the number of cells in the blastocysts in SCNT embryos derived from IVG–IVM oocytes were significantly higher for TSA-treated embryos compared with TSA-untreated embryos (40.6 versus 21.4% and 353.1 ± 59.1 versus 202.5 ± 54.6, P < 0.05). These results indicate that rabbit SCNT embryos using IVG–IVM oocytes have the developmental competence to reach the blastocyst stage.

Comparative expression analysis of embryonic development-related genes at different stages of parthenogenetic and in vitro fertilized embryos in caprine

Zygote ◽  
2013 ◽  
Vol 23 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Renu Singh ◽  
Kuldeep Kumar ◽  
R. Ranjan ◽  
Manish Kumar ◽  
T. Yasotha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Competence ◽  
Cell Stage ◽  
Developmental Abnormalities ◽  
Aberrant Gene Expression ◽  
Comparative Expression Analysis ◽  
Morula Stage ◽  
Aberrant Gene ◽  
Parthenogenetic Embryos

SummaryAberrant gene expression occurs in parthenogenetic embryos due to abnormal epigenetic modifications in the genome that probably diminish viability and enhance developmental abnormalities in these embryos. In the present study, five developmentally important genes (HPRT1, Cx43, Sox2, Mest and IGF2R) were analysed at different stages in parthenotes (haploid and diploid) and compared with similar stages in in vitro fertilized (IVF) embryos. The results indicated that in haploid parthenotes expression of HPRT1 was upregulated (P < 0.05) only at the 2–4-cell stage whereas Cx43 expression was significantly (P < 0.05) downregulated in all stages as compared with the control. However, expression of this gene was upregulated (P < 0.05) in 2–4-cell and morula stages of diploid parthenotes. Expression of Sox2 was significantly (P < 0.05) downregulated in morula stage haploid parthenotes, whereas it was upregulated (P < 0.05) in 8–16-cell stage diploid embryos. The expression of Mest was upregulated (P < 0.05) at the 2–4-cell stage of both haploid and diploid parthenotes, whereas it was downregulated in 8–16-cell stage diploid embryos as compared with control. IGF2R expression was upregulated (P < 0.05) only in morula stage haploid and diploid parthenote as compared with control. These results indicate that parthenogenetic embryos showed aberrant gene expression of developmentally important genes such as HPRT1, Cx43, Sox2, Mest and IGF2R in comparison with IVF embryos, this finding may be one of the major reasons for the poor developmental competence of parthenogenetic embryos.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

17 TREATMENT WITH MGCD 0103 IMPROVES THE IN VITRO DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2016 ◽  
Vol 28 (2) ◽  
pp. 138
Author(s):  
H.-Y. Zhu ◽  
L. Jin ◽  
Q. Guo ◽  
Y.-C. Zhang ◽  
X.-C. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Development ◽  
Single Donor ◽  
Or Groups ◽  
Humidified Air ◽  
Vitro Development

We use MGCD 0103 to test whether the treatment with this novel histone deacetylase inhibitor improves the in vitro development of porcine somatic cell NT (SCNT) embryos. Matured eggs were cultured in medium supplemented with 0.05 M sucrose and 0.4 μg mL–1 demecolcine for 1 h. Treated eggs with a protruding membrane were transferred to medium supplemented with 5 μg mL–1 cytochalasin B and 0.4 μg mL–1 demecolcine. Protrusions were then removed by aspirating with a 15-μm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each egg and electrically fused using 2 direct pulses of 150 V mm–1 for 50 μs in 0.28 M mannitol. Fused eggs cultured for 1 h were activated by 2 direct pulses of 100 V mm–1 for 20 μs and incubated with 2 mM 6-DMAP for 4 h. Subsequently, the cloned embryos were cultured in medium for 7 days at 38.5°C in 5% CO2 humidified air. In Experiment 1, after activation and treatment with 6-DMAP for 4 h, the SCNT embryos were cultured in medium supplemented with 0, 0.2, 2, or 20 μM MGCD 0103 for 24 h and then transferred to medium without MGCD 0103. In Experiment 2, SCNT embryos were cultured in medium supplemented with 0.2 μM MGCD 0103 for 0, 6, 24, or 48 h and then transferred to medium without MGCD 0103. As shown in Table 1, development to the blastocyst stage increased in SCNT embryos treated with 0.2 μM MGCD 0103 compared with the control or groups treated with 2 or 20 μM MGCD 0103 (25.51 v. 10.74, 3.53, 3.20%, respectively; P < 0.05). As shown in Table 1, treatment for 6 h with 0.2 μM MGCD 0103 significantly improved the rate of blastocyst formation compared with the control or groups treated for 24 or 48 h (21.17 v. 10.48, 19.23, 10.20%, respectively; P < 0.05). Our results suggested that 0.2 μM MGCD 0103 treatment for 6 h can improve in vitro developmental competence of porcine SCNT embryos. Table 1.In vitro development of pig SCNT embryos with different concentrations of MGCD 0103 for 24 h, and with 0.2 μM MGCD 0103 for different durations

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