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

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

76 PRODUCTION OF CLONED BOVINE TRANSGENIC EMBRYOS WITH VARIOUS TYPES OF MONO-COLONY CELLS AND OVUM PICKUP

2005 ◽  
Vol 17 (2) ◽  
pp. 188
Author(s):  
J.G. Zhao ◽  
X.Y. Yang ◽  
Y. Huang ◽  
H.F. Liu ◽  
H. Li ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Nuclear Transfer ◽  
Genetic Manipulation ◽  
Blastocyst Stage ◽  
High Tech ◽  
Cleavage Rate ◽  
Exogenous Dna ◽  
Developmental Potential ◽  
Reconstructed Embryos

The objective of this study was to determine the effects of genetic manipulation, cell type, and culture conditions on developmental potential of bovine nuclear transfer (NT) embryos. Ovum pickup (OPU) technology was developed to obtain the oocytes for NT. A total 4044 cumulus-oocyte complexes (COCs) were obtained during 492 OPU sessions, with an average of 8.2 COCs recovered each session. Cultured granulosa cells (CGC), bovine fetal (150 days) oviduct epidermic cells (FOEC), and adult ear skin fibroblasts (ASFC) were used as donor cells for NT and were transfected with the expression vector including human FIX coding sequence directed by goat β-casein promoter and neomycin gene. The cells were screened under 800 μg mL−1 G418 for 10–14 days until the apperance of a “mono-colony” of cells which were then picked. Each cell population was expanded by consecutive passage culture under 300 μg mL−1 G418 until used for NT, ensuring that the majority of cells were transgenic. 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 co-cultured with vero cells in B2 medium for 7 days. NT efficiency between primary granulosa cells (PGC) without in vitro culture and CGC, as well as among CGC, FOEC and ASFC that were transfected with exogenous DNA (named TCGC, TFOEC, TASFC, respectively), were compared (Table 1). Differences between groups were verified by chi-square test using SAS 6.12 (SAS Institute, Inc., Cary, NC, USA) program. CGCs presented a higher fusion rate (P < 0.01) for reconstructed embryos and higher development to the blastocyst stage for NT embryos than did PGC (67% vs. 54% and 41% vs. 21%, respectively). There were no significant differences (P > 0.05) in cleavage rate (65%, 71%, and 69%, respectively) and development to the blastocyst stage for NT embryos (36%, 30% and 40%, respectively) for TCGC, TFOEC, and TASFC. A total of 86 blastocysts were selected for transfer into uteri of 86 cows, resulting in 26 pregnancies (30%) at 60 days by ultrasound scanning. Among these, 12 cows remain pregnant and 14 have aborted. The results indicated that oocytes recovered from OPU can be successfully used for NT with development to the blasocyst stage. PGC, CGC, FOEC, and ASFC can all be used for generating transgenic cattle by NT, although this needs to be verified by the birth of live calves. Table 1. Nuclear transfer efficiency with various cell types This work was supported by the Chinese “863” High-Tech Plan Program (Grant No. 2002AA206201).

54 ESTABLISHMENT OF PREGNANCY BY OVINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED USING CAFFEINE-TREATED IN VITRO-MATURED OOCYTES AS CYTOPLAST RECIPIENTS

2006 ◽  
Vol 18 (2) ◽  
pp. 135 ◽  
Author(s):  
J.-H. Lee ◽  
M. Marfil ◽  
M. Panarace ◽  
M. Medina ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Animal Health ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Interferon Tau ◽  
Chi Square ◽  
Developmental Potential ◽  
Donor Nucleus ◽  
Chi Square Test

In embryos reconstructed by somatic cell nuclear transfer (SCNT), components of the oocyte cytoplasm are capable of reprogramming the somatic genome to control subsequent development. Although the mechanisms that control nuclear reprogramming are unknown, we have previously hypothesized that the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the donor nucleus are beneficial. In previous studies we have demonstrated that treatment of ovine oocytes with caffeine (10 mM), a protein phosphatase inhibitor, increased the activities of both MPF and MAPK in enucleated oocytes (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) and additionally resulted in a significant increase in the occurrence of NEBD and PCC in donor nuclei. Furthermore, SCNT embryos reconstructed following caffeine treatment had significantly increased cell numbers at the blastocyst stage. More recently we have demonstrated that the use of caffeine treated ovine oocytes as cytoplast recipients can regulate the expression of several developmentally important genes in SCNT embryos, including Oct-4 and interferon-tau (Choi et al. 2006 Reprod. Fertil. Dev. 18, in press). This study was designed to establish the developmental potential of NT embryos reconstructed using caffeine treated oocytes as cytoplast recipients. Ear skin fibroblast cells established from a Merino ram were quiesced in DMEM containing 0.1% fetal bovine serum (FBS) for 3 days. Oocyte maturation and embryo reconstruction and culture were performed as previously described (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) with the exception that ovaries from Merino � Romney Marsh cross ewes were stimulated with FSH sponge (Folltropin�-V; Bioniche Animal Health, Beltsville, Ontario, Canada) and were collected at slaughter on Day 13 following sponging. Blastocyst stage embryos were surgically transferred to the uterine horn of synchronized Merino � Romney Marsh cross recipients (three blastocysts per recipient). Recipient ewes were scanned by ultrasonography at Days 30, 60, and 90 following embryo transfer. All data were analyzed by chi-square test. There were no differences in fusion (145/167; 86.8% vs. 174/205; 84.9%), cleavage (123/145; 84.8% vs. 135/174; 77.6%), or the development to blastocyst (33/145; 22.8% vs. 34/174; 19.5%) between control SCNT embryos and caffeine treated SCNT embryos. However, although the frequency of pregnancy between control and caffeine-treated NT groups (5/15; 33.3% vs. 7/14; 50.0%) at 30 days was not significantly different, control SCNT embryos showed significantly lower pregnancies (1/15; 6.7%) than caffeine treated SCNT embryos (4/14; 28.6%) at both 60 and 90 days. In conclusion, embryos reconstructed using caffeine-treated cytoplasts can induce pregnancy at the same frequency as untreated controls; furthermore, the results suggest that SCNT embryos produced in this way are more able to maintain pregnancy.

55 PRELIMINARY STUDIES ON THE DEVELOPMENT OF RECONSTRUCTED EMBRYOS AFTER NUCLEAR TRANSFER IN DROMEDARY CAMEL (CAMELUS DROMEDARIUS)

2009 ◽  
Vol 21 (1) ◽  
pp. 128 ◽  
Author(s):  
N. A. Wani ◽  
J. A. Skidmore ◽  
U. Wernery
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Light Exposure ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Reconstructed Embryos

Experiments were conducted to study the in vitro development of reconstructed dromedary camel embryos after nuclear transfer by a modified zona-free method. Cumulus oocyte complexes, collected from slaughterhouse ovaries were cultured in TCM199 at 38.5°C in an atmosphere of 5% CO2 in air for 32 to 36 h. Matured oocytes were denuded of cumulus cells by repeated pipetting and the zona pellucida was removed by brief incubation in 5 mg mL–1 pronase dissolved in Ca- and Mg-free PBS. Zona-free oocytes were stained with 5 mg mL–1 Hoechst 33342 in H199 supplemented with 7.5 μg mL–1 cytochalasin B and 10% FCS. They were enucleated under constant UV-light exposure in H199 supplemented with cytochalasin B and 10% FCS. The granulosa cells at passage numbers 4 to 15 were used as nuclear donors. The zona-free cytoplasts were individually washed for a few seconds in 300 μg mL–1 of Phytohemagglutinin in H199, then quickly dropped on a single donor cell settled to the bottom of a drop of H199 with 0.5% FCS and pushed together with the mouth pipette. Couplets were electrically fused, at room temperature, with two DC pulses of 100 V cm–1 for 15 μs. Reconstructs were activated 2 h post-fusion, with 5 μm ionomycin for 3 min followed by culture in 6-diethylaminopurine for 4 h. The reconstructs were then cultured individually in either 5 μL drops under oil, in agar wells or in wells of wells (WOW) in a well of 4-well culture plate. Embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 μg mL–1 gentamycine, 1% insulin-transferrin-selenium (ITS), and 15% estrous dromedary serum. The number of oocytes that had cleaved was recorded on day 2, whilst those developing to morulae and blastocysts were recorded on day 7 of culture. For cell count, the blastocysts were stained with Hoechst and cells counted under a fluorescent microscope at ×400. Data obtained was analysed by chi-square test. About 92% (349/380) of the oocytes were successfully enucleated and 76% (259/340) fused with the attached cells. The cleavage rate was significantly lower (P < 0.05) in reconstructed embryos cultured in droplets (10/72, 14%) as compared with those cultured in agar wells (37/87, 42%) or WOW system (42/96, 44%). The proportions of cleaved embryos reaching morula stage were 0, 83, and 89% in droplets, agar wells, and WOW, respectively. However, only 8% and 5% of the cleaved embryos developed to the blastocyst stage in the agar well and WOW culture systems, respectively. No difference was observed in the cell number of blastocysts produced in agar wells (77.3 ± 8.02) or WOW (78.0 ± 4.2) culture system. To the best of our knowledge, this is the first report of embryo production up to the blastocyst stage after NT in camelids and it shows that NT can be successfully applied for embryo production in camelids. Further studies are needed to optimize the parameters and to improve the efficiency for production of transferable blastocysts in this species. This study was kindly sponsored by H.H. General Sheikh Mohammed bin Rashid Al Maktoum, Ruler of Dubai.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals 319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

2004 ◽  
Vol 16 (2) ◽  
pp. 279
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
F. Prati ◽  
G. Mari
Keyword(s):  
Polar Body ◽  
Basal Medium ◽  
Defined Medium ◽  
Chi Square ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Chi Square Test ◽  
The Media ◽  
Cytoplasmic Maturation

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P&lt;0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P&lt;0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media

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.

70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING

2006 ◽  
Vol 18 (2) ◽  
pp. 143
Author(s):  
D. Salamone ◽  
M. Catala ◽  
A. Gibbons ◽  
F. Pereyra Bonnet ◽  
M. Cueto
Keyword(s):  
Granulosa Cells ◽  
Room Temperature ◽  
Uv Light ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
First Passage ◽  
Chi Square ◽  
Donor Nucleus ◽  
Chi Square Test

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts. Table 1. Effect of non-plated granulosa and cumulus cells and first passage fibroblasts as donor nucleus oocytes in goat cloning

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

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

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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